helicobacter pylori and epstein-barr virus coinfection stimulates … · 2020. 11. 19. · 47...

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Title: Helicobacter pylori and Epstein-Barr virus coinfection stimulates the 1

aggressiveness in gastric cancer through the regulation of gankyrin 2

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Authors: Dharmendra Kashyapa, Budhadev Baral

a#, Nidhi Varshney

a#, Anil Kumar Singh

b, 5

Hem Chandra Jhaa*

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Affiliations: a = Discipline of Biosciences and Biomedical Engineering, Indian Institute of 9

Technology Indore, India 10

b= Biological Science and Technology Division, CSIR-North East Institute of Science and 11

Technology, India 12

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# Equal contributors 17

* Corresponding author 18

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Author for correspondence: Dr. Hem Chandra Jha, Infection Bio-engineering group, 23

Discipline of Biosciences and Biomedical Engineering, Indian Institute of Technology 24

Indore. Madhya Pradesh, PIN-453552, 0732-4306581 25

E-mail: [email protected] 26

Phone: +91-9971653189 27

Running title: Interplay between H. pylori and EBV through gankyrin 28

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.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under apreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for thisthis version posted November 20, 2020. ; https://doi.org/10.1101/2020.11.19.390807doi: bioRxiv preprint

https://doi.org/10.1101/2020.11.19.390807http://creativecommons.org/licenses/by-nc-nd/4.0/

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Abstract 31

Persistent coinfection of Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV) 32

promotes aggressive gastric carcinoma. The molecular mechanisms underlying the 33

aggressiveness in H. pylori and EBV coinfected gastric cancer is not well characterized. In 34

the current study, we investigated the molecular mechanism involved in the cooperation of H. 35

pylori and EBV-driven proliferation of gastric epithelial cells. Results showed that the 36

coinfections are significantly more advantageous to the pathogens to create a 37

microenvironment that favors the higher pathogen-associated gene expression. The EBV 38

latent genes EBNA1 and EBNA3C are highly overexpressed in the coinfections compared to 39

individual EBV infection at different time points (12 and 24 hrs). The H. pylori-associated 40

genes 16s rRNA, CagA, and BabA has also been highly overexpressed in coinfections 41

compared to H. pylori alone. Gankyrin is a small protein of 25 KDa involved in multiple 42

biological and physiological processes. The upregulation of gankyrin modulates the various 43

cell signaling pathways, leading to oncogenesis. The gankyrin shows a similar expression 44

pattern as EBNA3C at both transcript and protein levels, suggesting a possible correlation. 45

Further EBV and H. pylori create microenvironments that induce cell transformation and 46

oncogenesis by dysregulation of the cell-cycle regulator, GC marker, cell migration, DNA 47

response, and antiapoptotic genes in infected gastric epithelial cells by enhancing the 48

expression of gankyrin. Our study provides new insights into the molecular mechanism 49

where the interplay between two oncogenic agents (H. pylori and EBV) leads to the enhanced 50

carcinogenic activity of gastric epithelial cells through overexpression of oncoprotein 51

gankyrin. 52

Importance 53

In the present study, we have evaluated the synergistic effect of EBV and H. pylori infection 54

on gastric epithelial cells in various coinfection models. These coinfection models depict the 55

first exposures of gastric epithelial cells with EBV and then the H. pylori. While other 56

coinfection models narrated the first exposures of H. pylori followed by the infection of EBV. 57

This led to an enhanced oncogenic phenotype in gastric epithelial cells. We determined the 58

coinfection of EBV and H. pylori enhanced the expression of oncogenic protein gankyrin. 59

The interplay between EBV and H. pylori promotes the oncogenic properties of AGS cells 60

through the newly discovered oncoprotein gankyrin. EBV and H. pylori mediated 61

upregulation of gankyrin further dysregulates various cancer-associated hallmarks of genes 62

such as cell-migratory, gastric cancer marker, tumor suppressor, DNA damage response, and 63

proapoptotic genes. 64

Keywords: Helicobacter pylori, Epstein Barr Virus, Gankyrin, Gastric cancer, Coinfection 65

Introduction 66

Gastric cancer (GC) is the worlds' fourth leading cause of cancer-related deaths in both males 67

and females (1). Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV) are group1 68

carcinogens that potentially contribute to the development of GC (2). While doing so it can 69

alter gastric physiology and immunology (3). The prevalence of H. pylori in about half of the 70

global populations but it causes GC only in 3% of infected individuals (4, 5). The 71

discrepancy in H. pylori infection and GC might be influenced by its strain variability such as 72

expression of CagA, VacA, and BabA (6, 7, 8). EBV is the second most prominent cancer-73

associated pathogen involved in several types of lymphoid and epithelial cancers, including 74

GC (9). Latent and lytic genes expression associated with EBV such as EBNA-1, EBNA-2, 75

EBAN-3C, and BZLF-1 provokes the disruption of the host genes (9). The viral proteins also 76




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dysregulated the cell cycle, inflammation, angiogenesis, and tumor suppressor genes by hyper 77

methylation activities (10, 11). Earlier studies have shown the ability of EBV for oncogenic 78

transformation of primary gastric epithelial cells (12, 13). Thus, it is not only considered as a 79

passive carrier but also as an active oncogenic virus contributing to early events in the 80

development of GC (2). Lone infection of H. pylori or EBV is less severe in comparison to 81

coinfection and takes more time for the development of GC (2). 82

Gankyrin or PSMD-10 is a recently discovered small protein of 25 KDa known to be 83

overexpressed in various cancers (14). It is a novel oncogenic protein, involved in various 84

cellular processes such as cell cycle progression, apoptosis, and tumorigenesis (15). Gankyrin 85

negatively regulates the protein retinoblastoma (pRb) and tumor suppressor protein-53 86

(p53/TP53) to execute its oncogenic functions (16,17). Gankyrin was downregulated in GC 87

which leads to increased cell chemosensitivity to 5-fluorouracil and cisplatin was attained by 88

regulating cell cycle-related proteins, such as cyclin D1, cyclin E, and by activating the 89

PI3K/AKT pathway (18). 90

The mutualistic relationship which endures within the microbial niche could modulate host 91

gene expression, thus impact pathophysiology. We determined the molecular mechanism to 92

elucidate the unexplored strategy, which involves the participation of H. pylori in EBV-93

driven proliferation of gastric epithelial cells. We have developed five different infection 94

model including one uninfected control, using AGS human gastric epithelial cells, which is 95

an excellent system mimicking the human gastric epithelium. We elucidated that EBV and H. 96

pylori increased the gankyrin expression which led to the proliferation of infected cells in our 97

studied system. Furthermore, we determined the status of cell migratory, cell-cycle 98

regulatory, DNA repair, apoptosis, and tumor suppressor genes in coinfection models which 99

can be directly linked with aggressive carcinogenesis. 100

Results: 101

Coinfection of H. pylori and EBV synergistically regulates their genes expression in the 102

gastric epithelial cell after infection establishment 103

In this study, we evaluated the synergistic effect of H. pylori and EBV on gastric epithelial 104

cells. To understand the synergistic effect of these two pathogens we have developed the in-105

vitro coinfection models by using adenogastric carcinoma cell line (AGS). This cell line 106

mimics the gastric epithelial cells and provides the same microenvironment for the growth of 107

these two pathogens as the in-vivo conditions except pH (1.5-3.5). Before incubation of H. 108

pylori with AGS cells we have checked its morphology through the Gram staining, as 109

predicted we have observed the regular spiral shape of this bacterium (supplementary fig.1). 110

