幽门螺杆菌感染相关miRNA-148b在胃癌细胞增殖及侵袭中的分子机制研究
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摘要
目的研究幽门螺杆菌相关miRNA-148b在胃癌中过表达与幽门螺杆菌(H.pylori)感染的相关性及其在胃癌细胞增殖及侵袭中的分子机制。方法本研究首先建立稳定的H.pylori感染人胃上皮细胞模型;检测H.pylori感染前后胃上皮细胞的miRNAs表达谱变化,筛选出感染后miRNA-148b表达显著上调的为研究对象。选择胃癌细胞系AGS、HGC-27、SGC-7901及人正常胃上皮细胞GES-1为研究对象。设计合成miRNA-148b特异性siRNA及miRNA-148b的过表达质粒,抑制或过表达miRNA-148b的表达水平,通过细胞凋亡实验,Transwell小室细胞侵袭等实验,研究miRNA-148b在胃癌细胞增殖及侵袭中的作用。探讨miRNA-148b在胃癌细胞增殖侵袭中的分子作用机制。结果 realtime PCR检测,H.pylori在感染胃上皮细胞GES-1、胃癌细胞株AGS、HGC-27、SGC-7901后miR-148b表达升高,不同胃癌细胞株较正常胃黏膜miR-148b表达下降,在体外,发现miR-148b可以促进胃癌细胞株AGS、HGC-27、SGC-7901细胞的增殖,抑制胃癌细胞株SGC-7901细胞的凋亡;证实抑癌基因TP53INP1和癌基因PAI-1为miR-148b的靶基因,在胃癌细胞株和胃癌组织中miR-148b和PAI-1的表达水平呈显著负相关。结论通过H.pylori感染胃上皮细胞株前后miR-148b的表达检测,验证了先前的miRNAs芯片检测结果,在体外,过表达miR-148b促进胃癌细胞株增殖,抑制其凋亡,提示机体可能以这种方式减缓在H.pylori感染早期所诱导的胃上皮细胞的过度凋亡进程,从而发挥了相应的保护作用;相对于正常胃黏膜组织,在不同胃癌细胞株中miR-148b都表达下调暗示,在胃癌的发生发展过程中,miR-148b可能作为一个抑癌基因而被抑制,证实抑癌基因TP53INP1和癌基因PAI-1为miR-148b的靶基因,提示miR-148b在H.pylori感染和胃癌发生的不同时期功能不同。
OBJECTIVE To investigate the correlation between the overexpression of Helicobacter pylori related miRNA-148 bin gastric cancer and the H.pylori infections and observe the molecular mechanisms in proliferation and invasion of gastric cancer cells.METHODS A stable H.pylori infected human gastric epithelial cell model was firstly established in the study,the change of expression spectrum of miRNAs in gastric epithelial cells was detected before and after the H.pylori infection,and the study objects that showed remarkable up-regulated expression of miRNA-148 bafter the infection were screened out.Gastric cancer cell line AGS,HGC-27,SGC-7901 and human normal gastric epithelial cell line GES-1were selected as the research subjects.The over expression plasmids of miRNA-148 bspecific siRNA and miRNA-148 bwere designed and synthesized to inhibit or overexpress the level of expression of miRNA-148 b.The role of miRNA-148 bin the proliferation and invasion of the gastric cancer cells was studied by means of apoptosis through experiment and Transwell cell invasion experiment.The molecular mechanisms of miRNA-148 bin the proliferation and invasion of the gastric cancer cells were explored.RESULTS The results of real-time PCR detection showed that the level of expression of miR-148 bwas elevated after the gastric epithelial cells GES-1and gastric cancer cell lines AGS,HGC-27,and SGC-7901 were infected with H.pylori,the level of expression of miR-148 bwas lower in the gastric cancer cell lines than in the normal gastric mucosa,it was found that the miR-148 bin vitro could promote the proliferation of gastric cancer cell lines AGS,HGC-27,and SGC-7901 cell and inhibit the apoptosis of SGC-7901 cells of gastric cancer cell strain.It was concluded that the tumor suppressor gene TP53INP1 and oncogene PAI-1were the target genes of miR-148 b,and the expression levels of miR-148 band PAI-1in gastric cancer cell lines and gastric cancer tissues were significantly negatively correlated.CONCLUSIONThe detection of expression of miR-148 bin gastric epithelial cells before and after the infection with H.pylori has verified the result of the previous miRNAs chip test,the overexpression of miR-148 bin vitro can promote the proliferation of the gastric cancer cell strains and inhibit the apoptosis,indicating that the body may slow down the process of excessive apoptosis of gastric epithelial cells induced by the early H.pylori infection,thus,it plays the corresponding protective role.Compared with the normal gastric mucosa tissues,the down-regulated expression of miR-148 bin gastric cancer cell strains implies that the miR-148 bmay be inhibited as a tumor suppressor gene during the development of gastric cancer.It is confirmed that the tumor suppressor gene TP53INP1 and oncogene PAI-1are the target genets of miR-148 b,indicating that the function of miR-148 bvaries in different stages of H.pylori infections and gastric cancer;the specific mechanisms need to be further studied.
