let-7e suppresses dna damage repair and sensitizes...

MOLECULAR CANCER RESEARCH | GENOME MAINTENANCE

Let-7e Suppresses DNA Damage Repair and SensitizesOvarian Cancer to Cisplatin through Targeting PARP1 A C

Man Xiao, Jianfeng Guo, Lisha Xie, Chun Yang, Lanqing Gong, Zehua Wang, and Jing Cai

ABSTRACT◥

Increased DNA damage repair is one of the mechanisms impli-cated in cisplatin resistance. Our previous study indicated that thederegulation of let-7e promoted cisplatin resistance and that let-7ecould suppress DNA double-strand break repair in ovarian cancer.In this study, we further characterized the role of let-7e in DNAdamage repair and cisplatin resistance in ovarian cancer, andinvestigated the underlying mechanisms. The alkaline and neutralcomet assay indicated that let-7e impeded both DNA single- anddouble-strand break repairs through downregulating its target genePARP1. In vitro and in vivo experiments provided evidence that thelet-7e–PARP1–DNA repair axis was involved in the modulation ofcisplatin sensitivity in ovarian cancer. Contrary to let-7e, PARP1was overexpressed in cisplatin-resistant ovarian cancer tissues, and

patients with high PARP1 expression exhibited poor progression-free survival (PFS) and overall survival (OS). Multivariate logisticandCox regression analyses showed that let-7e and FIGO stagewereindependent prognostic factors for PFS and OS, whereas let-7e andPARP1 were able to independently predict chemotherapy response.Taken together, our results indicated that low expression of let-7epromoted DNA single- and double-strand break repairs and sub-sequently contributed to cisplatin resistance by relieving the sup-pression on PARP1 in ovarian cancer.

Implications: Targeting the let-7e–PARP1–DNA repair axis mightbe an effective strategy for the treatment of chemoresistant ovariancancer.

IntroductionThe tumors of the female genital tract represent a leading cause of

morbidity and mortality among women worldwide. Specifically, ovar-ian cancer is the most lethal gynecological malignancy. Primarycytoreductive surgery with platinum-based chemotherapy is the stan-dard of care for ovarian cancer. Although the last decades have seenimprovements in the diagnosis and treatment of ovarian cancer, the5-year survival rate still stands at less than 40% (1). Platinum resistanceremains a major factor accounting for the stubbornly high mortalityassociatedwith ovarian cancer (2). Platinum-based drugs bind toDNAand form intra- or interstrand cross-links, which causes DNA single-strand breaks (SSB) or double-strand breaks (DSB). To survive andmaintain genome integrity, the cellular DNA damage and repairsystems are triggered. Hence, enhanced DNA damage repair becomesa mechanism associated with platinum resistance (3). An enhancedactivity of DNA damage repair has been found in cisplatin-resistantovarian cancer cells (4).

Base excision repair (BER), nucleotide excision repair, and mis-match repair are the main pathways by which SSB are repaired. Bycatalyzing ADP-ribose units from nicotinamide adenine dinucleotide(NADþ) to various cellular factors, poly(ADP-ribose) polymerase 1

(PARP1) mainly participates in SSB repair (5). PARP1�/� cells showan impaired long patch repair subpathway, the polymerization step ofBER, resulting in enhanced sensitivity to the lethal effects of alkylatingagents and gamma irradiation (6). DSB are repaired through homol-ogous recombination (HR) or non-homologous end joining pathways.The nucleosomal histone H2AX is phosphorylated in response to theintroduction of DSB, and the number of gH2AX foci approximates thenumber of DSB (7). The downregulation of BRCA1/2 and Rad51, thecritical proteins for HR repair, can significantly enhance the efficacy ofplatinum complexes in killing ovarian and lung cancer cells (8, 9). Inaddition to SSB repair, the poly(ADP-ribosyl)ation (PAR) induced byPARP1 also plays a key role in DSB repairs through directly recog-nizing DSB ends and regulating the cell-cycle checkpoint activationand repair processes (10).

MicroRNAs are a group of noncoding small RNAs composed of17–24 nucleotides with regulatory functions through base pairing atthe 30-UTR of the target mRNA. In human, there are 13 different let-7familymembers, including let-7a-1, 7a-2, 7a-3, 7b, 7c, 7d, 7e, f7-1, 7f-2,7g, 7i, mir-98, andmir-202. Emerging data indicate that individual let-7 family members can have different activities. Even in the same cell,two let-7 family members could have different functions (11). Anexample of this is a study on malignant mesothelioma, where let-7b�

was found to be highly expressed but let-7e� was severely reduced (12).In ovarian cancer patients, the upregulation of let-7d-3p and let-7gwasassociated with the increased sensitivity to platinum-based chemo-therapy (13, 14), whereas miR-98-5p contributed to cisplatin resis-tance (15). Research about the implication of let-7e in cisplatinresistance is quite limited. In our previous research, we found thatlet-7e was decreased in chemoresistant ovarian cancer tissues and theoverexpression of let-7e increased the sensitivity of ovarian cancer cellsto cisplatin (16, 17), which might be partially attributed to its role insuppressing DSB repairs and inhibiting the expression of BRCA1 andRad51 (17). However, the direct target gene of let-7e is unknown. Ourprevious study indicated that PARP1 or IGF-1 might link let-7e toBRCA1 and Rad51 (17). Nevertheless, that remains to be furtherverified. Wielgos and colleagues’ study showed that let-7a coulddirectly target PARP1 in HER2-overexpressing breast tumors (18).

