circBACH1靶向miR-4500调控口腔鳞状细胞癌HSC3细胞增殖、迁移及侵袭的分子机制研究

Molecular mechanism of circBACH1 regulating the proliferation,migration and invasion of oral squamous cell carcinoma HSC3 cells by targeting miR-4500

目的:探讨circBACH1对口腔鳞状细胞癌HSC3细胞生物学行为的影响及其可能作用机制。方法:体外培养HSC3细胞,分为si-NC组、si-circBACH1组、miR-NC组、miR-4500组、si-circBACH1+anti-miR-NC组、si-circBACH1+anti-miR-4500组,分别转染si-NC、si-circBACH1、miR-NC、miR-4500 mimics、si-circBACH1与anti-miR-NC、si-circBACH1与anti-miR-4500。qRT-PCR检测口腔鳞状细胞癌组织、癌旁组织及各组HSC3细胞中circBACH1、miR-4500的表达量;CCK-8、平板克隆形成实验分别检测细胞增殖及克隆形成能力;划痕实验与Transwell小室实验分别检测细胞迁移及侵袭能力;双荧光素酶报告基因实验检测circBACH1对miR-4500的靶向调控作用;Western blotting检测E-cadherin、N-cadherin的蛋白表达量。结果:与癌旁组织比较,口腔鳞状细胞癌组织中circBACH1的表达量明显升高(P<0.01),miR-4500的表达量明显降低(P<0.01)。与si-NC组比较,si-circBACH1组HSC3细胞活力明显降低(P<0.01),细胞克隆形成数和侵袭细胞数均明显减少(P<0.01),划痕愈合率和N-cadherin蛋白水平均明显降低(P<0.01),E-cadherin蛋白水平明显升高(P<0.01)。与miR-NC组比较,miR-4500组HSC3细胞活力明显降低(P<0.01),细胞克隆形成数和侵袭细胞数均明显减少(P<0.01),划痕愈合率和N-cadherin蛋白水平均明显降低(P<0.01),E-cadherin蛋白水平明显升高(P<0.01)。WT-circBACH1与miR-4500 mimics共转染组HSC3细胞的荧光素酶活性明显低于WT-circBACH1与miR-NC共转染组(P<0.01)。qRT-PCR实验结果显示,与pcDNA组比较,pcDNA-circBACH1组miR-4500的表达量降低(P<0.05);与si-NC组比较,si-circBACH1组miR-4500的表达量升高(P<0.05)。与si-circBACH1+anti-miR-NC组比较,si-circBACH1+anti-miR-4500组HSC3细胞活力明显升高(P<0.01),细胞克隆形成数和侵袭细胞数均明显增多(P<0.01),划痕愈合率和N-cadherin蛋白水平均明显升高(P<0.01),E-cadherin蛋白水平明显降低(P<0.01)。结论:干扰circBACH1表达可通过负向调控miR-4500的表达从而抑制口腔鳞状细胞癌HSC3细胞增殖、克隆形成、迁移及侵袭。

Objective:To investigate the effect of circBACH1 on the biological behavior of oral squamous cell carcinoma HSC3 cells and its possible mechanism.Methods:HSC3 cells were cultured in vitro and divided into si-NC group,si-circBACH1 group,miR-NC group,miR-4500 group,si-circBACH1+anti-miR-NC group,si-circBACH1+anti-miR-4500 group,which was transfected into HSC3 cells with si-NC,si-circBACH1,miR-NC,miR-4500 mimics,si-circBACH1 plus anti-miR-NC,si-circBACH1 plus anti-miR-4500,respectively.qRT-PCR was used to detect the expression of circBACH1 and miR-4500 in oral squamous cell carcinoma tissue,its adjacent tissue and HSC3 cells in the groups.CCK-8 and plate clone formation assay were applied to determine cell proliferation and clone formation ability.Scratch assay and Transwell chamber assay were employed to analyze the cell migration and invasion ability.The dual luciferase reporter gene experiment was performed to evaluate the targeted regulation effect of circBACH1 on miR-4500.Western blotting was used to detect the protein expression of E-cadherin and N-cadherin.Results:Compared with the adjacent tissue,the expression of circBACH1 in oral squamous cell carcinoma tissue was significantly increased(P<0.01),and the expression of miR-4500 was significantly decreased(P<0.01).Compared with the si-NC group,the viability,number of cell clones and invaded cells,wound healing rate and protein level of N-cadherin of HSC3 cells in the si-circBACH1 group were significantly reduced(P<0.01),and the protein level of E-cadherin was significantly increased(P<0.01).Compared with the miR-NC group,the viability,number of cell clones and invaded cells,wound healing rate and protein level of N-cadherin of HSC3 cells in the miR-4500 group were significantly decreased(P<0.01),and the protein level of E-cadherin was significantly increased(P<0.01).The luciferase activity of HSC3 cells in the WT-circBACH1 plus miR-4500 mimics co-transfection group was significantly lower than that in the WT-circBACH1 plus miR-NC co-transfection group(P<0.01).The results of qRT-PCR showed that compared with the pcDNA group,the expression of miR-4500 in the pcDNA-circBACH1 group decreased(P<0.05);compared with si-NC group,the expression of miR-4500 in si-circBACH1 group increased(P<0.05).Compared with the si-circBACH1+anti-miR-NC group,the viability,number of cell clones and invaded cells,wound healing rate and protein level of N-cadherin of HSC3 cells in the si-circBACH1+anti-miR-4500 group were significantly increased(P<0.01),and the protein level of E-cadherin was significantly decreased(P<0.01).Conclusions:Interfering with the expression of circBACH1 can inhibit the proliferation,clone formation,migration and invasion of oral squamous cell carcinoma HSC3 cells by negatively regulating the expression of miR-4500.