We have also observed the interaction between the H. pylori and EBV and how their 111

synergistic interplay affects the pathogenic gene expression of these pathogens and 112

downstream host cellular physiology. Our results showed that the infection-III and Infection-113

IV are significantly more advantageous to the pathogens to create a microenvironment that 114

favors the higher pathogens associated gene expression as compared to individual infection at 115

a time (Fig2). The expression of pathogens associated with carcinogenic genes is multiple 116

folds higher in coinfection models in comparison to individual infection (Fig2 A&B). Both 117

H. pylori and EBV synergistically provided a suitable milieu for the growth of each other. 118

Subsequently, we have followed all the above models to understand the pathophysiology of 119

these two pathogens in GC progression and severity and how these two pathogens mutually 120

favor the infectivity of each other. Thus, after coinfection, we have collected the cells at 121




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various time points (12, 24, and 48 hrs) and checked the profiles of the pathogenic and host 122

gene expression. 123

After infection, we performed the qRT-PCR for the EBV-associated latent and lytic genes 124

such as EBNA1, EBNA3C, and BZLF1 respectively. Our results showed that the expression 125

of EBNA1 transcript is significantly higher in infection-III and IV at time point 12 (p=0.0027 126

and 0.054), 24 (p=0.0024 and 0.0021), and 48 (p=0.025 and 0.0021) hrs as compared to the 127

infection-II and I Fig2A (I). In this experiment, we have selected one uninfected negative 128

control. This mock confirmed to us that there was no cross-contamination of these two 129

pathogens and the mock was not compromised. Similarly, the nuclear antigen EBNA 3C 130

expression was significantly higher in infection-IV at 12hrs post-infection (p=0.0450 and 131

0.0022) as compared to infection-II and I whereas in the case of 24hrs of coinfected samples 132

the expression of EBNA3C was higher in infection III as compared to infection-II and I 133

showing the interplay between the EBV and H. pylori. However, the expression is 134

significantly dropped at 48hrs post-infection Fig2A (II). 135

Furthermore, the expression of BZLF-1 transcript is higher (~100-150 folds) in infection-II, 136

while decreasing in infection-III and IV at 12hrs post coinfection. The similar expression of 137

BZLF-1 was determined in Infection-II and III at 24 hrs post-infection as compared to 138

infection-IV. In 48hrs of post coinfection, there was a slightly higher expression of BZLF-1 139

in infection-II concerning infection-III and IV Fig2A (III). Meanwhile, when comparing the 140

infection-II, III, and IV with uninfected samples we observed that there was multiple fold 141

higher expression of EBV associated latent and lytic genes, which confirmed the persistence 142

of infection. 143

Additionally, we investigated the expression of H. pylori-associated genes' transcripts such as 144

16s-rRNA, pathogenic gene CagA, and BabA. Our finding showed that in the case of 145

coinfection the expression of 16s-rRNA transcripts is higher as compared to individual 146

infection of H. pylori (infection-I). The expression of 16s-rRNA transcripts is higher (~2000 147

folds) in infection-III while it has downregulated in infection-IV at 12hrs post-infection. 148

Moreover, the expression of this gene is about double in 24 and 48hrs as compared to 12hrs 149

post-infection in infection-III and IV. In addition to this, the expression of 16s-rRNA 150

transcripts increases successively from 12 (~2000 folds) to 48hrs (5000 folds) post-infection 151

Fig2B (I). 152

Further, we investigated the expression of well-known pathogenic genes such as CagA and 153

BabA. Following the qRT-PCR results, we determined that the expression of CagA 154

transcripts is significantly higher at 12 (p=0.0026 and 0.0020), 24 (p=0.0027, 0.0024), and 48 155

(p=0.0023 and 0.0004) hrs time point in infection-III and IV as compared to infection-I and II 156

Fig2B (II). Surprisingly, the expression of BabA is higher (~100-200 folds) at 12, 24, and 157

48hrs time point in infection-I while it significantly decreases in infection-II, III, and IV in all 158

the time points such as 12, 24, and 48 hrs post-infection Fig 2B (III). 159

EBV and H. pylori regulate the expression of gankyrin in gastric epithelial cells 160

In addition to pathogens' associated factors, we also determined the host factor that is 161

regulated by the coinfection of EBV and H. pylori. Elevated expression of ankyrin was 162

shown to be associated with several cancers. We investigated if this was also true for 163

pathogen mediated gastric cancer. As expected, the infection of H. pylori and EBV enhanced 164

the expression of host-associated oncogenic gene gankyrin at transcripts and protein levels. 165

The expression of gankyrin is slightly higher at 12 and 24 hrs in infection-III and IV as 166

compared to infection-I and II meanwhile, at 48 hrs post-infection there is significantly lower 167




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(p=0.0323, 0.0234, 0.0410 and 0.0415) expression of gankyrin in infection-III and IV 168

whereas we have observed higher expression in infection-I and II Fig3A (I). Additionally, we 169

have also observed a similar pattern of expression of gankyrin in western blot at protein level 170

except for infection-III & IV where we examined the less expression of gankyrin protein as 171

compared to infection-II after 12hrs of post-infection Fig3A (II) and (III). 172

Coinfection of EBV and H. pylori promotes the oncogenic properties of epithelial cells 173

by deregulating the expression of cell-cycle regulator and the tumor suppressor genes 174

qRT-PCR results showed that CCND-1 gene expression is significantly higher in infection-III 175

and IV at time point 12 (p=0.0231 and 0.0020) and 24 (p=0.0246 and 0.0349) hrs as 176

compared to individual infected samples such as infection-I and II Fig4A (I&II). 177

Surprisingly, we witnessed the significantly lower expression of CCND-1 at time point 48hrs 178

in the infection-II, III, and IV in comparison to infection-I (p = 0.0021) Fig4A (III). In the 179

case of DAPK3, after 12hrs post-infection the expression of this genes' transcripts was higher 180

in infection-I and III as compared to infection-II and IV Fig4B (I) while in 24hrs post 181

infected condition we observed the higher DAPK3 expression in infection-III and IV as 182

compared to infection-I and II Fig4B (II) whereas adverse expression pattern was observed at 183