Department of Obstetrics and Gynecology, Union Hospital, Tongji MedicalCollege, Huazhong University of Science and Technology, Wuhan, China.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

M. Xiao and J. Guo contributed equally to this article.

Corresponding Authors: Jing Cai, Union Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan, Hubei 430022, China.Phone/Fax: 86-27-85351649; E-mail: [email protected]; andZehua Wang, [email protected]

Mol Cancer Res 2020;18:436–47

doi: 10.1158/1541-7786.MCR-18-1369

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The current study characterized the involvement of let-7e in bothDNASSB and DSB repairs and identified PARP1 as a direct target of let-7e.In this work, we provided evidence supporting that let-7e inhibits therepair activity of cisplatin-induced DNA damage and subsequentlyimproves the susceptibility to cisplatin by regulating the expression ofPARP1 in ovarian cancer.

Materials and MethodsPatient tissue samples

The formalin-fixed paraffin-embedded tumor tissues from 88 ovar-ian cancer patients were analyzed. These patients were diagnosed andtreated atWuhanUnionHospital during the period fromAugust 2008to November 2015. The diagnosis of all patients was pathologicallyconfirmed, and all cases received a platinum-based chemotherapy aftersurgery. The clinicopathological parameters, including age, histolog-ical subtypes, International Federation of Gynecology and Obstetrics(FIGO) stage, tumor categories, and chemotherapy response of allcases were recorded. Patients who initially respond to platinum-basedchemotherapy and who subsequently relapse 6 months or more afterthe initial treatment were classified as chemotherapy sensitive. Patientswho relapse within 6 months after completing first-line therapy orprogress during the primary chemotherapy were defined as chemo-therapy resistance (19). The recurrence includes rising CA125, radio-logically or symptomatically relapse (19). In addition, up to 6 years ofclinical follow-up information involving progression-free survival(PFS) and overall survival (OS) of all patients were collected. Thisstudy was approved by the Ethical Review Committee of the UnionHospital, TongjiMedical College, HuazhongUniversity of Science andTechnology.Written informed consent was obtained from all patients.The procedures were carried out in accordance with the university'sscientific research guidelines and regulations.

Cell cultureHuman ovarian cancer cell lines A2780, SKOV3, and Caov3 were

purchased in 2010 from the China Center for Type Culture Collection(China). All three cell lines were passaged no more than 15 times andauthenticated by short tandem repeat analysis (Shanghai BiowingApplied Biotechnology Co.) in October 2016. Mycoplasma was testedandwas negative in all cells using the q-PCRmethod (YEASEN). All ofthe tumor cell lines were cultured in the Dulbecco's Modified EagleMedium with 10% fetal bovine serum at 37�C under 5% CO2

atmosphere.

Transient transfectionsThe let-7e agomir (B06003) and antagomir (B05005) were obtained

fromGenepharma. The short hairpin RNA (shRNA) targeting humanPARP1 (50-GCAGCTTCATAACCGAAGATT-30) and the negativecontrol shRNA (50-UUCUCCGAACGUGUCACGUTT-30) weredesigned, synthesized, and ligated into the GV248 vector by theGenechem Corporation, aiming to knock down the expression ofPARP1 mRNA. The full-length gene PARP1 (PARP1 cDNA) wasamplified by PCR reactions and cloned into the GV144 plasmid byGenechem Company to overexpress the PARP1 gene. Cells wereseeded in 6-well plates at a concentration of 4 � 105 cells per well.The transient transfection of 30 nmol/L let-7e agomir, 200 nmol/Llet-7e antagomir, shPARP1, PARP1 cDNA, and their negativecontrols was performed using PEI (Polysciences, 23966-1) accord-ing to the manufacturer's instructions. The total RNA and proteinsof cells were extracted 48 or 72 hours later to verify the interferentialefficiency.

Stable transfection of PARP1 shRNAThe PARP1 lentivirus was synthesized by Genechem, which includ-

ed above-mentioned GV248 vector (stably expressing shPARP1), amarker (GFP fusion protein), pHelper1.0 (gag/pol element), andHelper2.0 (VSVG element). After cellular adherence in 12-well platesat a concentration of 2 � 105 cells per well, the PARP1 lentiviralparticles were added at a multiplicity of infection of 20. Emptylentiviruswas used as a control. After 48 hours, the cells were incubatedwith a selective medium, which was supplemented with 5 mg/mL ofpuromycin, to kill untransfected cells. A limited dilution method wasused to establish the stable puromycin-resistant colonies. The inter-ferential efficiency was verified using qRT-PCR and western blotanalyses.

Colony-formation assayApproximately five hundred transfected cells per well were plated

into the six-well plates and incubated with cisplatin for 48 hours.After incubation with a fresh medium for 2 weeks, the survivingcolonies were stained with 0.1% crystal violet and quantifiedmanually.

MTT assayMTT assays were performed to evaluate the cytotoxicity of cisplatin

to cancer cells according to the methods described previously (17).Briefly, after transfection, cells were planted into the 96-well plates at aconcentration of 5,000 cells per well and incubated with a series ofcisplatin for 48 hours. Then, MTT reagent (Sigma) was added toevaluate cellular viability using Microplate Reader (Bio-Rad).

Quantitative real-time polymerase chain reaction (qRT-PCR)RNA was extracted using TRIzol (Sigma). To assess the expression

of let-7e, hairpin-it reverse transcription primers and specific PCRforward primers (RiboBio) were applied according to the manufac-turer's protocol. The small RNA U6 was used for normalization. ThecDNAofmRNAwas synthesized based on universal primers (Takara).b-Actin was used as an internal control. The PCR primers were asfollows: PARP1 upstream 50-CGAGTATTACTATTAGCCCTT-GGG-30 and downstream 50-AACATGGGAGCTCTTGAAATATG-30; b-actin upstream 50-CAGAGCCTCGCCTTTGCC-30 and down-stream 50-GTCGCCCACATAGGAATC-30.

