CircRNA-100395 基因通过启动子区甲基化调控miRNA-136-5p/Smad3 轴促进前列腺癌细胞增殖及侵袭的机制研究

目的　检测前列腺癌细胞中环状核糖核酸（circular RNAs, CircRNA）100395 基因启动子区甲基化状态及其表达水平，研究CircRNA-100395 基因去甲基化对前列腺癌细胞增殖、侵袭的影响及作用机制。方法　选择人正常前列腺上皮细胞（RWPE）和前列腺癌细胞系（LNCap，PC3 和DU145）进行研究。采用甲基化特异性聚合酶链式反应(methylationspecific polymerase chain reaction, MSP) 检测CircRNA-100395 基因启动子区甲基化状态。采用实时荧光定量聚合酶链反应(qRT-PCR) 法检测上述细胞中CircRNA-100395 mRNA 表达水平。应用去甲基药物5- 氮杂-2'- 脱氧胞苷（AZA）处理LNCap 细胞，检测AZA 去甲基化处理前后细胞中CircRNA-100395 表达水平。采用CCK-8 和Transwell 实验检测CircRNA-100395 基因去甲基化前后LNCap 细胞增殖和侵袭能力。构建CircRNA-100395 野生型和突变型质粒，通过双荧光素酶报告分析CircRNA-10039 与miRNA-136-5p 之间的靶向关系；利用qRT-PCR 检测CircRNA-10039 与miRNA-136-5p 表达水平，验证其调控关系。采用Westernblot 法检测AZA 去甲基化和miRNA-136-5p 过表达对Smad3，p-Smad3 蛋白表达的影响。结果　前列腺癌细胞中CircRNA-100395 呈高甲基化状态；LNCap，PC3 及DU145 细胞中CircRNA-100395 相对表达水平分别为0.39±0.08，0.65±0.14，0.62±0.10，显著低于RWPE（1.12±0.15）细胞中表达水平，差异有统计学意义（F=42.076，P ＜ 0.001）。经AZA 去甲基化处理后，LNCap 细胞中CircRNA-100395 表达水平为1.02±0.17，较未处理LNCap 细胞（0.42±0.05）明显升高，差异有统计学意义（t=5.808，P ＜ 0.01）。AZA 去甲基化处理显著抑制了LNCap 细胞的增殖能力（t=8.764~12.970，均P ＜ 0.001）、迁移能力（t=6.092，P ＜ 0.01）。miRNA-136-5 可能是CircRNA-100395 的下游靶基因。未经AZA 处理前LNCap 细胞中CircRNA-100395，miRNA-136-5p 表达水平分别为0.39±0.08，0.87±0.15，AZA 处理后两者表达水平分别为1.02±0.17，0.35±0.08，差异有统计学意义（t=5.808，5.298，均P ＜ 0.01）。AZA 处理后Smad3 和p-Smad3 蛋白表达明显升高，差异有统计学意义（t=7.394，11.889，均P ＜ 0.01）；miRNA-136-5p 过表达抑制了Smad3 和p-Smad3 蛋白表达，差异有统计学意义（t=4.996，5.422，均P ＜ 0.01）。结论　CircRNA-100395 基因启动子区的高甲基化状态可以抑制CircRNA-100395 在前列腺癌细胞中的表达水平，去甲基化处理后CircRNA-100395 表达恢复，并抑制前列腺癌细胞增殖和侵袭，其作用机制可能与CircRNA-100395 靶向调控miRNA-136-5p/Smad3 轴有关。

Mechanism of CircRNA-100395 Promoter Methylation Promoting Prostate Cancer Cells Proliferation and Invasion by Regulating miRNA-136-5p/Smad3 Axis

Objective　To investigate the relationship between the methylation status and expression level of CircRNA-100395 gene promoter in prostate cancer cells, and explore the effect and mechanism of CircRNA-100395 demethylation on prostate cancer cell proliferation and invasion. Methods　Human normal prostate epithelial cells (RWPE) and prostate cancer cell lines (LNCaP, PC3 and DU145) were selected for the study. The methylation status of the promoter region of CircRNA-100395 gene was detected by methylation-specific polymerase chain reaction (MSP). The mRNA expression level of CircRNA-100395 was detected by qRT-PCR .LNCaP cells were treated with 5-Aza-2’- deoxycytidine (AZA), a demethylating drug, and the expression level of CircRNA-100395 in cells before and after AZA demethylation was detected. CCK-8 and Transwell experiments were used to detect the proliferation and invasion of LNCaP cells before and after demethylation of CircRNA-100395 gene.The wildtype and mutant plasmids of circRNA-100395 were constructed, the targeting relationship between CircRNA-10039 and miRNA- 136-5p was analyzed by dual-luciferase reporter, and qRT-PCR was used to further confirm the relationship between CircRNA-10039 and miRNA-136-5p.The effects of AZA demethylation and miRNA-136-5p overexpression on the expression of Smad3 and p-smad3 proteins were detected by Western blot. Results　CircRNA-100395 was hypermethylated in prostate cancer cells, the difference was statistically significant. The relative expression levels of CircRNA-100395 in LNCaP, PC3 and DU145 cells were 0.39±0.08, 0.65±0.14 and 0.62±0.10, respectively, which were significantly lower than those in rwpe (1.12±0.15) cells，the difference was statistically significant (F=42.076，P<0.001).After AZA demethylation, the expression level of CircRNA-100395 in LNCaP cells was (1.02±0.17), which was significantly higher than that in untreated LNCaP cells (0.42±0.05), the difference was statistically significant(t=5.808, P<0.01).AZA demethylation significantly inhibited the proliferation (t=8.764~12.970, all P<0.001) and migration (t=6.092, P<0.01) of LNCaP cells.MiRNA-136-5 may be the downstream target gene of CircRNA-100395.The expression levels of CircRNA-100395 and miRNA-136-5p in LNCaP cells before AZA treatment were 0.39±0.08 and 0.87±0.15, respectively. After AZA treatment, the expression levels of the two were 1.02±0.17 and 0.35±0.08, respectively, with statistical significance (t=5.808, 5.298, all P<0.01).The expression of Smad3 and p-smad3 protein increased significantly after aza treatment (t=7.394, 11.889, all P<0.01). The overexpression of miRNA-136-5p inhibited the expression of Smad3 and p-smad3 proteins, and the difference was statistically significant (t=4.996, 5.422, all P<0.01). Conclusion　The abnormal hypermethylation state of the promoter region of CircRNA-100395 gene could inhibit the expression level of CircRNA-100395 in prostate cancer cells. After demethylation, it could restore the expression of CircRNA-100395 and inhibit the proliferation and invasion of prostate cancer cells. The mechanism may be related to the targeted regulation of miRNA-136-5p/Smad3 axis by circRNA-100395.