RESEARCH Open Access

MiR-203a-3p regulates the biologicalbehaviors of ovarian cancer cells throughmediating the Akt/GSK-3β/Snail signalingpathway by targeting ATMHong-Yun Liu1, Yu-Ying Zhang1, Bao-Lian Zhu2, Fu-Zhong Feng1, Hai-Tang Zhang1, Hua Yan1 and Bin Zhou3*

Abstract

Objective: To investigate whether miR-203a-3p can regulate the biological behaviors of ovarian cancer cells bytargeting ATM to affect the Akt/GSK-3β/Snail signaling pathway.

Methods: The expression levels of miR-203a-3p and ATM were detected by qRT-PCR, immunohistochemicalstaining and Western blotting in ovarian cancer tissues and adjacent normal tissues obtained from 152 subjects. Adual-luciferase reporter gene assay was performed to verify the relationship between miR-203a-3p and ATM.Human ovarian cancer cell lines (A2780 and SKOV3) were used to generate the Blank, miR-NC, miR-203a-3p mimic,Control siRNA, ATM siRNA, and miR-203a-3p inhibitor + ATM siRNA groups. The biological behaviors of ovariancancer cells were evaluated by CCK-8, wound healing, and Transwell invasion assays, annexin V-FITC/PI staining andflow cytometry. The levels of Akt/GSK-3β/Snail pathway-related proteins were assessed by Western blotting.

Results: Ovarian cancer tissues showed lower miR-203a-3p levels and higher ATM levels than adjacent normaltissues, both of which were associated with the FIGO stage, grade and prognosis of ovarian cancer. As confirmedby a dual-luciferase reporter gene assay, miR-203a-3p could target ATM. Furthermore, the miR-203a-3p mimic hadmultiple effects, including the inhibition of the proliferation, invasion and migration of A2780 and SKOV3 cells, thepromotion of cell apoptosis, the arrest of the cell cycle at the G1 phase, and the blockage of the Akt/GSK-3β/Snailsignaling pathway. ATM siRNA had similar effects on the biological behaviors of ovarian cancer cells, and theseeffects could be reversed by a miR-203a-3p inhibitor.

Conclusion: miR-203a-3p was capable of hindering proliferation, migration, and invasion and facilitating theapoptosis of ovarian cancer cells through its modulation of the Akt/GSK-3β/Snail signaling pathway by targetingATM, and therefore it could serve as a potential therapeutic option for ovarian cancer.

Keywords: Ovarian cancer, miR-203a-3p, ATM, Akt/GSK-3β/snail

IntroductionWorldwide, ovarian cancer is an extremely lethal cancerof the female reproductive system, and there are cur-rently limited therapeutic options for its treatment [1].Of all primary ovarian tumors, approximately 90% areepithelial ovarian cancer, which can be classified patho-logically into different types, such as serous, mucinous,

endometrioid, clear-cell and transitional-cell carcinomas[2]. In recent years, nearly 70% of patients have beendiagnosed with ovarian cancer when they are in the mid-dle or advanced stage due to the lack of effective screen-ing strategies [3], resulting in a relatively low5-yearsurvival rate [4]. Given the above, a thorough under-standing of the mechanism of ovarian cancer would bebeneficial for the early diagnosis and clinical treatmentof ovarian cancer.MicroRNAs (miRNA) are noncoding RNAs derived from

endogenous chromosomes that consist of approximately 22

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: zhouzhouzb@hotmail.com3Department of Rehabilitation Medicine, Linyi Central Hospital, No.17,Jiankang Road, Linyi 276400, Shandong, ChinaFull list of author information is available at the end of the article

Liu et al. Journal of Ovarian Research (2019) 12:60 https://doi.org/10.1186/s13048-019-0532-2

nucleotides [5]. Recently, there has been wide agreementthat a variety of miRNAs are expressed in an abnormalfashion and function as oncogenes or tumor suppressorgenesin ovarian cancer and are therefore potential targetsfor the diagnosis and treatment of tumors [6, 7]. MiR-203ais located in the human chromosome region 14q32, whichis an unstable region that contains approximately 12% of alldiscovered microRNA genes [8, 9]. A recent study reportedthat miR-203a-3p was poorly expressed in multiple mye-loma [10], gastric cancer [8], bladder cancer [11], but that itwas highly expressed in breast cancer [12], hepatocellularcarcinoma [13], and nasopharyngeal carcinoma [14]. How-ever, the expression of miR-203a-3p in ovarian cancerremains to be elucidated, and its mechanism also needs tobe clarified. To the best of our knowledge, miRNA couldplay a role based on its sequence complementarities withthe sequences of the 3′ UTRs in mRNAs from target genes,and the complementary pairing of specific bases causes thedegradation or inhibits the translation of target mRNA,thereby regulating gene expression at the posttranscrip-tional level [15]. Notably, miR-203a-3p was shown to haveregulatory effects in oral squamous cell carcinoma (OSCC)and glioma via targeting ATM [16, 17], which is a serine/threonine protein kinase located in chromosome region11q22–23 [18].In actuality, the abnormal expression of theATM gene has strong implications for the clinical diagnosisof ovarian cancer, and the overexpression of ATM has beendiscovered to be closely associated with poor prognosis inovarian cancer patients [19]. More importantly, ATM hasbeen revealed to mediate the Akt/GSK-3β/Snail signalingpathway to influence the metastasis of ovarian cancer [20].Nevertheless, it is still unknown whether miR-203a-3p cantarget the ATM-mediated Akt/GSK-3β/Snail pathway toaffect the onset and development of ovarian cancer. There-fore, we detected the expression of miR-203a-3p and ATMin ovarian cancer tissues and adjacent normal tissues fromclinical patients and then analyzed their correlations withthe clinicopathological characteristics and prognosis inthose patients. The A2780 cell line was selected for the invitro experiments to determine whetherand how miR-203a-3p can influence the biological characteristics ofovarian cancer cells through the modulation of the ATM-mediatedAkt/GSK-3β/Snail pathway.

Materials and methodsEthics statementAll patients in this study signed an informed consentform prior to the study, and all experiments gained theapproval of the Ethics Committee for Clinical Experi-ments at Linyi Central Hospital.

