| Objective:Colorectal cancer is one of the most common malignantgastrointestinal tumors throughout the world,with the3rd incidence rate andthe2nd mortality rate in all of the malignant diseases. How to prevent themetastasis and reduce the mortality of colorectal cancer is still the focus ofcurrent research. With the development of molecular biology technology, genetherapy is becoming a new therapeutic means and brings dawn for colorectalcancer. Through importing exogenous gene DNA or RNA fragments to thetarget cells or tissues, gene therapy enhances the therapeutic gene expression orinhibits the virulence gene expression to achieve the goal of treatment.Therefore identifying the molecular regulators,which play important roles forthe progression of cancer and can be used as gene therapy targets,is a majorgoal of colorectal cancer research. HMGA2gene is a member of the highmobility group AT-hook (HMGA) family genes, and encodes a smallnonhistone chromosomal protein, which can alter chromatin architecture toregulate the transcription of an ample number of genes and plays a critical rolein several cellular biological processes including cell transformation, growth,differentiation and cycle control. Normally, HMGA2is abundantly expressedduring embryogenesis, whereas its expression is low or absent in normal adultdifferentiated tissues. Over-expression of HMGA2was also observed in manykinds of malignant tumors of epithelial origin including colorectal cancer, andwas correlated with the carcinogenesis, invasion, and metastasis, as well aspoor patient survival. The results highlighted that HMGA2protein might playan important role in the disease progression of many types of epithelialmalignant tumors. However, the exact roles of HMGA2in colorectal cancer remain unclear. RNAi is a new technology for inhibiting gene expression inrecent years.Through blocking the expression of target genes in post-transcriptional level, it can lead the targeting gene expression partly orcompletely lost. RNA interference has become a powerful tool for the study ofgene function. Lentivirus-mediated RNAi is currently the most effective meansof the interference, which can achieve highly silencing efficiency and maintaintarget gene silencing affect for a longer period of time, so it profits thefollow-up research work and gets a general application. We successfullyinhibited the expression of HMGA2in the colorectal cancer cells HCT116byusing lentivirus-mediated RNAi technology. Then we observed biologicalbehavior of HCT116cell with HMGA2knockdown by a various ofexperiments in vivo and in vitro. The results provided theoretical basis forusing the HMGA2gene as a targeting to treat colorectal cancer.Materials and Methods:1. By applying immunohistochemical methods, the expression of HMGA2protein was detected in60specimens of colorectal cancer tissue, the correlationbetween the levels of HMGA2protein expression and the clinical pathologicalfeatures were analyzed.2. By Using Real-time PCR and Westernblotting,HMGA2expressionwere detected in five colorectal cancer cells, the cells highly expressedHMGA2was screened for RNAi experiment.3. Two pairs of shRNA sequences targeting HMGA2gene were designedand synthesised. By annealing and digesting, shRNA double-stranded templatewere inserted into the lentiviral vector. After transformation of E. colicompetent cells, screening of positive colonies and amplifying operation, theplasmids were extracted for DNA sequencing. Then the correct sequencingrecombinant lentiviral vector plasmids and lentiviral packaging plasmids weretransfected into293T cells. The supernatant that contains the virus particles was concentrated, and viral particles titer was determinated by limited dilution.After virus successfully infected the colorectal cancer HCT116cells,by usingRT-PCR,Western blotting and immunocytochemical staining methods, theshRNA target sequence that had the best interference effect was screened outfor the follow-up test.4. After silencing the expression of HMGA2, CCK-8assay and colonyformation assay were used to detect cell proliferation; Flow cytometry wasused to detect phase changes in the cell cycle; Trnaswell assay was used toassess the ability of cell invasion.5. The subcutaneous tumor model of nude mice was built. Tumor cellsemergence time, tumor volume differences and tumor weight difference weredeteced, and the impact of HMGA2implanted subcutaneously in nude micewas analysised.Results:1. The results of immunohistochemistry showed HMGA2was expressedin colorectal cancer tissues, and the expression degree was associated withmetastasis.2. HMGA2expression in the five colorectal cancer cells was detected.Results showed that HMGA2is expressed in HCT116cells, SW1116cells,SW480cells and SW620cells, but not in the HCT8cells. HCT116has thehighest expressed level of HMGA2in the four cells expressed HMGA2.3. The HMGA2shRNA lentiviral expression vector was successfullyconstructed and packaged. After virus infecting HCT116cells, the shRNAsequence for best silencing effect was screened out for subsequent trials.4. Silencing HMGA2expression significantly inhibited the proliferation ofHCT116cells, and led to G1arrested. The cell invasion ability compared withnon-transfected cells and transfected negative control group was significantlyreduced. 5. In the xenografts assays in nude mice,tumor formation time of theinterference group was significantly prolonged than that of control group andnegative control group,and the tumor volumes and the tumor weights of theinterference group were significantly samller than the control group andnegative control group.Conclusion:1. HMGA2protein was expressed in colorectal cancer tissues andcorrelated with the clinical aggressiveness of tumors.2. Lentivirus-mediated RNAi can effectively silence HMGA2expressionin the colorectal cancer HCT116cells. The HMGA2plays an importantregulatory role in the proliferation, cell cycle regulation and invasion ofcolorectal cancer. |
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