MiR-702-3p调控TGF-β1在纳米二氧化硅致肺上皮—间质转化中的作用机制研究
发布时间：2018-09-10 21:21
【摘要】：纳米二氧化硅(silicon dioxide,SiO_2)俗称白炭黑,因特殊的理化性.质被广泛应用于食品、涂料、橡胶、化妆品和医药卫生等许多领域,享有“工业味精”之美誉。中国是世界白炭黑生产大国之一,随着纳米SiO_2应用的日益广泛,相关行业人群接触的机会越来越多,其潜在危害不容忽视。动物实验研究表明,纳米SiO_2经呼吸道进入体内后可引起大/小鼠肺部炎症反应和肺组织纤维化等。纳米Si02致肺纤维化作用的机制目前尚未完全明了。上皮间充质转化(epithelial-to-messenchymal,EMT)是肺纤维化进程中的重要环节。已有研究报道miRNAs的异常表达与EMT的发生过程有关,Let-7d、miR-192、miR-124、miR-26a、miR-10b、miR-487a等可能通过TGF-β1信号通路在EMT的发生发展过程中发挥重要的调控作用。TGF-β1可以通过依赖Smads的方式诱导EMT,刺激肺上皮细胞转化为肺间质细胞,促进肺纤维化的发生。本研究在前期纳米Si02经一次性气管灌注大鼠染尘60、90天时主要表现为肺间质纤维化的基础上,以高通量测序显示miR-702-3p为纳米SiO_2致大鼠肺组织纤维化中差异表达上调的miRNA为前提,经生物功能学分析发现,miR-702-3p 可能与转化生长因子-β1(transforming growth factor-β1,TGF-β1)通路相关。miR-702-3p 可能通过预测靶基因骨形态发生蛋白-7(bone morphogenetic protein-7,BMP-7)调控TGF-β1信号通路发挥调节EMT的作用从而参与纳米SiO_2致肺纤维化的发生发展过程。为阐明miR-702-3p在纳米SiO_2致大鼠肺组织纤维化中的生物调节作用及明确miR-702-3p、TGF-β1信号通路、EMT三者之间的关系,研究从整体动物和细胞两个水平来探讨相关分子机制,实验结果如下:1.在线数据库TargetScan预测miR-702-3p的靶基因,其可能靶基因共136个。GO和KEGG通路分析这些靶基因的生物功能学,发现miR-702-3p对TGF-β信号通路和Smad蛋白信号转导中有调控作用,本研究将TGF-β超家族成员之一的BMP-7作为miR-702-3p重要的预测靶基因,探索miR-702-3p参与调控纳米SiO_2诱导EMT的相关机制。2.实时荧光定量PCR(qRT-PCR)检测结果显示,25mg/mL纳米Si02染尘60d后大鼠肺组织中miR-702-3p 表达是对照组的 2.04 倍(P0.05);BMP-7、TGF-β1、Smad3、E 钙粘蛋白(E-cadherin,E-cad)、α-平滑肌肌动蛋白(α-smooth muscle acti,α-SMA)、Ⅲ型胶原蛋白(Collagen type Ⅲ,COL3)mRNA 的表达分别为对照组的0.40、1.28、1.71、0.57、2.70、1.53倍,差异均有统计学意义(P0.05);蛋白免疫印迹法(Western Blot)检查结果表明,纳米SiO_2染尘组大鼠肺组织中BMP-7、TGF-β1、Smad3、E-cad、α-SMA、COL3蛋白表达量是对照组的0.60、1.48、1.24、0.65、1.43、1.23倍,差异均有统计学意义(P0.05)。双荧光素酶报告基因检测结果显示,将野生型BMP-7的3'UTR的荧光素酶报告质粒载体分别与miR-702-3p mimic/NC 共转染于人肺腺癌上皮细胞系(A549 细胞),miR-702-3p mimic 组与 miR-702-3p NC组的报告荧光值分别为0.73±0.010、1.00±0.005,差异有统计学意义(P0.05);将BMP-7突变位点的载体质粒再分别与miR-702-3p mimic/NC共转染于A549细胞,miR-702-3p mimic组与miR-702-3p NC组的报告荧光值分别为0.98±0.060、1.00±0.016,差异无统计学意义(P0.05)。结果提示纳米SiO_2致大鼠肺间质纤维化过程中发生了 EMT,miR-702-3p与BMP-7可通过CCACCCG-GGUGGGC的位点结合,靶向抑制BMP-7的表达并激活TGF-β1信号通路,促进肺上皮细胞转化为肺间质细胞,产生胶原蛋白增加,发生肺纤维化。3.纳米SiO_2对大鼠肺泡巨噬细胞(NR8383细胞)染尘24h,四甲基偶氮唑蓝法(MTT)检测得其IC_(50)为 0.761 mg/mL,95%可信区间为 0.001～3.844 mg/mL。50μg/mL 纳米 Si02 染毒 NR8383 细胞 24h 后细胞的存活率在85%以上,酶联免疫吸附(ELISA)法检测纳米SiO_2染毒NR8383细胞24h上清液中TGF-β1含量为1612.67±15.32pg/mL,与阴性对照组1072.67±60.10pg/mL比较,差异有统计学意义(P0.05);纳米SiO_2染毒组NR8383细胞中miR-702-3p的表达水平是对照组的3.11倍(P0.05),BMP-7 mRNA与蛋白表达水平分别是对照组的0.39和0.30倍(P0.05)。50μg/mL纳米Si02染毒NR8383细胞24h后的上清液与A549细胞共培养24h后,空白对照组、染毒NR8383细胞上清液组A549细胞的增殖率分别为(100±6)%、(101±5)%,差异无统计学意义(P0.05);染毒组A549细胞中miR-702-3p的表达是对照组的 1.22 倍(P0.05);BMP-7、TGF-β1、Smad3、E-cad、α-SMA、COL3 mRNA 表达水平分别是对照组的 0.25、1.33、1.53、0.99、6.75、2.68 倍,差异有统计学意义(P0.05,E-cad 的 P0.05)。纳米 Si02染毒组A549细胞中BMP-7、TGF-β1、Smad3、E-cad、α-SMA、COL3蛋白表达水平分别是对照组的0.48、1.88、2.20、0.40、1.91、3.1 倍,差异有统计学意义(P0.05)。miR-702-3p mimic/mimic NC、miR-702-3p inhibitor/inhibitor NC 和空白对照组分别转染 NR8383 细胞48h 后,各组增殖率分别为(101.40±3.45)%、(100.03±2.91)%、(100.76±0.96)%、(92.37±3.26)%、(100±3.89)%,差异无统计学意义(P0.05),镜下未观察到细胞有明显变化;转染48h后miR-702-3p mimic转染组的miR-702-3p的表达水平是阴性对照组的27.78倍(差异倍数≥2,P0.05),表明miR-702-3p mimic转染成功;miR-702-3p inhibitor转染NR8383细胞48h后miR-702-3p的表达水平是阴性对照组的0.19倍(P0.05),miR-702-3p mimic转染组NR8383细胞中BMP-7mRNA与蛋白的表达水平分别是阴性对照组的1.07、0.43 倍(P0.05),miR-702-3p inhibitor转染组NR8383 细胞中BMP-7mRNA与蛋白的表达水平分别是阴性对照组的2.02、1.32倍(P0.05);miR-702-3pmimic转染组NR8383细胞中TGF-β1mRNA与蛋白的表达水平分别是阴性对照组的1.23、1.58倍(P0.05),miR-702-3p inhibitor转染组NR8383细胞中TGF-β1 mRNA与蛋白的表达水平分别是阴性对照组的0.52、0.63倍(P0.05),提示转染miR-702-3p可通过抑制BMP-7的表达,促进TGF-β1生成增加。