The Experimental Study Of Let-7a Treatment On Gastric Carcinoma And Its Basic Molecule Mechanism

Cancer is a multi-step disease involving dynamic changes in the genome. However, studies on cancer genome so far have focused most heavily on protein-coding genes, and our knowledge on alterations of the functional noncoding sequences in cancer is largely absent. miRNA are a recently discovered class of short noncoding RNA genes that act posttranscriptionally as negative regulators of gene expression. A large body of research shows that animal miRNAs play fundamental roles in many biological processes, such as cell differentiation and cell proliferation. About 1,000 miRNA genes are thought to be encoded in the human genome. Although it is still difficult to identify accurately individual miRNA/target interactions, computational predictions of miRNA target genes indicate that as many as one third of all human protein-encoding genes may be regulated by miRNAs. This suggests that miRNAs could be involved in a wide variety of human diseases, including cancer. Indeed, as a result of intense research within the last 5 years, miRNA-mediated regulation of tumorigenesis is emerging as a new paradigm in the field of cancer biology. It has also been reported that these miRNA signatures may be a more robust tool than expression patterns of protein-encoding genes for distinguishing normal from tumor tissues. One particular miRNA, let-7, was discovered in the early 1990's humans and mice. It has been reported that let-7 as a novel candidate of tumor suppressor gene involving several cancers such as lung carcinoma and colon carcinoma. Gastric carcinoma, which is the second most prevalent cancer, is a complex, inadequately understood, and often fatal disease when not detected at early stages. Atypical hyperplasia is generally accepted as a precancerous lesion. Cancerization progression from normal gastric mucosa, chronic atrophic gastritis to gastric carcinoma has been well acknowledged. In this study, the expression of let-7a in normal gastric tissue, chronic atrophic gastritis (atypical hyperplasia)and gastric carcinoma was detected by in situ hybridization. It has been demonstrated that the lentivirus-delivered short hairpin RNAs (shRNAs) are capable of specific, highly stable and functional overexpression of let-7a gene expression both in vitro and in vivo, enabling rapid and efficient analysis of gene function. Then,analysis of different Proteins expressed in SGC-7901,SGC-7901/EV and SGC-7901/let-7a Cells by Proteomics was performed to explore the mechanism of let-7a and provide a new experimental and theoretical basis gene therapy for gastric cancer to guide further clinical application.Including the following five parts:Part 1 Study of in situ Expression of let-7a in The Gastric Mucosa Cancerization Tissue Array and Its Clinical SignificanceObjective To investigate the expression profile of novel candidate of tumor suppressor gene let-7a in gastric carcinoma, chronic atrophic gastritis (atypical hyperplasia) and normal gastric tissue, analyze the correlation between reduced let-7a and progression of gastric mucosa cancerization.Methods Human gastric carcinoma, chronic atrophic gastritis (atypical hyperplasia) and normal gastric tissure microarrays were constructed previously and tissue microarays combined with in situ hybridization were used to detect the expression of let-7a.Results Results showed that there was no significant difference of positive rates of let-7a among normal gastric tissue, chronic atrophic gastritis and gastric carcinoma (P>0.05).However, the degree of let-7a expression in the groups are closely related to the progression of gastric mucosa cancerization (P<0.05).Conclusions The results suggested that the gastric carcinoma had relatively lowered expression of let-7a. Reduced let-7a may be a fundamental factor in the formation of gastric carcinoma.Part 2 Construction and identification of the lentiviral Vector of human let-7a geneObjective To construct a lentiviral Vector of human let-7a gene,and use it in the following experiences.Methods Pwpxl-MOD2-let-7a eukaryotic expression plasmid was constructed by DNA recombinant method. Pwpxl-MOD2-let-7a was constructed along with pRsv-REV,pMD1g-pRRE,pMD2G into HEK 293 T to package lentivirus particles. According to the EGFP expression, the functional titer was determined by FCM after transduction into HEK 293 T cells.Results The eukaryotic expression vector Pwpxl-MOD2-let-7a has been successfully constructed. After cotransfection, lentiviral vector can be Packaged in HEK 293 T cell and high functional titer was detected.Conclusions Lentivirus vector of human let-7a gene has been constructed, which is the essential building block required for the let-7a related gene therapy research.Part 3 in vitro Study on the Effect of let-7a in Human Gastric Carcinoma Cell LineObjective To study the effects of the let-7a in human gastric carcinoma cell line by constructing a gastric cancer cell line SGC-7901 stably overexpressing let-7a gene.Methods The let-7a was transferred to the human gastric carcinoma cell lines SGC-7901 by recombinant lentiviruses which carrying let-7a. The infected cells were quantified by Real-Time RT-PCR for the expression level of let-7a, then characterized by cell proliferation assays, cell adhesion assays and cell invasion assays. Cell cycle was analysised by flow cytometry (FCM).Results (1) The efficiency of infection of the cell lines using the lentiviral vectors was almost 100 % allowing the stable expression of let-7a in nearly all cells. Over expression of let-7a was confirmed by quantitative Real-time PCR analysis. (2) SGC-7901 cells overexpressing let-7a grew slowly compared to SGC-7901 parental cells or cell transfected with empty vector.