Regulation of Docetaxel Sensitivity in Prostate Cancer Cells by hsa-miR-125a-3p via Modulation of Metastasis-Associated Protein 1 Signaling

Accepted Manuscript Title: Regulation of Docetaxel Sensitivity in Prostate Cancer Cells by HsamiR-125A-3P via Modulation of MTA1 Signaling Author: Jian-zhou Liu, Feng-yan Yin, Chang-you Yan, Hui Wang, Xiao-hui Luo PII: DOI: Reference:

S0090-4295(17)30013-4 http://dx.doi.org/doi: 10.1016/j.urology.2017.01.001 URL 20234

To appear in:

Urology

Received date: Accepted date:

8-10-2016 2-1-2017

Please cite this article as: Jian-zhou Liu, Feng-yan Yin, Chang-you Yan, Hui Wang, Xiao-hui Luo, Regulation of Docetaxel Sensitivity in Prostate Cancer Cells by Hsa-miR-125A-3P via Modulation of MTA1 Signaling, Urology (2017), http://dx.doi.org/doi: 10.1016/j.urology.2017.01.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Regulation of docetaxel sensitivity in prostate cancer cells by hsa-miR-125a-3p via modulation of MTA1 signaling Jian-zhou Liu1#, Feng-yan Yin1#, Chang-you Yan2, Hui Wang3, Xiao-hui Luo1* 1

Department of Urology, Baoji Central Hospital, Baoji 721008, Shaanxi Province,

China 2

Xi’an Health Management Service Center, Xi’an 710032, Shaanxi Province, China

3

Department of Medical Psychology, Fourth Military Medical University, Xi'an,

710032, Shaanxi Province, China #

These authors contribute equally to this work.

*Corresponding authors: Dr. Xiao-hui Luo, Department of Urology, Baoji Central Hospital, No. 8 Jiangtan Road, Baoji 721008, Shaanxi Province, China; Tel: 86-13892779082; E-mail address: [email protected]; Conflict of interest None. Acknowledgments The authors are indebted to Dr. Liang Yu (Department of Urology, 3rd Hospital of PLA, No. 45 Dongfeng Road, Bao Ji 721004, China) for his enthusiastic discussion during the preparation of the manuscript. Word counts for the Abstract: 209 Word counts for the manuscript text: 2,525 Keywords miR-125a; prostate cancer; chemoresistance; docetaxel; MTA1

Abstract OBJECTIVE To identify the potential down-stream targets of hsa-miR-125a-3p, a mature form of miR-125a, during the pathogenesis of chemoresistance in prostate cancer (PCa). 1

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METHODS Expression levels of hsa-miR-125a-3p was assessed in chemoresistant PCa tissues and experimentally-established chemoresistant cells using quantitative RT-PCR (qRT-PCR). The effect of hsa-miR-125a-3p knockdown or hsa-miR-125a-3p overexpression on the Dox-induced cell death was evaluated using apoptosis ELISA in chemosensitive PC-3 cells or in chemoresistant PC-3 cells (PC-3R). Finally, using multiple assays, the regulation of Metastasis-associated protein 1 (MTA1), an essential component of the Mi-2/nucleosome remodeling deacetylation (NuRD) complex, by hsa-miR-125a-3p was studied at both molecular and functional levels. RESULTS The expression of hsa-miR-125a-3p was significantly down-regulated in chemoresistant PCa tissues and cells. Inhibition of hsa-miR-125a-3p significantly increased docetaxel (Dox)-resistance in PC-3 cells, whereas, up-regulation of hsa-miR-125a-3p effectively reduced Dox-resistance in PC-3R, suggesting that this miRNA may act as tumor suppressor along the pathogenesis of drug resistance. Mechanistically, hsa-miR-125a-3p induced apoptosis and Dox-sensitivity in PCa cells through regulating MTA1. CONCLUSION Our results collectively indicate that miRNA-MTA1 can form a delicate regulatory loops to maintain a bistable state in the Dox chemosensitivity, and future endeavor in this filed should provide important clues to develop miRNA-based therapies that benefit advanced PCa patients through modulating the functional status of MTA1.