Study subjectsFrom January 2011 to January 2013, 152 ovarian cancerpatients were recruited into this study after pathological

diagnosis, and ovarian cancer tissues and adjacent nor-mal tissues were collected from them for subsequentexperiments. The age of patients was 55.84 ± 16.04 years(median = 55 years). There were 51 cases at stage I-IIAand 101 cases at stage IIB-IV according to the FIGOstaging system [21], and there were 26 cases at G1, 58cases at G2, and 68 cases at G3 based on the tumor dif-ferentiation grade [22]. None of the patients underwentradiotherapy, hormone therapy, or other therapies andwere prepared for surgery after complete examinationsup on admission. The selected tissues were preserved ina cryopreservation tube and stored in a liquid nitrogentank for later experiments.

qRT-PCRTotal RNA was extracted by TRIzol reagent, and theRNA concentration and purity was determined with aNanoDrop2000 (Thermo, Waltham, MA, USA), afterwhich the RNA was preserved at -80 °C. The primerswere designed by using Primer 5.0 according to se-quences published in the GenBank database and synthe-sized by Shanghai GenePharma Co., Ltd. (Shanghai,China). The ABI PRISM 7500 Real-Time PCR system(ABI, USA) and SYBR Green I Fluorescent Kit(DRR041A, Takara) were used to perform PCR. UsingU6/GAPDH as internal reference genes, the relativeexpression of the target genes was calculated using the2-△△Ct method. Independent experiments were con-ducted three times.

Immunohistochemical (IHC) stainingThe paraffin-embedded tissue sections were deparaffi-nized, cultured in 3%H2O2 at room temperature for 10min, and washed carefully with distilled water beforesoaking in PBS buffer 2 times for 5 min each. Then, thetissue sections were blocked in 10% normal goat serumdiluted in PBS for an appropriate duration and incu-bated at room temperature for 30 min.Then, the primaryantibodies were added overnight and incubated at 4 °C,followed by washing with PBS buffer 3 times for 5 mineach. Subsequently, these condary antibodies were addedfor 2 h of incubation at 37 °C, followed by washing withPBS buffer 3 times for 5 min each and development withDAB. The tissue sections were then rinsed with tapwater, lightly counterstained with hematoxylin, and sub-ject to dehydration, hyalinization and section mounting.The sections were observed and photographed under amicroscope. Two pathologists independently scored theIHC staining in a double-blind manner on the basis ofthe staining intensity and the number of positive cells.The staining intensity was scored as 0, 1, 2 or 3 to indi-cate absent, weak, moderate or strong staining, respect-ively, and the percentage of positive cells was graded as0, 1, 2, 3, 4 or 5 indicate 0%, 1–5%, 5–25%, 25–50%, 50–

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75% or 75–100% staining, respectively. The two scoreswere multiplied to evaluate the staining results.

Dual-luciferase reporter gene assayThe human ATM 3′-UTR fragment containing the bind-ing site of miR-203a-3p in the 3′-UTR region was ampli-fied and inserted into the TopMIR-Report vector toconstruct the wild-type plasmid ATM-WTand the mutantplasmid ATM-Mut. The combinations used for co-transfection were as follows: miR-NC/miR-203a-3pmimic/miR-203a-3p inhibitor + ATM-WT and miR-NC/miR-203a-3p mimic/miR-203a-3p inhibitor + ATM-Mut.The transfection of the human ovarian cancer cell lines(A2780 and SKOV3) was conducted in accordance withthe instructions included with the Lipofectamine 2000 kit(11668–027, Invitrogen, Carlsbad, CA, USA). Thelucifer-ase activity of the cells was determined by a dual-luciferase reporter gene assay (Promega, Madison, WI,USA).

Cell groupingsHuman ovarian cancer cell lines (A2780 and SKOV3)were obtained from the Cell Bank of the Chinese Acad-emy of Sciences and cultured in DMEM supplementedwith 10% fetal bovine serum and 100 units/ml penicillin-streptomycin in an incubator at 37 °C in 5% CO2. Whencells covered 80% of the visual field under amicroscope,they were digested with 0.25% trypsin and passaged. Then,the cells were divided and assigned to the Blank, miR-NC,miR-203a-3p mimic, Control siRNA, ATM siRNA, andmiR-203a-3p inhibitor + ATM siRNA groups. In thisstudy, the miR-203a-3p inhibitor (Catalog #: 4464084), themiR-203a-3p mimic (Catalog #: 4464066) and the miRNAmimic negative control (Catalog #:4464058) were pur-chased from Fisher Scientific (Ottawa, Ontario, Canada).The control siRNA (Catalog #:sc-37,007) and ATM siRNA(Catalog #:sc-29,761) were provided by Santa Cruz Bio-technology (Santa Cruz, CA, USA).

Western blot analysisThe proteins were extracted with a BCA kit (Pierce,Rockford, IL, USA),and their concentration was deter-mined. Then, loading buffer was added to the extractedproteins and heated for 10 min at 95 °C. Electrophoresiswas used for the separation of the proteins in a 10%polyacrylamide gel with a voltage of 80 V for concentra-tion and 120 V for separation. A wet transfer was usedtotransfer the proteins to a polyvinylidene fluoride (PVDF)membrane, which was placed in 5% BSA at roomtemperature for 1 h for blocking, followed by incubationwith the primary antibodies overnightat 4 °C. Next, themembrane was washed with TBST 3 times for 5 mineach. Later, the secondary antibodies were added andincubated for another 1 h, and the membrane was rinsed

again with TBST 3 times for 5 min each. Finally, chemi-luminescent detection was used for development. Theratio of the gray value of the target band to that of thereference band was used to measure the relative expres-sion level of the proteins, and GAPDH was used as theloading control. Image J software was used for the grayvalue analysis, and the experiment was repeated threetimes.

CCK-8 assayCells collected at the logarithmic growth phase weredigested with 0.25% trypsin, and the cell suspension wasinoculated into 96-well plates at104 cells/well (100 μl).The wells on the margin area were filled with sterilePBS, and the cells were incubated accordingly. Whenthe cell confluence reached 80%, the cells were dividedinto groups as described above before culture for 12 h,24 h, 36 h, 48 h and 72 h. Next, 10 μl CCK solution wasadded to each well, and cells were cultured at 37 °C for4 h in an incubator with 5%CO2. The optical density(OD) value was determined with a microplate reader ata wavelength of 450 nm.