Western blottingTotal proteins of cell samples were loaded on 10% SDS-

polyacrylamide gel for electrophoresis and then transferred onto apolyvinylidene difluoride (PVDF)membrane. The resulting blots wereblocked with 5% non-fat milk and probed overnight with rabbit anti-PARP1 monoclonal antibody (Abcam, ab32138), rabbit anti-Rad51(Santa Cruz Biotechnology, sc-8349), rabbit anti-BRCA1 (ProteintechGroup, 20649-1-AP), and mouse anti-b-actin (Epitomics, 1854-1).Protein bands were visualized via the Molecular Imager ChemiDocXRSþ system with Image Lab software from Bio-Rad.

Immunohistochemistry (IHC)After dehydration through a graded series of alcohols and antigen

retrieval in citrate buffer (PH 6.0) through microwave irradiation,the 4-mm sections were stained with rabbit monoclonal anti-PARP1(Abcam, ab32138) overnight at 4�C and biotinylated secondaryantibody for 30 minutes at room temperature subsequently. Neg-ative controls were treated with PBS instead of primary antibody. Atleast 300 tumor cells in three randomly high-power fields werecounted for each estimation. High expression of PARP1 was

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diagnosed when more than 54.8% (the median scores of PARP1expression) of the tumor cell nuclei were stained.

In situ hybridization (ISH)Expression of let-7e in ovarian cancer tissues was analyzed accord-

ing to the ISH staining procedures and score criteria describedpreviously (17). In short, after deparaffinization, slides were treatedwith 15 mg/mL proteinase-K at 37 �C for 7 minutes and subsequentlyhybridized with digoxigenin-labeled LNA probes (Exiqon) at 60�C for1 hour. The let-7e and a scramble probe were incubated at 20 nmol/L,and the positive control probe, U6, was hybridized at 1 nmol/L. Afterstringent washes with SSC buffers at 60�C, sections were incubatedwith blocking reagent, alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche; cat. 11093274910), NBT/BCIP sub-strate, and finally nuclear fast red (Vector Laboratories; cat.H-3403) counterstaining. The staining score (0–9) was quantified bymultiplying the intensity of the signal (0, negative; 1, light blue; 2, blue;3, dark blue) with the percentage of positive cells (0, 0%; 1, < 25%; 2,25%–50%; 3, > 50%).

ImmunofluorescenceThe cells were seeded on coverslips in 24-well plates and treated

with cisplatin. After incubation with a drug-free medium for theindicated time, cells were fixed with 4% paraformaldehyde and per-meabilized with 0.5% Triton X-100. Then, the cells were blocked with3% bovine serum albumin and immunostained overnight with anti-g-H2AX (Cell Signaling Technology, 9718). Subsequently, the cellswere incubated with a Cy3-conjugated secondary antibody and coun-terstained with DAPI (Roche). Images were captured using an A1-SiNikon confocal laser scanningmicroscope. Only those cells withfive ormore foci were considered g-H2AX foci positive.

Dual-luciferase reporter assayThe part of the 30-UTR of human PARP1 mRNA containing the

intact let-7e recognition sequence (at nucleotides 690–711 of 30-UTR)and the control sequences with let-7e binding site mutated wereamplified and subcloned into the dual-luciferase pmiR-RB-REPORT reporter by the RiboBio company. SKOV3 cells werecotransfected with the wild-type or mutated PARP1 vector and let-7e agomir/agomirNC using Lipofectamine 2000 (Invitrogen). After48 hours, the firefly and Renilla luciferase activities were measuredusing the Dual-Luciferase Reporter Assay System (Promega) as per themanufacturer's instructions. The Renilla luciferase activity was nor-malized to firefly luciferase activity for each well.

Single-cell gel electrophoresis (Comet assay)DNA lesions, including base damage, SSB, and DSB, were assessed

using single-cell gel electrophoretic assay (comet assay) under alkalineconditions, whereas DSB could only be detected under the neutralversion (20, 21). The majority of DNA breaks induced by H2O2 areSSB (22). Thus, cells were incubated with a low dose of H2O2 toevaluate SSB repairs in the present study. After transfection, cells weretreated with cisplatin or H2O2 and allowed to repair for a certain timebefore performing the alkaline and neutral comet assay. DamagedDNA from cancer cells was quantified by determining the tail moment(TM, a combination of both the tail length and intensity of fluores-cence in the tail) using the Comet Assay Software Project (CASP).

For the alkaline comet assay, fully frosted microscope slides weresequentially covered with 1% normal melting point agarose (NMP),0.5% low melting point agarose (LMP), and a mixture of 0.7% LMPagarose and the cell suspension. After the third layer solidified, the

slides were fully immersed in lysis buffer (2.5mol/LNaCl, 100mmol/LNa2EDTA, 10 mmol/L Tris base and 1% Triton X-100, pH 10) at 4�Cfor 1.5 hours. Then, slides were placed in the unwinding solution(300mmol/L NaOH, pH > 13) for 25minutes to unwind the DNA andconvert the alkali labeled sites into DNA breaks. Next, electrophoresis(1mmo/LNa2EDTA and 300mmo/LNaOH, pH > 13) was conductedat 25 V and 300 mA for 25 minutes on ice, and the neutralization wasperformed in 0.4 mol/L Tris base (pH 7.5) at 4�C for 10 minutes.Finally, the slides were stained with ethidium bromide and visualizedat 4 � magnification using a fluorescence microscope.

For the neutral comet assay, slides were directly immersed in neutralelectrophoresis buffer (300 mmo/L sodium acetate, 100 mmo/L Tris–HCl, pH 8.5) at 16 V and 50 mA (0.4 V/cm) for 1 hour without DNAunwinding after lysis. Then, the slides could be stained and visualizedwithout neutralization.