Prostate cancer (PCa), one of the most common cancers among males, is a leading cause of cancer-related male deaths in the United States, and its occurrence is increasing dramatically in China 1. After initial 2-3 years of successful treatment by androgen-ablation therapy, most of patients will eventually suffer castration-resistant prostate cancer (CRPC), which, as the lethal form of PCa, is often incurable nowadays 2

. So far, the patients who develop CRPC is usually subjected to Taxanes treatment

(docetaxel and paclitaxel). Docetaxel (Dox)-based regimens have been used as the best chemotherapy and are believed to offer symptomatic and survival benefits in men 2

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with metastatic hormone-refractory PCa. However, resistance to such chemotherapy will eventually develop and patients’ disease continually progresses. Resistance can be formed through a variety of mechanisms, both intrinsic and extrinsic, but the details remain largely unknown 3. MicroRNAs (miRNAs) are a class of 20-24 nt small non-coding RNAs that regulate a wide range of biological processes through changing the stability or translation of their target messenger RNA (mRNA) genes. Shortly after their identification, many miRNA genes have been found dysregulated in a variety of human cancers 4. Accumulated evidence reveals that aberrant expressions of miRNAs, which can act as tumor suppressors or oncogenes, are closely associated with the development of chemoresistance in various cancers including PCa 5. miR-125a, an anti-oncogene that plays a key role in multiple cancers, has been shown to be involved in the pathogenesis of chemoresistance including paclitaxel sensitivity in colon cancer 8

carcinoma

6

or cervical cancer

7

and cisplatin sensitivity in nasopharyngeal

. miR-125a has two mature forms, namely hsa-miR-125a-3p and

hsa-miR-125a-5p, and there are functional differences between these two mature miRNAs

9

. Of particular interest, recent advance in this field reveals that

hsa-miR-125a-3p is dramatically decreased in metastatic PCa and regulates proliferation and migration of PCa cells 10. The data are indicative of a unique role of hsa-miR-125a-3p in PCa. Metastasis-associated protein 1 (MTA1), an essential component of the Mi-2/nucleosome remodeling deacetylation (NuRD) complex, plays a pivotal role in signal regulation by modifying the acetylation status of crucial target genes. Deregulation of this important chromatin modifier has been shown to play a causative role in the maintenance and progression of more invasive phenotypes of many 3

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malignancies

11-13

. Our collaborators have demonstrated that MTA1 expression is

abnormally up-regulated in chemoresistant PCa and it acts as a novel Dox-resistance promoter in PC-3 cells via modulation of NR4A1 pathway, which indicates that MTA1 may potentially be used both as a prognostic marker and a potential therapy target in PCa 14. Given the fact that miRNAs can act as “rheostats” to fine-tune protein expression in subtle magnitude, it is intriguing to investigate whether certain miRNAs can function as the mediators to decide the functional mode of MTA1

15

. To this end,

emerging data have shown that MTA1 could be regulated by miR-661 in hepatocellular carcinoma

16

or by miR-30c in non-small cell lung cancer

17

. In this

study, we sought to determine the expression pattern and potential role of hsa-miR-125a-3p in PCa chemoresistance, and uncover the functional association between hsa-miR-125a-3p and MTA1. MATERIAL AND METHODS Patient samples Samples have been obtained after patients gave written informed consent. In detail, benign prostatic tissues from patients who underwent radical cystoprostatectomy due to bladder cancer (n = 30) and biopsies from chemosensitive PCa patients (n = 30) or from chemoresistant PCa patients (n = 14) were included. The chemoresistant PCa patients were defined as PCa patients with tumors clearly progressing on after 4 cycles of three-weekly first-line Dox-based chemotherapy (patients were treated with Dox chemotherapy 75 mg/m2 intravenously on day 1 repeated every three weeks along with prednisolone 10 mg daily) 14. Androgen suppression with LHRH analogues was continued throughout the duration of chemotherapy. The use of the human tissues in this study was approved by the local ethical committee. 4

