PIK3CA基因沉默对宫颈癌生物学特性影响及机制

EFFECT OF PIK3CA GENE SILENCING ON BIOLOGICAL CHARACTERISTICS OF CERVICAL CANCER AND ITS MECHANISM

目的探讨磷脂酰肌醇-4,5-二磷酸肌醇-3-激酶(PIK3CA)基因沉默对宫颈癌生物学特性的影响及其机制。方法选取188例宫颈癌病人癌组织及癌旁组织标本,采用SP免疫组化法检测PIK3CA蛋白表达阳性率。取宫颈癌细胞株并细胞转染分为6组:空白(Blank)组、阴性对照(NC)组、上调PIK3CA表达(HA-PIK3CA)组、沉默PIK3CA表达(PIK3CA-siRNA)组、信号通路拮抗剂(LY294002)组、信号通路拮抗剂+沉默PIK3CA表达(LY294002+PIK3CA-siRNA)组。采用实时定量PCR(qRT-PCR)和蛋白质印迹(Western blot)法分别检测转染后各组细胞中PIK3CA、磷脂酰肌醇3-激酶(PI3K)、PKB蛋白激酶(又称AKT)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)的mRNA和蛋白表达水平。应用噻唑蓝(MTT)法、流式细胞术分别检测各组细胞增殖及凋亡。结果与癌旁组织相比,癌组织PIK3CA蛋白阳性表达率显著升高,且与宫颈癌病人TNM分期、淋巴结转移和组织学分级均有关(χ^(2)=6.766~31.171,P均<0.05)。与Blank组和NC组相比,PIK3CA-siRNA组、LY294002组和LY294002+PIK3CA-siRNA组的PIK3CA、PI3K、AKT、N-cadherin和Vimentin的mRNA和蛋白表达量均明显降低,E-cadherin表达明显增加,细胞增殖活性降低,细胞凋亡增加,差异均有统计学意义(F_(mRNA)=58.5~185.1,F_(蛋白)=48.1~130.5,F_(增殖)=28.0~98.8,F_(凋亡)=213.9,P均<0.05);且LY294002+PIK3CA-siRNA组趋势更为显著(P均<0.05);而上述指标趋势在HA-PIK3CA组被逆转(P均<0.05)。结论沉默PIK3CA基因可能通过抑制PI3K/AKT信号通路的激活,进而抑制宫颈癌上皮间质转化,抑制细胞增殖并促进细胞凋亡。

Objective To investigate the effect of PIK3CA gene silencing on the biological characteristics of cervical cancer and its mechanism.Methods Cancer tissue specimens and adjacent tissue specimens were collected from 188 patients with cervical cancer,and SP immunohistochemistry was used to measure the positive rate of PIK3CA protein expression.Cervical cancer cell lines were collected,and the cells were transfected and divided into Blank group,negative control group(NC group),PIK3CA upregulation group(HA-PIK3CA group),PIK3CA silencing group(PIK3CA-siRNA group),signaling pathway antagonist(LY294002)group,and signaling pathway antagonist+PIK3CA silencing group(LY294002+PIK3CA-siRNA group).Quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression levels of PIK3CA,phosphoinositide 3-kinase(PI3K),protein kinase B(AKT),E-cadherin,N-cadherin,and Vimentin in each group of cells.MTT assay was used to measure cell proliferation,and flow cytometry was used to measure cell apoptosis.Results Compared with adjacent tissue,cancer tissue had a significant increase in the positive rate of PIK3CA,which was associated with TNM stage,lymph node metastasis,and histological grade of cervical cancer patients(χ^(2)=6.766-31.171,all P<0.05).Compared with the Blank group and the NC group,the PIK3CA-siRNA group,the LY294002 group,and the LY294002+PIK3CA-siRNA group had significant reductions in the mRNA and protein expression levels of PIK3CA,PI3K,AKT,N-cadherin,and Vimentin,a significant increase in the expression of E-cadherin,a significant reduction in cell proliferation activity,and a significant increase in cell apoptosis(F_(mRNA)=58.5-185.1,F_(protein)=48.1-130.5,F_(proliferation)=28.0-98.8,F_(apoptosis)=213.9,all P<0.05),and the LY294002+PIK3CA-siRNA group tended to have the most significant changes(all P<0.05).Changes in the above indices were reversed in the HA-PIK3CA group(all P<0.05).Conclusion PIK3CA gene silencing may inhibit cell proliferation and promote cell apoptosis by inhibiting activation of the PI3K/AKT signaling pathway and epithelial-mesenchymal transition in cervical cancer.