miR-15b-5p通过靶向CHRM3/YAP轴抑制前列腺癌去势抵抗生长

miR-15b-5p inhibits castration-resistant growth of prostate cancer by targeting CHRM3/YAP axis

摘要

目的：分析微小核糖核酸-15b-5p（miR-15b-5p）对前列腺癌去势抵抗性生长及毒蕈碱受体M3（CHRM3）/Yes相关蛋白（YAP）信号轴的影响，并分析其作用机制。方法：选取雄激素依赖型前列腺癌细胞系LNCaP及去势抵抗性细胞系PC3、PC3M，对LNCaP细胞使用活性炭剥离血清（CSS）和恩杂鲁胺（ENZ）模拟去势治疗，使用双荧光素酶报告基因检测及TargetScanHuman数据库分析LNCaP细胞系miRNA变化；PC3和PC3M细胞系均培养后随机分为对照组、卡巴胆碱组及转染组，每组11个样本，分别进行生理盐水干预、毒蕈碱激动剂卡巴胆碱（Carbachol）干预及卡巴胆碱+miR-15b-5p转染，观察RNA测序结果及双荧光素酶报告基因检测结果，比较各组结缔组织生长因子（CTGF）、半胱氨酸富集蛋白61（CYR61）的mRNA表达水平、YAP蛋白表达及细胞增殖检测结果。结果：与对照细胞相比，用CSS+ENZ处理的LNCaP细胞中有255个miRNA显著下调，111个miRNA显著上调，其中miR-15b-5p表达水平随时间的变化降低，CHRM3表达水平随时间升高（P<0.05）；CHRM3有两个预测的miR-15b-5p 3'UTR内的结合位点（核苷酸1464-1470和5174-5181）。PC3和PC3M细胞系共转染miR-15b-5p和克隆了含有miR-15b-5p结合位点的DNA序列的野生型载体后，荧光强度显著降低（P<0.05），共转染miR-15b-5p和缺乏miR-15b-5p结合位点的缺失型载体荧光强度无显著差异（P>0.05）；PC3和PC3M细胞系中卡巴胆碱组CTGF、CYR61 mRNA相对表达高于对照组及转染组，YAP蛋白相对表达低于对照组及转染组（P<0.05）；以对照组为标准，PC3和PC3M细胞系中卡巴胆碱组细胞增殖率高于对照组及转染组（P<0.05）。结论：miR-15b-5p是影响前列腺癌去势抵抗性生长miRNA之一，去势干预抑制了miR-15b-5p表达并增加了CHRM3表达。miR-15b-5p直接与CHRM3结合并抑制了CHRM3刺激诱导的YAP活化且miR-15b-5p转染消除了CHRM3刺激诱导的去势抵抗性前列腺癌细胞的生长，miR-15b-5p/CHRM3/YAP信号轴促进了前列腺癌的去势抵抗性生长。

Abstract

Objective: To analyze the effects of miR-15b-5p on the growth of castration-resistant prostate cancer and the cholinergic receptor muscarinic 3 (CHRM3)/Yes-associated protein (YAP) signaling axis, and to elucidate the underlying mechanisms. Methods: Androgen-dependent prostate cancer cell line LNCaP and castration-resistant cell lines PC3 and PC3M were employed. LNCaP cells were subjected to androgen deprivation using charcoal-stripped serum (CSS) combined with enzalutamide (ENZ) treatment to mimic castration therapy. Differential miRNA expression in LNCaP cells was screened using RNA sequencing, with bioinformatic target prediction performed using the TargetScanHuman database, and putative interactions were subsequently validated via dual-luciferase reporter assays. PC3 and PC3M cells were cultured and randomly allocated into three groups (n=11 replicates per group): control (vehicle), carbachol (muscarinic agonist), and carbachol+miR-15b-5p mimic transfection. Cells were treated with normal saline, carbachol, or carbachol combined with miR-15b-5p transfection, respectively. RNA sequencing and dual-luciferase reporter assays were performed to examine. Subsequently, mRNA expression levels of connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (CYR61), YAP protein expression, and cell proliferation rates were compared among the groups. Results: RNA sequencing results showed that, compared with control cells, 255 miRNAs were significantly down-regulated and 111 miRNAs were significantly up-regulated in LNCaP cells treated with CSS+ENZ. Among these, miR-15b-5p expression decreased over time, while CHRM3 expression increased (P<0.05). Bioinformatic analysis predicted two miR-15b-5p binding sites in the 3'UTR of CHRM3 (nucleotides 1464-1470 and 5174-5181). Dual-luciferase reporter assays confirmed that co-transfection of miR-15b-5p with the wild-type vector containing these binding sites significantly reduced fluorescence intensity in PC3 and PC3M cells (P<0.05), whereas no significant difference was observed with the deletion mutant vector lacking the binding sites (P>0.05). In the carbachol group, relative mRNA expression of CTGF and CYR61 was significantly higher, while YAP protein expression was lower than that respectively in the control and transfection groups (P<0.05). Similarly, cell proliferation rates in the carbachol group were higher than those in the control and transfection groups (P<0.05). Conclusion: miR-15b-5p is one of the miRNAs that affect castration-resistant growth of prostate cancer. Androgen deprivation inhibits miR-15b-5p expression and increases CHRM3 expression. miR-15b-5p directly binds to CHRM3 and suppresses CHRM3-induced YAP activation. Furthermore, miR-15b-5p transfection abrogates CHRM3-induced CRPC cell growth. The miR-15b-5p/CHRM3/YAP signaling axis regulates castration-resistant growth of prostate cancer.

关键词

miR-15b-5p / CHRM3/YAP / 前列腺癌 / 去势抵抗性生长

Key words

miR-15b-5p / CHRM3/YAP / Prostate Cancer / Castration-Resistant Growth

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基金资助

开封市科技发展计划项目(2303031)