抑制喉癌细胞瞬时受体电位M7表达对细胞生物学行为的影响及其机制

Effect and mechanism of inhibiting transient receptor potential M7 on biological behavior of laryngeal carcinoma cells

目的探讨干扰瞬时受体电位M7(TRPM7)对喉癌细胞增殖、凋亡、侵袭等生物学行为的影响及其机制。方法体外培养人喉癌TU212细胞,分别构建3组TRPM7-shRNA(TRPM7-shRNA1、TRPM7-shRNA2、TRPM7-shRNA3组)及阴性对照shRNA-NC(shRNA-NC组)质粒载体,并以脂质体转染法转染TU212细胞,以转染空载体的细胞作为Control组。采用ELISA法检测TU212细胞上清液中乳酸脱氢酶(LDH)含量,比色法检测上清液中丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性;使用CCK-8实验、克隆形成实验和Transwell实验检测敲低TRPM7表达对TU212细胞增殖、侵袭的影响;使用Western blot检测TU212细胞侵袭、凋亡相关蛋白表达。结果转染后,与Control组比较,TRPM7-shRNA1组、TRPM7-shRNA2组和TRPM7-shRNA3组的TRPM7 mRNA和蛋白表达水平均下调(P<0.05),且以TRPM7-shRNA1组下调最多(P<0.05)。功能实验中,与Control组比较,TRPM7-shRNA1组TU212细胞培养上清液中SOD水平降低(P<0.05),MDA、LDH水平升高(P<0.05);细胞增殖倍数减少、克隆形成率降低(P<0.05);细胞侵袭数减少(P<0.05),N-cadherin、Vimentin蛋白表达下调(P<0.05);线粒体膜电位降低(P<0.05),Bax/Bcl-2、cleaved caspase-3/caspase-3比值增加(P<0.05)。结论敲低TRPM7增加喉癌TU212细胞氧化应激水平,抑制TU212细胞增殖、侵袭,并通过线粒体途径诱导其凋亡。

Objective To explore the effects and mechanisms of interfering with transient receptor potential melastatin subfamily member 7(TRPM7)on biological behaviors(proliferation,apoptosis,invasion)of laryngeal carcinoma cells.Methods Human laryngeal carcinoma cells TU212 were cultured in vitro.TRPM7-shRNA plasmid vectors(TRPM7-shRNA1 group,TRPM7-shRNA2 group,TRPM7-shRNA3 group)and negative control shRNA-NC(shRNA-NC group)were constructed.TU212 cells were transfected by liposome transfection method.The cells transfected with empty vector were enrolled as Control group.The level of lactic dehydrogenase(LDH)in TU212 supernatant was detected by ELISA.The level of malondialdehyde(MDA)and activity of superoxide dismutase(SOD)in supernatant were detected by colorimetry.The effects of knocking-down TRPM7 on proliferation and invasion of TU212 cells were detected by CCK-8,clone formation assay and Transwell assay.The expressions of invasion and apoptosis-related proteins were detected by Western blot.Results After transfection,expression levels of TRPM7 mRNA and protein were down-regulated in TRPM7-shRNA1 group,TRPM7-shRNA2 group and TRPM7-shRNA3 group compared with Control group(P<0.05),and the decrease was the most significant in TRPM7-shRNA1 group(P<0.05).In functional experiments,SOD level in TRPM7-shRNA1 group decreased compared with Control group(P<0.05),while MDA and LDH levels increased(P<0.05).The cells proliferation rate and clone formation rate were decreased(P<0.05),the number of invasion cells was reduced(P<0.05),the expressions of N-cadherin and Vimentin proteins were down-regulated(P<0.05),mitochondrial membrane potentials were reduced(P<0.05),Bax/Bcl-2 and cleaved caspase-3/caspase-3 increased(P<0.05).Conclusion Knocking-down TRPM7 can increase oxidative stress level in laryngeal carcinoma cells TU212,inhibit their proliferation and invasion,and induce their apoptosis by mitochondrial pathways.