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Cells treatment Normal human prostate epithelial cells (PECs) were purchased from Clonetics (San Diego, CA, USA). LNCaP and PC-3 cells were purchased from ATCC (Manassas, VA, USA). Cells were maintained in RPMI-1640 medium including L-glutamine, supplemented with 10% (v/v) Fetal Bovine Serum and 1% (v/v) penicillin/streptomycin (100,000 U/l penicillin, 100 mg/l streptomycin), at 37°C in a humidified atmosphere containing 5% CO2. PC-3 resistant sub-lines (PC-3R) were generated according to our collaborates’ work

14

. Ten thousand cells/well were

cultured in a 96-well plate. 24 h later, cells were treated with different concentrations of Dox (Sigma–Aldrich, Shanghai, China) for different durations as indicated. Cells were then subjected to MTT assay for viability measurement. To manipulate the cellular levels of hsa-miR-125a-3p, cells were mixed with hsa-miR-125a-3p mimics (10nM) or antisense inhibitors (50 nM) (Sigma-Aldrich, Shanghai, China) in 100 μl culture medium without serum and antibiotics. Specific knockdown of MTA1 was achieved by transfecting cells with corresponding siRNA against MTA1 (sc-35981) or with a control siRNA (sc-37007) (Santa Cruz Biotechnology, CA, USA). 48 h after transfection, cells were collected and subjected to other experiments. quantitative RT-PCR (qRT-PCR) Total RNA was isolated from tissues and cells using RNeasy Mini Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer’s protocol

18

. For

hsa-miR-125a-3p and MTA1 expression analysis, total RNA was retro transcribed with microRNA-specific primers (3’-GATGCTCTACAGGTGAGGTTCTT-5’) and MTA1 primers using TaqMan microRNA and an mRNA reverse transcription kit (Applied biosystems, Foster City, CA), respectively. Amplification of human U6 snRNA and 18S RNA served as internal control. qRT-PCR was performed using 5

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Tagman microRNA and mRNA assays according to the manufacturer’s protocol. The relative abundance of each target transcript was quantified using the comparative △△Ct method. Assessment of apoptosis An apoptosis ELISA kit (Roche Diagnostics, Mannheim, Germany) was also used to

quantitatively

measure

cytoplasmic

histone-associated

DNA

fragments

(mononucleosomes and oligonucleosomes), as described previously 12,19. Promoter reporter assay LNCaP ells were transfected with 10 nM hsa- miR-125a-3p mimics or control miRNA mimics. Two days later, cells were transfected using Lipofectamine 2000 (Invitrogen) with a human MTA1 promoter reporter construct system (SwitchGear Genomics, Menlo Park, CA, USA) (pLightSwitch-3’UTR; Restriction pair: Nhe1 and Xho1). The pRL-TK Renilla reporter plasmid (Promega) was co-transfected to normalize the transfection efficiency

20

. Luciferase activity measurements were

performed using the dual-luciferase reporter assay system (Promega) per manufacturer’s protocol. Western blotting Western blotting was carried out according to our previous work

21,22

. Membranes

were incubated with different primary antibodies including goat anti-MTA1 and rabbit anti-TUBULIN (Santa Cruz Biotechnology, Shanghai, China). Statistical analysis Results are presented as mean ± S.E.M. from at least three independent experiments. Data normality was determined using normal probability plots and compared using Student’s t-test or one way analysis of variance (ANOVA) as appropriate. The correlation between hsa-miR-125a-3p expression and MTA1 mRNA 6

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level was assessed using qRT-PCR, followed by analysis using the Pearson Chi-Square test. A P-value of < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 15.0 software. RESULTS hsa-miR-125a-3p expression is down-regulated in chemoresistant PCa tissues and cells In the first attempt to study the potential involvement of hsa-miR-125a-3p in PCa chemoresistance, we examined its expression level in human PCa specimens. qRT-PCR analysis demonstrated that hsa-miR-125a-3p expression varied among groups: moderate in benign tissues (n = 30), and significantly decreased in chemosensitive PCa samples (n = 14) as well as in their chemoresistant counterparts (n = 30), with the minimal value being detected in the latter (Fig. 1A). We next studied the expression of hsa-miR-125a-3p in the Dox-induced cytotoxicity in PC-3 cells, by using experimentally-generated Dox-resistant sublines. PC-3-R exhibited higher apoptotic resistance (Fig. 1B) and faster growth (Fig. 1C) in the Dox-treated medium compared with PC-3. PEC and LNCaP cells showed the highest levels of hsa-miR-125a-3p, whereas the lower expression levels were found in PC-3 and PC-3R cells, with the lowest level observed in PC-3R cells (Fig. 1D). These data are indicative of a correlation of compromised hsa-miR-125a-3p expression with PCa chemoresistance. hsa-miR-125a-3p potentiates Dox-sensitivity in PC-3 cells We next explored the role of hsa-miR-125a-3p in the Dox-induced cytotoxicity in PC-3 and PC-3R cells. We transfected these two cell lines with hsa-miR-125a-3p inhibitors or mimics, respectively. The altered expression level of hsa-miR-125a-3p 7