Wound healing assayOnce cells had adhered to the wall and covered the bot-tom of the plate, a sterilized pipette tip was used to drawa line across the plate at the time recorded as 0 h. It isimportant to ensure a consistent width for each scratchand to mark the cover of the 6-well plate, which wasphotographed to facilitate the relocation of the same vis-ual field. After incubation at37°Cfor 24 h, the culturemedium was removed, and the plate was washed withPBS buffer three times. The cell debris produced duringscratching was washed away, followed by the addition ofserum-free culture medium and imaging at 24 h. Theimages were obtained with an Olympus inverted micro-scope (CKX31, Japan), and the experiment was repeatedindependently three times.

Transwell invasion assayMatrigel was melted and added into a Transwell cham-ber. Cell plates containing Transwell chambers were in-cubated at37°C for 30 min. Preheated media was addedto the cell culture plate, which was placed in an incuba-tor for 2 h of hydration at 37oC. Next, the liquid in theupper and lower chambers was carefully removed, and0.5 ml cell suspension at a density of 5 × 104cells/ml wasadded into each Transwell chamber for 24 h of incuba-tion at 37oC. Then, each chamber was removed to wipeaway cells on the upper side, which was then washedwith PBS buffer three times and soaked in precooledmethanol for 30 min. The cells that had migrated intothe lower chamber were fixed and incubated in 1% crys-tal violet solution for 10 min for staining. Running water

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was used to wash away the crystal violet solution, andthe chamber was removed for drying. The cells werestained for 10 min and washed with flowing water. Afterthorough rinsing, the cells were removed for drying. AnOlympus inverted microscope was used to observe andphotograph the cells that had migrated into the lowerchamber of the Transwell system. A statistical analysiswas carried out, and the experiment was repeated threetimes independently.

Annexin V-FITC/PI stainingFirst, the cell culture medium in a 6-well plate was re-moved, placed in a centrifuge tube, washed with PBSbuffer, and digested with 0.25% trypsin. Next, a singlecell suspension (1 × 106 cells/ml) was generated by re-placing the digestion solution, followed by 5 min of cen-trifugation at 12000 rpmat 4 °C. Then, 100 μl of cellsuspension allowed to warm to room temperature,mixed with propidium iodide (PI, 10 mg/ml) and RNaseA (10 mg/ml), and incubated for 30 min at 4 °C. Thesuspension was detected by a flow cytometer (BD Biosci-ences, San Diego, CA, USA) immediately after theaddition of 400 μl staining buffer, and Cell Quest soft-ware was used for the data analysis.The experiment wasrepeated at least three times.

Flow cytometryApproximately 48 h after transfection, the cells were col-lected, digested with 0.25% trypsin, and adjusted to adensity of 1 × 106 cells/mL. Then, the cell suspension (1mL) was centrifuged at 1500 rpm for 10 min, and thesupernatant was discarded, followed by the addition of 2mL PBS for each milliliter of cells. After centrifugation,the supernatant was discarded, and precooled 70% etha-nol was used to fix cells overnight at 4 °C. The next day,the cells were washed twice with PBS buffer,100 μL ofcell suspension was removed, and 50 μg of RNase-containing PI solution was added and incubated for 30min in the dark. Finally, the cells were filtered with a100-mesh nylon mesh, and a cell cycle analysis wasperformed with a flow cytometer (BD Biosciences, SanDiego, CA, USA).

Statistical analysisThe measurement data are presented as the mean ±standard deviation (SD) and were analyzed using thestatistical software SPSS21.0 (SPSS Inc. Chicago, IL,USA). One-way ANOVA was used for comparisonsamong multiple groups followed by Tukey’s multiplecomparison test. The expression of miR-203a-3p andATM in ovarian cancer tissues and adjacent normal tis-sues was analyzed by a paired-sample t-test, and theircorrelations with the clinicopathological characteristicsof patients was tested by Student’s t-test or a

nonparametric rank sum test. The correlation of miR-203a-3p expression with ATM expression was verifiedusing Pearson’s correlation analysis, and survival curvesfrom the different groups were compared using theKaplan-Meier method and the log-rank test.A Cox pro-portional hazards model was constructed to estimate theprognostic factors that influenced quality of life in ovar-ian cancer patients. Statistical significance was indicatedwhenP < 0.05.

ResultsExpression of miR-203a-3p and ATM in ovarian cancertissues and adjacent normal tissuesAccording to the results, ovarian cancer tissues exhibitedreduced miR-203a-3p expression and increased ATMexpression compared to adjacent normal tissues (bothP < 0.05, Fig. 1a). The correlation analysis showed anegative correlation for miR-203a-3p expression andATM expression (r = − 0.698, P < 0.001, Fig. 1b). Westernblotting was performed, which found the same expres-sion trend for ATM protein expression as was observedfor ATM mRNA expression (Fig. 1c-d). When we evalu-ated the positive ATM expression with immunohisto-chemical staining, we observed that the positive ATMexpression rate was higher in ovarian cancer tissues(58.55%, 89/152) than in adjacent normal tissues(13.82%, 21/152) (P < 0.05, Fig. 1e-f ).

Association of miR-203a-3p and ATM expression with theclinicopathological features of ovarian cancerThe expression of miR-203a-3p and ATM in ovariancancer tissues was not significantly different based onage or histology (all P > 0.05) but was closely associatedwith the FIGO stage and tumor grade in ovarian cancerpatients (all P < 0.05). The higher the FIGO stage wasand the less differentiation was found in the tumor, thelower the miR-203a-3p level would be and the highertheATM level would be (Table 1).

