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目的:探索调节TLR3/PI3K信号转导通路体外抑制宫颈癌细胞生长的作用。方法:成功构建携带HPV18 E6基因siRNA的慢病毒载体用于转染HeLa细胞,以RT-PCR和Western blot法检测宫颈癌HeLa细胞E6、TLR3及PI3K mRNA和蛋白表达水平的改变。MTT法检测宫颈癌细胞生长抑制率。结果:(1)LY294002组肿瘤细胞TLR3、PI3K mRNA表达水平为0.2863±0.0238、0.2269±0.0382,其蛋白表达分别为0.7042±0.0103、0.6326±0.0153,分别低于对照组,差异均有统计学意义(P<0.01;P<0.01),肿瘤细胞生长抑制率为(61.92±5.49)%;(2)慢病毒转染组肿瘤细胞HPV18 E6、TLR3、PI3K的mRNA表达水平分别为0.1360±0.0140、0.2367±0.0321、0.2013±0.0461,均低于对照组,差异有统计学意义(P<0.001;P<0.001;P=0.005);(3)慢病毒转染组肿瘤细胞HPV18 E6、TLR3、PI3K蛋白表达水平分别为0.1670±0.0290、0.6834±0.0173、0.5670±0.0440,均低于对照组,差异有统计学意义(P=0.005;P=0.002;P=0.005);(4)慢病毒转染组肿瘤细胞生长抑制率为(39.6±18.0)%,与对照组相比,转染组细胞生长受到明显抑制,差异有统计学意义(P=0.047)。结论:TLR3/PI3K信号转导通路在HPV18 E6介导的宫颈癌细胞转化及生长中发挥重要作用,抑制HPV18 E6表达能在mRNA和蛋白水平有效下调TLR3/PI3K表达,抑制宫颈癌HeLa细胞生长。TLR3信号转导通路有望成为治疗宫颈癌的新靶点。
Objective: To explore the role of TLR3 / PI3K signal transduction pathway in inhibiting the growth of cervical cancer cells in vitro. Methods: The lentiviral vector carrying HPV18 E6 gene siRNA was successfully constructed and transfected into HeLa cells. The expression of E6, TLR3 and PI3K mRNA and protein in cervical cancer HeLa cells were detected by RT-PCR and Western blot. The inhibition rate of cervical cancer cell growth by MTT assay. Results: (1) The expression of TLR3 and PI3K mRNA in LY294002 group was 0.2863 ± 0.0238,0.2269 ± 0.0382, the protein expression was 0.7042 ± 0.0103 and 0.6326 ± 0.0153, respectively, which were lower than those in control group P <0.01; P <0.01). (2) The mRNA expression levels of HPV18 E6, TLR3 and PI3K in tumor cells transfected with lentivirus were 0.1360 ± 0.0140,0.2367 ± 0.0321,0.2013 ± 0.0461, all of which were lower than the control group (P <0.001; P <0.001; P = 0.005). (3) The expression of HPV18 E6, TLR3 and PI3K in the lentiviral transfection group (0.167 ± 0.0290,0.6834 ± 0.0173,0.5670 ± 0.0440, respectively) were lower than those in the control group (P = 0.005; P = 0.002; P = 0.005). (4) The growth of tumor cells in lentivirus- The inhibition rate was (39.6 ± 18.0)%. Compared with the control group, the cell growth was significantly inhibited in the transfected group, with a significant difference (P = 0.047). CONCLUSION: TLR3 / PI3K signal transduction pathway plays an important role in HPV18 E6-mediated cervical cancer cell transformation and growth. Suppression of HPV18 E6 expression can effectively down-regulate TLR3 / PI3K mRNA and protein expression and inhibit the growth of cervical cancer HeLa cells. TLR3 signal transduction pathway is expected to become a new target for the treatment of cervical cancer.