慢病毒介导的RNAi沉默HMGA2基因表达对人结肠癌HCT116细胞生物学行为的影响
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摘要
HMGA2基因是HMGA基因家族（包括HMGA1和HMGA2）成员，编码含109个AA的完整蛋白产物，可通过其特有的三个AT钩结构结合到众多基因DNA上的AT富集区域，通过改变这些被调节基因DNA构象，增强它们的转录活性。大量研究显示HMGA2在多种恶性肿瘤的演进过程中起重要作用，参与调控肿瘤细胞增殖、周期、转移等生物学行为，显示出HMGA2对恶性肿瘤疾病具有重要的研究价值，同时也是一个基因治疗的良好靶点。但目前对HMGA2在结直肠癌演进过程中所起的确切作用，尚缺乏研究。RNAi通过对目的基因的表达在转录后水平上进行阻断，使其功能部分或完全丧失，现已成为一种强有力的工具被广泛应用于对基因功能及肿瘤治疗的研究中。慢病毒介导的RNAi是目前最为有效的一种沉默目的基因表达的方法，可达到高水平的沉默效率，并能维持较长时间的对目的基因的沉默影响，得到普遍的应用。本实验通过免疫组化染色方法对HMGA2在人结肠癌组织中的表达情况及其与临床病理特征间关系进行了研究，结果显示结直肠癌组织中存在着HMGA2的异常表达，并且表达程度与远处转移呈正相关，显示HMGA2可能在结直肠癌侵袭及转移过程中具有重要的调控作用。在此基础上，我们设计针对HMGA2的siRNA序列并构建HMGA2shRNA慢病毒表达载体，成功包装成病毒感染高表达HMGA2的结肠癌HCT116细胞，将HCT116细胞中HMGA2的表达沉默。通过研究沉默HMGA2表达后HCT116细胞生物学行为的改变，发现HMGA2可促进结直肠癌的增殖、调控细胞周期进程、促进肿瘤细胞的侵袭。本研究为进一步深入研究HMGA2基因在结直肠癌演进过程中作用机制及应用HMGA2作为基因治疗靶点提供理论依据。
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.
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.
引文
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