Effect of miR-203a-3p and ATM expression on theprognosis of ovarian cancerKaplan-Meier survival analysis demonstrated that the prog-nosis of ovarian cancer patients was closely related to FIGOstage and grade but not to age and histology (Fig. 2). Inaddition, patients with high miR-203a-3p expression (≥0.390) had longer survival times than those with low miR-203a-3p expression (< 0.390), and the mean survival timeswere 48.77 ± 1.85months and 37.99 ± 2.10months, respect-ively (χ2 = 12.690, P = 3.68E-4), for patients with high andlow miR-203a-3p expression. In addition, the survival timeof patients with low ATM expression (≥3.721) was longerthan that of patients with low ATM expression (< 3.721)(50.36 ± 1.60months vs. 36.54 ± 2.18months, respectively,χ2 = 19.520, P = 9.95E-6). Multivariate Cox regression

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Fig. 1 Expression of miR-203a-3p and ATM in ovarian cancer tissues and adjacent normal tissues. Note: a The expression levels of miR-203a-3pand ATM in ovarian cancer tissues and adjacent normal tissues detected by qRT-PCR; b The correlation between miR-203a-3p expression and ATMexpression in ovarian cancer tissues; c-d ATM protein expression levels in ovarian cancer tissues and adjacent normal tissues examined byWestern blot; e-f, Positive ATM expression in ovarian cancer tissues and adjacent normal tissues detected by immunohistochemical staining

Table 1 Association of miR-203a-3p and ATM with clinicopathological features of ovarian cancer patientsCharactersitics N miR-203a-3p P ATM P

Age (years)

≤ 55 75 0.39 ± 0.03 3.73 ± 0.31

> 55 77 0.39 ± 0.03 0.528 3.67 ± 0.31 0.227

FIGO stage

Early (I-IIA) 51 0.41 ± 0.02 3.58 ± 0.29

Advanced (IIB-IV) 101 0.38 ± 0.02 5.14E-13 3.76 ± 0.30 0.001

Histology

serous 76 0.39 ± 0.02 3.70 ± 0.28

non-serous 76 0.39 ± 0.03 0.740 3.70 ± 0.34 0.970

Grade

1 26 0.43 ± 0.02 3.32 ± 0.22

2 58 0.40 ± 0.01* 3.72 ± 0.21*

3 68 0.37 ± 0.02*# 1.80E-39 3.83 ± 0.29*# 2.08E-14

Note: *, P < 0.05 compared with Grade 1; #, P < 0.05 compared with Grade 2

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analysis showed that ATM expression was an independentrisk factor for the prognosis of ovarian cancer (HR: 2.579,95%CI: 1.581~4.207, P = 1.49E-04, Table 2).

MiR-203a-3p could target ATMBased on the information generated by the bioinformaticsanalysis tools miRDB (http://mirdb.org/cgi-bin/target_de-tail.cgi?targetID=3441169) and microRNA.org (http://www.microrna.org/microrna/getMrna.do?gene=472&utr=14588

&organism=9606#hd), ATM was a downstream target geneof miR-203a-3p (Fig. 3a-b). As confirmed by the dual-luciferase reporter gene assay, within the ATM-WT groups,the miR-203a-3p mimic group showed decreased luciferaseactivity, and the miR-203a-3p inhibitor group demonstratedincreased luciferase activity in comparison with the miR-NC group (all P < 0.05). Nevertheless, no significantdifference was observed in luciferase activity among theATM-Mut groups (all P > 0.05,Fig. 3c).

Fig. 2 The prognostic analysis of 152 ovarian cancer patients. Note: a Age; b Histology; c FIGO stage; d Grade; e miR-203a-3p; f ATM expression

Table 2 Univariate and multivariate Cox regression models to analyze the prognostic factors of ovarian cancer patients

Charactersitics Univariate analysis Multivariate analysis

HR 95%CI P HR 95%CI P

Age

> 55 vs. ≤ 55 0.877 0.568~1.354 0.555 1.046 0.673~1.625 0.841

Histology

Serous vs. Non-serous 0.933 0.605~1.438 0.753 0.855 0.548~1.334 0.489

FIGO stage

Advanced vs. Early 1.654 1.021~2.678 0.041 1.157 0.609~2.198 0.656

Grade

G2 vs.G1 1.762 0.803~3.867 0.158 1.060 0.447~2.511 0.895

G3 vs.G1 3.428 1.614~7.280 0.001 2.082 0.762~5.688 0.153

miR-203a-3p expression

Low vs. High 2.189 1.401~3.419 0.001 1.060 0.470~2.389 0.889

ATM expression

High vs. Low 2.655 1.685~4.183 2.55E-05 2.579 1.581~4.207 1.49E-04

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Comparison of miR-203a-3p and ATM expression inovarian cancer cellsAs shown in Fig. 4, the miR-203a-3p mimic group hadincreased miR-203a-3p and decreased ATM, while themiR-203a-3p inhibitor + ATM siRNA group exhibiteddecreased miR-203a-3p compared with the miR-NCgroup (all P < 0.05). When compared to the controlsiRNA group, the ATM siRNA group presented reducedATM (P < 0.05), but no observable difference was foundin miR-203a-3p expression (P > 0.05). However, therewas no significant changein ATM expression among themiR-NC, Control siRNA, and miR-203a-3p inhibitor +ATM siRNA groups (all P > 0.05).

Effects of miR-203a-3p on proliferation, apoptosis and thecell cycle in ovarian cancer cells via its targeting of ATMCompared with that observed in the miR-NC group andthe Control siRNA group, the miR-203a-3p mimic andthe ATM siRNA reduced the proliferation and promotedthe apoptosis of A2780 and SKOV3 cells (all P < 0.05).

However, there was no obvious difference among theBlank group, miR-NC group, Control siRNA group, andmiR-203a-3p inhibitor + ATM siRNA group in terms ofthe proliferation and apoptosis rate of cells (all P > 0.05,Fig. 5). Furthermore, the numbers of G1 phase cells weresignificantly increased in the miR-203a-3p mimic groupand the number of S phase cells was remarkably de-creased in the ATM siRNA group (all P < 0.05). Whencompared with the ATM siRNA group, the number ofG1 phase cells declined and the number of S phase cellsincreased in the miR-203a-3p inhibitor + ATM siRNAgroup (all P < 0.05, Fig. 6).

Effect of the targeting of ATM by miR-203a-3p on themigration and invasion of ovarian cancer cellsAccording to the results of the wound healing assay andthe Transwell invasion assay (Fig. 7), the cells in themiR-NC group showed no significant changes in termsof migration distance or invasive cell numbers comparedwith those in the Blank group (both P > 0.05), while

Fig. 3 Targeted regulation of ATM by miR-203a-3p. Note: a-b, The binding sites of miR-203a-3p in ATM as predicted by the public databases miRDB(a) and microRNA.org (b); c-d, miR-203a-3p regulates ATM expression, as confirmed by dual-luciferase reporter gene assays in A2780 cells (c) andSKOV3cells (d). The same letters indicate nonsignificant differences, P > 0.05, and the different letters indicate statistically significant differences, P < 0.05

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those in the miR-203a-3p mimic group and the si-ATMgroup showed increased migration distances and de-creased invasive cell numbers (all P < 0.05). However,when compared with those in the miR-203a-3p inhibitorgroup, the cells in the miR-203a-3p inhibitor + ATMsiRNA group showed clear reductions in cell migrationand invasion (both P < 0.05).

