层粘连蛋白γ1基因沉默对膀胱癌细胞增殖及细胞周期的影响

Effects of LAMC1 gene silencing on proliferation and cell cycle of bladder cancer cells

目的探讨层粘连蛋白γ1(LAMC1)在膀胱癌细胞中的表达情况,以及沉默LAMC1基因表达对膀胱癌细胞增殖和细胞周期的影响。方法选取河北省邯郸市中心医院2019年1月至2021年12月收治的48例膀胱癌患者的癌及癌旁组织。采用RT-qPCR法检测UMUC3、T24、5637及J82细胞中LAMC1的m RNA表达水平。将LAMC1-si RNA质粒转染至5637细胞中,设置LAMC1沉默组和阴性对照组,RT-qPCR和Western blot验证转染效率;采用CCK-8法、克隆形成实验、流式细胞术检测LAMC1基因沉默对膀胱癌细胞增殖、克隆形成能力及细胞周期的影响,Western blot检测细胞周期相关蛋白cyclin A、cyclin D1、p16和p21的表达水平。结果5637细胞中LAMC1 m RNA表达水平较UMUC3、T24、J82细胞高(P<0.05)。癌旁组织与膀胱癌组织中LAMC1阳性表达率比较,差异无统计学意义(P>0.05)。LAMC1沉默组中LAMC1 m RNA和蛋白表达水平低于阴性对照组(P<0.01)。LAMC1沉默组中细胞增殖活力和克隆形成能力低于阴性对照组(P<0.01)。LAMC1沉默组中细胞中S期细胞所占百分比低于阴性对照组(P<0.01),G1期细胞所占百分比高于阴性对照组(P<0.01)。LAMC1沉默组中的cyclin D1和cyclin A蛋白的表达水平低于阴性对照组(P<0.01),而p16和p21蛋白的表达水平高于阴性对照组(P<0.01)。结论沉默LAMC1基因表达能够抑制细胞周期相关蛋白的表达从而抑制膀胱癌5637细胞增殖能力,提示其可能是膀胱癌潜在的治疗靶点。

Objective To investigate the expression of recombinant laminin gamma 1(LAMC1)in bladder cancer cells and the effect of silencing lamc1 gene expression on the proliferation and cell cycle of bladder cancer cells.Methods Cancer and adjacent tissues of 48 patients with bladder cancer admitted to Handan Central Hospital,Hebei Province from January 2019 to December 2021 were selected.RT-qPCR was used to detect the expression of LAMC1 mRNA in UMUC3,T24,5637,and J82 cells.LAMC1-siRNA plasmid was transfected into 5637 cells,LAMC1 silencing group and negative control group were set up,and the transfection efficiency was verified by RT-qPCR and Western blot.CCK-8 assay,clonogenesis assay and flow cytometry were used to detect the effect of LAMC1 gene silencing on the proliferation,clonogenesis ability,and cell cycle of bladder cancer cells.Western blot was used to detect the expression levels of cyclin A,cyclin D1,p16,and p21.Results The expression level of LAMC1 mRNA in 5637 cells was higher than that in UMUC3,T24,and J82 cells(P<0.05).There was no significant difference in LAMC1 positive expression rate between bladder and paracancer tissues(P>0.05).LAMC1 mRNA and protein expression levels in LAMC1 silencing group were lower than those in negative control group(P<0.01).Cell proliferation and clonogenesis in LAMC1 silencing group were lower than those in negative control group(P<0.01).The percentage of S phase cells in LAMC1 silencing group was higher than that in negative control group(P<0.01),and the percentage of G1 phase cells was higher than that in negative control group(P<0.01).The expression levels of cyclin D1 and cyclin A proteins in LAMC1 silencing group were highly lower than those in negative control group(P<0.01),while the expression levels of p16 and p21 proteins were higher than those in negative control group(P<0.01).Conclusion Silencing LAMC1 gene expression can inhibit the expression of cell cycle related proteins,thus inhibiting the proliferation of bladder cancer 5637 cells,suggesting that LAMC1 may be a potential therapeutic target for bladder cancer.