TGF-β1、TGF-β1+TGF-β1受体阻断剂分别诱导A549细胞48h后,对照组、TGF-β1+RB组与TGF-β1组的细胞增殖率分别为(100±5.04%)、(106.28±1.38)%、(108.99±6.51)%,差异无统计学意义(P0.05);5ng/mL的TGF-β1诱导A549细胞48h后,观察到细胞从上皮样逐渐转变为成纤维细胞样,qRT-PCR检测结果显示,TGF-β1 诱导组 A549 细胞中 miR-702-3p、BMP-7、Smad3、E-cad、α-SMA、COL3mRNA 的表达水平分别是对照组的1.37、0.39、0.57、0.18、3.14、2.64倍(P0.05),TGF-β1诱导组A549细胞中BMP-7、Smad3、E-cad、α-SMA、COL3 蛋白的表达量分别是对照组的 0.41、1.82、0.72、1.61、14.51 倍(P0.05);TGF-β1受体阻断剂诱导3h后再给与TGF-β1诱导48h后A549细胞的形态未发生明显改变,BMP-7蛋白表达增加(1.68倍)外miR-702-3p、Smad3、E-cad、α-SMA、COL3mRNA与蛋白表达水平均未出现明显变化(P0.05)。提示TGF-β1对miR-702-3p有上调作用,促使EMT的发生。综上所述,纳米SiO_2致大鼠肺间质纤维化过程中发生了 EMT,BMP-7是miR-702-3p的靶基因。miR-702-3p、TGF-β1信号通路与EMT过程密切相关,一方面miR-702-3p与BMP-7可通过CCACCCG-GGUGGGC的位点结合,抑制靶基因BMP-7的表达,活化TGF-β1信号通路,促使肺上皮细胞向肺间质细胞转化,胶原蛋白分泌增多,促进肺纤维化;另一方面TGF-β1可上调miR-702-3p的表达,促进EMT过程。
[Abstract]:Nano-silica (SiO_2), commonly known as silica, is widely used in food, coatings, rubber, cosmetics and medical hygiene and many other fields, enjoying the reputation of "industrial monosodium glutamate". Animal studies have shown that nano-SiO_2 can cause pulmonary inflammation and fibrosis in rats and mice after it enters the body through the respiratory tract. The mechanism of pulmonary fibrosis induced by nano-SiO_2 has not been fully understood. It has been reported that the abnormal expression of microRNAs is related to the occurrence of EMT. Let-7d, microRNAs-192, microRNAs-124, microRNAs-26a, microRNAs-10b, microRNAs-487a may play an important regulatory role in the development of EMT through TGF-beta 1 signaling pathway. TGF-beta 1 can induce EMT and stimulate EMT by relying on Smads. Pulmonary epithelial cells were transformed into pulmonary interstitial cells to promote the development of pulmonary fibrosis. On the basis of the primary manifestation of pulmonary interstitial fibrosis in rats exposed to nano-Si02 by one-off tracheal perfusion for 60,90 days, the high-throughput sequencing showed that microRNAs-702-3p were differentially expressed and up-regulated in rat pulmonary fibrosis induced by nano-SiO_2. Biological function analysis revealed that microRNAs-702-3p may be involved in the transforming growth factor-beta 1 (TGF-beta 1) pathway. MicroRNAs-702-3p may play a role in regulating EMT by predicting the target gene bone morphogenetic protein-7 (BMP-7) regulating the TGF-beta 1 signaling pathway. To elucidate the role of microRNAs-702-3p in the regulation of pulmonary fibrosis induced by nano-SiO_2 in rats and to clarify the relationship between microRNAs-702-3p, TGF-beta 1 signaling pathway and EMT, we studied the molecular mechanism of microRNAs-702-3p at both animal and cellular levels. The results were as follows: 1. TargetS online database. Mi-702-3p target genes were predicted by can. A total of 136 probable target genes were analyzed by