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 immunoprecipitation 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 expression 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 Bioengineering Co., Ltd., Beijing, China) in the following order: BLACAT1 methyl- miR-361 Expression ated (M), BLACAT1 non-methylated (U) positive RNeasy Mini Kit (Qiagen, Valencia, CA, and negative methylation; BLACAT1 M and BLA- USA) was used for total RNA extraction from CAT1 U partially methylated; BLACAT1 M and both cell lines and tissues. qRT-PCR assays were BLACAT1 U positive and negative non-methylat- performed using miScript Reverse Transcription ed. Table II lists all primer sequences. kit (Qiagen, Valencia, CA, USA) and miScript SYBR® Green PCR kit (Qiagen, Valencia, CA, Western Blotting USA). PCR parameters: 95°C for 1 min; 40 x 95°C Transfected cells were harvested in radio im- for 10 s; 55°C for 30 s; 70°C for 30 s. miRNA ex- munoprecipitation assay (RIPA) buffer, and pro- pression was normalized to U6 and calculated via teins were resolved on 8-15% sodium dodecyl the 2-ΔΔCT method. sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) gels and transferred to polyvinylidene di- Assessment of Methylation Status fluoride (PVDF) membrane (Amersham Pharma- DNA isolation kit (Zymo, Irvine, CA, USA) cia, Piscataway, NJ, USA). The PVDF membrane was used for complementary DNA (cDNA) isola- was washed in Tris-buffer saline (TBS), blocked tion. EZ DNA Methylation-Gold kit (Zymo, Irvine, with 5% skimmed milk in TBS, and was probed CA, USA) was used to produce sulfated DNA, 2 μ with anti-EZH2, anti-HDAC1, anti-MMP-2, an- of which was added with Zymo Taq PerMix (12.5 ti-MMP-9, anti-Bax, anti-Bcl-2 and anti-ATCB μl) (Zymo, Irvine, CA, USA), water (8.5 μl) and primary antibodies (Abcam, Cambridge, MA, upstream and downstream primers (1 μl of each). USA). Membranes were then washed and labeled

Table II. Primer sequences of the methylated BLACAT1 gene. Gene Primer Product

BLACAT1,M-MSP Forward: 5’-CGTAGCTACTGCTATTATCGTTC-3’ 162 bp Reverse: 5’-TTATGCCTTGGGCTACGTACT-3’ BLACAT1,U-MSP Forward:5’-GGTACGTATGCTTGGGCTTATG-3’ 162bp Reverse: 5’-ATTGGCGCTTAGCTAGGCTA-3’

M, methylated; MSP, methylation-specific polymerase chain reaction; U, unmethylated.