Effect of the targeting of ATM by miR-203a-3p on theAkt/GSK-3β/snail signaling pathwayCompared with the Blank group, the miR-NC group andAkt/GSK-3β/Snail group showed no significant differencesin the expression of Akt/GSK-3β/Snail pathway-relatedproteins (all P > 0.05). After cells were transfected with themiR-203a-3p mimic or ATM siRNA, the expression levelsof p-AKT, p-GSK-3β and Snail were all remarkably de-creased (all P < 0.05). In addition, when compared to theATM siRNA group, the miR-203a-3p inhibitor + ATMsiRNA group exhibited upregulated expression of p-AKT,p-GSK-3β and Snail (all P < 0.05, Fig. 8).

DiscussionMiR-203, as the first keratinocyte-specific miRNA thatwas discovered [23], was proven in a recent study toexert a significant influence on the occurrence and pro-gression of bladder cancer [24], and its epigenetic silen-cingwas also observed in hematopoietic malignancies[9]. To date, accumulating evidence has revealed thatmiR-203 can effectively regulate chemotherapeutic re-sistance to cisplatin [25], cell invasiveness [26], cell pro-liferation [27] and metastasis [28], which could also

allow it to function as a biomarker to predict prognosisin certain diseases [29].An important finding of this study was that miR-203a-

3p expression in ovarian cancer tissues was lower thanthat in adjacent normal tissues, and it had an importanteffect on the FIGO stage, tumor grade and prognosis ofpatients. A similar study by Liu Y et al. also revealed thatthe downregulation of miR-203a was closely correlatedto the TNM stage, the degree of pathological differenti-ation and lymph node metastasis in esophageal squa-mous cell carcinoma (ESCC) [30]. In addition, miR-203adeletion indicated a poor prognosis for both hepatocellu-lar carcinoma (HCC) and gastric cardia adenocarcinoma(GCA) patients [31, 32]. As previously reported, the pro-moter region of miR-203a contains a CPG island that is875 bp in length [33] and is located in the proximalpromoter region [30]. In addition, the methylation ofmiR-203a has been found to be tumor-specific, sincemiR-203a methylation was only observed in cancer cellsbut not in normal cells [9]. The hypermethylation ofmiR-203a can promote the proliferation of chronic mye-logenous leukemia (CML) cells by inhibiting the carcino-genic BCR-ABL fusion protein, suggesting that miR-203a hypermethylation is carcinogenic in CML [34]. Ithas been proven that hypermethylation of the proximalpromoter of miR-203a can affect its transcriptional activ-ity [30], and the methylation rate of CpG islands in miR-203 was significantly negatively correlated with theexpression of miR-203 [32, 35]. In one particular in-stance, the hypermethylation of miR-203a was appar-ently associated with the invasion and metastasis of

Fig. 4 Comparison of miR-203a-3p and ATM expression in ovarian cancer cells. Note: a-b, The expression of miR-203a-3p and ATM in A2780 cells(a) and SKOV3 cells (b) detected by qRT-PCR; c-d, ATM protein expression in ovarian cancer cells (A2780 and SKOV3) detected by Western blot;1,Blank group; 2, miR-NC group; 3, miR-203a-3p mimic group; 4, Control siRNA group; 5, ATM siRNA group; 6, miR-203a-3p inhibitor + ATM siRNAgroup. The same letters indicate nonsignificant differences, P > 0.05, and the different letters indicate statistically significant differences, P < 0.05

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ovarian cancer, as suggested by LoginovVI et al. [36].However, we did not detect the methylation of miR-203a-3p in ovarian cancer tissues in this study due tolimitations in time and funding, but this will be exploredin future experiments. Currently, accumulating evidencealso highlights the protective role of the overexpressionof miR-203a in various tumors. For instance, in bladdercancer, the overexpression of miR-203a contributed toincreases in cell proliferation, invasion, and migration,the inhibition of EMT, the arrest of the cell cycle, and anincrease in apoptosis in cells [11]. Similarly, the findingsof our study also demonstrated that the miR-203a-3pmimic could effectively inhibit ovarian cancer cell prolif-eration, invasion, and migration, accelerate cell

apoptosis, and cause cell cycle arrest at the G1 phase,which further confirmed the anti-oncogenic function ofmiR-203a-3p in ovarian cancer.ATM is a protein consisting of 3056 amino acids that

has a molecular weight of 350 kDa [37]. Its activationnot only has an important effect on responses to DNAdouble-strand breakages in cells but also shows a closeassociation with the occurrence and development oftumors. For example, the mRNA and protein levels ofATM were found to be dramatically higher in nasopha-ryngeal carcinoma tissues thanin adjacent normal tissues[38]. In addition, the upregulation of ATM may be amajor cause of therapeutic resistance in ovarian cancer[19]. In our study, high ATM expression was correlated

A

C

D E

B

Fig. 5 Targeting of ATM by miR-203a-3p modulates the proliferation and apoptosis of ovarian cancer cells. Note: A-B, CCK-8 assays detected theproliferation of A2780 cells (a) and SKOV3 cells (b) in each group; c-e, Annexin V-FITC/PI staining evaluated the apoptosis of A2780 cells (c, d) andSKOV3 cells (c, e) in each group. The same letters indicate nonsignificant differences, P > 0.05, and the different letters indicate statisticallysignificant differences, P < 0.05

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with clinicopathological features and poor prognosis ofovarian cancer patients, whereas the inhibition of ATMexpression is expected to become a potential therapy forpatients with ovarian cancer. Another study reportedthat the ATM inhibitor KU-55933 can hinder cancer cellproliferation by inducing G1 blockade and therebytriggering apoptosis in cancer cells [39]. Liu Ret al. alsorevealed that ATM deletion could dramatically decreasethe proliferation, migration and invasion of colon cancercells [40], which could also lead to a remarkable reduc-tion in migration and epithelial-mesenchymal transition(EMT) in prostate cancer cells [41]. In our experiment,

