KLF4通过Wnt信号通路抑制甲状腺癌细胞EMT及侵袭迁移的实验研究

Experimental study on inhibition of KLF4 on EMT,invasion and migration of thyroid cancer cells by Wnt signaling pathway

目的探讨Kruppel样因子4(KLF4)对甲状腺癌细胞上皮间质转化(EMT)、侵袭和迁移的影响及机制。方法免疫组织化学(IHC)检测45份甲状腺癌标本中KLF4的表达情况。常规体外人甲状腺乳头状癌细胞株KTC1培养,将其分为空白对照组(不作任何处理)、阴性对照组(转染LipofectamineTM 2000)、KLF4过表达组(转染LipofectamineTM 2000+KLF4)、siRNA-NC组(转染LipofectamineTM 2000+NC-siRNA)和KLF4-siRNA组(转染LipofectamineTM 2000+KLF4-siRNA)。免疫荧光检测细胞中KLF4与β-catenin的分布和共表达;Transwell检测癌细胞侵袭和迁移的能力,实时荧光定量PCR(qRT-PCR)和Western blot检测EMT和Wnt信号通路相关的基因和蛋白质表达水平。蛋白质免疫共沉淀(Co-IP)检测KLF4和β-catenin间的互作情况。结果免疫荧光标记显示,KLF4和β-catenin在KTC1细胞质中共定位。qRT-PCR结果显示,KLF4过表达组上皮型钙黏蛋白(E-cad)mRNA表达明显高于其他组(P<0.05),神经型钙黏蛋白(N-cad)和基质金属蛋白酶7(MMP7)mRNA表达水平明显低于其他组(P<0.05);沉默干扰KLF4后,与其他组相比,KLF4-siRNA组中的E-cad mRNA明显降低(P<0.05),N-cad和MMP7 mRNA明显升高(P<0.05)。Western blot结果显示,KLF4过表达组中E-cad表达较其他组明显升高(P<0.05),而N-cad、MMP7表达则明显低于其他组(P<0.05);当沉默干扰KLF4时,上皮标志物减少(P<0.05),间质标志物增加(P<0.05)。Transwell结果显示,过表达KLF4可以抑制KTC1细胞的迁移和侵袭,干扰沉默KLF4可增强KTC1细胞的迁移和侵袭。Co-IP结果显示,KLF4与β-catenin之间存在相互作用。KLF4在甲状腺癌及癌旁组织中的阳性表达率分别为24.44%(11/45)和71.11%(32/45),二者比较差异有统计学意义(χ2=19.639,P<0.001)。45份甲状腺癌标本中,伴淋巴结转移18份标本中KLF4表达4份阳性,而不伴淋巴结转移27份标本中,KLF4表达15份阳性,二者比较差异有统计学意义(22.22%vs.55.56%,χ2=4.919,P=0.027)。结论KLF4通过Wnt信号通路调节相关下游靶基因的表达,从而影响了甲状腺癌的侵袭和迁移能力。

Objective To investigate the effect and mechanism of Kruppel like factor 4(KLF4)on epithelial mesenchymal transition(EMT),invasion and migration of thyroid cancer cells.Methods The immunohistochemstry(IHC)was used to detect the KLF4 expression level in 45 thyroid cancer samples.The human thyroid papillary carcinoma cells(KTC1)were routinely cultured in vitro and divided into the blank control group(without any treatment),negative control group(transfected with LipofectamineTM 2000),KLF4 overexpression group(transfected with LipofectamineTM 2000 and KLF4),siRNA NC group(transfected with LipofectamineTM 2000 and NC-siRNA)and KLF4 siRNA group(transfected with LipofectamineTM 2000 and KLF4 siRNA).Furthermore,the immunofluorescence was used to detect the distribution and co-expression of KLF4 andβ-catenin in cells.The migration and invasion ability of cancer cells was examined by Transwell.TheEMT and Wnt signaling pathways related genes and protein expression levels were detected by Western blot and qRT-PCR,respectively.The protein co-immunoprecipitation test was employed to determine the potential interaction between KLF4 andβ-catenin.Results The immunofluorescence labeling showed that KLF4 andβ-catenin were co-localized in the cytoplasm of KTC1.The qRT-PCR results showed that E-cadherin(E-cad)mRNA expression level in the KLF4 overexpression group was significantly higher than that in the other groups(P<0.05),the N-cadnerin(N-cad)and MMP7 mRNA expression levels were significantly lower than those in the other groups(P<0.05);after silent interference on KLF4,compared with other groups,the E-cad mRNA in the KLF4-siRNA group was significantly decreased(P<0.05),while N-cad and MMP7 mRNA were significantly increased(P<0.05).The Western blot results showed that the E-cad expression in the KLH4 overexpression group was significantly increased compared with the other groups(P<0.05),while the N-cad and MMP7 expressions were significantly lower than those in the other groups(P<0.05);when silent interference on KLF4,the epithelial markers were decreased(P<0.05),while the mesenchymal markers were increased(P<0.05).The Transwell results showed that the KLF4 overexpression could inhibit the migration and invasion of KTC1 cells.Interfering and silencing KLF4 could enhance the migration and invasion of KTCI cells.The Co-IP results demonstrated that the interaction existed between KLF4 andβ-catenin.The positive expression rates of KLF4 in thyroid carcinoma and paracancerous tissues were 24.44%(11/45)and 71.11%(32/45)respectively,the difference between them was statistically significant(χ2=19.639,P<0.001).Among 45 cases of thyroid cancer samples,4 cases in 18 cases of lymph node metastasis were KLF4 expression positive,while 15 cases in 27 cases of non-lymph node metastasis were KLF4 expression positive(22.22%vs.55.56%,χ2=4.919,P=0.027).Conclusion KLF4 regulates the expression of related downstream target gene by Wnt signal pathway,thus affect the invasion and migration abilities of thyroid cancer.