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was validated using qRT-PCR (Fig. 2A and 2B). The subsequent apoptotic analysis revealed

that

down-regulation

of

hsa-miR-125a-3p

significantly

increased

Dox-resistance in PC-3 cells, whereas, up-regulation of hsa-miR-125a-3p effectively reduced Dox-resistance in PC-3R cells (Fig. 2C and 2D). Additionally, the hsa-miR-125a-3p-overexpressing cells exhibited a round shape that resembled epithelial cells rather than mesenchymal cells (Fig. 2E). Collectively, these data indicate that accumulation of hsa-miR-125a-3p may enhance the chemosensitive in PCa cells. MTA1 is a direct target of hsa-miR-125a-3p in PCa cells In an effort to search for the potential mechanism underlying the role of hsa-miR-125a-3p in chemoresistance in PC-3 cells, we found that MTA1 might be a potential down-stream target gene for hsa-miR-125a-3p, by using miRanda (mirSVR; http://www.microrna.org/microrna/home.do) (Fig. 3A). Interestingly, transfection in PC-3R cells with hsa-miR-125a-3p mimic resulted in a significant decrease of MTA1 protein level, but failed to inspire the MTA1 mRNA level, as reflected by western blotting (Fig. 3B) and qRT-PCR analyses (Fig. 3C), respectively. We then measured changes after hsa-miR-125a-3p mimics co-transfection in luciferase activity from a MTA1 3′UTR reporter. The reporter assay revealed that hsa-miR-125a-3p markedly reduced luciferase activity of the pLightSwitch-3’UTR-MTA1 reporter (Fig. 3D), suggesting that hsa-miR-125a-3p may directly inhibit MTA1 post-transcriptional expression via binding of 3'-UTR. MTA1 knockdown attenuates hsa-miR-125a-3p deficiency-induced chemoresistance in PC-3R cells Since MTA1 is a direct target gene of hsa-miR-125a-3p, we then investigate 8

Page 8 of 16

whether attenuation of MTA1 can abolish the effect of hsa-miR-125a-3p deficiency in PCa cells. MTA1 knockdown by siRNA treatment was verified using western blotting analysis (Fig. 4A). We then incubated the cells with Dox (20 nM) for 24 h. Apoptosis ELISA method revealed that inhibition of MTA1 expression, as well as supplement with hsa-miR-125a-3p mimic, could both effectively increase the percentage of apoptotic cells induced by Dox treatment (Fig. 4B). Additionally, by using Pearson Chi-Square test, it was found that the relative hsa-miR-125a-3p expression level was inversely correlated to MTA1 mRNA expression in PCa tissues from patients who received Dox-based chemotherapy (Fig. 4C). Thus, hsa-miR-125a-3p may induce apoptosis and Dox-sensitivity in prostate cancer cells through negatively regulating the MTA1 pathway. DISCUSSION Our research collaborates have previously reported that MTA1 is up-regulated in chemoresistant prostate cancer tissues compared to their chemosensitive counterparts and benign prostatic tissues, and exerts a chemoresistance-enhancer role mainly through the transcriptional regulation of NR4A1 molecular

mechanism

underlying

the

14

. Then, we questioned for the

modulation

of

MTA1

in

prostate

chemoresistance focusing on miRNAs. Actually, deregulation of miRNA expression is closely linked to chemoresistance and represents nowadays the actual challenge in both diagnostic and therapy for biomedical purposes 3. Using bioinformatic analysis, we have identified several miRNAs predicted to target MTA1. Among them, we concentrated on hsa-miR-125a-3p since it was one of the most down-regulated miRNAs in PCa tissues with high Gleason score