48hrs post-infection. Further, we have observed lower expression of DAPK3 in all infected 184

models in contrast to uninfected control Fig4B (III) 185

The expression of another gene PCNA was higher in 12, 24, and 48hrs post infected samples 186

concerning uninfected control, while a lower expression of this gene was examined at 48hrs 187

in comparison to 12 and 24hrs post infected conditions in infection-III and IV Fig4C (I, II & 188

III). The expression of another important gene Akt is comparatively higher in all infected 189

models at 12 and 24hrs post-infection for 48hrs post infected samples Fig4D (I, II & III). 190

Further, in the case of 24hrs, we witnessed the significantly higher expression of these genes' 191

transcripts in infection-IV in comparison of infection-I and II (p= 0.0023, 0.0002, 0.0021 and 192

0.0008) Fig4D (II) 193

Tumor suppressor genes are the defense system of the body that helps to control the abnormal 194

growth and proliferation of cells through various mechanisms such as promotes the DNA 195

repair system, apoptosis, or modulates cell signaling. Expression of one of the important 196

tumor suppressor gene PTEN is significantly higher in infection-I and II in comparison to 197

infection-III and IV at 12 and 24hrs time points Fig4E (I & II). The expression of this gene 198

is downregulated in all infection models as compared to uninfected control at 48hrs of post-199

infection Fig4E (III). 200

Additionally, APC is another tumor suppressor gene which performs their function through 201

the interaction of cell adhesion molecules E-cadherin and negatively regulates the expression 202

of beta-catenin. We have evidence that the expression of APC is significantly downregulated 203

in the case of 12hrs post-infection in all the four infection models (p= 0.0231, 0.0248, 0.0349, 204

and 0.0423) Fig4F (I). Unexpectedly, the expression of APC is upregulated in 24hrs post-205

incubation of these two pathogens in infection-I, II, III, and IV while higher expression of 206

this gene was observed in infection-I and II in comparison of infection-III and IV Fig4F (II). 207

Moreover, in 48hrs post infected samples, we determined the higher expression of APC in 208

infection-II followed by infection-I and IV Fig4F (III). 209

Furthermore, we noticed the expression of the transcripts of another important tumor 210

suppressor gene p53 was higher in infection-I and II in comparison to infection-III and IV at 211

12 and 24hrs post infected conditions Fig4G (I & II). While in 48hrs post-infection, the 212




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expression of this gene was higher in infection-II concerning infection-I, III, and IV Fig4G 213

(III). 214

Similarly, pRb, a tumor suppressor gene, was showing its higher expression in infection-I in 215

contrast to infection-II, III & IV at 12 and 24 hrs post-infection Fig4H (I & II). Meanwhile, 216

the opposite expression pattern was observed in 48hrs of the infection Fig4H (III). 217

H. pylori and EBV linked with the abrupt expression of gastric cancer marker and cell 218

migratory genes by interfering with the oncogenic assessment of epithelial cells 219

The expression of gastric cancer marker gene Abl-1 is significantly higher in infection-III 220

(p=0.0210) as compared to infection-I and II at 12hrs post-infection whereas we witnessed 221

the downregulation in case of infection-IV as compared to infection-I and II Fig5A (I). 222

Further, its expression is higher in all infected models in comparison to uninfected control 223

after 24 hrs of post-co-infection Fig5A (II). Surprisingly, the expression of Abl-1 is 224

downregulated in the case of infection-II, III, and IV as compared to infection-I Fig5A (III). 225

Adversely, the higher expression of TFF-2 was observed in infection-I and II in comparison 226

with uninfected control and infection-III and IV Fig5B (I). In the case of 24hrs post-227

infection, we determined the significantly higher expression of TFF-2 in infection-I followed 228

by the infection-III and IV Fig 5B (II). Besides these, in 48hrs post, infected samples the 229

expression of TFF-2 increased successively from infection-I to IV while the expression of 230

this gene is comparatively alleviated in respect to 12 and 24hrs of post-infection Fig5B (III). 231

Expression of another gastric cancer marker gene CDX-2 is higher in infection-I and III in 232

comparison to infection-II and III at 12hrs of post infected samples Fig5C (I). CDX-2 233

expression was slightly higher in infection-I as compared to other infection models in 24hrs 234

post infected samples Fig5C (II). In addition to the above results it was significantly 235

decreased in infection-I, II, III, and IV in 48hrs post-infected samples Fig5C (III). 236

The upregulation of cell migratory genes is a hallmark for cancer cell metastasis. We 237

witnessed the expression of MMP3, another cell migratory gene that helps the cells to 238

metastasize, was also significantly upregulated in infection-III and IV in comparison of 239

infection-I and II in 12, and 24hrs post infected samples Fig5D (I & II). Additionally, after 240

48hrs post-infection of EBV and H. pylori the expression of MMP3 is moderately higher in 241

infection-I while in infection-II, III, and IV the expression of these gene transcripts relatively 242

similar to uninfected control Fig5D (III). Although the results revealed that expression of 243

MMP7 is upregulated in infection-III and IV in contrast to infection-I and II at 12 and 24hrs 244

of post-infection Fig5E (I, II), while we observed the adverse expression in 48hrs of post-245

infection Fig5E (III). 246

EBV and H. pylori-associated pathogenic gene expression could cause GC by regulating 247

DNA damage response genes and anti-apoptotic gene Bcl-2 248

The effective function of DNA damage response genes in humans is to provide stability and 249

maintain its genome integrity. Moreover, cancer-causing infectious agents such as EBV and 250

H. pylori dysregulated the status of DNA damage response transcripts by changing the milieu 251

of the surrounding gastric epithelial cells. Interestingly, we determined the expression of 252

DNA damage response gene Apaf-1 expression is slightly higher in infection-I in 12hrs post 253

infected samples. A quite similar expression was observed in infection-II, III, and IV in 254

comparison to uninfected control Fig5F (I). Furthermore, the expression of this gene at 24hrs 255

is similar to the 12 hrs post infected samples in all the studied models except infection II 256

where we observed higher expression of Apaf-1 Fig5F (II). Meanwhile, the expression of 257




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Apaf-1 in 48hrs post infected samples successively increases from the infection-I to IV Fig5F 258

(III). 259

In addition to Apaf-1, the expression of Bax is significantly downregulated in 12hrs post 260

infected samples in all the conditions except in infection-II Fig5G (I). While the expression 261

of Bax is successively downregulated in all the four infections in 24 hrs post infected samples 262

Fig5G (II). Furthermore, this gene showed higher expression in infection-I, II, III, and IV in 263

comparison of uninfected control in 48hrs time point, whereas, in infection-III and IV the 264

expression of Apaf-1 was about 1.5-fold higher than the infection-I, II Fig5G (III). 265