GO and KEGG pathways. It was found that Mi-702-3p could regulate the TGF-beta signaling pathway and signal transduction of Smad protein. BMP-7, one of the members of TGF-beta superfamily, was used as an important predictive target gene of Mi-702-3p in this study. Real-time fluorescence quantitative PCR (qRT-PCR) results showed that the expression of microRNAs-702-3p in lung tissues of rats exposed to 25 mg/mL nano-Si02 for 60 days was 2.04 times higher than that of control group (P 0.05); BMP-7, TGF-beta 1, Smad3, E-cadherin, alpha-smooth muscle actin (alpha-smooth muscle, alpha-smooth muscle, alpha-smooth muscle actini, alpha-smooth muscle actin) were detected. The expression of SMA and COL3 mRNA was 0.40, 1.28, 1.71, 0.57, 2.70 and 1.53 times higher than that of the control group (P 0.05). The expression of BMP-7, TGF-beta 1, Smad3, E-cad, alpha-SMA and COL3 protein in lung tissue of rats exposed to nano-SiO_2 was significantly higher than that of the control group (P 0.05). The results of double luciferase reporter gene test showed that the luciferase reporter plasmid vector of wild type BMP-7 was co-transfected with Mi-702-3p mimic/NC into human lung adenocarcinoma epithelial cell line (A549 cells), Mi-702-3p mic group and Mi-702-3, respectively. The reported fluorescence values of P NC group were 0.73 (+ 0.010) and 1.00 (+ 0.005) respectively, and the difference was statistically significant (P 0.05); the vector plasmid of BMP-7 mutation site was transfected into A549 cells with Mi-702-3p mimic/NC, respectively. The reported fluorescence values of Mi-702-3p mic group and Mi-702-3p NC group were 0.98 (+ 0.060) and 1.00 (+ 0.016), respectively, with no significant difference (P 0.05). The results suggest that EMT occurs in the process of pulmonary interstitial fibrosis induced by nano-SiO_2 in rats. Mi-702-3p and BMP-7 can bind to the site of CCACCCG-GGUGGC, inhibit the expression of BMP-7 and activate the TGF-beta 1 signaling pathway, promote the transformation of pulmonary epithelial cells into pulmonary interstitial cells, increase the production of collagen and induce pulmonary fibrosis in rats. Alveolar macrophages (NR8383 cells) were exposed to dust for 24 hours, and their IC_ (50) was 0.761 mg/mL by MTT assay. The 95% confidence interval was 0.001-3.844 mg/mL. The survival rate of NR8383 cells exposed to nano-Si02 was above 85% after 24 hours. The supernatant of NR8383 cells exposed to nano-SiO_2 was detected by enzyme linked immunosorbent assay (ELISA) for 24 hours. The content of TGF-beta 1 in the solution was 1612.67+15.32 pg/mL, which was significantly different from that in the negative control group (P 0.05). The expression of microRNA-702-3p in NR8383 cells of nano-SiO_2 group was 3.11 times higher than that of the control group (P 0.05), and the expression of BMP-7 mRNA and protein were 0.39 and 0.30 times higher than that of the control group (P 0.05). The proliferation rate of A549 cells in NR8383 cell supernatant group was (100 65 The expression levels of BMP-7, TGF-beta 1, Smad3, E-cad, alpha-SMA, COL3 in A549 cells were 0.48, 1.88, 2.20, 0.40, 1.91, 3.1 times higher than those in control group (P 0.05, P 0.05 for E-cad). 