Mice modelThe animal experiments were supervised and approved by the

Animal Care andUse Center of the TongjiMedical College, HuazhongUniversity of Science and Technology. SKOV3 cells with PARP1 stablyknockdown were transfected with 200 nmol/L let-7e antagomir/antagomirNC according to the following groups: shNC þ antago-mirNC, shNCþ let-7e antagomir, shPARP1þ let-7e antagomir. After48 hours of transfection, 4 � 106 viable cells in 200 mL PBS wereinjected subcutaneously into the right flank of 6-week-old female nudemice (5 mice/group), which were purchased from the Hunan SLACLaboratory Animal Co., Ltd. The tumor size was measured with acaliper every 2 to 3 days, and the tumor volume was calculated asvolume ¼ length � (width)2/2. After confirming the successfulmaturation of xenografts and the average volume of tumors reached50 mm3, cisplatin was given via intraperitoneal injection at a dose of3 mg/kg of body weight every other day. Three days after the firstadministration of cisplatin, xenografts were intratumorally injectedwith let-7e antagomir/antagomirNC at a dose of 2.5 OD/mice accord-ing to the above-mentioned groups once a week for 2 weeks.

On the other hand, an equal quantity (4 � 106) of SKOV3 cells orSKOV3 cells transfected with 30 nmol/L let-7e agomir was subcuta-neously injected into the nude mice. After the average tumor sizereached 50 mm3, the mice were intraperitoneally injected with cis-platin at a dose of 3mg/kg every other day or with olaparib at 50mg/kgevery day according to the following groups: SKOV3, SKOV3 þcisplatin, SKOV3 þ let-7e agomir þ cisplatin, SKOV3 þ olaparibþ cisplatin. Three days after the first administration of cisplatin orolaparib, xenografts were intratumorally injected with let-7e agomir ata dose of 0.67OD/mice according to the above-mentioned groups oncea week for 2 weeks.

All mice were sacrificed 1 week after the final treatment. Then, thexenograft tumors were excised for paraffin-embedding, and the sec-tions were used for H&E staining and IHC of PARP1 (Abcam; cat.#ab32138), BRCA1 (Affinity; cat. #AF6289), Rad51 (Santa CruzBiotechnology; cat. #sc-8349), g-H2AX (Cell Signaling Technology;cat. # 9718), cleaved caspase-3 (Cell Signaling Technology; cat. #9664),IGF1R (Proteintech; cat. 20254-1-AP), and p-Akt (Cell SignalingTechnology, 4060S) according to the procedures described above.

Statistical analysisThe SPSS 20.0 was used to carry out the statistical analyses in this

work. The differences between groupswere assessed by Student t test orMann–Whitney test. Fisher exact test was used to analyze the rela-tionship between PARP1 expression and the clinicopathological char-acteristics of ovarian cancer patients. Survival curves were drawn by

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the Kaplan–Meier method and compared with the log-rank test. Coxand logistic regression analyses were performed to identify the inde-pendent factors that could predict the chemoresponse and prognosis ofovarian cancer. A P value of < 0.05 was considered significant.

ResultsLet-7e impairs DNA damage repair

To explore the potential role of let-7e in the repair activity ofcisplatin-induced total DNA damage, an alkaline comet assay wasperformed. The unrepaired damaged DNA after a recovery period wasused to evaluate the repair capacity of cancer cells in the present work.The damaged DNA began to recover 4 hours after cisplatin treatmentin the control A2780 and SKOV3 cells, whereas an increased let-7eexpression resulted in a delay in DNA repair (Fig. 1A–C). Conversely,inhibition of let-7e in A2780 and SKOV3 cells led to an acceleration inDNA repair (Fig. 1D–F).

DSB represents a form of lethal DNA damage (23). Using theneutral comet assay, which only detects DSB, we have demonstratedthat let-7e could suppress the repair of cisplatin-induced DSB anddownregulate the expression of BRCA1 and Rad51, two importantfactors implicated inDSB repair (17). Next, we examined the impact oflet-7e on SSB repair. Themajority of DNAbreaks induced byH2O2 areSSB (22). Thus, cells were incubated with a low dose of H2O2 toevaluate SSB repairs in the present study (22). We found that the timeneeded for SSB repairs in A2780 and SKOV3 cells was delayed whenthe expression of let-7e was increased (Fig. 1G–I), whereas dramaticincreases in SSB repairs were seenwhen let-7e expressionwas inhibitedin A2780 and SKOV3 cells (Fig. 1J–L). Altogether, these findingssuggest that overexpression of let-7e inhibits the repair of cisplatin-induced DNA damage in ovarian cancer.

PARP1 is a direct target of let-7eTo address the mechanisms by which let-7e influences DNA

damage repair, we searched the miRNA database (http://www.microrna.org/microrna/getMirnaForm.do) andmiRWalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/) to predict the target genes oflet-7e. Among the searched genes, PARP1 attracted our attentionbecause of its involvement in DNA damage repair (10, 24). Weperformed real-time PCR andWestern blot assays to confirm whetherPARP1 was regulated by let-7e in ovarian cancer cells. The resultsindicated that overexpression of let-7e could substantially decrease themRNA and protein levels of PARP1 (Fig. 2A and B). Conversely,downregulation of let-7e greatly increased the expression of PARP1(Fig. 2C andD). The miRNA target prediction indicated the 30UTR ofPARP1 mRNA contains a putative let-7e binding site (Fig. 2E). Toverify whether let-7e could directly target PARP1 via the binding site,we cloned wild-type and mutated PARP1 30UTR into the pmiR-RB-REPORT vector and performed a dual-luciferase reporter assay. Wefound that let-7e agomir significantly decreased the luciferase activityof the construct in which the luciferase gene was fused with PARP130UTR. However, mutation of the binding site abolished the inhibitoryeffect of let-7e on luciferase activity (Fig. 2F). Taken together, theseresults revealed that PARP1 is a direct target of let-7e.

