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Long Non-Coding RNA BLACAT1 Inhibits Prostate Cancer Cell Proliferation Through Sponging Mir-361

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Long Non-Coding RNA BLACAT1 Inhibits Prostate Cancer Cell Proliferation Through Sponging Mir-361 European Review for Medical and Pharmacological Sciences 2020; 24: 74-85 Long non-coding RNA BLACAT1 inhibits prostate cancer cell proliferation through sponging miR-361 H.-Y. LI1, F.-Q. JIANG1, L. CHU2, X. WEI1 1Department of Urology, China-Japan Union Hospital of Jilin University, Changchun, People's Republic of China 2Department of Bidding and Purchase, China-Japan Union Hospital of Jilin University, Changchun, People's Republic of China Abstract. – OBJECTIVE: LncRNAs play a key Introduction role in the development and progression of prostate cancer. In this study, the effects of the Globally, prostate cancer remains a leading lncRNA BLACAT1 in prostate cancer were in- cause of mortality1,2. Prostate cancer is the 3rd vestigated. PATIENTS AND METHODS: most common cancer across the world, and it Real-time PCR 3 was used to detect the expression of BLACAT1 typically affects males aged over 55 years . The and miR-361 in prostate cancer tissues and lack of early symptoms makes prostate cancer adjacent normal tissues (n=25). The function diagnosis challenging4. Magnetic resonance im- of BLACAT1 was detected through proliferation aging (MRI) is the most effective way to identify assay and apoptosis assay. The interaction be- prostate cancer, with detection rates of 85-100%. tween BLACAT1 and miR-361 in prostate cancer Prostate specific antigen (PSA) is a protein pro- was studied by luciferase assay, RNA immuno- precipitation assay and chromatin immunopre- duced in the prostate gland, elevated levels of cipitation analysis were performed to detect the which can be suggestive of prostate cancer. In the BLACAT1 binding proteins. The xenograft mice clinic, elevated PSA levels frequently lead to un- experiment was performed to further confirm necessary biopsies. Despite intense research ef- the functional significance of lncRNA BLACAT1 forts, the mechanisms of prostate cancer develop- in vivo. ment remain poorly characterized5 and effective RESULTS: In patient samples and prostate cancer cell lines, BLACAT1 was down-reg- therapeutic treatment regimens are lacking. ulated and inversely proportional to DNMT1, Long non-coding RNAs (lncRNAs) are frequent- HDAC1, EZH2, MDM2 and miR-361 expression. ly dysregulated in an array of cancers6 including Treatment with 5-azacytidine and chidamide prostate cancer7. For example, HCG11 is poorly ex- enhanced BLACAT1 expression and decreased pressed in prostate cancer cells and associated with the levels of miR-361. The BALCAT1 promoter poor survival in afflicted patients8. Previous studies was methylated in prostate cancer tissue and revealed that the expression of prostate cancer as- found to interact with miR-361 via luciferase assays. BLACAT1 bound to EZH2, DNMT1 and sociated lncRNA transcript 1 (PCAT-1) inhibits cell 9 HDAC1. ChIP-seq analysis revealed that HDAC1 proliferation through miR-145-5p sponging . Ln- interacts with STAT3, while EZH2 interacts with cRNA bladder cancer associated transcript 1 (BLA- the mitogen-activated protein kinase (MAPK) CAT1) was firstly found in bladder cancer10, and the promoter. silencing of BLACAT1 prevents tumor growth and CONCLUSIONS: Two regulatory axes of BLA- promotes cancer cell death11. Conversely in colorec- CAT1-EZH2-MAPK and BLACAT1-HDAC1-STAT3 were identified to be associated with the pro- tal cancer, BLACAT1 expression is suppressed, high- gression of prostate cancer. Both chidamide lighting its differential effects according to cancer and 5-azacytidine represent promising thera- tissue/status12. In prostate cancer cells, the cellular peutic options in prostate cancer treatment. roles of BLACAT1 and its potential involvement in prostate cancer development have not been defined. Key Words: As a form of small non-coding RNAs, mi- Prostate cancer, 5-azacytidine, Chidamide, EZH2, croRNAs (miRNAs) target specific cellular HDAC1. mRNA sequences to either suppress or sustain 74 Corresponding Author: Xin Wei, MD; e-mail: [email protected] Long non-coding RNA BLACAT1 inhibits prostate cancer cell proliferation through sponging miR-361 the expression of cellular genes13. MiR-361 shows Cell Culture pro-oncogenic effects in a range of human can- Human prostate cancer cell lines PC3 and cers. Competing endogenous RNAs (ceRNA) act DU145 cells and the normal prostate cell line as sponges that upregulate or downregulate target RWPE-1 cells (Shanghai Hong Shun Biotechnol- miRNAs. The roles of miR-361 in prostate cancer ogy Co., Ltd., Shanghai, China) were cultured in cells and its interaction with lncRNAs have not complete Dulbecco’s modified Eagle’s medium been defined. (DMEM) (Invitrogen, Carlsbad, CA, USA) (sup- Epigenetics represents