5. After 48 hours of transfection, the proliferation rates of NR8383 cells were (101.40 (3.45)%, (100.03 (2.91)%, (100.76 (0.96)%, (92.37 (3.26)%, (100 (3.89)%, respectively. There was no significant difference (P 0.05). After 48 hours of transfection, no significant changes were observed in NR8383 cells. The expression level of microRNAs-702-3p in the microRNAs-702-3p mimic transfection group was 27.78 times higher than that in the negative control group (P 0.05), indicating that the microRNAs-702-3p mimic transfection was successful; the expression level of microRNAs-702-3p inhibitor in NR8383 cells 48 hours after microRNAs-702-3p inhibitor transfection was 0.19 times higher than that in the negative control group (P 0.05), BMP-7 mRNA and BMP-7 mRNA in NR8383 cells of the microRNAs-702-3p mic transfection group were 0.19 times higher than those in the negative control group (P 0. The expression levels of BMP-7 mRNA and protein in NR8383 cells transfected with miR-702-3p inhibitor were 2.02 and 1.32 times higher than those in the negative control group (P 0.05), and the expression levels of TGF-beta 1 mRNA and protein in NR8383 cells transfected with miR-702-3pmic were 1.23 times higher than those in the negative control group (P 0.05). TGF-1 mRNA and protein expression levels in NR8383 cells transfected with microRNAs-702-3p inhibitor were 0.52 and 0.63 times higher than those in the negative control group (P 0.05), suggesting that microRNAs-702-3p could inhibit the expression of BMP-7 and promote the production of TGF-beta 1. TGF-1, TGF-beta 1 + TGF-beta 1 receptor blockers induced A549 cells 48 hours later in the control group, respectively. The cell proliferation rates of GF-beta 1+RB group and TGF-beta 1 group were (100+5.04%), (106.28+1.38)%, (108.99+6.51)%. There was no significant difference between them (P 0.05). After 48h induction of A549 cells by 5 ng/ml TGF-beta 1, the cells gradually transformed from epithelial to fibroblast-like cells. The results of qRT-PCR showed that the cells of TGF-beta 1 induced A549 cells were microRNA702-3p, 3 P. The expression levels of BMP-7, Smad3, E-cad, alpha-SMA, COL3 mRNA were 1.37, 0.39, 0.57, 0.18, 3.14, 2.64 times as high as those in the control group (P 0.05). The expression levels of BMP-7, Smad3, E-cad, alpha-SMA, COL3 protein in A549 cells induced by TGF-beta 1 receptor blocker were 0.41, 1.82, 0.72, 1.61, 14.51 times as high as those in the control group (P 0.05). The expression of BMP-7 protein increased (1.68 times) and the mRNA and protein levels of extracellular microarray-702-3p, Smad3, E-cad, alpha-SMA, COL3 did not change significantly (P 0.05). These results suggest that TGF-beta 1 can up-regulate the expression of microarray-702-3p and promote the occurrence of EMT in rat pulmonary fibrosis induced by nano-SiO_2. Mi-702-3p, TGF-beta 1 signaling pathway is closely related to EMT process. On the one hand, Mi-702-3p and BMP-7 can bind to the site of CCACCCG-GGUGGC, inhibit the expression of target gene BMP-7, activate TGF-beta 1 signaling pathway, promote the transformation of lung epithelial cells into pulmonary interstitial cells, increase the secretion of collagen, and promote the proliferation of lung epithelial cells. Pulmonary fibrosis; on the other hand, TGF- beta 1 can increase the expression of miR-702-3p and promote the EMT process.