Mechanism of androgen receptor promoting invasion of non-small cell lung cancer cells via the let-7c-5p/transmembrane protease serines 2 pathway
Objective To explore the mechanism of androgen receptor(AR)facilitating the inva-sion of non-small cell lung cancer(NSCLC)cells.Methods After the AR was overexpressed or knocked down in A549 and H1533 cells,the effect of AR on the invasive ability of A549 and H1533 cells was stud-ied using the Transwell assay.The genes closely related to the invasion of NSCLKC cells were screened,and the downstream genes potentially modulated by AR were identified.The regulatory link between the tar-get gene and AR was confirmed using quantitative polymerase chain reaction(qPCR)and Western blotting,and the Transwell method was employed to assess the impact of AR on cell invasion.Bioinformatic analysis was employed to pinpoint the microRNA(miRNA)regulating the transmembrane protease serines 2(TMPRSS2)gene.The regulatory link between the miRNA and TMPRSS2 was identified using qPCR and Western blotting,while the effect of miRNA on cellular invasion was verified using the Transwell method.Finally,the impact of these genes on tumor cell invasion was observed in nude mice,andt test was used to compare the differences among the groups.Results The overexpression of AR(oeAR)increased the inva-sion ability of lung cancer cells(H1355-oeAR and A549-oeAR)(0.75±0.08 vs.1.84±0.25,0.54± 0.04 vs.1.39±0.30,t=36.360,5.362,P＜0.05),and the known-down of AR(shAR)decreased the invasion ability of lung cancer cells(H1355-shAR and A549-shAR)(0.83±0.08 vs.0.22±0.03,0.61±0.07 vs.1.90±0.29,t=45.178,P＜0.01).The bioinformatic analysis and qPCR results re-vealed that AR could modulate the expression of TMPRSS2(0.55±0.04 vs.1.67±0.37,0.46±0.04 vs.1.67±0.29,t=23.152,43.228,P＜0.01).The expression of TMPRSS2 in both H1355-oeAR and A549-oeAR groups was significantly elevated as compared with the control group(1.28±0.18 vs.0.29± 0.04,1.16±0.15 vs.0.32±0.03,t=7.152,P＜0.05;t=4.298,P＜0.05).The knock-down of TMPRSS2 could partially reduce cell invasive ability in both H1355-oeAR and A549-oeAR groups(0.78± 0.05 vs.0.25±0.01;0.82±0.06 vs.0.36±0.01,t=6.358,14.298,P＜0.05),and similarly,the down-regulation of TMPRSS2 could partially decrease the AR levels in both H1355-oeAR and A549-oeAR groups(2.25±0.44 vs.1.10±0.14;2.32±0.53 vs.1.15±0.23,t=8.258,12.538,P＜0.05).Bioinformatic analysis and validation studies revealed that let-7c-5p could suppress the expression of TMPRSS2(1.27±0.11 vs.0.48±0.08;1.35±0.33 vs.0.56±0.08,t=47.110,P＜0.01).The overexpression of let-7c-5p could partially reverse the AR-induced invasion of H1355 and AQ549 cells(1.77±0.34 vs.0.94±0.10;2.35±0.33 vs.1.10±0.11,t=33.116,21.108,P＜0.01),and sim-ultaneously reduce the protein expression of TMPRSS2 induced by AR overexpression(1.09±0.13 vs.0.55±0.08;1.36±0.21 vs.0.76±0.10,t=3.760,10.105,P＜0.05).ChIP results indicated that let-7c-5p could bind to AREs in TMPRSS2 3'promoter region in H1355 and A549 cells(0.23±0.02 vs.1.32±0.20;0.34±0.03vs.1.10±0.10,t=7.110,5.175,P＜0.05),ande double luciferase tests revealed let-7c-5p could suppress the luciferase in wild-type TMPRSS2 3'untranslated regions(3'UTR)structure but not in mutant TMPRSS2 3'UTR structure(1.25±0.14 vs.0.44±0.03;0.58±0.03 vs.0.66±0.08,t=12.510,P＜0.05;t=1.175,P＞0.05);By assessing the tumors in nude mice by IVIS,it was found that the tumor cell invasion rate in the oeAR+luc group was significantly higher than in the Ctrl+luc group(0.33±0.04 vs.0.78±0.08,t=7.566,P＜0.05),that in the oeAR+TMSS2+luc group was lower than in oeAR+luc group(0.19±0.04 vs.0.78±0.18,t=8.56,P＜0.05).Conclusion AR can facilitate the invasion of lung cancer cells(A549 and H1533)through modifications in the let-7c-5p/TMPRSS2 signaling pathway.
Androgen receptorTransmembrane serine proteinase 2Non-small cell lung cancer
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