OBJECTIVE To investigate the correlation between the overexpression of Helicobacter pylori related miRNA-148 bin gastric cancer and the H.pylori infections and observe the molecular mechanisms in proliferation and invasion of gastric cancer cells.METHODS A stable H.pylori infected human gastric epithelial cell model was firstly established in the study,the change of expression spectrum of miRNAs in gastric epithelial cells was detected before and after the H.pylori infection,and the study objects that showed remarkable up-regulated expression of miRNA-148 bafter the infection were screened out.Gastric cancer cell line AGS,HGC-27,SGC-7901 and human normal gastric epithelial cell line GES-1were selected as the research subjects.The over expression plasmids of miRNA-148 bspecific siRNA and miRNA-148 bwere designed and synthesized to inhibit or overexpress the level of expression of miRNA-148 b.The role of miRNA-148 bin the proliferation and invasion of the gastric cancer cells was studied by means of apoptosis through experiment and Transwell cell invasion experiment.The molecular mechanisms of miRNA-148 bin the proliferation and invasion of the gastric cancer cells were explored.RESULTS The results of real-time PCR detection showed that the level of expression of miR-148 bwas elevated after the gastric epithelial cells GES-1and gastric cancer cell lines AGS,HGC-27,and SGC-7901 were infected with H.pylori,the level of expression of miR-148 bwas lower in the gastric cancer cell lines than in the normal gastric mucosa,it was found that the miR-148 bin vitro could promote the proliferation of gastric cancer cell lines AGS,HGC-27,and SGC-7901 cell and inhibit the apoptosis of SGC-7901 cells of gastric cancer cell strain.It was concluded that the tumor suppressor gene TP53INP1 and oncogene PAI-1were the target genes of miR-148 b,and the expression levels of miR-148 band PAI-1in gastric cancer cell lines and gastric cancer tissues were significantly negatively correlated.CONCLUSIONThe detection of expression of miR-148 bin gastric epithelial cells before and after the infection with H.pylori has verified the result of the previous miRNAs chip test,the overexpression of miR-148 bin vitro can promote the proliferation of the gastric cancer cell strains and inhibit the apoptosis,indicating that the body may slow down the process of excessive apoptosis of gastric epithelial cells induced by the early H.pylori infection,thus,it plays the corresponding protective role.Compared with the normal gastric mucosa tissues,the down-regulated expression of miR-148 bin gastric cancer cell strains implies that the miR-148 bmay be inhibited as a tumor suppressor gene during the development of gastric cancer.It is confirmed that the tumor suppressor gene TP53INP1 and oncogene PAI-1are the target genets of miR-148 b,indicating that the function of miR-148 bvaries in different stages of H.pylori infections and gastric cancer;the specific mechanisms need to be further studied.
引文
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[2]Lee RC,Feinbaum RL,Ambros V.The C.elegans heterochronic gene lin-4encodes small RNAs with antisense complementarity to lin-14[J].Cell,1993,75(5):843-854.
[3]Lewis BP,Burge CB,Bartel DP.Conserved seed pairing,often flanked by adenosines,indicates that thousands of human genes are microRNA targets[J].Cell,2005,120(1):15-20.
[4]Calin GA,Sevignani C,Dumitru CD,et al.Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers[J].Proc Natl Acad Sci U S A,2004,101(9):2999-3004.
[5]Saito Y,Suzuki H,Hibi T.The role of microRNAs in gastrointestinal cancers[J].J Gastroenterol,2009,44(19):18-22.
[6]刘文忠.幽门螺杆菌研究进展[M].上海:上海科学技术文献出版社,2001.
[7]Knipp U,Birkholz S,Kaup W,et al.Partial characterization of a cell proliferation-inhibiting protein produced by Helicobacter pylori[J].Infect Immun,1996,64(9):3491-3496.
[8]Yu F,Deng H,Yao H,et al.Mir-30reduction maintains self-renewal and inhibits apoptosis in breast tumor-initiating cells[J].Oncogene,2010,29(29):4194-4204.
[9]Wszolek MF,Rieger-Christ KM,Kenney PA,et al.A MicroRNA expression profile defining the invasive bladder tumor phenotype[J].Urol Oncol,2011,29(6):794-801.
[10]Lu Y,Ryan SL,Elliott DJ,et al.Amplification and overexpression of Hsa-miR-30b,Hsa-miR-30d and KHDRBS3 at8q24.22-q24.23in medulloblastoma[J].PLoS One,2009,4(7):e6159.
[11]Wagner S,Beil W,Westermann J,et al.Regulation of gastric epithelial cell growth by Helicobacter pylori:evidence for a major role of apoptosis[J].Gastroenterology,1997,113(6):1836-1847.
[12]Matsui T,Matsukawa Y,Sakai T,et al.Effect of ammonia on cell-cycle progression of human gastric cancer cells[J].Eur J Gastroenterol Hepatol,1995,7(Suppl 1):S79-S81.
[13]Wienholds E,Kloosterman WP,Miska E,et al.MicroRNA expression in zebrafish embryonic development[J].Science,2005,309(5732):310-311.