LNMAT1/CADM1调控轴对前列腺癌侵袭和免疫抑制因子的影响

目的研究非编码RNA淋巴结转移相关转录本1/细胞黏附分子1(LNMAT1/CADM1)调控轴对前列腺癌(prostate adenocarcinoma,PRAD)细胞侵袭和免疫抑制因子表达水平的影响。方法收集临床PRAD和癌旁(TAC)的组织。构建LNMAT1的慢病毒短发夹RNA(shRNA)载体和阴性对照(NC-shRNA)、CADM1 siRNA(si-CADM1)和阴性对照(NC-siRNA)并转染人高转移性前列腺癌细胞PC-3M。PC-3M细胞处理分组为:NC-shRNA组、LNMAT1 shRNA组、LNMAT1 shRNA+si-CADM1组、LNMAT1 shRNA+NC-siRNA组。实时荧光定量PCR(RT-qPCR)检测PRAD组织、TAC组织、人前列腺上皮细胞RWPE-1、人前列腺癌细胞DU145、PC-3M中的LNMAT1和CADM1的表达。RT-qPCR检测PC-3M分组中LNMAT1、CADM1、MMP-2,MMP-9,E-cadherin和N-cadherin的水平。蛋白免疫印迹法检测PC-3M分组中CADM1、TGF-β、IL^-10、VEGF-A、FasL、HLA-G的水平。伤口愈合检测PC-3M细胞的迁移率,Transwell法检测细胞侵袭率。结果 LNMAT1在PC-3M细胞和有淋巴转移的PRAD组织中上调(均为P<0.05);沉默LNMAT1不仅抑制PC-3M细胞侵袭和迁移能力(均为P<0.05),下调MMP-2、MMP-9、N-cadherin的mRNA水平(均为P<0.05),而且上调E-cadherin的mRNA(P<0.05)。另外,沉默LNMAT1抑制PC-3M细胞免疫抑制因子TGF-β、IL^-10、VEGF-A、FasL、HLA-G的蛋白水平(均为P<0.05);LNMAT1负调控PC-3M细胞中CADM1的表达(P<0.05);沉默CADM1显著逆转PC-3M细胞中LNMAT1对侵袭和免疫抑制因子的抑制能力(均为P<0.05)。结论本研究阐明LNMAT1/CADM1调控轴可影响高转移性PRAD细胞的侵袭和免疫抑制因子。

Effects of LNMAT1/CADM1 axis on prostate adenocarcinoma invasion and immunosuppressive factors expression

This study was designed to study the effect of LNMAT1 regulatory axis on prostate adenocarcinoma(PRAD)cell invasion and immunosuppressive factor expression levels.Clinical PRAD and adjacent cancer(TAC)tissues were collected.Then,LNMAT1 lentiviral short hairpin RNA(shRNA)vector and its negative control(NCshRNA),CADM1 siRNA(si-CADM1)and its negative control(NC-siRNA)were constructed and transfected into human highly metastatic prostate cancer cell PC-3M.PC-3M cells were grouped into NC-shRNA group,LNMAT1 shRNA group,LNMAT1 shRNA+si-CADM1 group,and LNMAT1 shRNA+NC-siRNA group.Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression of LNMAT1 and CADM1 in PRAD tissue,TAC tissue,human prostate epithelial cells RWPE-1,human prostate cancer cells DU145 and PC-3M;RTqPCR also used to detect the levels of LNMAT1,CADM1,MMP-2,MMP-9,E-cadherin and N-cadherin in the PC-3M groups.Western blot was used to detect the levels of CADM1,TGF-β,IL^-10,VEGF-A,FasL,HLA-G in PC-3M groups;wound healing was used to detect the migration rate of PC-3M cells,and Transwell method was used to detect the cell invasion rate.Data showed that LNMAT1 was up-regulated both in PC-3M cells and lymphatic metastatic PRAD tissues(both P<0.05);Silencing LNMAT1 not only inhibited the invasion and migration of PC-3M cells(all P<0.05),down-regulated the mRNA levels of MMP-2,MMP-9,and N-cadherin(all P<0.05),but also up-regulated the mRNA of E-cadherin(P<0.05).In addition,silencing LNMAT1 inhibited the protein levels of TGF-β,IL^-10,VEGF-A,FasL,and HLA-G in PC-3M cells(all P<0.05).LNMAT1 negatively regulated the expression of CADM1 in PC-3M cells(P<0.05).Silencing CADM1 significantly reversed the inhibitory effects of LNMAT1 on the invasion and immunosuppressive factors in PC-3M cells(both P<0.05).In conclusion,the present study clarifies that the regulatory axis of LNMAT1/CADM1 can affect the invasion and immunosuppressive factors of highly metastatic PRAD cells.