10

and has been recently shown to

function as a tumor-suppressor as well as a chemosensitivity-promoter in breast cancer 23. 9

Page 9 of 16

Our

quantitative

analysis

revealed

that

hsa-miR-125a-3p

was

notably

down-regulated in chemoresistant PCa tissues and cells, which is compatible with former studies that indicated down-regulation of hsa-miR-125a-3p in human gastric 24 and lung cancer

25

. The available data suggest that this miRNA might serve as a

potential tumor suppressor and this tumor-suppressing effect appears to be relatively conserved. Although the mechanisms whereby hsa-miR-125a-3p expression is inhibited in chemoresistant PCa remain to be defined, two factors have been so far reported to determine the level of hsa-miR-125a-3p expression. Several mechanisms contributing to abnormal silencing of miRNAs have been discovered, repression of tumor

suppressor-miRNA

by

epigenetic

mechanism

such

as

promoter

hypermethylation and/or inhibitory histone modification has emerged as a major cause

26

. Of particular interest, it has been observed that hsa-miR-125a-3p level in

multiple myeloma was significantly increased in the presence of demethylating agent 5′azacytidine

27

. Therefore, deregulation of promoter hypermethylation status may

have a causative role in the down-regulation of hsa-miR-125a-3p in chemoresistant PCa. Furthermore, MTA1 is a component of the Mi-2/nucleosome remodeling deacetylation (NuRD) complex, and regulates divergent cellular pathways by modifying the acetylation status of crucial target genes. More importantly, MTA1 may exert its regulatory effects at different levels by acting both as a target and as a component of certain pathway 28. So, the coordinated existence of MTA1 along with hsa-miR-125a-3p may help to integrate the complicated regulatory network in chemoresistant PCa and also warrants further investigation. Ectopic expression of hsa-miR-125a-3p impairs cell cycle and viability in PC-3 cells 10. It is therefore a logical hypothesis that this miRNA may directly participate in PCa pathology. Our results demonstrated that down-regulation of hsa-miR-125a-3p 10

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significantly increased Dox-resistance in PC-3 cells, while in good contrast, up-regulation of hsa-miR-125a-3p effectively reduced Dox-resistance in PC-3R cells (Fig. 2C and 2D). These data support our emerging view that hsa-miR-125a-3p probably serves as a key post-transcriptional regulator during the pathogenesis of chemoresistance in PC-3 cells. Of note, development of chemoresistance in cancer cells usually leads to a mesenchymal motility mode that involves dynamic changes in the rearrangement of actin cytoskeleton and its interplay with focal adhesion proteins that induces membrane protrusions mandatory for further invasiveness 29. In our study, the hsa-miR-125a-3p-overexpressing cells exhibited a round shape that resembled epithelial cells rather than mesenchymal cells (Fig. 2E), implying that high levels of hsa-miR-125a-3p might interfere with epithelial to mesenchymal transition (EMT) in PCa cells. This intriguing possibility is currently under investigation in our lab. Our collaborators have established that hsa-miR-125a-3p regulates breast cancer chemoresistance by directly targeting breast cancer early onset gene 1 (BRCA1). However, since a single miRNA may encompass multiple targets, alternative pathways should be further explored 3. Emerging literature establishes MTA1 to be a valid DNA-damage responsive protein with a significant role in maintaining the optimum DNA-repair activity in mammalian cells exposed to genotoxic stress. Of particular interest, it has been shown that repression of NR4A1 transcription by MTA1-HDAC2 may represent a novel mechanism contributing to the pathogenesis of PCa chemoresistance

14

. However, the transcriptional or posttranscriptional

mechanisms responsible for controlling MTA1 expression in response to DNA damage are poorly understood. Our findings extend these understanding by identifying hsa-miR-125a-3p as a potent upstream regulator of MTA1 expression. The 11