76 Long non-coding RNA BLACAT1 inhibits prostate cancer cell proliferation through sponging miR-361 with conjugated secondary antibodies. Protein pared to the reference genome coverage and an bands were visualized on a BioSpectrum Imaging in-depth statistical analysis of the genome loci System (UVP, LLC, Upland, CA, USA). was established. Furthermore, gene functional element distributions, gene enrichment and path- Luciferase Assays way enrichment analysis were performed. LncRNA BLACAT1 (lncRNA-BLACAT1-wt) and its mutated 3’-untranslated region (3’-UTR) Xenograft Models (lncRNA-BLACAT1-mu) were cloned into pRL- Athymic male mice (Chinese Academy of Sci- TK (Promega, Madison, WI, USA). Plasmids ence, Shanghai, China) were provided free access were co-transfected with miRNA mimics or to water and food in laminar flow cabinets under negative control mimics and pGL3 as firefly lu- pathogen-free conditions. To produce the mod- ciferase control vector (Promega, Madison, WI, els, stably transfected PC3 cells (control vector or USA). Dual reporter assays were performed using LV-BLACAT1 expressing) were xenografted into the Promega system and assessed in a Modulus BALB/c male nude mice. Each week, both the tu- Single-tube Multimode Reader (Promega, Madi- mor volumes and weights were measured. After son, WI, USA). pRL-TK values were normalized 35 d of tumor growth, tumors were collected from to pGL3. Assays were performed on a minimum sacrificed mice for further analyses. The experi- of three independent occasions. ments involving animals were approved by The Animal Committee of Jilin university. RNA Pull-Down Assay For the detection of KCNK15-AS1 binding Statistical Analysis proteins, Pierce Magnetic RNA-Protein Pull- Data are expressed as the mean ± SD and an- Down Kit (Thermo Fisher Scientific, Waltham, alyzed using SPSS 19.0 (IBM Corporation, Ar- MA, USA) was utilized. BLACAT1 lncRNAs monk, NY, USA). Group data were compared (both sense and antisense) were synthesized and using a one-way ANOVA (for multiple compar- added to the PC3 cell lysates. The protein was isons to the mean) or a Student’s t-test for single then collected for mass spectrometry analysis. group comparisons. A chi-squared test was used to compare rates. p < 0.05 indicated significant RNA Immunoprecipitations (RNA-IPs) differences between groups. Magna RIP kit (EMD Millipore, Burlington, MA, USA) was used for RNA-IPs. Beads were resuspended, mixed with the samples at 4°C over- Results night. IPs products were washed for at least 6 times and RNA was extracted to determine its abundance. BLACAT1-Binding Proteins in PC3 Cells Recent studies on lncRNA-binding proteins Chromatin Immunoprecipitation (ChIP) have established that the proteins that interact Cells were crosslinked with formaldehyde with lncRNAs regulate disease progression. RNA and then harvested. Large DNA fragments (10- pull-downs were performed to reveal novel BLA- 500 bp) were disrupted by sonification, and the CAT1-binding proteins in PC3 cells, and the total HDAC1 and EZH2 antibodies/beads were added number of BLACAT1-binding proteins was de- for protein-DNA precipitations. Cross-linking tected by mass spectrometry. The results showed solution and sodium chloride were added at 65°C that 1,823 unique proteins bound to BLACAT1. to dissociate the DNA and proteins. QIAGEN The results were first subjected to species selec- PCR kit (Qiagen, Hilden, Germany) was used for tion; the polymorphic gene sequences were then DNA and chromatin purifications and subsequent compared with sequences in protein database. sequencing. Initial read outputs were filtered to After choosing a homology higher than 99%, the remove potential contaminants and samples were results showed that EZH2, DNMT1 and HDAC1 sequenced to obtain clean data. Short oligonu- interacted with BLACAT1. cleotide alignment program 2 (SOAP2) was em- ployed to compare the reads with reference ge- Increased Expression of DNA nomes according to each individual library read. Modifying Genes and Proteins Quality control (QC) assessments were performed in Prostate Cancer Tissues to assess the quality of the sequencing according Following the results of the RNA pull-down to known QC standards. All sequences were com- results, the mRNA levels of BLACAT1, DNMT1,

77 H.-Y. Li, F.-Q. Jiang, L. Chu, X. Wei

HDAC1, EZH2, MDM2 and MIR361 were deter- regulated in the tumor tissues (Figure 1A). Con- mined by using RT-PCR. BLACAT1 expression sistent with the gene expression data, DNMT1, was significantly reduced in tumor vs. healthy HDAC1, EZH2 and MDM2 showed elevated tissue, while the gene expressions of DNMT1, levels of expression in cancer tissues vs. adjacent HDAC1, EZH2, MDM2, and MIR361 were up- non-cancerous tissues (Figures 1B-C).

B Figure 1. mRNA expression in normal and tumor tissue determined by RT-qPCR. A, mRNA expressions of BLACAT1, DNMT1, HDAC1, EZH2, MDM2, and MIR-361 in tumor tissue vs. normal tissue (◇☆★▼▲▽p<0.05). B, Protein levels of the genes in both tissues (★▲▽☆, p<0.05). C, β-actin was probed as a loading con- C trol.