(P<0.01). (3)The adhesion ability of SGC-7901 cells overexpressing let-7a reduced when compared to their parental or cell transfected with empty vector. (4) Cell invasion assays indicated that let-7a can decrease the capability of SGC-7901. (5)Flow cytometry based cell cycle assays showed that overexpression of let-7a can arrest SGC-7901 cells (P< 0.01).Conclusion The use of lentiviral vectors allowed the efficient transfection of let-7a in nearly 100 % of target cells. Overxpression of the let-7a resulted in a decrease of cell proliferation, adhesion, invasion and arrest cell cycle progression in SGC-7901 cells. It suggests that as a novel tumor suppressor gene let-7a can effectively inhibit gastric carcinoma.Part 4 Lentiviral vector-mediated up-regulation of let-7a therapy for human gastric carcinoma xenogeneic graft in nude mouse subcutaneousObjective By forming subcutaneous xenograft tumors in nude mouse to observe affection of let-7a on human gastric carcinoma.Method Gastric carcinoma cells SGC-7901 stably overexpressing let-7a(SGC-7901/let-7a) were injected in nude mouse subcutaneous. SGC-7901/EV and parental SGC-7901 cells were used as the controls. After 6 weeks of observation, the weight and value of xenografts among the three groups were surveyed.Result The xenografts were successfully formed in the three groups. On the 42th day after inoculation all the mice were sacrificed. The tumor masses were weighted and their volumes were measured. The average tumor weight of SGC-7901/let-7a, SGC-7901/NS and parental SGC-7901 were 1250±20mg,1330±32mg,1350±31mg respectively. Their average tumor volume were 1100±50mm3,1300±68mm3,1340± 55mm3.The tumor weight and volume of SGC-7901/let-7a were significantly lower than SGC-7901/NS and parental SGC-7901(P< 0.05),while there is no significant difference between the laters (P> 0.05).Conclusion Overexpression of let-7a can inhibit tumor growth of transplanted human gastric cancer in nude mice and has a curative effect on gastric carcinoma as genetic therapy.Part 5 Analysis of different proteins expression in SGC-7901,SGC-7901/EV and SGC-7901/let-7a cells by proteomicsObjective In order to better understand the basic mechanisms of let-7a influncing gastric carcinoma, the different protein expression were investigated in SGC-7901,SGC-7901/EV and SGC-7901/let-7a cells.Mathods The total proteins of SGC-7901,SGC-7901/EV and SGC-7901/let-7a cells were separated by immobilized pH gradient (IPG)-based two-dimensional gel electrophorsis (2-DE),the 2-DE maps were established for SGC-7901,SGC-7901/EV and SGC-7901/let-7a cells respectively. The different expression protein spots were analyzed with Image Master 2D Elite4.01 analysis software. The peptide mass fingerprinting of different expressed proteins were verified by MALDI-TOF-MS. The function of the different expressed proteins were identified through swiss-port database searching. Western blot analysis was used to confirm the differential expression levels of the Partial Proteins.Rsult We established the 2-DE maps of SGC-7901,SGC-7901/EV and SGC-7901/let-7a cells respectively.79 different protein spots were found by image software. Among the total,6 overexpressed proteins and 4 underexpressed proteins of SGC-7901/let-7a cells were recognized with MALDI-TOF MS. The biological information of these proteins was identified through Swiss-port database search. The overexpressed proteins including Antioxidant protein 2, Insulin-like growth factor binding protein 2, Protein disulfide isomerase A2, C-1-tetrahydrofolate synthase, Cyclin-dependent kinase inhibitorl and Rho-GTPase activating protein 4 so on. The underexpressed proteins were consisted of S-phase kinase-associated protein 2, Platelet membrane glycoprotein, Fibronectin and Cksl protein. Some of those proteins were related to cell cycle control, cellular metabolism invasion and metastasis of tumor. The differential expression levels of the Partial Proteins (Cyclin-dependent kinase inhibitorl, S-phase kinase-associated protein 2, Fibronectin) were verified by western blot analysis.Conclusion Many different expressed proteins among SGC-7901,SGC-7901/EV and SGC-7901/let-7a cells were identified. These different expressed proteins perhaps have closed relation to the function of let-7a in genesis and development of gastric carcinoma through influence of the cell cycle control, invasion, metastasis and cellular metabolism. Investigating these proteins is helpful to explore the basic mechanisms of let-7a in gastric carcinoma.We could better understand the function of let-7a in gastric carcinoma and enrich its theory.