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available data broaden our understanding of the role of hsa-miR-125a-3p/MTA1 cascade in PCa apoptosis, and also provide a mechanistic explanation for MTA1 function in the pathogenesis of chemoresistance. Additionally, knockdown of MTA1 expression could only partially reverse the hsa-miR-125a-3p deficiency-elicited Dox-resistance (Fig. 4B), indicative of a role for hsa-miR-125a-3p in regulating Dox-induced apoptosis, possibly, in part, via a MTA1-dependent manner. Future endeavor should be concentrated on looking for other potential targets of this important miRNA. CONCLUSION The data presented here allow us to propose a model wherein the expression of hsa-miR-125a-3p is progressively reduced during the pathogenesis of PCa chemoresistance, and the loss of hsa-miR-125a-3p, in turn allows MTA1 expression to be evoked as the post-transcriptional repression of MTA1 3’-UTR is compromised. These results raise the possibility that hsa-miR-125a-3p regulates Dox-resistance by targeting MTA1 signaling and thereby it may be developed as a therapeutic target for down-regulating the expression of MTA1, which is widely up-regulated in chemoresistant PCa. Our systematic analysis should provide central pieces of information to better understand the biological functions of hsa-miR-125a-3p/MTA1 cascade in PCa progression. References 1. 2. 3.

Pang C, Guan Y, Li H, Chen W and Zhu G: Urologic cancer in China. Jpn J Clin Oncol. 46: 497-501, 2016. Ning P, Zhong JG, Jiang F, Zhang Y, Zhao J, Tian F and Li W: Role of protein S in castration-resistant prostate cancer-like cells. Endocr Relat Cancer. 23: 595-607, 2016. Kopczynska E: Role of microRNAs in the resistance of prostate cancer to docetaxel and paclitaxel. Contemp Oncol (Pozn). 19: 423-7, 2015. 12

Page 12 of 16

4. 5. 6. 7. 8.

9. 10.

11.

12. 13.

14. 15. 16. 17. 18. 19.

20.

Bertoli G, Cava C and Castiglioni I: MicroRNAs as Biomarkers for Diagnosis, Prognosis and Theranostics in Prostate Cancer. Int J Mol Sci. 17: 421, 2016. Kojima S, Goto Y and Naya Y: The roles of microRNAs in the progression of castration-resistant prostate cancer. J Hum Genet, 2016. Chen J, Chen Y and Chen Z: MiR-125a/b regulates the activation of cancer stem cells in paclitaxel-resistant colon cancer. Cancer Invest. 31: 17-23, 2013. Fan Z, Cui H, Yu H, Ji Q, Kang L, Han B, Wang J, Dong Q, Li Y, Yan Z et al.: MiR-125a promotes paclitaxel sensitivity in cervical cancer through altering STAT3 expression. Oncogenesis. 5: e197, 2016. Chen JJ, Liu SX, Chen MZ and Zhao ZY: HasmiR125a and 125b are induced by treatment with cisplatin in nasopharyngeal carcinoma and inhibit apoptosis in a p53dependent manner by targeting p53 mRNA. Mol Med Rep. 12: 3569-74, 2015. Tang H, Li RP, Liang P, Zhou YL and Wang GW: miR-125a inhibits the migration and invasion of liver cancer cells via suppression of the PI3K/AKT/mTOR signaling pathway. Oncol Lett. 10: 681-686, 2015. Ninio-Many L, Grossman H, Levi M, Zilber S, Tsarfaty I, Shomron N, Tuvar A, Chuderland D, Stemmer SM, Ben-Aharon I et al.: MicroRNA miR-125a-3p modulates molecular pathway of motility and migration in prostate cancer cells. Oncoscience. 1: 250-61, 2014. Zhang C, Lai JH, Hu B, Zhang S, Zhao J and Li W: A chromatin modifier regulates Sertoli cell response to mono-(2-ethylhexyl) phthalate (MEHP) via tissue inhibitor of metalloproteinase 2 (TIMP2) signaling. Biochim Biophys Acta. 1839: 1170-82, 2014. Liang Y, Dong Y, Zhao J and Li W: YES1 activation elicited by heat stress is anti-apoptotic in mouse pachytene spermatocytes. Biol Reprod. 89: 131, 2013. Li W, Wu ZQ, Zhao J, Guo SJ, Li Z, Feng X, Ma L, Zhang JS, Liu XP and Zhang YQ: Transient protection from heat-stress induced apoptotic stimulation by metastasis-associated protein 1 in pachytene spermatocytes. PLoS One. 6: e26013, 2011. Yu L, Su YS, Zhao J, Wang H and Li W: Repression of NR4A1 by a chromatin modifier promotes docetaxel resistance in PC-3 human prostate cancer cells. FEBS Lett. 587: 2542-51, 2013. Zhang Y and Wang XF: Post-transcriptional regulation of MTA family by microRNAs in the context of cancer. Cancer Metastasis Rev. 33: 1011-6, 2014. Reddy SD, Pakala SB, Ohshiro K, Rayala SK and Kumar R: MicroRNA-661, a c/EBPalpha target, inhibits metastatic tumor antigen 1 and regulates its functions. Cancer Res. 69: 5639-42, 2009. Xia Y, Chen Q, Zhong Z, Xu C, Wu C, Liu B and Chen Y: Down-regulation of miR-30c promotes the invasion of non-small cell lung cancer by targeting MTA1. Cell Physiol Biochem. 32: 476-85, 2013. Ding J, Wang H, Wu ZB, Zhao J, Zhang S and Li W: Protection of murine spermatogenesis against ionizing radiation-induced testicular injury by a green tea polyphenol. Biol Reprod. 92: 6, 2015. Li W, Fu J, Zhang S, Zhao J, Xie N and Cai G: The proteasome inhibitor bortezomib induces testicular toxicity by upregulation of oxidative stress, AMP-activated protein kinase (AMPK) activation and deregulation of germ cell development in adult murine testis. Toxicol Appl Pharmacol. 285: 98-109, 2015. Zhang S, Li W, Zhu C, Wang X, Li Z, Zhang J, Zhao J, Hu J, Li T and Zhang Y: Sertoli cell-specific expression of metastasis-associated protein 2 (MTA2) is 13