DNA damage response genes perform their function through its anti-apoptotic properties 266

hence, the overexpression of anti-apoptotic genes such as Bcl-2 helps in cell survival and 267

proliferation. Pathogens such as EBV and H. pylori modulate the expression of this type of 268

host protein and promote the severity of gastric cancer. Coinfection of EBV and H. pylori 269

upregulates the expression of anti-apoptotic gene Bcl-2 in 12hrs post-infection in all the four 270

infection models compared to uninfected control whereas, we determined significantly higher 271

expression of this gene in infection-IV Fig5H (I). In addition to 12hrs, the similar expression 272

of Bcl-2 is observed in 24hrs post infected samples except for the infection-III in which we 273

have detected the higher expression of Bcl-2 Fig5H (II). While in 48hrs post infected 274

samples the expression of Bcl-2 is still higher compared to uninfected control. Moreover, the 275

expression of this gene successively decreases from the infection-I to IV Fig 5H (III). 276

Coinfection of EBV and H. pylori promotes GC through the upregulation of oncogenic 277

protein gankyrin in a time-dependent manner 278

Coinfection of EBV and H. pylori enhances the expression of oncogenic protein gankyrin in a 279

time-dependent manner. As in the above-mentioned results, we revealed that the EBV and H. 280

pylori could cause gastric cancer through the upregulation of gankyrin transcripts. To further 281

support our qRT-PCR results we performed the immunostaining of gankyrin after coinfection 282

of EBV and H. pylori by following the above-mentioned infection models (infection-I, II, III, 283

and IV). Surprisingly, we obtained the similar expression pattern of gankyrin in 284

immunostaining as observed at the transcript level. While, the higher expression of protein 285

gankyrin is observed in all the four infectious models moreover, in infection-III and IV we 286

evident that there is a significantly higher expression of gankyrin in 12hrs post-infected 287

samples Fig6A (I&II). Interestingly, in 24hrs post infected samples the expression of 288

gankyrin remains higher in infection-III and IV in comparison of infection-I and II Fig6B 289

(I&II). Furthermore, we determined the significantly downregulated expression of gankyrin 290

at 48hrs post infected samples in all four studied models in comparison to 12 and 24hrs post 291

infected samples Fig6C (I&II). 292

Endogenous and exogenous expression of gankyrin promotes the cell proliferation by 293

dysregulating the cell migration, cell-cycle regulator, protooncogene, gastric cancer 294

marker, and tumor suppressor gene in AGS cells 295

Protein blot image showed the exogenous gradient expression of gankyrin in AGS cells after 296

48hrs of post-transfection of the plasmid containing gankyrin gene Fig7 (A) and B. The 297

result revealed a concentration-dependent increase in gankyrin expression. Moreover, to 298

support our hypothesis that EBV and H. pylori cause gastric cancer via the upregulation of 299

gankyrin we exogenously overexpressed gankyrin in AGS cells and performed cell 300

proliferation assay. Surprisingly, results showed that the exogenous expression of gankyrin 301

promotes cell proliferation Fig7 (C). Furthermore, to validate our previous results the 302

coinfection of EBV and H. pylori upregulated the expression of oncogenic protein gankyrin 303




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which in turn changes the cell microenvironment and promotes cell proliferation we 304

performed the cell proliferation assay by following all the four studied infection models. 305

Interestingly, we observed that the rate of cell proliferation is comparatively significantly 306

higher than the uninfected mock. Besides this in 12, 24, 36, and 48hrs post infected samples 307

the number of viable cells is higher in infection-II, III, and IV in comparison to infection-I 308

and mock Fig7 (D) 309

Interestingly, exogenous overexpression of gankyrin results showed that the oncogene 310

gankyrin play their role via the interaction of various host cellular physiological pathway as it 311

acts differentially on a various cellular mechanism such as cell migration, cell-cycle 312

regulator, proto-oncogenes, gastric cancer marker, and tumor suppressor genes. Exogenous 313

overexpression of gankyrin significantly upregulated the cell migratory gene MMP7 and 314

MMP3 Fig7 (E) and the cell cycle regulator transcripts of Akt and CCND-1 Fig7 (F). Fig7 315

(G) Showed the ectopic expression of gankyrin which enhances the expression of Hurp. 316

Interestingly, the expression of tumor suppressor genes PTEN, APC, and pRB significantly 317

downregulated in exogenously overexpressed samples Fig7 (H). Furthermore, the ectopic 318

overexpression of gankyrin increases the expression of gastric cancer marker gene CCL8, 319

TFF-2, and CDX-2 while it downregulated the expression gastrin Fig7 (I). 320

Coinfection of EBV and H. pylori promotes the cell migration linked with the gankyrin 321

We further investigated whether the cellular physiological changes elicited by EBV and H. 322

pylori could contribute to cell migration using a wound-healing assay. This assay 323

demonstrated that EBV and H. pylori stimulated motility in the AGS cells. As expected, we 324

observed the higher cell migration in infection-III and IV (p = 0.0025, 0.0023, 0.0022, and 325

0.0027) Fig8A (I) in all the selected time points. Furthermore, we counted the migratory cell 326

and represented in graph Fig8A (II) 327

Coinfection and ectopic overexpression of gankyrin promotes the oncogenic properties 328

of AGS cells 329

We performed the clonogenic assay to select the EBV positive cells for the understanding of 330

oncogenic transformation efficiency of coinfected samples in comparison to individual 331

infection. The pore size of the transwell is smaller than the H. pylori only allowed the H. 332

pylori secretory molecules to reach the AGS cells, not the H. pylori. The experimental 333

models are depicted in Fig9 (A). The lower number of colonies was observed in uninfected 334

and H. pylori-infected AGS cells. Besides these the density of colonies is more in EBV 335

infected AGS cells followed by the significantly higher number of colonies were determined 336

in coinfected samples (B). Further, we quantified these colony numbers into the density and 337

observed that the density of colonies was higher in EBV and H. pylori coinfected samples 338

(C). As we speculated, we observed that the secretory molecules which are secreted by H. 339

pylori may be linked with the increased EBV virion in AGS cells. To further validate our 340

finding the coinfection of EBV and H. pylori promote the oncogenic properties of AGS cells 341

through the upregulation of gankyrin. We exogenously overexpressed the gankyrin in AGS 342

cells and determined that the ectopic expression of gankyrin enhanced the clonogenic 343

properties of AGS cells (D). We quantified the densities of colonies and represented them in 344

graph (E). 345

Discussion 346

Chronic or acute infection of pathogens that destroy the immune system is a severe threat to 347

human health and can cause diseases like Alzheimer's, meningitis, cancer, etc (19-21). 348




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Infectious agents not only induced the carcinogenesis but it is also involved in the promotion 349

of its aggressiveness (22, 23). About one-fifth of total human oncogenesis is associated with 350

infectious agents. Pathogens modulate the cellular milieu by dysregulation the gene 351

expression, fit for the potential carcinogenesis (24, 25). Studies suggested that H. pylori and 352

