PKCι在宫颈癌YAP1信号通路中的作用及与预后的关系

Role of PKC ι in the YAP1 signaling pathway in cervical cancer and its relationship with prognosis

目的 探讨蛋白激酶Cι(protein kinase Cι,PKCι/PRKCI)在宫颈癌YAP1信号通路中的作用及其与预后的关系。方法 选择2013年1月至2015年1月全子宫术后宫颈正常组织(normal cervical tissue, NCT)、宫颈低级别鳞状上皮内病变组织(low-grade squamous intraepithelial lesions, LSIL)、宫颈高级别鳞状上皮内病变组织(high-grade squamous intraepithelial lesions, HSIL)以及早期宫颈鳞状细胞癌组织(squamous cell carcinoma, SCC),每组80例。采用免疫组织化学(immunohistochemistry, IHC)法检测4组组织中PKCι以及YAP1表达水平并进行相关性、临床病理以及预后分析。将PRKCI基因过表达转染人宫颈癌SiHa细胞系,通过倒置荧光显微镜观察细胞内荧光数量及强度评判转染效率,PRKCI抑制剂aurothiomalate处理,采用CCK-8检测存活率,Transwell实验检测细胞迁移。通过实时荧光定量逆转录聚合酶链反应(real-time quantitative reversetranscription polymerase chain reaction, qRT-PCR)检测PRKCI基因表达,并予以转录组测序以及qRT-PCR验证。结果 NCT、LSIL、HSIL以及SCC患者PKCι、YAP1阳性率差异有统计学意义(P<0.05),且级别越高,PKCι、YAP1阳性率越高(P<0.05)。宫颈病变级别与PKCι以及YAP1阳性表达均呈正相关(P<0.05)。在SCC中PKCι以及YAP1阳性表达相互之间的均呈正相关(P<0.05)。PKCι以及YAP1阳性表达在SCC不同的分化程度以及有无脉管瘤栓之间均有统计学差异(P<0.05)。SCC患者生存预后分析中单因素分析显示,各蛋白表达、分化程度、淋巴结是否转移以及脉管瘤栓情况是影响总生存预后的独立因素(P<0.05);SCC患者各蛋白表达以及淋巴结是否转移是影响患者5年生存率的独立因素,其中PKCι、YAP1蛋白高表达以及淋巴结转移为危险因素(P<0.05)。SiHa细胞中PKCι转染组PKCιmRNA表达水平均明显升高,与空载脂质体组比较差异有统计学意义(P<0.05)。结论 宫颈病变患者常伴有PKCι以及YAP1表达异常,相互之间可能存在协同作用,影响SCC的发生发展及其预后,促进SCC细胞增殖以及迁移,为SCC的发病机制研究及其诊治判断预后提供可能的依据。

Objective To explore the role of protein kinase cι(PKCι/PRKCI)in the yes-associated protein 1(YAP1)signaling pathway in cervical cancer and its relationship with prognosis.Methods Normal cervical tissues(NCTs,n=80),low-grade squamous intraepithelial lesions(LSILs,n=80),high-grade squamous intraepithelial lesions(HSILs,n=80)and early cervical squamous cell carcinoma(SCC,n=80)were collected during the total hysterectomy from January 2013 to January 2015.Expression levels of PKCιand YAP1 were analyzed by immunohistochemistry(IHC),and their correlation with clinical pathology and prognosis was analyzed.Human cervical cancer SiHa cells were transfected with overexpression plasmid of PKCι,followed by the assessment of transfection efficiency via the observation of fluorescence quantity and intensity using an inverted fluorescence microscope.After the induction of PRKCI inhibitor aurothiomalate,cell viability and migration were measured by CCK-8 and Transwell assay,respectively.Expression level of PRKCI was detected by real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR),followed by transcriptome sequencing and qRT-PCR for verification.Results There were significant differences in the positive rates of PKCιand YAP1 in NCTs,LSILs,HSILs and SCC(P<0.05),which were higher in those with a higher pathological grade(P<0.05).Pathological grade of cervical lesions was positively correlated with both positive expressions of PKCιand YAP1(P<0.05).Positive expression of PKCιwas positively correlated with that of YAP1 in SCC(P<0.05).There were significant differences in the positive expressions of PKCιand YAP1 in SCC with varying differentiation levels and those either with blood vessel invasions or not(P<0.05).Univariate analysis in survival analysis of SCC patients showed that protein expressions,differentiation,lymph node metastasis and blood vessel invasion were independent factors for the overall prognosis(P<0.05).Protein expressions and lymph node metastasis were independent risk factors for the 5-year survival of SCC,and higher protein expressions of PKCιand YAP1 and lymph node metastasis were risk factors(P<0.05).The expression level of PKCιin SiHa cells transfected with the overexpression plasmid of PKCιwas significantly higher than those transfected with a negative control(P<0.05).Conclusion Patients with cervical lesions are often accompanied by abnormal expressions of PKCιand YAP1,which may have synergistic effects between each other to affect the occurrence and development of SCC and its prognosis,and promote the proliferation and migration of SCC cells.Our findings provide a possible basis for the pathogenesis study of SCC and its diagnosis,treatment and prediction of the prognosis.