PARP1 confers DSB repair in ovarian cancerIn addition to the role in SSB repair, recent studies suggest that

PARP1 also participates in DSB repair (10). Hegan and colleaguesshowed that inhibition of PARP1 could suppress homology-dependent DSB repair through stimulating repressive E2F4/p130complexes to bind to the promoters of BRCA1 and Rad51 in colon

and lung cancer cell lines (25).We then investigated the role of PARP1in DSB repair in ovarian cancer. Using the neutral comet assay, whichmainly detects DSB, we revealed that the rate of DSB repairs wasdecreased when PARP1 expression was inhibited in SKOV3 cells(Fig. 3A). Additionally, knockdown of PARP1 led to a remarkableincrease in the formation of gH2AX foci after recovery from cisplatintreatment, further confirming the decreased DSB repair in this con-dition (Fig. 3B). When PARP1 was overexpressed in Caov3 cells, thecapacity of DSB repairs was accelerated (Fig. 3C). Western blotanalyses showed that decreased PARP1 inhibited the expression ofBRCA1 and Rad51, whereas increased PARP1 exerted the oppositeeffects (Fig. 3D and E). These results indicated that PARP1 promotedDSB repairs and positively regulated the expression of BRCA1 andRad51 in ovarian cancer.

Let-7e inhibits DNA damage repair by downregulating PARP1Given that PARP1 is an integral component of SSB and DSB repair,

and a direct target gene of let-7e, we wondered whether let-7e–mediated inhibition of DNA repair was achieved by downregulatingPARP1. First, the alkaline comet assay was carried out to measure SSBrepair in cells treated with a low dose of H2O2. As shown in Fig. 4A,enhanced SSB repairs caused by let-7e suppression in SKOV3 cellscould be counteracted to some extent by downregulating PARP1. Onthe other hand, the inhibitory effect of let-7e on H2O2-induced SSBrepair was blocked by the exogenous expression of PARP1 in Caov3cells (Fig. 4B). Next, the neutral comet assay was performed to assessDSB repairs. In SKOV3 cells, the increased DSB repairs and elevatedexpression of BRCA1 and Rad51 induced by let-7e depletion wererelieved by PARP1 knockdown (Fig. 4C and E). In Caov3 cells,overexpression of let-7e suppressed DSB repairs and decreased theexpression of BRCA1 and Rad51, whereas these phenomena wererescued by the reintroduction of PARP1 (Fig. 4D and F).

Because the inhibitory effect of let-7e on both SSB and DSB repairswas achieved to some extent by targeting PARP1, we examined theimpact of the let-7e/PARP1 axis on the repair of total DNA damagecaused by cisplatin using the alkaline comet assay. We found thatcotransfection with let-7e antagomir and shPARP1 could partiallyreverse the increased PARP1 expression and the consequent acceler-ation of DNA repair induced by let-7e downregulation in SKOV3 cells(Fig. 4E and G). Additionally, let-7e suppression could decrease theformation of gH2AX foci after recovery from cisplatin treatment inSKOV3 cells, whereas cotransfection with shPARP1 increased gH2AXfoci formation to some extent, indicating that DNA damage repair wasrepressed in the combination group compared with let-7e inhibitionalone (Fig. 4I and J). In Caov3 cells, cotransfection with let-7e agomirand PARP1 cDNA could partially rescue the suppressed PARP1expression and subsequently delayed total DNA damage repair causedby let-7e overexpression (Fig. 4F and H). Collectively, these findingsindicated that the inhibitory impact of let-7e onDNA damage repair ispartially achieved by downregulating PARP1 in ovarian cancer.

Let-7e–mediated PARP1 suppression enhances cisplatinsensitivity

DNA repair–deficient cells are sensitive to chemotherapeuticdrugs (3, 26). Because let-7e–mediated downregulation of PARP1inhibited DNA damage repair, we wondered whether the let-7e/PARP1 axis could influence the cytotoxicity of cisplatin in ovariancancer. First, using MTT and colony-formation assays, we found thatthe resistance to cisplatin caused by let-7e depletionwas reversedwhendecreasing the levels of PARP1 in SKOV3 cells (Fig. 5A and B). Theincreased sensitivity to cisplatin induced by let-7e overexpression was





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Figure 1.

Let-7e impairs DNAdamage repair.A–F,Total DNAstrandbreakagewasquantified in let-7eupregulated anddownregulated cells by the alkaline comet assay.A2780and SKOV3 cellswere respectively incubatedwith 3 mmol/L or 5mmol/L cisplatin for 8 hours, followed by incubation in a drug-freemedium for 0, 8, and 24 hours. TheTM valuesmeasured at 0 hourswere set at 100% and served as a control, the unrepairedDNAdamage at 8 and 24 hours after cisplatin incubationwere expressed as apercentageof that at 0 hours. Representative images (A andD) and themean� SDof the TMvalues (B–C andE–F) for each group are shown.G–L, The alkaline cometassay detected SSB repairs in cells treated with a low dose of H2O2. A2780 and SKOV3 were respectively treated with 10 mmol/L or 25 mmol/L H2O2 on ice for 20minutes and allowed to recover for 0, 1, and 2 hours subsequently. The TM valuesmeasured at 0 hourswere set at 100% and served as control; the unrepaired SSB at 1and 2 hours after H2O2 incubationwere expressed as a percent of that at 0 hours. Representative images (G and J) and themean� SDof the TM values for each group(H–I and K–L) are shown. Three separate experiments were done. Student t test: � , P < 0.05.

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relieved when upregulating the expression of PARP1 in Caov3 cells(Fig. 5C and D).

Next, we established subcutaneous xenografts in nude mice usingSKOV3 cells. As shown in Fig. 5E, the tumors in the let-7e–inhibitedmice grewmore quickly compared with control tumors. This might bepartially attributed to the amplified IGF-1/IGF1R signaling and sub-sequent activation of the Akt pathway, indicated by the augmentedexpression of IGF1R and p-Akt in let-7e–inhibited tumors (Supple-mentary Fig. S1).However, the tumor growthwas retardedwhen let-7eand PARP1 were downregulated simultaneously in the combinationgroup, suggesting that PARP1 is essential for let-7e repression-mediated tumor growth promotion. Activated PARP1 expression inlet-7e–inhibited tumors and repressed PARP1mediated by a lentiviralvector were validated by IHC. Moreover, we found that BRCA1 andRad51 were highly expressed in the let-7e–inhibited tumors, but thiseffect did not exist in thosewith simultaneous PARP1 depletion. In linewith the alterations in tumor growth, IHC showed a decrease ingH2AX and cleaved caspase-3, the markers for DSB and apoptosis,respectively, in the let-7e–repressed group, whichwere not observed inthe combination group (Fig. 5F). Together, these findings providedevidence that the role of let-7e in cisplatin sensibilization was partiallymediated by targeting PARP1 in ovarian cancer.

