【摘要】：目的:探索下調(diào)T細(xì)胞因子3(TCF3)抑制非小細(xì)胞肺癌細(xì)胞增殖和遷移的分子機(jī)制。方法:運(yùn)用Lipofectamine 2000轉(zhuǎn)染法將siTCF3和陰性對(duì)照siRNA(NCsiRNA)轉(zhuǎn)染非小細(xì)胞肺癌A549和H1299細(xì)胞;運(yùn)用real-time PCR和Western blot分別測(cè)定TCF3的mRNA和蛋白水平;運(yùn)用螢光素酶報(bào)告基因?qū)嶒?yàn)測(cè)定TCF3的轉(zhuǎn)錄活性;MTT、克隆形成實(shí)驗(yàn)、Transwell實(shí)驗(yàn)和Annexin V-FITC/PI染色聯(lián)合流式細(xì)胞術(shù)分別測(cè)定細(xì)胞的活力、克隆形成能力、轉(zhuǎn)移能力及細(xì)胞凋亡率;Western blot檢測(cè)Wnt、c-Myc、基質(zhì)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(dá)水平。結(jié)果:與NCsiRNA轉(zhuǎn)染組的細(xì)胞比較,siTCF3顯著抑制A549細(xì)胞和H1299細(xì)胞中TCF3的mRNA和蛋白水平(P0.01)。TCF3轉(zhuǎn)錄活性和c-Myc蛋白表達(dá)水平明顯低于NCsiRNA細(xì)胞(P0.05)。MTT實(shí)驗(yàn)結(jié)果顯示,培養(yǎng)24 h、48 h、72 h和96 h的A549-siTCF3和H1299-siTCF3細(xì)胞活力均顯著低于NCsiRNA細(xì)胞(P0.05)。與NCsiRNA細(xì)胞相比,siTCF3顯著抑制A549細(xì)胞和H1299細(xì)胞的克隆形成能力(P0.01)。Transwell實(shí)驗(yàn)結(jié)果顯示A549-siTCF3和H1299-siTCF3細(xì)胞遷移數(shù)顯著低于A549-NCsiRNA和H1299-NCsiRNA組細(xì)胞(P0.05)。流式細(xì)胞術(shù)分析結(jié)果顯示A549-siTCF3細(xì)胞和H1299-siTCF3細(xì)胞的凋亡率顯著高于A549-NCsiRNA和H1299-NCsiRNA細(xì)胞(P0.01)。Western blot實(shí)驗(yàn)結(jié)果顯示,下調(diào)TCF3表達(dá)能抑制Wnt蛋白的表達(dá),MMP-9和MMP-13的蛋白表達(dá)明顯降低,TIMP-1的蛋白表達(dá)增高。結(jié)論:siTCF3顯著抑制A549細(xì)胞和H1299細(xì)胞的增殖和遷移能力,并誘導(dǎo)細(xì)胞凋亡,其分子機(jī)制可能通過(guò)下調(diào)Wnt通路活性以及調(diào)控MMP家族關(guān)鍵成員的表達(dá)而實(shí)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(hào)】：R730.23

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