78 Long non-coding RNA BLACAT1 inhibits prostate cancer cell proliferation through sponging miR-361

DU145 cells vs. RWPE-1 cells (Supplementary Figure 1A). LV-BLACAT1 was then transfected into PC3 and DU145 cells, and increased BLA- A CAT1 levels were verified by RT-PCR (Supple- mentary Figure 1B). We next performed MTT viability assays to assess the influence of BLA- CAT1 on cell growth and apoptosis. Transfection B with BLACAT1 slowed the growth of both prostate cancer cell lines at 72 h and 96 h, respective- ly (Figure 4A). In vivo assessments showed that the overexpression of BLACAT1 slowed tumor growth at 28 days and 35 days (Figure 5). We performed Annexin-PI staining to determine the influence of BLACAT1 on PC3 and DU145cell apoptosis. BLACAT1 overexpression led to apop- C totic induction in both cell lines (Figure 4B). We further analyzed the expression of apoptotic as- Figure 2. Methylation status of BLACAT1 determined sociated proteins including Bcl-2, Bax, matrix by MSP. A, BLACAT1 was methylated in tumor tissue. B, BLACAT1 was non-methylated in normal tissue. C, BLA- metallopeptidase 2 (MMP-2), and matrix metal- CAT1 was methylated in both prostate cancer cell lines but lopeptidase 9 (MMP-9) following the exogenous became non-methylated following drug treatment. expression of BLACAT. The results showed that high BLACAT1 levels suppressed Bcl-2, MMP- The BLACAT1 Promoter is Methylated 2, and MMP-9, but increased Bax expression in in Prostate Cancer PC3 and DU145 cells, underliying the roles of Methylation-specific PCR (MSP) was performed these proteins in the BLACAT mediated effects on the BLACAT1 promoter in prostate cancer tissue on prostate cancer cell survival (Figures 4C-D). vs. adjacent non-cancerous tissue. Prostate tumor tissues showed that the levels of BLACAT1 pro- BLACAT1 Interacts with miR-361 moter methylation (Figure 2A) were high in tumor CeRNAs are lncRNAs that produce a sponge tissue and absent in adjacent non-cancerous tissues. effect through their binding to miRNAs that block BLACAT1 promoter methylation was confirmed their subsequent effects on gene expression. We in vitro in the prostate cancer cell lines. Following investigated if BLACAT1 acts via this mechanism treatment with chidamide and 5-azacytidine, BLA- through luciferase reporter assay, which assessed CAT1 methylation was absent (Figure 2C). the binding of BLACAT1 to miR-361. The relative luciferase activity showed no differences between Chidamide and 5-Azacytidine Inhibit miR-361 control and mimics groups after the the Expression of DNA Modifying Genes transfection of the mutant 3’-UTR of BLACAT1. We next assessed epigenetic regulation in the However, luciferase activity declined in the miR- presence of chidamide and 5-azacytidine. In the 361 mimic groups compared to miR-361 control experiments that followed, PC3 and DU145 cells group after transfection of wild 3’-UTR of BLA- were exposed to a range of concentrations of each CAT1 (Figure 6A). Using the RNA up algorithm, compound in which BLACAT1 mRNA expres- significant complementary regions were predict- sion increased, while the expression of DNMT1, ed, suggesting that miR-361 binds to BLACAT1 HDAC1, EZH2, MDM2, and MIR361 decreased (Figure 6B). RIP results showed that the fold en- (Figures 3A-B). These effects on gene expression richment of lncRNA BLACAT1 was elevated in were confirmed by Western blot assessments of EZH2 and MDM2 groups vs. the IgG alone group protein expression (Figures 3C-D). (Figure 5C). Furthermore, the overexpression of BLACAT1 led to a loss of miR-361 in both PC3 BLACAT1 Overexpression Slows and DU145 prostate cancer cells (Figures 6D-E). Cells Growth and Leads to Apoptotic Induction Knockdown of miR-361 Slows Prostate BLACAT1 mRNA expression was initial- Cancer Growth and Promotes Cell Death ly assessed in PC3, DU145 and RWPE-1 cells. To reveal the effects of miR-361 in prostate BLACAT1 mRNA expression was lower in PC3/ cancer cells, its expression was detected in PC3

79 H.-Y. Li, F.-Q. Jiang, L. Chu, X. Wei

Figure 3. Gene and protein expressions following treatment with chidamide or 5’azacytidine. A, mRNA expressions of BLACAT1, DN- MT1, HDAC1, EZH2, MDM2 and MIR361 following drug treatment in PC3 cells (▲△▽▼☆◆, p<0.05). B, mRNA levels of the aforementioned A genes following drug treatments in DU145 cells (▲▽◇★◆△, p<0.05). C, DNMT1, HDAC1, EZH2 and MDM2 expressions at the protein level following 5-azacytidine or- chidamide treatment in PC3 cells (▲▼▽△◇◆■□, p<0.05). β-actin was probed as a loading control. D, Expressions of DNMT1, HDAC1, EZH2 and MDM2 at the protein level following 5-azacyt- B idine or chidamide treatment in DU145 cells (▽△▲▼★☆■□, p<0.05).

and DU145, and found to be significantly higher cell viability and proliferation were assessed via than that of RWPE-1 cells (Supplementary Fig- MTT assays. The inhibition of miR-361 expres- ure 1A). To further analyze the roles of miR-361, sion caused a delayed cell growth 72 h post-trans- miR-361 inhibitors were transfected into PC3 and fection (Figure 7A). We further performed flow DU145 cells (Supplementary Figure 1C) and cytometry to investigate the effects of miR-361 on

80 Long non-coding RNA BLACAT1 inhibits prostate cancer cell proliferation through sponging miR-361

Figure 4. A, PC3 and DU145 proliferation assays following the overexpression of BLACAT1 (☆▽▲, p<0.05). B, Apoptosis assessments in cells overexpressing BLACAT1. C, MMP2, MMP-9, Bax, and Bcl-2 expressions in PC3 cells following BLA- CAT1 overexpression (☆▽★▲▼△◇★, p<0.05). D, MMP2, MMP-9, Bax, and Bcl-2 expressions in DU145 cells following BLA- CAT1 overexpression (▽◆★◇▼▽★◇, p<0.05).