Considering the inhibition of let-7e to PARP1 expression and thesuppression of PARP1 to DNA damage repair, we next evaluated theinfluence of let-7e agomir and PARP inhibitor on the cytotoxicity ofcisplatin, respectively. The xenograft growth curves showed that the

mice treated with cisplatin had less tumor burden compared withcontrol, whereas the effect was more remarkable when accompaniedby the let-7e agomir or PARP inhibitor (olaparib; Fig. 5G and H). Inline with the above results, IHC staining of tumor sections revealed anincrease in the expression of gH2AX and cleaved caspase-3 in thegroups with let-7e agomir or olaparib (Fig. 5I). We have also per-formed the in vitro MTT assays to detect the cytotoxicity of cisplatinalone, olaparib alone, and cisplatin þ olaparib treatments (Supple-mentary Fig. S2). The results showed that the combination of cisplatin(10 mmol/L) and olaparib (10 mmol/L) was able to inhibit cell growthmore potently than a single drug. These results indicated that both let-7e agomir and PARP inhibitor could sensitize ovarian cancer tocisplatin.

Let-7e and PARP1 are predictive of response to platinum-basedchemotherapy

Our previous study discovered that let-7e was decreased in che-moresistant ovarian cancer tissues compared with chemosensitivecases (17). In contrast to let-7e, IHC showed an increased expressionof PARP1 in chemoresistant tissues (Fig. 6A andB).Moreover, PARP1was negatively correlated with let-7e in ovarian cancer specimens(Fig. 6C and D). Analyzing the correlation of PARP1 and theclinicopathological characteristics of ovarian cancer patients, we foundthat high expression of PARP1 was closely related with chemotherapyresistance and an advanced FIGO stage other than age, tumorcategories, or histological subtypes (Supplementary Table S1). The

Figure 2.

Let-7e directly targets PARP1.A–D,Three ovarian cancer cellswere transfectedwith let-7e agomir or antagomir. PCRandWestern blot assays showed the changes ofmRNA and protein levels of PARP1. E, MiRNA and miRWalk predict that the 30UTR of PARP1 contains a putative let-7e binding site. F, Dual-luciferase reporter geneassay shows that let-7e directly targets PARP1. SKOV3 cells were cotransfected with RB-PARP1-wt/RB-PARP1-mut vector and let-7e agomir/NC. The Renillaluciferase activity was assessed 48 hours later and normalized to the internal firefly luciferase activity. The mean � SD of the three biological replicates are shown.Student t test: � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






Kaplan–Meier analyses and log-rank tests indicated that patients withhigh expression of PARP1 exhibited poor PFS and OS (Fig. 6E and F),which were consistent with the observations in the Gene Expression-Omnibus (GEO) database (Fig. 6G and H). These results wereconfirmed by the univariate logistic and Cox regression analyses,which showed that high PARP1 was significantly associated with poorPFS/OS and chemoresistance in ovarian cancer patients (Supplemen-tary Table S2). Finally, we performed a multivariate logistic and Coxregression model to analyze all variables that were significant in theunivariate analyses (Supplementary Table S2), including let-7e, FIGOstage, BRCA1, Rad51 (17), and PARP1. The results showed that let-7eand FIGO stagewere independent prognostic factors for PFS andOS inovarian cancer, whereas let-7e and PARP1 were able to independentlypredict chemotherapy response (Table 1).

DiscussionChemoresistance has been a significant problem exercising the

minds of oncologists. Increasing evidence demonstrates that miRNAs

play important roles in the development of chemoresistance. In thisstudy, we identified the DNA damage repair gene PARP1 as a directtarget of let-7e and showed that low expression of let-7e promotedDNA SSB and DSB repairs and subsequently contributed to cisplatinresistance through relieving the suppression on PARP1 in ovariancancer. Multivariate regression analyses indicated that let-7e was anindependent factor that could predict prognosis and chemotherapyresponses, providing a potential therapeutic vulnerability node forovarian cancer.

To date, a number of miRNA modulators have been identified toregulate almost every aspect of the cellular response to DNAdamage, including damage sensing, signal transduction, and dam-age repair (27). Patients with an impaired repair capacity areespecially sensitive to DNA damage agents (28). Upregulation ofmiR-24 during postmitotic differentiation of hematopoietic celllines reduced DNA repair and boosted damage sensitivity throughtargeting H2AX (29). Pre-miR-630 blocked the phosphorylation ofthe ataxia-telangiectasia mutated (ATM) kinase and two ATMsubstrates, H2AX and p53, resulting in reduced proliferation rates

Figure 3.

PARP1 promotes DNA DSB repair in ovarian cancer. A and C, The neutral comet assay detected DSB repairs. After transfection with shPARP1 and PARP1 cDNA,respectively, SKOV3 and Caov3 cells were treatedwith 20 mmol/L cisplatin for 2 hours, followed by incubation in a drug-freemedium for 0, 8, and 24 hours. Similar tothe methodmentioned above, the TM values analyzed at 0 hours were set at 100% and served as control, and values measured at 8 and 24 hours were expressed asthe percentage of that at 0 hours. Representative images and the mean � SD of the TM values are shown. B, After incubation with 20 mmol/L cisplatin for 2 hours,gH2AX foci in SKOV3 cells transfected with shPARP1 were examined 8 hours after recovery. Representative images and the mean � SD for the two groups wereshown.D and E, Expression of PARP1, BRCA1, andRad51 in conditionswhere shPARP1 or PARP1 cDNAwere transfected. Three experimentswere done. Student t test:� , P < 0.05; ��, P < 0.001.

