摘要
目的观察多形性腺瘤基因样因子2(PLAGL2)在胃癌组织和胃癌细胞株中的表达,探讨其对胃癌细胞侵袭、上皮-间充质转化(EMT)的影响及分子机制。方法采用实时定量聚合酶链反应(Real-time PCR)和Western blot方法检测PLAGL2 mRNA和蛋白在胃癌及相应癌旁组织和人胃癌MKN-28、BGC-823、NCI-N87、SGC-7901细胞株中的表达。将PLAGL2小干扰RNA(siRNA)转染SGC-7901细胞,Real-time PCR、Western blot检测PLAGL2 mRNA和蛋白表达,Transwell侵袭实验检测敲低PLAGL2对细胞侵袭的影响,Western blot检测敲低PLAGL2对EMT相关分子标志物N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、E-钙黏蛋白(E-cadherin)及Wnt/β-连环蛋白(β-catenin)信号通路相关分子表达的影响。分别采用Wnt/β-catenin信号通路激活剂SKL2001和抑制剂XAV939处理转染PLAGL2-siRNA的SGC-7901细胞,Transwell侵袭实验检测细胞侵袭变化。结果PLAGL2 mRNA和蛋白在胃癌组织中的表达量(mRNA:1.11±0.27;蛋白:0.98±0.26)高于癌旁组织(mRNA:0.37±0.10;蛋白:0.28±0.11;t=-20.941,P<0.01;t=-20.186,P<0.01)。PLAGL2 mRNA和蛋白在胃癌细胞株MKN-28、BGC-823、NCI-N87、SGC-7901中的表达量高于人胃黏膜正常细胞GES-1(mRNA:t=-12.327,P<0.01;t=-19.812,P<0.01;t=-13.080,P<0.01;t=-15.218,P<0.01;蛋白:t=-11.816,P<0.01;t=-21.162,P<0.01;t=-14.517,P<0.01;t=-15.899,P<0.01)。转染后,SGC-7901的PLAGL2 mRNA和蛋白表达(mRNA:2.10±0.29;蛋白:1.96±0.27)较Control siRNA组(mRNA:6.61±0.73;蛋白:6.46±0.71)显著降低(t=-1.000,P<0.01;t=-1.000,P<0.01)。转染24、48、72 h后PLAGL2-siRNA组侵袭细胞数[(30.75±5.74)、(48.75±6.90)、(64.50±4.80)个]较Control siRNA组[(51.00±4.97)、(75.75±4.92)、(132.50±5.57)个]显著减少(t=-1.000,P<0.05;t=-1.000,P<0.01;t=-1.000,P<0.01),同时N-cadherin、Vimentin、β-catenin、基质金属蛋白酶(MMP)-7、c-Myc蛋白表达显著降低,E-cadherin蛋白表达显著增高(t=-1.000,P<0.01;t=-1.000,P<0.01;t=-1.000,P<0.01;t=-1.000,P<0.01;t=-1.000,P<0.01;t=-8.205,P<0.01)。此外,SKL2001能够逆转敲低PLAGL2对SGC-7901细胞侵袭的抑制作用,XAV939则能够进一步加强以上作用。结论胃癌组织PLAGL2表达上调,siRNA干扰PLAGL2可通过抑制Wnt/β-cateninn信号通路的激活和EMT抑制胃癌侵袭。
Objective To detect the expression of pleomorphic adenoma gene like-2 (PLAGL2) in human gastric cancer and cell lines, and its role in cell invasion and epithelial-mesenchymal transition (EMT) and the molecular mechanism involved. Methods Fifteen cases of pathologically confirmed gastric cancer tissues and their adjacent normal tissues were collected during operation in our hospital from Jan. 10, 2016 to Mar. 20, 2018. Gastric cancer cell lines MKN-28, BGC-823, NCI-N87 and SGC-7901 were cultured. The expression levels of PLAGL2 in gastric cancer tissue samples and MKN-28, BGC-823, NCI-N87 and SGC-7901 cells were determined by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. After successful transfection with PLAGL2-small interfering RNA (siRNA) in SGC-7901 cells, the expression levels of PLAGL2 mRNA and protein were detected by Real-time PCR and Western blotting respectively. Cell invasion was detected by Transwell assay. The expression levels of N-cadherin, Vimentin and E-cadherin, which were EMT related markers, and Wnt/β-catenin related proteins were detected by Western blotting. After PLAGL2-siRNA SGC-7901 cells were treated with Wnt pathway agonist SKL2001 and inhibitor XAV939, the changes of cell invasion ability were retested by Transwell assay. Results The expression levels of PLAGL2 mRNA (1.11±0.27) and protein (0.98±0.26) in gastric cancer tissues were significantly higher than those in the adjacent tissues (mRNA: 0.37±0.10;protein: 0.28±0.11;t=-20.941, P<0.01;t=-20.186, P<0.01). The expression levels of PLAGL2 mRNA and protein in MKN-28, BGC-823, NCI-N87 and SGC-7901 were significantly higher than GES-1 cells (mRNA: t=-12.327, P<0.01;t=-19.812, P<0.01;t=-13.080, P<0.01;t=-15.218, P<0.01;protein: t=-11.816, P<0.01;t=-21.162, P<0.01;t=-14.517, P<0.01;t=-15.899, P<0.01). The expression levels of PLAGL2 mRNA and protein in SGC-7901 cells were significantly decreased after PLAGL2-siRNA transfection (mRNA: 2.10±0.29;protein: 1.96±0.27) as compared with control siRNA group (mRNA: 6.61±0.73;protein: 6.46±0.71;t=-1.000, P<0.01;t=-1.000, P<0.01). The number of invasive cells at 24, 48, 72 hin PLAGL2-siRNA transfection group [(30.75±5.74),(48.75±6.90) and (64.50±4.80) cells] was significantly less than in control siRNA group [(51.00±4.97),( 75.75±4.92) and (132.50±5.57) cells;t=-1.000, P<0.05;t=-1.000, P<0.01;t=-1.000, P<0.01]. The expression of N-cadherin, Vimentin,β-catenin, matrix metalloproteinase (MMP)-7 and c-Myc proteins was significantly down-regulated, and that of E-cadherin protein was significantly up-regulated (t=-1.000, P<0.01;t=-1.000, P<0.01;t=-1.000, P<0.01;t=-1.000, P<0.01;t=-1.000, P<0.01;t=-8.205, P<0.01). SKL2001 could reverse the inhibitory effect on EMT and invasion of SGC-7901 cells after PLAGL2-siRNA transfection, while XAV939 enhanced the above effects of PLAGL2-siRNA. Conclusion PLAGL2 is highly-expressed in gastric cancer. Inhibition of PLAGL2 gene by siRNA can inhibit the invasion of gastric cancer cells probably by down-regulating the Wnt/β-catenin signaling pathway.
作者
王啸飞
路夷平
Wang Xiaofei;Lu Yiping(Department of Oncology Surgery, Beijing Traditional Chinese Medicine Hospital, Capital Medical University, Beijing 100010, China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2019年第3期432-435,共4页
Chinese Journal of Experimental Surgery