ATM was confirmed to be the downstream target geneof miR-203a-3p through bioinformatics prediction and adual-luciferase reporter gene assay. At the same time, wedid finda negative relationship between ATM and miR-203a-3p in our clinical investigation, and the higherexpression of ATM predicted a poor prognosis in ovar-ian cancer patients according to a univariate Cox regres-sion model, highlighting the possible protective role ofmiR-203a-3p in ovarian cancer via its mediation ofATM. However, the results of the multivariate analysisshowed that ATM was associated with prognosis, whilemiR-203a-3p was not associated with prognosis. The

Fig. 6 MiR-203a-3pmodulates the cell cycle of A2780 cells (a-b) and SKOV3cells (c-d) by targeting ATM

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possible reason for this finding might be the small sam-ple size used in this study, the limited clinical features,and the activity of other potential regulators of ATM ortargets of miR-203a-3p. Notably, activated ATM can

cause the binding of HSP90 to HER2 and then block theubiquitination and degradation of HER2 to maintain itsstability, and it can eventually regulate itsdownstreamAkt activity to affectthe malignancy of breast cancer

Fig. 7 Effect of miR-203a-3p on the migration and invasion of A2780 cells (a-c) and SKOV3 cells (d-e) by targeting ATM. Note: The same lettersindicate nonsignificant differences, P > 0.05, and the different letters indicate statistically significant differences, P < 0.05

Fig. 8 Effect of miR-203a-3p on Akt/GSK-3β/Snail signaling pathway via its targeting of ATM in A2780 cells (a-b) and SKOV3 cells (c-d). Note: Thesame letters indicate nonsignificant differences, P > 0.05, and the different letters indicate statistically significant differences, P < 0.05

Liu et al. Journal of Ovarian Research (2019) 12:60 Page 11 of 13

cells [42]. Moreover, in malignant glioma cells, theATM-Akt signaling pathway could mediate cell migra-tion, as discovered by Golding et al. [43].There was alsoevidence that a specific inhibitor of ATM (KU55933)could effectively inhibit cell migration induced by sorafe-nib, a chemotherapeutic agent, in HCC cells [44]. Onthe other hand, GSK-3β, which is a downstream gene inthe AKT signaling pathway, can lead to the phosphoryl-ation of the Snail transcription factor, thus regulatingEMT and participating in tumorinvasion and metastasis[45–47].Additionally, Mianen and colleagues also dem-onstrated that ATM was highly activated in breast can-cer tissues with advanced lymph node metastasis, andATM was associated with the positive expression ofSnail, which is an EMT-related molecule [48]. Their invitro experiments confirmed that activated ATM couldphosphorylate Snail and lead HSP90 to bind to Snail tomaintain its stability, thereby promoting EMT trans-formation and lymph node metastasis.Through Westernblot analysis, we found that the expression levels of p-AKT, p-GSK-3β and Snail were all decreased dramaticallyafter transfection with the miR-203a-3p mimic or ATMsiRNA. However, those cells that were cotransfected withthe miR-203a-3p inhibitor and ATM siRNA showed nosignificant differences when compared to nontransfectedcells in terms of those indicators, suggesting that miR-203a-3p can target ATM to mediate the Akt/GSK-3β/Snailsignaling pathway in ovarian cancer.

ConclusionIn conclusion, miR-203a-3p was downregulated andATM was upregulated in ovarian cancer tissues. Inaddition, because the biological behaviors of ovariancancer cells can be modulated by miR-203a-3p throughits effects on the Akt/GSK-3β/Snail signaling pathwayvia ATM, miR-203a-3p upregulation is expected to pro-vide a beneficial strategy for the clinical treatment ofovarian cancer.

AcknowledgmentsWe thank all the reviewers for their helpful suggestions and commentsabout our study.

Authors’ contributionsHYL and YYZ conceived and designed the study. BLZ performed theexperiments. FZF interpreted the data and corrected the manuscript. HTZwrote the manuscript. HY corrected the manuscript and critically revised themanuscript. BZ participated in the revision of the manuscript. All authorsapproved the final manuscript.

FundingNone.

Availability of data and materialsAll data generated and analyzed during this work were included in thisarticle.

Ethics approval and consent to participateAll the patients in this study signed the informed consent form prior to thestudy, and all experiments gained the approval of the Ethics Committee forClinical Experiments at Linyi Central Hospital.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Obstetrics and Gynecology, Linyi Central Hospital, Linyi276400, Shandong, China. 2Department of Infection, Linyi Central Hospital,Linyi 276400, Shandong, China. 3Department of Rehabilitation Medicine, LinyiCentral Hospital, No.17, Jiankang Road, Linyi 276400, Shandong, China.

Received: 6 January 2019 Accepted: 12 June 2019

References1. Agostini A, Panagopoulos I, Davidson B, Trope CG, Heim S, Micci F. A novel

truncated form of HMGA2 in tumors of the ovaries. Oncol Lett. 2016;12:1559–63.

2. Prat J. Ovarian carcinomas: five distinct diseases with different origins,genetic alterations, and clinicopathological features. Virchows Arch. 2012;460:237–49.

3. Ni X, Zhang W, Huang KC, Wang Y, Ng SK, Mok SC, Berkowitz RS, Ng SW.Characterisation of human kallikrein 6/protease M expression in ovariancancer. Br J Cancer. 2004;91:725–31.

4. Teng Z, Han R, Huang X, Zhou J, Yang J, Luo P, Wu M. Increase of incidenceand mortality of ovarian Cancer during 2003-2012 in Jiangsu Province,China. Front Public Health. 2016;4:146.

5. Wilson KD, Hu S, Venkatasubrahmanyam S, Fu JD, Sun N, Abilez OJ, BaughJJ, Jia F, Ghosh Z, Li RA, Butte AJ, Wu JC. Dynamic microRNA expressionprograms during cardiac differentiation of human embryonic stem cells:role for miR-499. Circ Cardiovasc Genet. 2010;3:426–35.

6. Zheng H, Zhang L, Zhao Y, Yang D, Song F, Wen Y, Hao Q, Hu Z, Zhang W,Chen K. Plasma miRNAs as diagnostic and prognostic biomarkers forovarian cancer. PLoS One. 2013;8:e77853.