PC3 and DU145 cell apoptosis. MiR-361 silenc- MMP-2 and MMP-9. MiR-361 silencing led to a ing enhanced the apoptotic rates of both prostate loss of Bcl-2, MMP-2 and MMP-9 expressions at cancer cell lines (Figure 7B). These effects were both the mRNA and protein levels, while Bax ex- confirmed through the expressions of Bcl-2, Bax, pression was enhanced (Figures 7C-D).

81 H.-Y. Li, F.-Q. Jiang, L. Chu, X. Wei

Figure 5. Tumor sizes in BLACAT1 overexpression groups vs. the control group (▼☆p<0.05).

Figure 6. A, miR-361 mimics and the miR-361 control groups after transfection of mutant or wild 3’-UTR of BLACAT1. B, miR-361 binds to BLACAT1 through complementary sequences in the indicated regions, as predicted by the RNA up algorithm C, Fold enrichment of lncRNA BLACAT1 was elevated in EZH2 and MDM2 groups vs. the IgG group. D, miR-361 ex- C D E pression following BLACAT1 overexpression in PC3 cells. E. miR-361 expression following BLACAT1 overexpression in DU145 cells.

82 Long non-coding RNA BLACAT1 inhibits prostate cancer cell proliferation through sponging miR-361

Figure 7. A, PC3 and DU145 cell proliferation assay following miR- 361 inhibition (◇△p<0.05). B, Apoptosis assessment in the prostate cancer cell lines following miR- 361 inhibition. C, MMP2, MMP- 9, Bax and Bcl-2 expressions following miR-361 inhibition in PC3 cells (▽◆▲◇▲☆▼▽, p<0.05). D, MMP2, MMP-9, Bax and Bcl-2ex- A pressions following miR-361 inhibition in DU145 cells (◇★☆▲P☆★◇▽, p<0.05).

EZH2 and HDAC1 Interacting Partners disease progression. Despite the intense research Following sequencing, we identified 26,347,096 efforts, the mechanisms of prostate cancer devel- original reads which. After filtering, it was re- opment remain poorly understood and effective vealed that the mitogen-activated protein kinase chemotherapeutic treatment regimens are lacking. (MAPK) can interact with EZH2. After sequenc- To improve prostate cancer therapies, a clear un- ing, 69,837,024 original reads were obtained. Upon derstanding of the main cancer drivers is required. filtering, it was revealed that HDAC1 interacted Here, we have identified two regulatory axes with the STAT3 promoter region. namely BLACAT1-EZH2-MAPK and BLA- CAT1-HDAC1-STAT3 that are associated with prostate cancer progression. MiR-361, the target Discussion miRNA of BLACAT1, was additionally detected to be associated with prostate cancer progression Prostate cancer remains a leading cause of due to its effects on PC3 and DU145 cell lines. death across the globe, most commonly in elder- Chidamide and 5-azacytidine were further re- ly males. To-date, prostate cancer diagnostics are vealed as promising prospects for prostate cancer lacking due to the lack of early symptoms during treatment.

