超声微泡造影剂携带pcDNA-STC1转染人宫颈癌细胞HeLa的研究

Transfection of human cervical cancer cell HeLa by ultrasound microbubble contrast agent carrying pcD⁃NA⁃STC1

目的 观察超声微泡造影剂携带pcDNA-STC1转染人宫颈癌细胞HeLa后增殖、转移、侵袭,血管生成以及PI3K/AKT信号通路变化。方法 利用GEPIA数据库分析宫颈癌组织STC1的表达水平、患者的预后生存期。选择2019年1-10月在我院诊治的宫颈癌患者80例作为研究对象,收集癌组织和癌旁组织,qRT-PCR检测STC1 m RNA的表达,并分析STC1 mRNA表达与患者临床病理因素的关系;HeLa细胞分组:空白对照组:HeLa细胞正常培养,不做任何处理。阴性对照组(HeLa-pcDNA-NC):5%微泡浓度+2μg空载质粒+无血清DMEM培养基。实验组(HeLa-pcDNA-STC1):5%微泡浓度+2μg pcDNA-STC1质粒+无血清DMEM培养基。MTT法及癌细胞单克隆形成数目检测细胞增殖能力;划痕实验检测细胞迁移能力;Transwell小室实验检测细胞侵袭能力;Matrigel体外成管实验检测细胞的血管生成能力;Western blot法检测p-PI3K、p-AKT、基质金属蛋白酶-2(MMP-2)/基质金属蛋白酶-9(MMP-9)、血管内皮生长因子(VEGF)、血管生成素2(Ang2)的表达。结果 GEPIA数据库显示宫颈癌组织STC1的表达水平低于正常组织,且预后生存期与STC1的表达水平呈负相关;与癌旁正常宫颈组织相比,宫颈癌组织和STC1 mRNA下调,且miR-574-5p水平与STC1水平呈负相关,与肿瘤分化程度、FIGO分期、淋巴结转移有明显的相关性,与空白对照组和阴性对照组相比,实验组48 h、72 h、96 h OD值降低,细胞克隆数极少,迁移率降低,细胞侵袭数减少,血管生成能力下降,MMP-2、MMP-9、VEGF、Ang2、p-PI3K、p-AKT蛋白表达下调(P <0.05)。结论 超声微泡造影剂携带pcDNA-STC1转染能够明显抑制人宫颈癌细胞HeLa增殖、转移、侵袭和血管生成,其作用机制可能是通过抑制PI3K/AKT信号通路实现的。

Objective To observe the proliferation,metastasis,invasion,angiogenesis and PI3K/AKT signal pathway changes of human cervical cancer cell HeLa transfected with pcDNA⁃STC1 by ultrasound microbub⁃ble contrast agent.Methods GEPIA database was used to analyze the expression level of STC1 in cervical cancer tissues and the prognosis and survival in patients.80 patients with cervical cancer who had been treated in our hos⁃pital from January 2019 to October 2019 were selected as research objects.The cancer and adjacent tissues were collected.Expression of STC1 mRNA was detected by qRT⁃PCR,and the relationship between STC1 mRNA expres⁃sion and clinical pathological factors was analyzed.HeLa cells were divided into three groups.Blank control group:HeLa cells were normally cultured without any treatment;negative control group(HeLa⁃pcDNA⁃NC):5%micro⁃bubble concentration plus 2μg empty plasmid plus serum⁃free DMEM medium;and study group(HeLa⁃pcDNA⁃STC1):5%microbubble concentration plus 2μg pcDNA⁃STC1 plasmid plus serum⁃free DMEM medium.MTT assay and the number of cancer cell monoclones were used to detect cell proliferation.Scratch test was used to de⁃tect cell migration ability;Transwell chamber test was used to detect the invasion ability of cells.Matrigel tube forming experiment in vitro was used to detect the angiogenesis ability of cells.The expressions of p⁃PI3K,p⁃AKT,MMP⁃2,MMP⁃9,VEGF,and angiopoietin⁃2(Ang2)were detected by Western blot.Results GEPIA database showed that the expression level of STC1 in cervical cancer tissues was lower than that in normal tissues,and the prognosis and survival were negatively correlated with the expression level of STC1.As compared with the normal cervical tissues adjacent to cancer,the expression of STC1 mRNA in cervical cancer tissue and STC1 was down⁃regulated,and the level of miR⁃574⁃5p was negatively correlated with STC1 level,and significantly correlated with tumor differentiation,FIGO stage and lymph node metastasis.As compared with the blank control and nega⁃tive control groups,the OD value in the study group decreased at 48,72 and 96 hours.Few cell clones was pro⁃duced.The migration rate and the number of cell invasion decreased.The angiogenesis ability decreased,and the expressions of MMP⁃2,MMP⁃9,VEGF,Ang2,p⁃PI3K and p⁃AKT were down⁃regulated(P<0.05).Conclu⁃sions Ultrasound microbubble contrast agent carrying pcDNA⁃STC1 transfection can obviously inhibit the prolifer⁃ation,metastasis,invasion and angiogenesis of human cervical cancer cell HeLa,and its mechanism may be in⁃volved in inhibiting PI3K/AKT signal pathway.