【摘要】：目的:探索下調T細胞因子3(TCF3)抑制非小細胞肺癌細胞增殖和遷移的分子機制。方法:運用Lipofectamine 2000轉染法將siTCF3和陰性對照siRNA(NCsiRNA)轉染非小細胞肺癌A549和H1299細胞;運用real-time PCR和Western blot分別測定TCF3的mRNA和蛋白水平;運用螢光素酶報告基因實驗測定TCF3的轉錄活性;MTT、克隆形成實驗、Transwell實驗和Annexin V-FITC/PI染色聯(lián)合流式細胞術分別測定細胞的活力、克隆形成能力、轉移能力及細胞凋亡率;Western blot檢測Wnt、c-Myc、基質金屬蛋白酶(MMP)-9、MMP-13、金屬蛋白酶組織抑制物(TIMP)-1的蛋白表達水平。結果:與NCsiRNA轉染組的細胞比較,siTCF3顯著抑制A549細胞和H1299細胞中TCF3的mRNA和蛋白水平(P0.01)。TCF3轉錄活性和c-Myc蛋白表達水平明顯低于NCsiRNA細胞(P0.05)。MTT實驗結果顯示,培養(yǎng)24 h、48 h、72 h和96 h的A549-siTCF3和H1299-siTCF3細胞活力均顯著低于NCsiRNA細胞(P0.05)。與NCsiRNA細胞相比,siTCF3顯著抑制A549細胞和H1299細胞的克隆形成能力(P0.01)。Transwell實驗結果顯示A549-siTCF3和H1299-siTCF3細胞遷移數(shù)顯著低于A549-NCsiRNA和H1299-NCsiRNA組細胞(P0.05)。流式細胞術分析結果顯示A549-siTCF3細胞和H1299-siTCF3細胞的凋亡率顯著高于A549-NCsiRNA和H1299-NCsiRNA細胞(P0.01)。Western blot實驗結果顯示,下調TCF3表達能抑制Wnt蛋白的表達,MMP-9和MMP-13的蛋白表達明顯降低,TIMP-1的蛋白表達增高。結論:siTCF3顯著抑制A549細胞和H1299細胞的增殖和遷移能力,并誘導細胞凋亡,其分子機制可能通過下調Wnt通路活性以及調控MMP家族關鍵成員的表達而實現(xiàn)。
[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.
【作者單位】： 山東省菏澤市立醫(yī)院胸外科;
【分類號】：R730.23

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