Page 13 of 16

21.

22. 23. 24.

25. 26. 27. 28.

29.

required for transcriptional regulation of the follicle-stimulating hormone receptor (FSHR) gene during spermatogenesis. J Biol Chem. 287: 40471-83, 2012. Dong YS, Hou WG, Li Y, Liu DB, Hao GZ, Zhang HF, Li JC, Zhao J, Zhang S, Liang GB et al.: Unexpected requirement for a binding partner of the syntaxin family in phagocytosis by murine testicular Sertoli cells. Cell Death Differ. 23: 787-800, 2016. Li W, Zeng Y, Zhao J, Zhu CJ, Hou WG and Zhang S: Upregulation and nuclear translocation of testicular ghrelin protects differentiating spermatogonia from ionizing radiation injury. Cell Death Dis. 5: e1248, 2014. He H, Xu F, Huang W, Luo SY, Lin YT, Zhang GH, Du Q and Duan RH: miR-125a-5p expression is associated with the age of breast cancer patients. Genet Mol Res. 14: 17927-33, 2015. Hashiguchi Y, Nishida N, Mimori K, Sudo T, Tanaka F, Shibata K, Ishii H, Mochizuki H, Hase K, Doki Y et al.: Down-regulation of miR-125a-3p in human gastric cancer and its clinicopathological significance. Int J Oncol. 40: 1477-82, 2012. Huang B, Luo W, Sun L, Zhang Q, Jiang L, Chang J, Qiu X and Wang E: MiRNA-125a-3p is a negative regulator of the RhoA-actomyosin pathway in A549 cells. Int J Oncol. 42: 1734-42, 2013. Yu JJ and Xia SJ: Novel role of microRNAs in prostate cancer. Chin Med J (Engl). 126: 2960-4, 2013. Bi C, Chung TH, Huang G, Zhou J, Yan J, Ahmann GJ, Fonseca R and Chng WJ: Genome-wide pharmacologic unmasking identifies tumor suppressive microRNAs in multiple myeloma. Oncotarget. 6: 26508-18, 2015. Chen S, Dong Y, Xu C, Jiang L, Chen Y, Jiang C, Hou W and Li W: Involvement of a chromatin modifier in response to mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury: probably an indirect action via the regulation of NFkappaB/FasL circuitry. Biochem Biophys Res Commun. 440: 749-55, 2013. Li F and Mahato RI: MicroRNAs and drug resistance in prostate cancers. Mol Pharm. 11: 2539-52, 2014.