EBV promote oncogenesis by reframing the expression of cellular genes at the level of 353

transcription and translation in infected cells (2). Reprogramming of expression of transcript 354

and protein could promote the intrusiveness of these lesions. In this study, AGS cell lines 355

were used which mimic the primary gastric epithelial cells except for adherent’s junction. It 356

is well established that H. pylori and EBV infect the AGS cells and NCI-N87 in equal 357

efficiency (2, 25). Thus, all these above reasons elicited us to investigate the synergistic 358

effect of H. pylori exposed AGS cells for the successful infection of EBV and further how the 359

microenvironment of EBV infected AGS cells favour the fortunate infection of H. pylori. 360

Various studies reported that H. pylori exposed cellular environmental conditions are suitable 361

for the enhanced EBV infection and vice-versa (2, 25). Moreover, we also observed the 362

enhanced EBV-GFP fluorescence in fluorolog analysis at various time intervals in all the 363

coinfection models except in infection-I (data not shown). In a study by Pandey et. al. and 364

Sonkar et. al, reported that these pathogens dysregulated the cellular genes’ transcript 365

expression which led to the aggressive GC (2). Moreover, till yet it is an enigma whether the 366

prior exposures of H. pylori create a suitable niche for the EBV infection or anterior windage 367

of EBV creates a niche that favors the aggressive growth of H. pylori and further gastric 368

carcinoma. Interestingly, we got similar results as being speculated, coinfection of EBV and 369

H. pylori enhanced the pathogenic gene expression such as EBNA-1, EBNA3C, and BZLF-1 370

in various time intervals. Various studies reported that EBNA-1 and EBNA-3C enhanced the 371

G1/S phase transition, cell invasiveness, and metastasis through the upregulation of cell 372

signaling genes such as slug, snail, vimentin, and E-cadherin (23, 26-29). In addition to these 373

functions, it is also known to regulate tumor suppressor genes like p53, pRb which play an 374

important role in oncogenesis and cancer progression. 375

In this study, we determined the expression of EBV associated latent genes (EBNA-1 and 376

EBNA3C) can significantly promote the replication and spread of the virus in coinfected 377

conditions up to 48hrs then the individual infection. The gene expression pattern in H. pylori 378

and EBV coinfections revealed that H. pylori contributed to the increased replication and 379

transcription of EBV pathogenic genes and virion production. Surprisingly, we observed that 380

another latent gene EBNA3C expression is significantly correlated with the expression of 381

oncogenic protein gankyrin at 12, 24, and 48hrs post infected samples. To support this 382

finding, we performed the western blot analysis in which we observed the same expression 383

pattern of gankyrin as we observed in qRT-PCR. These results revealed that the EBV-384

associated EBNA3C may be directly linked with the expression of host oncogenic protein 385

gankyrin as the expression of EBNA3C and gankyrin was alleviated in the only presence of 386

EBV. The current study suggested that EBNA3C could directly regulate the host oncogenic 387

protein gankyrin expression. These results supported that EBNA3C may cause gastric cancer 388

through the regulation of host oncogenic protein gankyrin. It is an established oncoprotein 389

which is found to be overexpressed in various cancers and involved in various biological 390

processes, encompassing transformations of normal cells into cancerous one (15). Abnormal 391

expression of gankyrin observed in human liver cancer, pancreatic cancer, oesophageal 392

cancer, cervical, lung cancer, breast cancer, and glioma (16). Although there is no report 393

available till which suggests that infectious agents caused the GC through the upregulation of 394

oncogene gankyrin? 395




10

This is the first study demonstrating that the exposure of AGS cells with EBV before H. 396

pylori infection and vice-versa increased the expression of gankyrin which led to aggressive 397

gastric carcinogenesis. The expression pattern of bacterial signature gene 16s-rRNA indicated 398

the establishment of infection in AGS cells. H. pylori-associated secretory antigen CagA 399

encoded by the CagA-pathogenicity island (CagA-PAI) present in about 89% of H. pylori 400

strains caused morphological dysplastic alteration in epithelial cells (30), (31). Dysplastic 401

changes are important and contribute to the initiation of carcinogenesis. Furthermore, H. 402

pylori encoding CagA is associated with hustle inflammation, peptic ulcers, gastritis, and GC 403

(24). 404

In the present study, we determined that the human gastric epithelial cells have enhanced 405

susceptibility to EBV infection in the presence of CagA positive H. pylori strain (I10). 406

Pandey et al. showed that a CagA mutant strain of H. pylori has a significant reduction in 407

viral titer production (2). This study was suggested that instead of CagA mutant, wild type H. 408

pylori contributed significantly to viral replication and subsequently cancer progression and 409

aggressiveness. H. pylori loaded with type-IV bacterial secretion system that helps in the 410

injection of its associated secretory protein into the host-derived cells. Alternative mode of 411

uptake of CagA through integrins mediated rearrangements of actin filaments and further 412

endocytosis. Endocytosis of CagA further changed the intracellular milieu and led to the 413

morphological alteration in the gastric epithelial cells. Here, our results supported the 414

hypothesis that the type-IV secretory mechanism was used by this bacterium, as the direct 415

incubation of wild type H. pylori showed enhanced infectivity. The expression of signature 416

sequence 16s-rRNA, CagA were higher in coinfected samples which concluded that the virus 417

could also help synergistically to increase the expression of H. pylori associated factors. 418

Expression of 16s-rRNA inferences that enhanced replication and multiplication of H. pylori 419

genome in presence of EBV. BabA transcripts are known to be highly expressed in peptic 420

ulcer and gastric cancer rather than asymptomatic colonization. Similar observations were 421

made with the BabA transcript in infection-I which showed that the expression of BabA is 422

important for enhanced infectivity. Further, the higher BabA expression early point suggested 423

the increased infectivity of H. pylori as it helps in the attachment of H. pylori with host cells. 424

In addition to the above results, the recruitment of gankyrin by the coinfection dysregulated 425

the downstream genes which were known to be directly influenced by upregulation of this 426

protein. These genes are studied under six categories such as cell cycle regulator genes, GC 427

marker, cell migratory, tumor suppressor, DNA damage response, and antiapoptotic gene. 428

Moreover, we investigated that EBNA-3C and gankyrin can further change the status of 429

various cellular proteins which have played a significant role in gastric carcinogenesis. 430

Although, experiments need to be conducted to validate the one-to-one relation of EBNA3C 431

and gankyrin. Results of the present study suggested that the differential expression of cell 432

cycle regulatory genes such as CCND-1, DAPK3, PCNA, and Akt in all the infectious 433

models at various time points could be linked with the increased oncogenesis. Gankyrin 434

upregulated the CCND-1 and PCNA in pancreatic and gastric cancer respectively (32). 435

Besides these genes, the expression of gankyrin upregulates the Akt at transcript and protein 436

level in hepatocellular, liposarcoma, and gastric cancer (33–35). The upregulation of Akt 437

protein through the action of gankyrin enhanced cell proliferation, metastasis, stem cell self-438

renewal capacity, autophagy, and chemoresistance (18, 36). The upregulation of death-439

associated protein kinase domain-3 (DAPK-3) is linked with enhanced apoptosis. While 440

coinfection of these two oncogenic pathogens downregulated this, pro-apoptotic which 441

supported the accumulation of AGS gastric epithelial cells over the period. Results showed a 442

significantly lower expression of gankyrin in 12 and 24hrs post-infection in comparison of 443