Xiao et al.





and diminished sensitivity to cisplatin in A549 cells (30). ThemiRNA-506 and miRNA-103/107 sensitized cells to cisplatin-induced DSB by directly targeting Rad51 (31, 32). In the presentstudy, we investigated the role of let-7e in DNA damage repair andfound that let-7e impeded cisplatin-induced SSB and DSB repairsby targeting PARP1. The discovery of the let-7e–PARP1–DNArepair axis supports the potential therapeutic strategy of combininglet-7e with cisplatin, which might benefit chemoresistant ovariancancer management.

Although platinum-based agents have obtained improved out-comes in the last decades (33), there are still ovarian cancer patientswho exhibit intrinsic or acquired resistance to cisplatin (19). Treat-ment regimens for these patients are different from those of cisplatin-sensitive group (34). Therefore, it is essential to identify patients whowill or will not benefit from platinum-based therapy prior to thetreatment or in the early stage of chemotherapy. Several miRNAs havebeen shown to be well-known biomarkers for treatment therapydecisions in several studies. For example, chronic myeloid leukemia

Figure 4.

Let-7e inhibits DNA damage repair viatargeting PARP1. The analytical data ofthe comet assay were drawn as thefraction (%) of DNA damage remainingat the indicated time points. A–B, SSBrepairs were detected using the alka-line comet assay in SKOV3 and Caov3cells treatedwith 10mmol/LH2O2 on icefor 20 minutes and allowed to recoverfor 0, 1, and 2 hours subsequently.C–D, DSB repairs were measured bythe neutral comet assay in SKOV3 andCaov3 cells incubated with 20 mmol/Lcisplatin for 2 hours and allowed torecover for 0, 8, and 24 hours subse-quently. E, Protein levels of BRCA1,Rad51, and PARP1 in SKOV3 cellscotransfected with let-7e antagomirand shPARP1. F, Expression of BRCA1,Rad51, and PARP1 in Caov3 cellscotransfected with let-7e agomir andPARP1 cDNA. G–H, The alkaline cometassay detected total DNA damagerepair in SKOV3 and Caov3 cells incu-bated with 20 mmol/L cisplatin for2 hours followed by recovery for 0, 8,and 24 hours subsequently. I–J, Theformation of gH2AX foci in SKOV3 cellsafter incubation with 20 mmol/L cis-platin for 2 hours and recovery for8 hours. Representative images and themean � SD for different groups wereshown. Each experiment was per-formed in triplicate.






is characterized by the BCR-ABL rearrangement, which decreases overtime with imatinib treatment. In addition, miR-451 is inverselycorrelated with BCR-ABL levels at the time of diagnosis and ontreatment. Thus, miRNA-451 could be a putative predictor markerof imatinib therapy response in chronic myeloid leukemia (35). Insmall cell lung cancer, miR-92a-2� may have an application inscreening patients at risk for chemoresistance (36). The work pre-sented here suggested that let-7e could be an independent markerpredicting the prognosis and response to cisplatin treatment in ovarian

cancer. Additionally, our study indicated that let-7e inhibition accel-erated ovarian cancer tumor growth through promoting the IGF1/IGF1R/Akt signaling pathway. This was consistent with the result ofPouria Samadi1 and Zhenjun Li, which demonstrated that let-7etargeted IGF1R and modulated the proliferation, migration, andradiosensitivity of colorectal cancer cells (37, 38). Future investigationsshould strive to incorporate let-7e detection into clinical trials toevaluate the value of let-7e for stratification of patients with risk todevelop chemoresistance and to relapse.

Figure 5.

Let-7e–mediated regulation of PARP1affects cisplatin sensitivity in ovariancancer. A–B, Cellular viability wasassessed byMTT and clonogenic survivalassays after downregulating the expres-sion of let-7e and PARP1 simultaneouslyin SKOV3 cells. C–D, MTT and colony-formation assays of Caov3 cells with let-7e and PARP1 expression upregulated.E, The tumor volumes and growth curvesof xenografts transfected with let-7eantagomir alone or in combination withshPARP1. The vertical axes of the growthcurves represent the relative tumor vol-ume, which is in accordance with eachvolume measurement from the secondonwards minus the first volume exceptfor the volume deviations. F, H&E stain-ing and immunohistochemical analysesof xenografts. The expression of PARP1,BRCA1, Rad51, gH2AX and cleaved cas-pase-3 in the three groups were exam-ined. G–H, Tumor volumes and thegrowth curves of nudemice treated withcisplatin alone and in combination withlet-7e agomir or olaparib. I, H&E stainingand immunohistochemical analyses ofxenografts.

Xiao et al.





PARP1 was originally implicated in SSB repair because of its directrole in base excision repair, which form the basis for the use of PARPinhibitors (PARPi) in the treatment of cancers with deficiencies inHR-dependent repair pathways (39). In recent years, PARP1 has beenreported to be involved in DSB repairs. However, the role of PARP1 inDSB repairs is controversial. Favaudon and colleagues establishedPARP1�/� and PARP1þ/þmouse 3T3 fibroblasts to determine the roleof PARP1 in DSB repairs and discovered that the rejoining kinetics ofneocarzinostatin-induced DSB were identical in the two cells, suggest-ing that PARP1 is not a major determinant of DSB recovery (40).Thereafter, Wang and colleagues showed the recruitment of PARP1for DSB repairs in the absence of Ku and non-DSB lesions in irradiatedcells (41). Hegan and colleagues also presented evidence that chemicalinhibitors and siRNA targeting PARP1 suppressed homology-dependent repair by inhibiting BRCA1 and Rad51 (42). In fact,increasing evidence has demonstrated that the poly(ADP-ribosyl)ation mediated by PARP plays a pivotal role in DSB repairs (10). Ourstudy showed that PARP1 promoted the repair of cisplatin-inducedDSB and positively regulated the expression of BRCA1 and Rad51 in

ovarian cancer cells. Furthermore, in vivo and in vitro experimentsshowed that PARPi could boost the toxicity of cisplatin, indicating thatPARPi might play a role in platinum resistance.

