下调TCF3抑制非小细胞肺癌细胞的增殖和迁移能力
发布时间：2019-04-19 16:58
【摘要】：目的:探索下调T细胞因子3(TCF3)抑制非小细胞肺癌细胞增殖和迁移的分子机制。方法:运用Lipofectamine 2000转染法将siTCF3和阴性对照siRNA(NCsiRNA)转染非小细胞肺癌A549和H1299细胞;运用real-time PCR和Western blot分别测定TCF3的mRNA和蛋白水平;运用萤光素酶报告基因实验测定TCF3的转录活性;MTT、克隆形成实验、Transwell实验和Annexin V-FITC/PI染色联合流式细胞术分别测定细胞的活力、克隆形成能力、转移能力及细胞凋亡率;Western blot检测Wnt、c-Myc、基质金属蛋白酶(MMP)-9、MMP-13、金属蛋白酶组织抑制物(TIMP)-1的蛋白表达水平。结果:与NCsiRNA转染组的细胞比较,siTCF3显著抑制A549细胞和H1299细胞中TCF3的mRNA和蛋白水平(P0.01)。TCF3转录活性和c-Myc蛋白表达水平明显低于NCsiRNA细胞(P0.05)。MTT实验结果显示,培养24 h、48 h、72 h和96 h的A549-siTCF3和H1299-siTCF3细胞活力均显著低于NCsiRNA细胞(P0.05)。与NCsiRNA细胞相比,siTCF3显著抑制A549细胞和H1299细胞的克隆形成能力(P0.01)。Transwell实验结果显示A549-siTCF3和H1299-siTCF3细胞迁移数显著低于A549-NCsiRNA和H1299-NCsiRNA组细胞(P0.05)。流式细胞术分析结果显示A549-siTCF3细胞和H1299-siTCF3细胞的凋亡率显著高于A549-NCsiRNA和H1299-NCsiRNA细胞(P0.01)。Western blot实验结果显示,下调TCF3表达能抑制Wnt蛋白的表达,MMP-9和MMP-13的蛋白表达明显降低,TIMP-1的蛋白表达增高。结论:siTCF3显著抑制A549细胞和H1299细胞的增殖和迁移能力,并诱导细胞凋亡,其分子机制可能通过下调Wnt通路活性以及调控MMP家族关键成员的表达而实现。
[Abstract]:Aim: to explore the molecular mechanism of down-regulation of T cytokine 3 (TCF3) on proliferation and migration of non-small cell lung cancer cells. Methods: siTCF3 and negative control siRNA (NCsiRNA) were transfected into A549 and H1299 non-small cell lung cancer cells by Lipofectamine 2000 transfection method, and the mRNA and protein levels of TCF3 were measured by real-time PCR and Western blot, respectively. The transcriptional activity of TCF3 was measured by luciferase reporter gene assay, MTT, clone formation test, Transwell assay and Annexin V-FITC/PI staining combined with flow cytometry were used to determine the cell viability, clone forming ability, metastasis ability and apoptosis rate. The expression levels of Wnt,c-Myc, matrix metalloproteinase (MMP)-9, MMP-13 and tissue inhibitor of metalloproteinase-1 (TIMP)-1) were detected by Western blot. Results: compared with NCsiRNA transfected cells, siTCF3 significantly inhibited mRNA and protein levels of TCF3 in A549 cells and H1299 cells (P0.01). TCF3 transcriptional activity and c-Myc protein expression level were significantly lower than those in NCsiRNA cells (P0.05), and the results of MTT assay showed that TCF3 transcription activity and c-Myc protein expression were significantly lower in A549 cells and H1299 cells than in NCsiRNA cells (P0.05). The viability of A549-siTCF3 and H1299-siTCF3 cells cultured for 24 h, 48 h, 72 h and 96 h was significantly lower than that of NCsiRNA cells (P0.05). Compared with NCsiRNA cells, siTCF3 significantly inhibited the clonal formation of A549 cells and H1299 cells (P0.01). Transwell assay showed that the migration number of A549-siTCF3 and H1299-siTCF3 cells was significantly lower than that of A549-NCsiRNA and H1299-NCsiRNA cells (P0.05). Flow cytometry analysis showed that the apoptosis rate of A549-siTCF3 cells and H1299-siTCF3 cells was significantly higher than that of A549-NCsiRNA and H1299-NCsiRNA cells (P0.01). Western blot test showed that down-regulation of TCF3 expression could inhibit the expression of Wnt protein. The protein expression of MMP-9 and MMP-13 decreased significantly, while the protein expression of TIMP-1 increased. Conclusion: siTCF3 can significantly inhibit the proliferation and migration of A549 and H1299 cells and induce apoptosis. Its molecular mechanism may be achieved by down-regulating the activity of Wnt pathway and regulating the expression of key members of MMP family.
【作者单位】： 山东省菏泽市立医院胸外科;
【分类号】：R730.23
本文编号：2461121
[Abstract]:Aim: to explore the molecular mechanism of down-regulation of T cytokine 3 (TCF3) on proliferation and migration of non-small cell lung cancer cells. Methods: siTCF3 and negative control siRNA (NCsiRNA) were transfected into A549 and H1299 non-small cell lung cancer cells by Lipofectamine 2000 transfection method, and the mRNA and protein levels of TCF3 were measured by real-time PCR and Western blot, respectively. The transcriptional activity of TCF3 was measured by luciferase reporter gene assay, MTT, clone formation test, Transwell assay and Annexin V-FITC/PI staining combined with flow cytometry were used to determine the cell viability, clone forming ability, metastasis ability and apoptosis rate. The expression levels of Wnt,c-Myc, matrix metalloproteinase (MMP)-9, MMP-13 and tissue inhibitor of metalloproteinase-1 (TIMP)-1) were detected by Western blot. Results: compared with NCsiRNA transfected cells, siTCF3 significantly inhibited mRNA and protein levels of TCF3 in A549 cells and H1299 cells (P0.01). TCF3 transcriptional activity and c-Myc protein expression level were significantly lower than those in NCsiRNA cells (P0.05), and the results of MTT assay showed that TCF3 transcription activity and c-Myc protein expression were significantly lower in A549 cells and H1299 cells than in NCsiRNA cells (P0.05). The viability of A549-siTCF3 and H1299-siTCF3 cells cultured for 24 h, 48 h, 72 h and 96 h was significantly lower than that of NCsiRNA cells (P0.05). Compared with NCsiRNA cells, siTCF3 significantly inhibited the clonal formation of A549 cells and H1299 cells (P0.01). Transwell assay showed that the migration number of A549-siTCF3 and H1299-siTCF3 cells was significantly lower than that of A549-NCsiRNA and H1299-NCsiRNA cells (P0.05). Flow cytometry analysis showed that the apoptosis rate of A549-siTCF3 cells and H1299-siTCF3 cells was significantly higher than that of A549-NCsiRNA and H1299-NCsiRNA cells (P0.01). Western blot test showed that down-regulation of TCF3 expression could inhibit the expression of Wnt protein. The protein expression of MMP-9 and MMP-13 decreased significantly, while the protein expression of TIMP-1 increased. Conclusion: siTCF3 can significantly inhibit the proliferation and migration of A549 and H1299 cells and induce apoptosis. Its molecular mechanism may be achieved by down-regulating the activity of Wnt pathway and regulating the expression of key members of MMP family.
【作者单位】： 山东省菏泽市立医院胸外科;
【分类号】：R730.23
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