48hrs. Furthermore, this finding supported that H. pylori and EBV infection mediated higher 444




11

expression of gankyrin led to aggressive GC through the dysregulation of cell-cycle 445

regulatory genes. 446

EBV and H. pylori can hijack one of the GC markers non-receptor tyrosine kinase (Abl-1) 447

signaling to remodel the host cytoskeleton for multiple purposes such as late phase 448

autophagy, cell motility, and cell adhesion. Trefoil factor-2 (TFF-2) is another gene that is 449

known to be overexpressed in GC. It is a structural component of gastric mucosa and inhibits 450

gut acid secretion and its motility. Inhibition of acid secretion provides the platform for the 451

aggressive growth of H. pylori which can further enhance the EBV reactivation and 452

replication (37, 38). Homeobox protein (CDX-2) preferentially binds to methylated DNA and 453

is involved in multiple cellular processes such as transcriptional regulation of genes of 454

epithelial cells. In the present study, profiles of GC marker genes suggested that H. pylori 455

changed the status of GC related marker genes for their growth and development of 456

oncogenesis. It was not only involved in the dysregulation of host oncogenic factors but also 457

changed the expression of genes that provided a microenvironment for its growth and 458

replication. 459

Matrix metalloproteinase-3 and 7 (MMP-3 & MMP-7) is an enzyme that increases the 460

expression of pro-collagenase and degrades the collagen layer, fibronectin, and laminin. The 461

overexpression of this protein in infection models indicates the probable role of EBV and H. 462

pylori in gankyrin mediated aggressiveness of GC through the recruitment of such kinds of 463

cell migratory genes. 464

Downregulated form of the phosphatase and tensin homolog (PTEN) is associated with 465

several types of cancers and functions as a negative regulator of the PI3-kinase/AKT pathway 466

by downregulating PIP3 (phosphatidylinositol 3,4,5-triphosphate) levels. The protein 467

gankyrin downregulated the tumor suppressor protein-16 (p-16), protein-53 (p-53), and 468

protein retinoblastoma (pRB) through the direct interaction with its various domains (17). 469

The loss of activity of these tumor suppressor genes is the key marker for carcinogenesis. 470

This demonstrates that the association of infection of EBV and H. pylori in gankyrin 471

upregulation, cellular genome, and dysregulation of cellular signaling was important for 472

oncogenesis. 473

Apaf-1 and Bax are mitochondrial enzymes that lead to the release of cytochrome c and 474

CASP3 and efficiently initiate the process of apoptosis. Apoptotic protease activating-factor-475

1 (Apaf-1) and apoptosis regulator (Bax) profiles after coinfection suggested that the 476

coinfection of EBV and H. pylori downregulated the proapoptotic genes via upregulating the 477

oncoprotein gankyrin. Furthermore, EBV and H. pylori infection mediated upregulated 478

gankyrin downregulates the anti-apoptotic B-cell lymphoma-2 (BCL-2). 479

The current study demonstrated that EBV and H. pylori-associated factors specifically 480

enhanced the expression of gankyrin. Upregulated gankyrin mediated dysregulation of 481

expression of the cell-cycle regulator, GC marker, cell migration, DNA response, and 482

antiapoptotic genes in infected gastric epithelial cells. EBV and H. pylori create intracellular 483

and extracellular milieu which potentially favor gastric epithelial cell proliferation and 484

carcinogenesis. It provided the H. pylori-EBV tie, which forms a suitable tumor 485

microenvironment for gastric epithelial cell proliferation and carcinogenesis. Further 486

gankyrin might be playing a central role in this transformation process by dysregulation of 487

multiple associated genes Fig 10. 488

489




12

Materials and methods 490

Cell culture: 491

The gastric cell lines AGS were obtained from the National Center for Cell Science (NCCS) 492

Pune India. AGS cell line which is an EBV negative cells, cultured in Dulbecco’s modified 493

Eagle’s medium (DMEM; Himedia, Mumbai, India) containing 10% fetal bovine serum 494

(FBS; BIOWEST, South America origin) and 100 U/mL penicillin/streptomycin (Himedia, 495

Mumbai India) under conditions of 5% CO2 and humidified air at 37°C (Eppendorf India). 496

Virus (EBV) culture and isolation: 497

HEK293T cells containing GFP-EBV (obtained as a gift from the Erle S. Robertson’s 498

laboratory, University of Pennsylvania, USA) were cultured and used for EBV amplification. 499

Cultured the virus and purified as described previously (2, 39, 25) 500

Bacterial (H. pylori) culture: 501

H. pylori strain (I10) was a kind gift by Dr. Asish Kumar Mukhopadhyay (National Institute 502

of Cholera and Enteric Diseases, ICMR, Kolkata, India) H. pylori was grown according to the 503

conditions described earlier (25). 504

Coinfection of EBV and H. pylori with AGS cells: 505

For infection, we took 0.25 million AGS cells and cultured in a 6-well plate. Observed the 506

cells up to the 40-50% confluencies and then incubated the MOI-100 of H. pylori and 25μl of 507

EBV. We optimized the EBV dose by incubating the increasing dose of EBV such 25, 50, 508

100, 150, and 200 μl with AGS cells and observed 25 μl of EBV culture was sufficient for a 509

successful infection. For this study, we have developed five independent approaches for the 510

construction of coinfection models by using H. pylori and EBV. In the first approach, 511

uninfected AGS cells were taken as the negative control (Fig.1A control). In our second and 512

third approaches, we have incubated the H. pylori and EBV with AGS cells (Fig.1B 513

infection-I and C Infection-II) respectively. Moreover, in the fourth approach, we have 514

incubated the H. pylori with AGS cells for six hours thereafter given the infection of EBV 515

and incubated for 12, 24, and 48 hours (Fig.1D infection-III). In the fifth approach, the 516

infection of EBV is given first for six hours followed by the infection of H. pylori (Fig.1E 517

Infection-IV). 518

RNA isolation and qRT-PCR: 519

Fixed (MOI-100) of H. pylori (I10) and EBV (25μl) were incubated with AGS cells under 520

specific conditions (5% CO2, 37 °C) for 12, 24, and 48 hrs. RNA isolated and qRT-PCR was 521

executed as described earlier (25). The pathogens and host genes were analyzed. 522

Western Blot: 523

Western blot experiments were carried out as described earlier (40). The lysates were 524

analyzed by Western blots using an anti-gankyrin polyclonal primary antibody (cell 525

signaling) and HRP-tagged secondary antibody. Blots were observed under the gel doc 526

system (ChemiDoc™ XRS+ System with Image Lab™ Software #1708265) 527

Immunofluorescence Assay 528




13

An immunofluorescence assay was performed as described earlier (40) by using an anti-529

gankyrin primary antibody and specific signals were detected with secondary antibodies 530

conjugated with Alexa Fluor 488 (cell signaling). The cell's nucleus was counterstained with 531