the study of heritable plemented with 10% fetal bovine serum (FBS) alterations in gene copy numbers in the absence (Thermo Fisher Scientific, Waltham, MA, USA) of genetic mutations14. DNA methylation has and penicillin-streptomycin (Thermo Fisher Sci- emerged as a critical driver of tumor development, entific, Waltham, MA, USA)) under standard most thoroughly characterized in nasopharyngeal culture conditions (37°C, 5% CO2). For all exper- carcinoma15. The methylation of LncRNAs has iments, cells (5×105/well) were treated with pre- gained intense interest in cancer genetics through defined EC50 of 5-azacytidine and chidamide. the identification of its ability to regulate the ex- pression of epithelial splicing regulatory protein Lentiviral miR-361 Inhibitors and Mimics 2 (ESRP2) in breast cancer and maternally ex- Lentiviral vectors were used for all overex- pressed 3 (MEG3) in lung cancer16. pression and/or depletion experiments. The BLA- CpG methylation, in addition to methyla- CAT1 overexpression vector was synthesized by tion-related genes, regulates cancer occurrence Invitrogen (Carlsbad, CA, USA). All miR-361 re- and development. DNA methylation is regu- gents and clones were purchased from Invitrogen lated by DNA methyltransferase (cytosine-5) 1 (Carlsbad, CA, USA). Lipofectamine 3000 (Carls- (DNMT1) in chronic myeloid leukemia and he- bad, CA, USA) was used for the transfection of patocellular carcinoma17,18. The histone N-methyl- LV-BLACAT1, LV-controls, miR-361 controls or transferase enzyme, enhancer of zeste homolog 2 miR-361 inhibitors. (EZH2), is implicated in many important cancer types. Histone modification and histone deacety- Cell Proliferation Assays lase 1 (HDAC1) is also involved in the progres- Transfected PC3 and DU145in 96-well plates sion of breast cancer, pancreatic cancer and renal (10000 cells/well) were treated with MTT cell carcinoma19, and HDAC inhibitors (HDACis) (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- have obtained promising results in the clinic. Chi- zolium bromide) (10 µl) (Sigma-Aldrich, St. Lou- damide is a novel HDACi that has been used in is, MO, USA) for 4 h. Absorbance values were lymphoma treatment20,21. However, the regulation read at 490 nm as per standard protocols as a mea- of chidamide on BLACAT1 and miR-361expres- sure of cell viability. sion in prostate cancer are poorly characterized. Here, we have performed a range of experi- Apoptosis Assays ments to dissect the role of BLACAT1 in prostate To assess the number of apoptotic cells per cancer progression and assess the potential roles sample, transfected cells were analyzed by flow of chidamide and 5-azacytidineas in cancer treat- cytometry after staining with Annexin V-FITC ments. (fluorescein isothiocyanate) and propidium iodide (PI) (BD Biosciences, San Jose, CA, USA). Patients and Methods Real-Time Polymerase Chain Reaction (RT-PCR) Analysis Prostate Cancer Samples For RT-PCR, total RNA was extracted via Prostate cancer tumor tissue and healthy tissue TRIzol lysis (Thermo Fisher Scientific, Waltham, were obtained from 25 patients treated at our lo- MA, USA) and reverse transcription reactions cal hospital between May 2015 and March 2016. were performed to produce cDNA at 42°C for 60 Tissues were collected and stored at −80°C prior min; 25°C for 5 min; and 70°C for 5 min. The to experimental analysis. All patients completed qPCR mixture (20 µl) consisted of 1 µl cDNA, 0.5 informed consent forms and all study protocols µl of each primer, 10 µl SYBR Green Mix (Invi- were approved by the Institutional Ethical Com- trogen, Carlsbad, CA, USA), and 8 µl of diethyl mittee of China Japan Union Hospital of Jilin pyrocarbonate (DEPC)-treated water. The qRT- University. PCR conditions: 95°C for 5 min; 45 x 95°C for 15 75 H.-Y. Li, F.-Q. Jiang, L. Chu, X. Wei Table I. Primer sequences for RT-PCR. Gene Primer Product BLACAT1 Forward: 5’-CTCCCCTTCTAGCGCTCACG-3’ 154 bp Reverse: 5’-CTAGCCGCCGTCTATACTACCGGCT-3’ DNMT1 Forward: 5’-AGACACTCGCTCAGCTTCTTG-3’ 106 bp Reverse: 5’-CAATTGCTGCTGGGATTCATC-3’ HDAC1 Forward: 5’-CTGCTTTGTATTCCCTTTTGCA-3’ 131 bp Reverse: 5’-TTGATTTCTCCTGGCTGTCTC-3’ EZH2 Forward: 5’-CCTCCCGAGTGAAGTCATCGTGG-3’ 142 bp Reverse: 5’-GGACAGGTGCTTCATCAGCTCG-3’ MDM2 Forward: 5’-ACCACACGTTCCTAAGCTGG-3’ 119 bp Reverse: 5’-TCCCTGCACGCAGAGATTTT-3’ Bax Forward:5’-TCCCTGCACGCAGAGATTTT-3’ 131bp Reverse: 5’-TTGATTTCTCCTGGCTGTCTC-3’ bcl-2 Forward: 5’-AACCCTGCTGTTTGGCTTAAC-3’ 127 bp Reverse: 5’-CGTACTTGCTGTACTCGCTCTTC-3’ ACTB Forward: 5’-GAGCTACGAGCTGCCTGAC-3’ 121 bp Reverse: 5’-GGTAGTTTCGTGGATGCCACAG-3’ s; 60°C for 35 s; and 72°C for 20 s. The relative PCR parameters: 95°C (10 min); 35 x 30 s at 95°C; gene expression was calculated using the 2-ΔΔCT 54°C (45 s); 72°C (45 s); extension at 72°C (7 min). method (n = 3 reactions per sample). Primer se- Products were run on agarose gel (GoldView; quences are shown in Table I. Saibaisheng
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