7. Nagaraj AB, Joseph P, DiFeo A. miRNAs as prognostic and therapeutic toolsin epithelial ovarian cancer. Biomark Med. 2015;9:241–57.

8. Wang Z, Zhao Z, Yang Y, Luo M, Zhang M, Wang X, Liu L, Hou N, Guo Q,Song T, Guo B, Huang C. MiR-99b-5p and miR-203a-3p function as tumorsuppressors by targeting IGF-1R in gastric Cancer. Sci Rep. 2018;8:10119.

9. Bueno MJ, Perez de Castro I, Gomez de Cedron M, Santos J, Calin GA,Cigudosa JC, Croce CM, Fernandez-Piqueras J, Malumbres M. Genetic andepigenetic silencing of microRNA-203 enhances ABL1 and BCR-ABL1oncogene expression. Cancer Cell. 2008;13:496–506.

10. Yang M, Zhang L, Wang X, Zhou Y, Wu S. Down-regulation of miR-203a bylncRNA PVT1 in multiple myeloma promotes cell proliferation. Arch Med Sci.2018;14:1333–9.

11. Na XY, Shang XS, Zhao Y, Ren PP, Hu XQ. MiR-203a functions as a tumorsuppressor in bladder cancer by targeting SIX4. Neoplasma. 2018;66:211-21.

12. Gomes BC, Martins M, Lopes P, Morujao I, Oliveira M, Araujo A, Rueff J,Rodrigues AS. Prognostic value of microRNA-203a expression in breastcancer. Oncol Rep. 2016;36:1748–56.

13. Huo W, Du M, Pan X, Zhu X, Gao Y, Li Z. miR-203a-3p.1 targets IL-24 tomodulate hepatocellular carcinoma cell growth and metastasis. FEBS OpenBio. 2017;7:1085–91.

14. Jiang Q, Zhou Y, Yang H, Li L, Deng X, Cheng C, Xie Y, Luo X, Fang W, Liu Z.A directly negative interaction of miR-203 and ZEB2 modulates tumorstemness and chemotherapy resistance in nasopharyngeal carcinoma.Oncotarget. 2016;7:67288–301.

15. Pei Y, Wang X, Zhang X. Predicting the fate of microRNA target genesbased on sequence features. J Theor Biol. 2009;261:17–22.

16. Shieh TM, Huang YT, Yu CC, Hsia SM, Shih YH, Wang TH. P0028Isoliquiritigenin causes DNA damage and inhibits ATM expression in oralsquamous cell carcinoma. Eur J Cancer. 2015;51:e8–9.

Liu et al. Journal of Ovarian Research (2019) 12:60 Page 12 of 13

17. Yang CH, Wang Y, Sims M, Cai C, He P, Hacker H, Yue J, Cheng J, Boop FA,Pfeffer LM. MicroRNA203a suppresses glioma tumorigenesis through an ATM-dependent interferon response pathway. Oncotarget. 2017;8:112980–91.

18. Filippi AR, Franco P, Galliano M, Ricardi U. Peripheral blood completeremission after splenic irradiation in mantle-cell lymphoma with 11q22-23deletion and ATM inactivation. Radiat Oncol. 2006;1:35.

19. Abdel-Fatah TM, Arora A, Moseley P, Coveney C, Perry C, Johnson K, Kent C,Ball G, Chan S, Madhusudan S. ATM, ATR and DNA-PKcs expressionscorrelate to adverse clinical outcomes in epithelial ovarian cancers. BBA Clin.2014;2:10–7.

20. Yin S, Wang P, Yang L, Liu Y, Wang Y, Liu M, Qi Z, Meng J, Shi TY, Yang G,Zang R. Wip1 suppresses ovarian cancer metastasis through the ATM/AKT/snail mediated signaling. Oncotarget. 2016;7:29359–70.

21. Zeppernick F, Meinhold-Heerlein I. The new FIGO staging system forovarian, fallopian tube, and primary peritoneal cancer. Arch GynecolObstet.2014;290:839–42.

22. Rosen DG, Yang G, Liu G, Mercado-Uribe I, Chang B, Xiao XS, Zheng J, XueFX, Liu J. Ovarian cancer: pathology, biology, and disease models. FrontBiosci (Landmark Ed). 2009;14:2089–102.

23. Sonkoly E, Wei T, Janson PC, Saaf A, Lundeberg L, Tengvall-Linder M,Norstedt G, Alenius H, Homey B, Scheynius A, Stahle M, Pivarcsi A.MicroRNAs: novel regulators involved in the pathogenesis of psoriasis? PLoSOne. 2007;2:e610.

24. Gottardo F, Liu CG, Ferracin M, Calin GA, Fassan M, Bassi P, Sevignani C,Byrne D, Negrini M, Pagano F, Gomella LG, Croce CM, Baffa R. Micro-RNAprofiling in kidney and bladder cancers. Urol Oncol. 2007;25:387–92.

25. Ru P, Steele R, Hsueh EC, Ray RB. Anti-miR-203 upregulates SOCS3expression in breast Cancer cells and enhances cisplatin Chemosensitivity.Genes Cancer. 2011;2:720–7.

26. Chen T, Xu C, Chen J, Ding C, Xu Z, Li C, Zhao J. MicroRNA-203 inhibitscellular proliferation and invasion by targeting Bmi1 in non-small cell lungcancer. Oncol Lett. 2015;9:2639–46.

27. Hailer A, Grunewald TG, Orth M, Reiss C, Kneitz B, Spahn M, Butt E. Loss oftumor suppressor mir-203 mediates overexpression of LIM and SH3 protein1 (LASP1) in high-risk prostate cancer thereby increasing cell proliferationand migration. Oncotarget. 2014;5:4144–53.

28. Taipaleenmaki H, Browne G, Akech J, Zustin J, van Wijnen AJ, Stein JL, HesseE, Stein GS, LianJB. Targeting of Runx2 by miR-135 and miR-203 impairsprogression of breast Cancer and metastatic bone disease. Cancer Res.2015;75:1433–44.

29. Imaoka H, Toiyama Y, Okigami M, Yasuda H, Saigusa S, Ohi M, Tanaka K,Inoue Y, Mohri Y, Kusunoki M. Circulating microRNA-203 predictsmetastases, early recurrence, and poor prognosis in human gastric cancer.Gastric Cancer. 2016;19:744–53.