4′, 6′-diamidino-2-phenylindole (DAPI). The results were analyzed with confocal microscopy 532

(Olympus IX83) at 60X and zoom two times in triplicates. 533

Cell proliferation assay 534

An assay of cell proliferation was accomplished through trypan blue exclusion and crystal 535

violet staining methods as described earlier (2, 40). 536

537

Colony formation assay 538

The AGS cells were infected with EBV and H. pylori and EBV positive cells selected with 539

puromycin (2ug/ml). We selected the ectopically expressed gankyrin positive AGS cells 540

through geneticin (G148). The image was taken by using ChemiDoc™ XRS+ System with 541

Image Lab™ Software #1708265 (40). 542

Statistical Analysis 543

Data were statistically analyzed using a two-tailed Student's t-test. All the results were 544

derived from triplicate experiments P-values by using the Graph Pad Prism version 6 of 545

14

H. pylori (Helicobacter pylori); EBV (Epstein Barr Virus); GC (Gastric cancer); EBNA1 567

(Epstein Barr Nuclear antigen-1); EBNA3C (Epstein Barr Nuclear antigen-3C); EBNA2 568

(Epstein Barr Nuclear antigen-2); BZLF-1 (BamHI Z fragment leftward open reading frame 569

1); 16s rRNA (16s Ribosomal RNA); CagA (cytotoxin-associated gene A); BabA (Blood 570

group antigen binding adhesin A); VacA (Vacuolating toxin A); pRb (protein 571

retinoblastoma); P53 (Tumor suppressor protein 53); AGS (Adenogastric carcinoma cell); 572

CCND-1 (Cyclin D1); DAPK3 (Death associated protein kinase domain-3); PCNA 573

(Proliferating cell nuclear antigen); Akt (Protein kinase B); PTEN (Phosphatase and tensin 574

homolog); APC (Adenomatous polyposis coli); Abl-1 (Tyrosine-protein kinase ABL1); TFF2 575

(Trefoil factor-2); CDX-2 (Homeobox protein CDX-2); MMP-3 (Matrix metalloproteinase-576

3); MMP-7 (Matrix metalloproteinase-7); Apaf-1 (Apoptotic protease activating-factor-1); 577

Bax (Apoptosis regulator BAX); Bcl-2 (B-cell lymphoma-2); CCL8 (Chemokine ligand 8); 578

CagA-PAI (CagA-pathogenicity island); PIP3 (phosphatidylinositol 3,4,5-triphosphate) 579

580

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692

693

694

Figures and figure legends 695

696

Fig.1 Coinfection models of H. pylori and EBV depicts the deep into the pathophysiology 697 of gastric cancer. Illustration of all the studied infectious models. AGS cells were cultured in 698

6-well plates, and directly incubated the H. pylori and EBV with these cells (A) Uninfected 699

AGS control cells (B) Infection-I portrayed the infection of H. pylori (I10) (C) Infection-700

II AGS were infected with EBV only. (D) Infection-III AGS cells were infected 701

sequentially, first exposing the cells with EBV for 6 hrs then these exposed cells incubated 702

with H. pylori. (E) Infection-IV AGS cells were firstly exposed with the H. pylori then 703

incubated with EBV. All the infected cells were further incubated for 12, 24 and 48 hrs. 704

These infection models were used for the whole of this study except colony formation assay. 705




18

706

Fig.2 Interplay between H. pylori and EBV synergistically increases the transcript of its 707 associated pathogenic genes. (A) Transcript expression of EBV associated latent and lytic 708




19

genes. (I) & (II) Quantification of transcript of latent gene EBNA-1 & EBNA-3C respectively 709

(III) qRT-PCR quantification of lytic gene BZLF-1 (B) Transcript quantification of H. pylori 710

associated signature 16S-rRNA (I) and its associated pathogenic gene cytotoxin associated 711

gene A (CagA) (II) and blood group antigen binding adhesin A (BabA) (III) in all the 712

infection model for 12, 24 and 48 hrs post infected samples. The experiment performed for 713

two biological and two technical replicates and the results are shown as the mean SD of two 714

independent experiments. 715

716

Fig.3 Coinfection of H. pylori and EBV upregulates the oncogenic protein gankyrin at 717 both transcripts and protein levels (A) Transcript expression of gankyrin in coinfection 718

models (I), (II) & (III) depict the expression of gankyrin at 12, 24 and 48 hrs respectively. (B) 719

(I), (II) & (III) western blot image of protein gankyrin for 12, 24 and 48 hrs post infected 720

samples respectively. (C) (I), (II) & (III) Illustrate the quantification of blot image by Image 721

J. software and representative graph presented in terms of fold changes for 12, 24 and 48 hrs 722

post infected samples respectively. The significantly higher expression of gankyrin observed 723

at 12 and 24 hrs post infected samples. While the expression of gankyrin is significantly 724

downregulated in infection-II, III and IV in 48 hrs post infected samples. The experiment was 725

performed for two biological and one technical replicate. 726




20

727

728




21

Fig.4 Coinfection of H. pylori and EBV promotes gastric cancer through the 729 dysregulations of cell-cycle regulator and the expression of tumor suppressor gene (A) I, 730

II & III represents the cyclin D1 (CCND-1) profiles in coinfection at 12, 24 and 48 hrs 731

respectively (B) I, II & III depicts the death associated protein kinase-3 (DAPK3) expression 732

pattern at 12, 24 and 48 hrs respectively (C) I, II & III demonstrate the transcript expression 733

profiles of proliferating cell nuclear antigen (PCNA) 12, 24 and 48 hrs respectively (D) I, II 734

& III depicts the expression profiles of AKR thymoma (Akt) coinfected samples at 12, 24 and 735

48 hrs of post infection of EBV and H. pylori. (E) I, II & III illustrate the downregulation of 736

phosphatase and tensin homolog (PTEN) in 24 and 48 hrs post infection, while it is slightly 737

upregulated in 12 hrs post infection. (F) I, II & III demonstrate the expression profiles of 738

adenomatous polyposis coli (APC) in EBV and H. pylori coinfected samples for 12, 24 and 739

48 hrs time points. (G) & (H) I, II & III represents the expression of p53 and pRB 740

respectively in 12, 24 and 48 hrs post infected samples. The experiment has performed for 741

two biological and two technical replicates and the results are shown as the mean SD of two 742


744




22

745

Fig.5 Coinfection of H. pylori and EBV changes oncogenic assessment of gastric 746

epithelial cells by dysregulating gastric cancer marker, cell migratory and antiapoptotic 747




23

genes. (A) I, II & III depicts the status of tyrosine protein kinase Abl-1 gene expression in 748