83 H.-Y. Li, F.-Q. Jiang, L. Chu, X. Wei

Consistent with the literature, we found that Conclusions BLACAT1 is downregulated in prostate cancer tissue due to its methylation confirmed by MSP. We identified two regulatory axes of BLACAT1- This highlighted the high methylation rates of EZH2-MAPK and BLACAT1-HDAC1-STAT5, BLACAT1 as a potentially key event during which were associated with prostate cancer pro- breast cancer prognosis. gression. Chidamide and 5-azacytidine might pro- Histone deacetylation and DNA methylation vide new ideas in treating prostate cancer. both influence promoter activity and subsequent gene expression14,22,23. CpG island methylation is strongly regulated by members of the DNA meth- Authors' Contributions yltransferase (DNMT) family including DNMT124, Hong-yan Li, Fu-quan Jiang and Lin Chu carried out the while histone modifications are regulated by the experiments and Xin Wei prepared the manuscript. HDAC family25. The expressions of DNMT1, HDAC1, MDM2 and EZH2 were at high levels in prostate cancer tissue. It was therefore speculated Conflict of Interests that DNMT1, HDAC1, MDM2 and EZH2 could act The Authors declare that they have no conflict of interests. as key regulators of prostate cancer development. Exposing in vitro prostate cancer lines to chidamide and 5-azacytidine led to enhanced levels of References BLACAT1. This phenomenon revealed that BLA- CAT1 expression mediates histone acetylation in 1) Kim HL, Halabi S, Li P, Mayhew G, Simko J, Nixon addition to DNA methylation. AB, Small EJ, Rini B, Morris MJ, Taplin ME, George D. A molecular model for predicting overall sur- We further revealed that miR-361 is a cellular vival in patients with metastatic clear cell renal target for BLACAT1. BLACAT1 overexpression carcinoma: results from CALGB 90206 (Alliance). and miR-361 silencing reduced cell growth and en- EBioMedicine 2015; 2: 1814-1820. hanced the apoptotic rates of prostate cancer cells. 2) Zhang D, Tang DG. "Splice" a way towards neu- Further analysis of the expression of key mediators roendocrine prostate cancer. EBioMedicine 2018; 35: 12-13. of cellular apoptotic pathways revealed that these 3) Munkley J, Vodak D, Livermore KE, James K, Wilson effects were mediated by a loss of Bcl-2 expression BT, Knight B, Mccullagh P, Mcgrath J, Crundwell M, and increased levels of Bax. Thus, both BLACAT1 Harries LW, Leung HY, Robson CN, Mills IG, Rajan overexpression and miR-361 silencing slowed cell P, Elliott DJ. Glycosylation is an androgen-regu- cycle progression and promoted apoptotic cell death lated process essential for prostate cancer cell viability. EBioMedicine 2016; 8: 103-116. through downregulating the expression of Bcl-2 in 4) Lee AR, Gan Y, Tang Y, Dong X. A novel mech- prostate cancer cells. The overexpression of both anism of SRRM4 in promoting neuroendocrine MMP-2 and MMP-9 have been implicated in an ar- prostate cancer development via a pluripotency ray of lethal human cancers including cervical, ovar- gene network. EBioMedicine 2018; 35: 167-177. 5) Wang L, Wang J, Xiong H, Wu F, Lan T, Zhang Y, ian, hepatocellular, head, neck and thyroid carcino- uo ang aleem iang u eng 26,27 G X, W H, S M, J C, L J, D mas, in addition to squamous cell carcinomas . In Y. Co-targeting hexokinase 2-mediated Warburg this study, BLACAT1 overexpression and miR-361 effect and ULK1-dependent autophagy suppress- silencing led to a loss of these oncogenic proteins es tumor growth of PTEN- and TP53-deficien- in prostate cancer cells, revealing new insights into cy-driven castration-resistant prostate cancer. the roles of these host factors in the progression and EBioMedicine 2016; 7: 50-61. 6) Munschauer M, Nguyen CT, Sirokman K, Hartigan development of prostate cancer. Furthermore, RNA CR, Hogstrom L, Engreitz JM, Ulirsch JC, Fulco CP, pulldown and RIP analysis showed that DNMT1, Subramanian V, Chen J, Schenone M, Guttman M, EZH2 and HDAC1 interacted with BLACAT1, Carr SA, Lander ES. The NORAD lncRNA assem- suggesting that these proteins regulate BLACAT1 bles a topoisomerase complex critical for genome expression. ChIP-seq was performed to detect stability. Nature 2018; 561: 132-136. 7) Luo Z, Rhie SK, Lay FD, Farnham PJ. A prostate HADC1 and EZH2 expressions in prostate cancer cancer risk element functions as a repressive cells, which revealed that EZH2 interacted with the loop that regulates HOXA13. Cell Rep 2017; 21: MAPK promoter while HDAC1 interacted with the 1411-1417. promoter of STAT3. It was therefore speculated that 8) Zhang Y, Zhang P, Wan X, Su X, Kong Z, Zhai MAPK regulates EZH2 while STAT3 regulates Q, Xiang X, Li L, Li Y. Downregulation of long non-coding RNA HCG11 predicts a poor prog- HDAC1, implicating a role for both genes in the reg- nosis in prostate cancer. Biomed Pharmacother ulation of BLACAT1. 2016; 83: 936-941.

84 Long non-coding RNA BLACAT1 inhibits prostate cancer cell proliferation through sponging miR-361

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