30. Liu Y, Dong Z, Liang J, Guo Y, Guo X, Shen S, Kuang G, Guo W. Methylation-mediated repression of potential tumor suppressor miR-203a and miR-203bcontributes to esophageal squamous cell carcinoma development. TumourBiol. 2016;37:5621–32.

31. Liu D, Wu J, Liu M, Yin H, He J, Zhang B. Downregulation of miRNA-30c andmiR-203a is associated with hepatitis C virus core protein-induced epithelial-mesenchymal transition in normal hepatocytes and hepatocellularcarcinoma cells. Biochem Biophys Res Commun. 2015;464:1215–21.

32. Liu W, Dong Z, Liang J, Guo X, Guo Y, Shen S, Kuang G, Guo W.Downregulation of potential tumor suppressor miR-203a by promotermethylation contributes to the invasiveness of gastric cardiaadenocarcinoma. Cancer Investig. 2016;34:506–16.

33. Shibuta T, Honda E, Shiotsu H, Tanaka Y, Vellasamy S, Shiratsuchi M, UmemuraT. Imatinib induces demethylation of miR-203 gene: an epigenetic mechanismof anti-tumor effect of imatinib. Leuk Res. 2013;37:1278–86.

34. Bueno MJ, Perez de Castro I, Gomez de Cedron M, Santos J, Calin GA,Cigudosa JC, Croce CM, Fernandez-Piqueras J, Malumbres M. Genetic andEpigenetic Silencing of MicroRNA-203 Enhances ABL1 and BCR-ABL1Oncogene Expression. Cancer Cell. 2016;29:607–8.

35. Hao M, Zhao W, Zhang L, Wang H, Yang X. Low folate levels are associatedwith methylation-mediated transcriptional repression of miR-203 and miR-375 during cervical carcinogenesis. Oncol Lett. 2016;11:3863–9.

36. Loginov VI, Burdennyy AM, Filippova EA, Pronina IV, Kazubskaya TP,Kushlinsky DN, Ermilova VD, Rykov SV, Khodyrev DS, Braga EA.Hypermethylation of miR-107, miR-130b, miR-203a, miR-1258 genesassociated with ovarian Cancer development and metastasis. Mol Biol(Mosk). 2018;52:801–9.

37. Okumura N, Yoshida H, Kitagishi Y, Nishimura Y, Iseki S, Matsuda S. Againstlung Cancer cells: to be, or not to be, that is the problem. Lung Cancer Int.2012;2012:659365.

38. Wang M, Liu G, Shan GP, Wang BB. In vivo and in vitro effects of ATM/ATRsignaling pathway on proliferation, apoptosis, and Radiosensitivity ofnasopharyngeal carcinoma cells. Cancer BiotherRadiopharm. 2017;32:193–203.

39. Li Y, Yang DQ. The ATM inhibitor KU-55933 suppresses cell proliferation andinduces apoptosis by blocking Akt in cancer cells with overactivated Akt.Mol Cancer Ther. 2010;9:113–25.

40. Liu R, Tang J, Ding C, Liang W, Zhang L, Chen T, Xiong Y, Dai X, Li W, Xu Y,Hu J, Lu L, Liao W, Lu X. The depletion of ATM inhibits colon cancerproliferation and migration via B56gamma2-mediated Chk1/p53/CD44cascades. Cancer Lett. 2017;390:48–57.

41. Zhang L, Xu LJ, Zhu J, Li J, Xue BX, Gao J, Sun CY, Zang YC, Zhou YB, YangDR, Shan YX. ATMJAKPDL1 signaling pathway inhibition decreases EMT andmetastasis of androgenindependent prostate cancer. Mol Med Rep. 2018;17:7045–54.

42. Stagni V, Manni I, Oropallo V, Mottolese M, Di Benedetto A, Piaggio G, FalcioniR, Giaccari D, Di Carlo S, Sperati F, Cencioni MT, Barila D. ATM kinase sustainsHER2 tumorigenicity in breast cancer. Nat Commun. 2015;6:6886.

43. Golding SE, Rosenberg E, Valerie N, Hussaini I, Frigerio M, Cockcroft XF,Chong WY, Hummersone M, Rigoreau L, Menear KA, O'Connor MJ, PovirkLF, van Meter T, Valerie K. Improved ATM kinase inhibitor KU-60019radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvivalsignaling, and inhibits migration and invasion. Mol Cancer Ther. 2009;8:2894–902.

44. Fujimaki S, Matsuda Y, Wakai T, Sanpei A, Kubota M, Takamura M, YamagiwaS, Yano M, Ohkoshi S, Aoyagi Y. Blockade of ataxia telangiectasia mutatedsensitizes hepatoma cell lines to sorafenib by interfering with Akt signaling.Cancer Lett. 2012;319:98–108.

45. Lu LL, Chen XH, Zhang G, Liu ZC, Wu N, Wang H, Qi YF, Wang HS, Cai SH,Du J. CCL21 facilitates Chemoresistance and Cancer stem cell-like propertiesof colorectal Cancer cells through AKT/GSK-3beta/snail signals. OxidativeMed Cell Longev. 2016;2016:5874127.

46. Wang S, Wang X, Li J, Meng S, Liang Z, Xu X, Zhu Y, Li S, Wu J, Xu M, Ji A,Lin Y, Liu B, Zheng X, Xie B, Xie L. c-Met, CREB1 and EGFR are involved inmiR-493-5p inhibition of EMT via AKT/GSK-3beta/snail signaling in prostatecancer. Oncotarget. 2017;8:82303–13.

47. Lan Y, Han J, Wang Y, Wang J, Yang G, Li K, Song R, Zheng T, Liang Y, PanS, Liu X, Zhu M, Liu Y, Meng F, Mohsin M, Cui Y, Zhang B, Subash S, Liu L.STK17B promotes carcinogenesis and metastasis via AKT/GSK-3beta/snailsignaling in hepatocellular carcinoma. Cell Death Dis. 2018;9:236.

48. Sun M, Guo X, Qian X, Wang H, Yang C, Brinkman KL, Serrano-Gonzalez M,Jope RS, Zhou B, Engler DA, Zhan M, Wong ST, Fu L, Xu B. Activation of theATM-snail pathway promotes breast cancer metastasis. J Mol Cell Biol. 2012;4:304–15.

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