EBV and H. pylori coinfection. (B) I, II & III demonstrate the expression profiles of trefoil 749

factor-2 (TFF-2) at 12, 24 and hrs time points. (C) I, II & III illustrate the transcription factor 750

CDX-2 in coinfected samples for 12, 24 and 48 hrs of post infection. (D) I, II & III depicts 751

the expression pattern of matrix metalloprotease-3 (MMP-3) in 12, 24 and 48 hrs post 752

infected samples. (E) I, II & III represents the expression status of matrix metalloprotease-7 753

(MMP-7) (F) I, II & III illustrate the downregulation of Apaf-1 (G) I, II & III represents the 754

decrease in amount of Bax transcripts in coinfected samples at 12, 24 and 48 hrs of post 755

infected samples. (H) I, II & III represents the expression pattern of BCL-2 in which at 12 756

and 24 hrs post infection the expression of antiapoptotic gene is elevated as the expression of 757

gankyrin is also several folds upregulated in 12 and 24 hrs post infected samples in above 758

mentioned results. While the expression of BCL-2 follows the same pattern at 48hrs post 759

infected as the expression of gankyrin was observed. The experiment has performed for two 760

biological and two technical replicates and the results are shown as the mean SD of two 761





24

763

764




25

Fig.6 Coinfection of EBV and H. pylori causes gastric cancer through the upregulation 765

of oncogenic protein gankyrin. 766

Immunofluorescence results of oncogenic gene gankyrin validate our previous transcripts and 767

western blot results. Graphical representation of quantified immunoblotting image through 768

the Image J. software. After coinfection of EBV and H. pylori we observed elevated protein 769

expression of gankyrin in all the time points for all studied infection except 48 hrs post 770

infection in which observed downregulation of gankyrin in infection-II, III and IV. (A) I, (B) 771

I & (C) I Representative immunoblotting image of gankyrin represents the protein expression 772

of gankyrin after 12, 24 and 48 hrs of post infection respectively. Graphical representation of 773

quantified immunoblotting image through the Image J. software. First panel showed an 774

uninfected mock. Second and third panel illustrate the gankyrin protein after I10 and EBV 775

infection. While the fourth and fifth panel shows the intensity of gankyrin expression after 776

EBV-I10 and I10-EBV post infection. (A) II Approximately 600-700-fold increased 777

expression of gankyrin observed in EBV-I10 and I10-EBV coinfected samples as compared 778

to individual infection of I10 and EBV after 12 hrs post infection. B (II) The expression 779

pattern of gankyrin in 24 hrs post infected samples is about 100 to 200-fold higher in EBV-780

I10 and I10-EBV coinfection. C (III) The expression of oncogenic protein gankyrin after 48 781

hrs of post infection is about 3-32 higher in infection III and IV in comparison of infection-I. 782

The expression of gankyrin in infection-II is about equal to infection III while the 25-30-fold 783

lower expression of gankyrin was observed in infection IV. The experiment has performed 784

for two biological and three technical replicates and the results are shown as the mean SD of 785

two independent experiments. 786

787




26

788

Fig.7 Coinfection mediated upregulation of gankyrin and ectopic expression of gankyrin 789

promotes the rate of cell proliferation by interfering with the expression of various 790 cellular genes. (A) Western blot image represents the ectopic expression of oncogenic 791

protein gankyrin. (B) Graphical representation of increased concentration gradient of 792

gankyrin quantified by the image J software of western blot image. (C) Shows the ectopic 793

expression of gankyrin enhances the cell proliferation in trypan blue cell exclusion method of 794

cell counting (D) Moreover, coinfection of EBV and H. Pylori also enhances the rate of cell 795

proliferation in coinfected samples and may be linked with the increased expression of 796

gankyrin. (E) Represents the ectopic expression of gankyrin significantly enhanced the 797

expression of cell migratory gene MMP-7 and MMP-3. (F) & (G) Representative graph 798

shows the elevated expression of cell-cycle regulatory gene Akt and CCND-1 and proto-799

oncogenes hepatoma upregulated protein (Hurp) respectively. Moreover, overexpression of 800

gankyrin significantly alleviates the expression of tumor suppressor gene PTEN, APC and 801




27

pRB shown in (H). While the expression of gastric cancer marker gene gastrin expression is 802

slightly lower followed by the upregulation of C-C motif chemokine ligand-8 (CCL-8), TFF-803

2 and CDX-2 (I). The experiment has performed for two biological and two technical 804

replicates and the results are shown as the mean SD of two independent experiments. 805

806

807

Fig.8 Coinfection of EBV and H. pylori enhanced the efficiency of cell migration of AGS 808 cells. (A) I Representative image of scratch wound healing assay. First panel shows the 809

uninfected AGS cells. Second, third, fourth and fifth panel shows the infection-I, II, III and 810

IV respectively for the 0, 6, 12, 24 and 48 hrs of post infection. (A) II Graphical 811

representation of the number of cells migrated in each infection and uninfected mock. 812

813




28

814

Fig.9 Coinfection and ectopic overexpression of gankyrin can be linked with the 815 elevated oncogenic properties of AGS cells. A (I) Uninfected AGS cells. (II) AGS cells 816

with transwell and H. pylori infection. (III) AGS cell only with EBV infection. (IV) First 817

infection of H. pylori for 6 hrs followed by second infection of EBV. (V) First infection of 818

EBV for 6 hrs followed by second infection of H. pylori. Selection of EBV positive colonies 819

for 14 days. (B) Representative image of foci formation after coinfection. (C) 820

Quantification and graphical representation of density of foci through the Image J. software. 821

(D) Further we have validated foci formation results through the ectopic expression of 822

gankyrin in AGS cells and selected the gankyrin positive cells through the geneticin (G-418) 823

for 14 days. Representative image of foci in exogenously overexpressed AGS cells. (E) 824

Quantification and graphical representation of density of foci in exogenously overexpressed 825

AGS cells through the Image J. software. The experiment has been performed three times 826

and the results are shown as the mean SD of two independent experiments. 827




29

828

Figure 10: A model illustrating the association of gankyrin in EBV and H. pylori 829 mediated gastric cancer. This association drives the progression of EBV and H. pylori 830

mediated gastric cell transformation. Oncogenic activity of gankyrin is accelerated in the 831

presence of pathogen which targets the cell cycle regulators, cell migratory genes, gastric 832

cancer marker, anti-apoptotic, DNA damage response and tumor suppressor genes thus 833

caused the aggressive oncogenesis. 834

835

Supplementary Figure and figure legend 836

837

838

Supplementary Fig.1 Gram staining of H. pylori (I10) showing the regular spiral shape of 839

bacteria 840

841






https://doi.org/10.1101/2020.11.19.390807http://creativecommons.org/licenses/by-nc-nd/4

helicobacter pylori and epstein-barr virus coinfection stimulates … · 2020. 11. 19. · 47...

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