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R135.2
[Abstract]:Nano-silica (SiO_2), commonly known as silica, is widely used in food, coatings, rubber, cosmetics and medical hygiene and many other fields, enjoying the reputation of "industrial monosodium glutamate". Animal studies have shown that nano-SiO_2 can cause pulmonary inflammation and fibrosis in rats and mice after it enters the body through the respiratory tract. The mechanism of pulmonary fibrosis induced by nano-SiO_2 has not been fully understood. It has been reported that the abnormal expression of microRNAs is related to the occurrence of EMT. Let-7d, microRNAs-192, microRNAs-124, microRNAs-26a, microRNAs-10b, microRNAs-487a may play an important regulatory role in the development of EMT through TGF-beta 1 signaling pathway. TGF-beta 1 can induce EMT and stimulate EMT by relying on Smads. Pulmonary epithelial cells were transformed into pulmonary interstitial cells to promote the development of pulmonary fibrosis. On the basis of the primary manifestation of pulmonary interstitial fibrosis in rats exposed to nano-Si02 by one-off tracheal perfusion for 60,90 days, the high-throughput sequencing showed that microRNAs-702-3p were differentially expressed and up-regulated in rat pulmonary fibrosis induced by nano-SiO_2. Biological function analysis revealed that microRNAs-702-3p may be involved in the transforming growth factor-beta 1 (TGF-beta 1) pathway. MicroRNAs-702-3p may play a role in regulating EMT by predicting the target gene bone morphogenetic protein-7 (BMP-7) regulating the TGF-beta 1 signaling pathway. To elucidate the role of microRNAs-702-3p in the regulation of pulmonary fibrosis induced by nano-SiO_2 in rats and to clarify the relationship between microRNAs-702-3p, TGF-beta 1 signaling pathway and EMT, we studied the molecular mechanism of microRNAs-702-3p at both animal and cellular levels. The results were as follows: 1. TargetS online database. Mi-702-3p target genes were predicted by can. A total of 136 probable target genes were analyzed by GO and KEGG pathways. It was found that Mi-702-3p could regulate the TGF-beta signaling pathway and signal transduction of Smad protein. BMP-7, one of the members of TGF-beta superfamily, was used as an important predictive target gene of Mi-702-3p in this study. Real-time fluorescence quantitative PCR (qRT-PCR) results showed that the expression of microRNAs-702-3p in lung tissues of rats exposed to 25 mg/mL nano-Si02 for 60 days was 2.04 times higher than that of control group (P 0.05); BMP-7, TGF-beta 1, Smad3, E-cadherin, alpha-smooth muscle