Fig. 1. hsa-miR-125a-3p expression is down-regulated in chemoresistant prostate caner (PCa) tissues and cells. (A) Expression of hsa-miR-125a-3p in human prostate tissues. Total RNA was isolated from benign, chemosensitive and chemoresistant human prostate tissues and subjected to quantitative reverse transcription-PCR (qRT-PCR) as described in Materials and methods. Amplification of human U6 snRNA served as internal control. All data are presented as the mean ± S.E.M. of triplicate experiments. (B) 14

Page 14 of 16

Parental PC-3 cells and Dox-resistant PC-3R cells were incubated with Dox (20 nM or 80 nM) for 24 h and the extent of apoptosis was then quantitatively measured using apoptosis ELISA. (C) Parental PC-3 cells and Dox-resistant PC-3R cells were treated with Dox (5 nM) for different durations and cell viability was recorded using MTT assay. (D) Relative expression levels of hsa-miR-125a-3p in normal human prostate epithelial cells (PECs), LNCaP, PC-3 and PC-3R cells were quantified using qRT-PCR. Fig. 2. Effect of inhibition/overexpression of hsa-miR-125a-3p on Dox-elicited cell death. To manipulate the cellular levels of hsa-miR-125a-3p, PC-3 cells (A) or PC-3R cells (B) were mixed with hsa-miR-125a-3p antisense inhibitors (50 nM), hsa-miR-125a-3p mimics (10nM) or their corresponding negative controls (NC) in 100 μl culture medium without serum and antibiotics. 48 hr later, the relative expression level of hsa-miR-125a-3p was determined using qRT-PCR. All data are presented as the mean ± S.E.M. of triplicate experiments. 48 hr after

treatment

with

hsa-miR-125a-3p

antisense

inhibitors

(50

nM),

hsa-miR-125a-3p mimics (10nM) or their corresponding negative controls (NC), cells were incubated with Dox (20 nM) for 24 h and the extent of apoptosis was then quantitatively measured using apoptosis ELISA in PC-3 cells (C) or PC-3R cells (D), respectively. (E) Representative image showing that the hsa-miR-125a-3p-overexpressing PC-3R cells exhibited a round shape that resembled epithelial cells rather than mesenchymal cells. Fig. 3. hsa-miR-125a-3p regulates PCa apoptosis by targeting MTA1. (A) Prediction of MTA1-targeting sites of hsa-miR-125a-3p by using miRanda (mirSVR; http://www.microrna.org/microrna/home.do). PC-3R cells were 15

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incubated with hsa-miR-125a-3p mimics (10nM) or the corresponding negative controls (NC) for 48 hr, followed by Western blotting (B) or qRT-PCR (C) analyses of MTA1/MTA1 expression levels. (D) LNCaP ells were transfected with 10 nM hsa-miR-125a-3p mimics or control miRNA mimics. Two days later, cells were transfected using Lipofectamine 2000 with a human MTA1 promoter reporter construct system (pLightSwitch-3’UTR; Restriction pair: Nhe1 and Xho1), as well as the pRL-TK Renilla reporter plasmid (Promega). After 48 h, cells were harvested and luciferase activity was measured. Luciferase reporter assay was performed in triplicate. Fig. 4. Effect of MTA1 knockdown on hsa-miR-125a-3p deficiency-induced chemoresistance. (A) Effect of MTA1 knockdown by siRNA treatment was evaluated using Western blotting. (B) 48 hr after siRNA treatment or transfection with hsa-miR-125a-3p mimics/the corresponding negative controls (NC), cells were incubated with Dox (20 nM) for 24 h and the extent of apoptosis was then quantitatively measured using apoptosis ELISA. (C) A statistically significant inverse correlation between hsa-miR-125a-3p and MTA1 mRNA levels in PCa tissues.

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