CLDN6对胰腺癌细胞增殖的影响及其在胰腺癌组织中的表达和临床意义

Effect of CLDN6 on the proliferation of pancreatic cancer cells and study on the expression and clinical significance of CLDN6 in pancreatic cancer

目的分析紧密连接蛋白6(CLDN6)在人胰腺癌组织中的表达及其与胰腺癌患者临床病理特征的关系,探究其对人胰腺癌细胞增殖及相关上皮细胞-间充质转化(EMT)蛋白表达的影响。方法选取2014年1月至2018年6月在郑州大学附属洛阳中心医院行手术切除的50例胰腺癌组织及癌旁组织,免疫组织化学法及qRT-PCR法检测50例胰腺癌组织及对应癌旁组织中CLDN6的表达情况,分析其与患者临床病理特征及预后的相关性;qRT-PCR法检测胰腺癌细胞株(AsPC-1、BxPC-3、Capan-1、PANC-1、HPAC、Hs 766T、MIA Paca-2)及正常胰腺导管上皮细胞株HPDE中CLDN6的表达。胰腺癌细胞株脂质体转染CLDN6 siRNA沉默基因,qRT-PCR检测转染情况;MTT法、平板克隆实验检测各胰腺癌细胞增殖及克隆形成能力;Western blotting检测上皮型钙黏蛋白(E-cadherin)、神经型钙黏蛋白(N-cadherin)、波形纤维蛋白(VIM)及纤维连接蛋白(FN)的表达水平。结果胰腺癌组织中CLDN6表达水平为(19.44±1.22),明显高于癌旁组织的(1.35±0.71),差异有统计学意义(P=0.001),其表达水平与患者年龄、性别、肿瘤大小、病理学类型、肿瘤部位及CA19-9水平无关(P>0.05),而与TNM分期、肿瘤分化程度及淋巴结转移有关(P<0.05)。CLDN6阳性表达胰腺癌患者的中位生存期为8.993个月,短于阴性表达者的18.402个月,差异有统计学意义(P<0.05)。各胰腺癌细胞株中CLDN6表达量均高于HPDE细胞株(均P<0.05),成功构建沉默CLDN6的胰腺癌细胞AsPC-1-si-CLDN6、BxPC-3-si-CLDN6,转染CLDN6 siRNA沉默质粒的AsPC-1及BxPC-3细胞的增殖能力及克隆形成能力较转染前明显降低,E-cadherin表达明显增高,而N-cadherin、VIM及FN表达明显降低(均P<0.05)。结论CLDN6在胰腺癌中高表达,且与患者临床特征及预后相关,其可促进人胰腺癌细胞增殖及克隆形成,机制可能与其促进上皮细胞-间充质转化有关。

Objective To analysis the expression of claudin 6(CLDN6)in human pancreatic cancer tissues and its relationship with clinical factors,and to explore its effect on human pancreatic cancer cell proliferation.Methods The expression of CLDN6 in 50 cases of pancreatic cancer and its adjacent tissues was detected by immunohistochemistry and qRT-PCR,and analyzed the correlation between CLDN6 and clinicopathological factors and prognosis.The expression of CLDN6 in different pancreatic cancer cell lines(AsPC-1,BxPC-3,Capan-1,PANC-1,HPAC,Hs 766T,MIA Paca-2)and normal pancreatic ductal epithelial cell line HPDE was detected by qRT-PCR.CLDN6 siRNA silencing gene was transfected with liposomes in pancreatic cancer cell lines,and the transfection was detected by qRT-PCR.MTT method and plate cloning test were used to detect the proliferation and clonogenesis of the cells.Western blotting method was used to detect the expression of E-cadherin,N-cadherin,VIM and FN.Results The expression level of CLDN6 in pancreatic cancer tissues was significantly higher than that in adjacent normal tissues(19.44±1.22 vs.1.35±0.71,t=9.288,P=0.001),which was not related to age,gender,tumor size,pathological type,tumor site and CA19-9 level(P>0.05),but was related to TNM stage,tumor differentiation and lymph node metastasis(P<0.05).Median Survival Time(MST)of pancreatic cancer patients with positive expression of CLDN6 was significantly shorter than that of patients with negative expression(8.993 months vs.18.402 months)(P<0.05).The expression of CLDN6 in each pancreatic cancer cell line was higher than that in HPDE cell line(all P<0.05).The successful construction of CLDN6-silenced pancreatic cancer cells with AsPC-1-si-CLDN6 and BxPC-3-si-CLDN6 showed a significant decrease in proliferation and cloning when compared with it before transfection,and the expression of E-cadherin was significantly increased,while the expression of N-cadherin,VIM and FN were significantly decreased(P<0.05).Conclusions CLDN6 is highly expressed in pancreatic cancer,and it is related to the clinical characteristics and prognosis of patients.It can promote the proliferation and clone formation of human pancreatic cancer cells,and the mechanism may be related to the promotion of epithelial to mesenchymal transformation.