actin (alpha-smooth muscle, alpha-smooth muscle, alpha-smooth muscle actini, alpha-smooth muscle actin) were detected. The expression of SMA and COL3 mRNA was 0.40, 1.28, 1.71, 0.57, 2.70 and 1.53 times higher than that of the control group (P 0.05). The expression of BMP-7, TGF-beta 1, Smad3, E-cad, alpha-SMA and COL3 protein in lung tissue of rats exposed to nano-SiO_2 was significantly higher than that of the control group (P 0.05). The results of double luciferase reporter gene test showed that the luciferase reporter plasmid vector of wild type BMP-7 was co-transfected with Mi-702-3p mimic/NC into human lung adenocarcinoma epithelial cell line (A549 cells), Mi-702-3p mic group and Mi-702-3, respectively. The reported fluorescence values of P NC group were 0.73 (+ 0.010) and 1.00 (+ 0.005) respectively, and the difference was statistically significant (P 0.05); the vector plasmid of BMP-7 mutation site was transfected into A549 cells with Mi-702-3p mimic/NC, respectively. The reported fluorescence values of Mi-702-3p mic group and Mi-702-3p NC group were 0.98 (+ 0.060) and 1.00 (+ 0.016), respectively, with no significant difference (P 0.05). The results suggest that EMT occurs in the process of pulmonary interstitial fibrosis induced by nano-SiO_2 in rats. Mi-702-3p and BMP-7 can bind to the site of CCACCCG-GGUGGC, inhibit the expression of BMP-7 and activate the TGF-beta 1 signaling pathway, promote the transformation of pulmonary epithelial cells into pulmonary interstitial cells, increase the production of collagen and induce pulmonary fibrosis in rats. Alveolar macrophages (NR8383 cells) were exposed to dust for 24 hours, and their IC_ (50) was 0.761 mg/mL by MTT assay. The 95% confidence interval was 0.001-3.844 mg/mL. The survival rate of NR8383 cells exposed to nano-Si02 was above 85% after 24 hours. The supernatant of NR8383 cells exposed to nano-SiO_2 was detected by enzyme linked immunosorbent assay (ELISA) for 24 hours. The content of TGF-beta 1 in the solution was 1612.67+15.32 pg/mL, which was significantly different from that in the negative control group (P 0.05). The expression of microRNA-702-3p in NR8383 cells of nano-SiO_2 group was 3.11 times higher than that of the control group (P 0.05), and the expression of BMP-7 mRNA and protein were 0.39 and 0.30 times higher than that of the control group (P 0.05). The proliferation rate of A549 cells in NR8383 cell supernatant group was (100 65 The expression levels of BMP-7, TGF-beta 1, Smad3, E-cad, alpha-SMA, COL3 in A549 cells were 0.48, 1.88, 2.20, 0.40, 1.91, 3.1 times higher than those in control group (P 0.05, P 0.05 for E-cad). 