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目的探讨Krüppel样因子5(KLF5)和肿瘤坏死因子受体超家族成员11a(TNFRSF11a)在宫颈癌组织中的表达及对宫颈癌细胞增殖、迁移和侵袭的作用。方法利用基因芯片筛查宫颈组织中细胞应答炎症反应的相关基因mRNA的表达。采用实时荧光定量PCR对芯片检测的结果进行验证。免疫双荧光染色检测宫颈组织中KLF5和TNFRSF11a的共表达。在人宫颈癌He La细胞中,采用脂质体转染特异性小分子干扰RNA分别敲低KLF5和TNFRSF11a的表达,构建KLF5超表达腺病毒,感染细胞过表达KLF5。Western blot检测细胞内相关蛋白水平变化。采用双荧光素酶报告基因检测转录因子KLF5对TNFRSF11a的表达调控作用。CCK8和Transwell实验检测细胞增殖和迁移侵袭情况。临床分析TNFRSF11a的mRNA表达与宫颈癌临床病理参数的关系。结果基因芯片结果证实宫颈鳞癌组织中基因TNFRSF11a、KLF5较正常宫颈组织表达上调(P=0.002,P=0.045),实时荧光定量PCR结果证实与正常宫颈组织相比,宫颈上皮内瘤变(CIN)Ⅰ、CINⅡ-Ⅲ、宫颈鳞癌组织中KLF5和TNFRSF11a的mRNA表达结果均上调(KLF5:F=32.79,P=0.018,P=0.014,P=0.011;TNFRSF11a:F=36.72,P=0.013,P=0.010,P=0.009)。免疫双荧光染色结果证实与正常宫颈组织相比,CINⅠ、CINⅡ-Ⅲ、宫颈鳞癌组织中KLF5和TNFRSF11a的蛋白表达结果均上调(KLF5:F=42.38,P=0.014,P=0.008,P=0.002;TNFRSF11a:F=35.42,P=0.021,P=0.012,P=0.004)。体外实验证实KLF5靶向调控TNFRSF11a的表达并促进宫颈癌细胞的增殖和迁移侵袭。临床分析显示TNFRSF11a的mRNA表达与肿瘤病理分级、临床分期、肌层浸润深度、淋巴结转移正相关(P<0.05)。结论 KLF5和TNFRSF11a与宫颈癌相关;KLF5通过上调TNFRSF11a的表达促进宫颈癌细胞的增殖和侵袭、转移。
Objective To investigate the expression of KLF5 and tumor necrosis factor receptor superfamily member 11a (TNFRSF11a) in cervical cancer and its effect on the proliferation, migration and invasion of cervical cancer cells. Methods Gene chips were used to screen the mRNA expression of related genes in response to inflammation in cervical tissues. Real-time PCR was used to verify the results of the chip test. Double immunofluorescence staining was used to detect the coexpression of KLF5 and TNFRSF11a in cervical tissue. In human cervical cancer HeLa cells, lipofectamine was used to knock down the expression of KLF5 and TNFRSF11a, respectively, to construct KLF5 overexpression adenovirus. The infected cells overexpressed KLF5. Western blot detection of intracellular related protein levels. Dual luciferase reporter gene detection of KLF5 transcription factor TNFRSF11a expression regulation. CCK8 and Transwell assay of cell proliferation and migration invasion. Clinical analysis of TNFRSF11a mRNA expression and clinicopathological parameters of cervical cancer. Results Gene microarray results confirmed that the expression of TNFRSF11a and KLF5 in cervical squamous cell carcinoma was significantly higher than that in normal cervical tissues (P = 0.002, P = 0.045). Real-time PCR results confirmed that cervical intraepithelial neoplasia (CIN (P = 0.014, P = 0.011; TNFRSF11a: F = 36.72, P = 0.013, P = 0.013, P = P = 0.010, P = 0.009). Immunofluorescence double staining showed that the protein expression of KLF5 and TNFRSF11a in CINⅠ, CINⅡ-Ⅲ and cervical squamous cell carcinoma were all up-regulated compared with normal cervical tissue (KLF5: F = 42.38, P = 0.014, P = 0.008, P = 0.002; TNFRSF11a: F = 35.42, P = 0.021, P = 0.012, P = 0.004). In vitro experiments confirmed KLF5 targeted regulation of TNFRSF11a expression and promote cervical cancer cell proliferation and migration invasion. Clinical analysis showed that TNFRSF11a mRNA expression was positively correlated with tumor grade, clinical stage, myometrial invasion depth and lymph node metastasis (P <0.05). Conclusion KLF5 and TNFRSF11a are associated with cervical cancer. KLF5 promotes the proliferation, invasion and metastasis of cervical cancer cells by up-regulating the expression of TNFRSF11a.