摘要
目的研究Kirsten大鼠肉瘤病毒癌基因同源物(KRAS)基因沉默介导MAPK1/MAPK3信号通路对乳头状甲状腺癌细胞上皮间质转化的调控机制。方法收集80例乳头状甲状腺癌组织及相应癌旁组织,免疫组化法分析KRAS蛋白阳性表达率;购买人乳头状甲状腺癌细胞株TPC-1,分为空白组、阴性对照组、siRNA-KRAS组、SCH772984组、茴香霉素(Anisomycin)组、siRNA-KRAS+Anisomycin组,采用qRT-PCR和Western blotting法检测癌组织、癌旁组织和各组细胞中KRAS、C-jun氨基末端激酶(JNK)、细胞外信号调节蛋白激酶1(ERK1)、细胞外信号调节蛋白激酶2(ERK2)、p-JNK、p-ERK1、p-ERK2、E-钙黏蛋白(E-cadherin)、波形纤维蛋白(Vimentin)及E盒结合锌指蛋白1(ZEB1)的mRNA和蛋白表达水平。采用光学显微镜观察各组细胞上皮间质转化状态。结果癌组织的KRAS阳性表达率高于癌旁组织(63.75%vs.36.25%);与癌旁组织比较,癌组织中E-cadherin表达下调,KRAS、JNK、p-JNK、ERK1、p-ERK1、ERK2、p-ERK2、Vimentin和ZEB1表达上调(均P<0.05)。在细胞实验中,沉默KRAS和抑制MAPK1/MAPK3信号通路能够抑制JNK、p-JNK、ERK1、p-ERK1、ERK2、p-ERK2、Vimentin和ZEB1的表达,激活E-cadherin的表达(均P<0.05),同时抑制上皮间质转化。激活MAPK1/MAPK3信号通路能够激活JNK、p-JNK、ERK1、p-ERK1、ERK2、p-ERK2、Vimentin和ZEB1的表达,抑制E-cadherin的表达(均P<0.05),同时激活上皮间质转化。而沉默KRAS表达并Anisomycin处理,可逆转沉默造成的上述功能改变,与空白组和阴性对照组无明显差异。结论KRAS基因在乳头状甲状腺癌中高表达,沉默KRAS基因能抑制MAPK1/MAPK3信号通路,从而抑制乳头状甲状腺癌的上皮间质转化。
Objective To explore the regulatory mechanism of Kirsten rat sarcoma viral oncogene homolog(KRAS)gene silencing mediating MAPK1/MAPK3 signaling pathway on epithelial-mesenchymal transition of papillary thyroid cancer cells.Methods The papillary thyroid cancer and corresponding paracancerous tissues were collected from 80 cases,and the positive rate of KRAS protein expression was analyzed by immunohistochemistry.Human papillary thyroid cancer cell line TPC-1 was purchased and divided into blank group,NC group,siRNA-KRAS group,Anisomycin group,siRNA-KRAS+Anisomycin group and SCH772984 group.Meanwhile,the mRNA and protein expressions of KRAS,c-Jun N-terminal kinase(JNK),extracellular signal-regulated protein kinase 1(ERK1),extracellular signal-regulated protein kinase 2(ERK2),p-JNK,p-ERK1,p-ERK2,E-cadherin,Vimentin and zinc-finger E-box-binding homeobox 1(ZEB1)were detected by qRT-PCR and Western blotting.The epithelial-mesenchymal transition of cells in each group was observed under light microscope.Results The positive rate of KRAS expression in cancer tissues was higher than that in adjacent tissues(63.75%vs.36.25%);compared with paracancerous tissues,E-cadherin expression was down-regulated,KRAS,JNK,p-JNK,ERK1,p-ERK1,ERK2,p-ERK2,Vimentin and ZEB1 were up-regulated,while E-cadherin expression was down-regulated in cancer tissues(all P<0.05).In cell experiment,silencing KRAS and inhibiting MAPK1/MAPK3 signaling pathway can inhibit the expression of JNK,p-JNK,ERK1,p-ERK1,ERK2,p-ERK2,Vimentin and ZEB1,activate the expression of E-cadherin(all P<0.05),and inhibit epithelial-mesenchymal transformation.Silence of KRAS expression combined with Anisomycin treatment reversed the above-mentioned functional changes(all P<0.05),and there was no significant difference compared with Blank group and NC group(all P>0.05).Conclusions KRAS gene is highly expressed in papillary thyroid cancer.Silencing KRAS gene can inhibit the MAPK1/MAPK3 signaling pathway,thereby inhibiting epithelial-mesenchymal transformation of papillary thyroid cancer.
作者
张芬
余建琴
张怀念
ZHANG Fen;YU Jianqin;ZHANG Huainian(Department of Pathology,Zhongnan Hospital of Wuhan University,Wuhan 430000,China;Department of Medicine,Wuhan Union Hospital,Wuhan 430022,China)
出处
《中国肿瘤外科杂志》
CAS
2020年第5期473-479,共7页
Chinese Journal of Surgical Oncology
