NMHC Ⅱ A在前列腺癌转移中的作用及lncRNA调控轴筛选

The role of Nonmuscle myosin heavy chain Ⅱ A in prostate cancer metastasis and screening of lncRNA regulatory axis

目的研究NMHCⅡA在前列腺癌转移中的作用并筛选其相关lncRNA调控轴。方法使用Oncomine数据库分析前列腺癌中NMHCⅡA编码基因MYH9的mRNA表达水平。通过Western bolt检测良性前列腺增生细胞系BPH-1、前列腺癌细胞系PC3及其高转移型亚细胞系PC3M中NMHCⅡA的蛋白表达水平。构建沉默NMHCⅡA的PC3M细胞,筛选稳定感染细胞株,使用Western bolt及细胞免疫荧光染色鉴定沉默效果。CCK-8检测法测定细胞增殖能力,Transwell迁移和侵袭实验检测细胞迁移和侵袭能力。R语言筛选TCGA数据库中前列腺癌转录组数据,得到异常表达的lncRNA和miRNA并配对;miRcode、miRDB和Targetscan数据库在线预测靶向MYH9的miRNA,比对在线预测结果与TCGA数据库筛选结果,得到共有的miRNA,并根据lncRNA与miRNA配对结果明确miRNA上游的lncRNA,进而推测lncRNA/miRNA/MYH9调控轴。结果Oncomine数据库分析显示,MYH9在前列腺癌组织中的表达高于正常前列腺组织(P<0.05)。Western bolt结果证明与BPH-1相比,NMHCⅡA在前列腺癌细胞PC3以及PC3M细胞中表达增高(P<0.05)。shMYH9 PC3M细胞的NMHCⅡA表达水平显著低于无感染组和空载组(P<0.05)。shMYH9 PC3M细胞增殖能力显著低于无感染组和空载组(P<0.05),迁移和侵袭能力也明显低于空载组与无感染组(P<0.05)。数据库筛选结果显示,KIAA0087/miR-133b/MYH9、HNF1A-AS1/miR-133b/MYH9和CACNA1C-IT3/miR-133b/MYH9轴可能在前列腺癌中发挥调节NMHCⅡA的作用。结论NMHCⅡA可能通过靶向前列腺癌细胞的增殖、迁移和侵袭能力调节前列腺癌的转移;KIAA0087/miR-133b/MYH9、HNF1A-AS1/miR-133b/MYH9和CACNA1C-IT3/miR-133b/MYH9这3条调控轴可能调控NMHCⅡA。

Objective To study the role of NMHCⅡA in prostate cancer metastasis and screen its related lncRNA regulatory axis.Methods Oncome database was used to analyze the mRNA expression level of NMHCⅡA coding gene MYH9 in prostate cancer.The protein expression level of NMHCⅡA in the benign prostatic hyperplasia cell line BPH-1,prostate cancer cell line PC3,and its highly metastatic subcellular line PC3M was detected by Western blot.PC3M cells silencing NMHCⅡA were constructed,stable infected cell lines were screened,and the silencing effect was identified by Western blot and cellular immunofluorescence staining.CCK-8 assay was used to detect cell proliferation,and the Transwell migration and invasion assay was used to detect cell migration and invasion.R language screened the transcriptome data of prostate cancer in the TCGA database,obtained the abnormally expressed lncRNA and miRNA,and matched them;The miRcode,miRDB,and Targetscan databases predict the miRNA targeting MYH9 online,compare the online prediction results with the screening results of the TCGA database,obtain the shared miRNA,and determine the lncRNA upstream of the miRNA according to the matching results of lncRNA and miRNA,and then speculate the lncRNA/miRNA/MYH9 regulatory axis.Results Oncome database analysis showed that the expression of MYH9 in prostate cancer tissue was higher than that in normal prostate tissue(P<0.05).Western blot results showed that compared with BPH-1,the expression of NMHCⅡA in PC3 and PC3M in prostate cancer cells was higher(P<0.05).The NMHCⅡA expression level in shMYH9 PC3M cells was significantly lower than that in the non-infection and empty groups(P<0.05).The proliferation,migration,and invasion ability of shMYH9 PC3M cells was significantly lower than that of the non-infection group and empty group(P<0.05).The database screening results show that KIAA0087/miR-133b/MYH9,HNF1A-AS1/miR-133b/MYH9 and CACNA1C-IT3/miR-133b/MYH9 axes may play a role in regulating NMHCⅡA in prostate cancer.Conclusion NMHCⅡA may regulate prostate cancer metastasis by targeting the proliferation,migration,and invasion of prostate cancer cells;KIAA0087/miR-133b/MYH9,HNF1A-AS1/miR-133b/MYH9,and CACNA1C-IT3/miR-133b/MYH9 may regulate NMHCⅡA.