Despite the fact that PARPi can partially target tumor cells withimpairedHR repair, there is also a substantial fraction of tumors that donot respond to or eventually develop resistance to PARPi. Manyongoing studies aim to understand the fundamentals as well as thedetails of this phenomenon. Epigenetic reexpression of BRCA1, BRCA1deletion isoforms, genetic reversion of BRCA, or replicationmachinerystabilization may partially restore the HR molecular pathway andmediate PARPi resistance. Additionally, researchers showed that sev-eral miRNAs could induce PARPi resistance via balancing NHEJ andDSB repair pathways or by suppressing HR repair proteins (43). Here,we demonstrated that let-7e decreased the expression of BRCA1 andRad51 and suppressed DSB repairs through targeting PARP1. Next, wewill further investigate whether let-7e could influence the sensitivity ofovarian cancer to PARPi via inhibiting HR-dependent DNA repair.

Our previous data indicated that the CpG island adjacent to let-7e ishypermethylated, which might contribute to let-7e silencing and the

Figure 6.

The expression and clinical values of PARP1in ovarian cancer tissues. A, Immunohis-tochemistry assays detecting the expres-sion of PARP1 in ovarian cancer tissues.B, Semiquantification of PARP1 in chemore-sistant versus chemosensitive specimens.C, Expression of PARP1 in let-7e high andlow expression cohort. D, Negative corre-lation between the percentage of PARP1-positive cells and the let-7e staining score inovarian cancer tissues (Spearman correla-tion test). E–F, Kaplan–Meier survivalcurves displaying the relationship betweenPARP1 and PFS/OS in ovarian cancerpatients. Log-rank test. G–H, Associationsbetween PARP1 and the prognosis of ovar-ian cancer patients in the GEO database.MannWhitney test, � P <0.05; �� P <0.001.






development of cisplatin resistance in ovarian cancer (16). Neverthe-less, the methylation-mediated silencing of let-7e was not complete,suggesting that other mechanisms may account for the regulation oflet-7e simultaneously in ovarian cancer. Apart from DNA methyla-tion, long noncoding RNA (lncRNA) are reported to influencemiRNAexpression. H19 lncRNA is implicated inmodulating the availability ofthe let-7 family members (not including let-7e) by acting as a molec-ular sponge. Based on the potential let-7 binding sites, overexpressionof H19 specifically sequestered the endogenous let-7 levels, therebyaffecting the expression of let-7 targets (e.g., Dicer and HMGA2;ref. 44). Bioinformatics analyses revealed putative complementarysequences for let-7e in human HOTAIR, whose elevated expressionhas been shown to correlate with the chemoresistance of ovariancancer through activating Wnt/b-catenin signaling (45, 46). Wetherefore wish to investigate whether HOTAIR acts as a molecularsponge for let-7e and collectively contribute to cisplatin resistance inovarian cancer in further studies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors’ ContributionsConception and design: M. Xiao, J. Guo, Z. Wang, J. CaiDevelopment of methodology: M. Xiao, J. Guo, L. Xie, J. CaiAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): C. Yang, L. GongAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. XiaoWriting, review, and/or revision of the manuscript: M. Xiao, Z. Wang, J. CaiAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): M. Xiao, C. Yang, L. GongStudy supervision: Z. Wang, J. Cai

AcknowledgmentsWewould like to thank the support of the National Natural Science Foundation of

China (No. 81101961, 81572572, 81672573, and 81602277).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 27, 2018; revised May 26, 2019; accepted November 7, 2019;published first November 13, 2019.

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Table 1. Multivariate regression analysis of the significant prediction factors on the PFS, OS, and chemotherapy response in ovariancancer patients.

PFSa OSa Chemotherapy responseb

VariablesCaseNo. (%) HR (95% CI) P HR (95% CI) P OR (95% CI) P

FIGO stageI–II 20 (28.2) 0.380 (0.164–0.883) 0.024 0.351 (0.122–1.006) 0.051 — —

III–IV 51 (71.8) 1 1let-7e expression

Low 27 (38.0) 2.245 (1.180–4.272) 0.014 2.345 (1.127–4.880) 0.023 12.776 (2.605–62.654) 0.002High 44 (62.0) 1 1 1

BRCA1 expressionLow 25 (35.2) 0.787 (0.342–1.811) 0.573 — — 0.624 (0.074–5.248) 0.664High 46 (64.8) 1

Rad51 expressionLow 34 (47.9) 0.665 (0.298–1.484) 0.319 0.814 (0.350–1.894) 0.633 0.239 (0.032–1.764) 0.161High 37 (52.1) 1 1 1

PARP1 expressionLow 33 (46.5) 0.636 (0.328–1.233) 0.180 0.588 (0.263–1.312) 0.194 0.107 (0.018–0.650) 0.015High 38 (53.5) 1 1 1

Abbreviations: CI, confidence interval; FIGO, International Federation of Gynecology and Obstetrics; HR, hazards ratio; OR, odds ratio; OS, overall survival; PFS,progression-free survival.aMultivariate Cox proportional hazards regression analysis.bMultivariate logistic regression analysis.

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2020;18:436-447. Published OnlineFirst November 13, 2019.Mol Cancer Res Man Xiao, Jianfeng Guo, Lisha Xie, et al. Cancer to Cisplatin through Targeting PARP1Let-7e Suppresses DNA Damage Repair and Sensitizes Ovarian

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