5. After 48 hours of transfection, the proliferation rates of NR8383 cells were (101.40 (3.45)%, (100.03 (2.91)%, (100.76 (0.96)%, (92.37 (3.26)%, (100 (3.89)%, respectively. There was no significant difference (P 0.05). After 48 hours of transfection, no significant changes were observed in NR8383 cells. The expression level of microRNAs-702-3p in the microRNAs-702-3p mimic transfection group was 27.78 times higher than that in the negative control group (P 0.05), indicating that the microRNAs-702-3p mimic transfection was successful; the expression level of microRNAs-702-3p inhibitor in NR8383 cells 48 hours after microRNAs-702-3p inhibitor transfection was 0.19 times higher than that in the negative control group (P 0.05), BMP-7 mRNA and BMP-7 mRNA in NR8383 cells of the microRNAs-702-3p mic transfection group were 0.19 times higher than those in the negative control group (P 0. The expression levels of BMP-7 mRNA and protein in NR8383 cells transfected with miR-702-3p inhibitor were 2.02 and 1.32 times higher than those in the negative control group (P 0.05), and the expression levels of TGF-beta 1 mRNA and protein in NR8383 cells transfected with miR-702-3pmic were 1.23 times higher than those in the negative control group (P 0.05). TGF-1 mRNA and protein expression levels in NR8383 cells transfected with microRNAs-702-3p inhibitor were 0.52 and 0.63 times higher than those in the negative control group (P 0.05), suggesting that microRNAs-702-3p could inhibit the expression of BMP-7 and promote the production of TGF-beta 1. TGF-1, TGF-beta 1 + TGF-beta 1 receptor blockers induced A549 cells 48 hours later in the control group, respectively. The cell proliferation rates of GF-beta 1+RB group and TGF-beta 1 group were (100+5.04%), (106.28+1.38)%, (108.99+6.51)%. There was no significant difference between them (P 0.05). After 48h induction of A549 cells by 5 ng/ml TGF-beta 1, the cells gradually transformed from epithelial to fibroblast-like cells. The results of qRT-PCR showed that the cells of TGF-beta 1 induced A549 cells were microRNA702-3p, 3 P. The expression levels of BMP-7, Smad3, E-cad, alpha-SMA, COL3 mRNA were 1.37, 0.39, 0.57, 0.18, 3.14, 2.64 times as high as those in the control group (P 0.05). The expression levels of BMP-7, Smad3, E-cad, alpha-SMA, COL3 protein in A549 cells induced by TGF-beta 1 receptor blocker were 0.41, 1.82, 0.72, 1.61, 14.51 times as high as those in the control group (P 0.05). The expression of BMP-7 protein increased (1.68 times) and the mRNA and protein levels of extracellular microarray-702-3p, Smad3, E-cad, alpha-SMA, COL3 did not change significantly (P 0.05). These results suggest that TGF-beta 1 can up-regulate the expression of microarray-702-3p and promote the occurrence of EMT in rat pulmonary fibrosis induced by nano-SiO_2. Mi-702-3p, TGF-beta 1 signaling pathway is closely related to EMT process. On the one hand, Mi-702-3p and BMP-7 can bind to the site of CCACCCG-GGUGGC, inhibit the expression of target gene BMP-7, activate TGF-beta 1 signaling pathway, promote the transformation of lung epithelial cells into pulmonary interstitial cells, increase the secretion of collagen, and promote the proliferation of lung epithelial cells. Pulmonary fibrosis; on the other hand, TGF- beta 1 can increase the expression of miR-702-3p and promote the EMT process.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R135.2
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