| Background:Hepatocellular carcinoma (HCC) is one of the most common malignant gastrointestinal tumors in China.Tumor molecular markers for clinical guide diagnosis,treatment and prognosis evaluation,but it could not provide HCC early diagnosis.Therefore,a new HCC gene looking for cancer genes is clearly great significance from the genetic level study of the cancer mechanism.Many studies have reported about microRNAs and HCC related genes,miRNA expression increased the role of the oncogene in HCC,but downregulation from the tumor suppressor gene is reported.Foreign scholars shudies have shown that expression of miR-10b gene in hepatocelluar carcinoma and relatively non HCC tissue is significantly higher,similar domestic literature has not yet seen the report.We used bioinformatics methods to predict target genes of miR-10b HOXD10, and real-time quantitative RT-PCR method to detect its expression in HCC cell lines based on primary liver cancer tissue,and line establishment process has not changed its biological characteristics.HepG2cells in nude medium-range poor tumorigenicity of SMMC-7721allogeneic transplant success rate is higher Huh7. and HepG2, contain no HBV. With the development of molecular biology techniques, the researchers designed to transform the original cell lines transfected with the virus through artificial or genes into cells, thereby establishing both the specific biological characteristics of hepatocellular carcinoma cell line containing the target gene. HOXD10gene located on chromosome2, play an important role in embryonic development and cell differentiation process. HOXD10gene and tumor research is currently at a preliminary stage, mainly in terms of breast cancer, blood disease, esophageal cancer, glioma, studies have reported. In China is still no reports of HOXD10gene and liver cancer. This study on the three hepatoma cell lines Huh7, HepG2, SMMC27using SYBR GREEN Real-time RT-PCR), technology, and established a new method detection HOXD10mRNA expression, and analysis of the HOXD10gene liver cancer cells Huh7, HepG2SMMC27, and expression.The PCR amplification process in Real-time RT-PCR, real-time detection and continuous analysis of the amplified fluorescence signal, with the reaction, monitoring the fluorescence signal changes can be drawn into a wire,. Used to define the sample threshold cycle number (Ct). In this study, the method of choice for fluorescence embedded in legitimate, the use of SYBR Green fluorescence dye, a number of housekeeping genes (such as beta-actin,3-phosphate dehydrogenase, GAPDH,18sRNA gene etc.). Relative quantification is mainly used in mRNA expression parsing, we use the melting curve analysis to determine the specificity of the amplification products.Transfection technology is exogenous molecules such as DNA/RNA into eukaryotic cells technology。A class of transient transfection and a class of stable transfection. Former outside source of DNA/RNA is not integrated into the host chromosome, so a host cell can exist in multiple copy number, resulting in a high level of expression, but usually only lasted a few days, and used for the analysis of the promoter and other regulatory elements.Matrigel is extracted from the EHS sarcoma matrix components containing LN, The membrane structure can not freely pass through cell must secrete hydrolytic enzymes and deformation movement to get through this shop Matrigel membrane, which is similar to the in vivo situation. Transwell chamber membrane invasion of reconstruction matrix analysis.Aim Objective To investigate the miR-10b and potential target genes HOXD10expression in hepatocellular carcinoma three cell lines Huh7, of HepG2SMMC-7721features research miRNA10b by inhibiting the translation or degradation of mRNA to regulate expression of target genes HOXD10and HepG2invasive function.Method (1) cultured three cell lines Huh7, HepG2, SMMC-7721.(2) by Trizol extraction of total RNA extracted RNA using M-MLV reverse transcriptase kit reverse transcriptase first strand cDNA synthesis.(3)With the bioinformatic analysis and prediction of miR-10b of potential target genes HOXD10the the GeneBank retrieval the human18srRNA and HOXD10gene full sequence, oligo6.0primer design software the to design18srRNA and HOXD10gene primers.(4) cDNA as a template, the18srRNA and HOXD10gene fragments were amplified by PCR reaction, standard sample preparation relative quantitative reverse transcription of cDNA as a template for polymerase chain reaction (RT-PcR) detection of three hepatoma cell lines in HOXD10expression screening high expression cell lines.(5) the establishment of the normal control group (cell group) negative control (NC) group and experimental group (miR-10b) the instantaneous liposome2000transfected cells, overexpression mir10b, and genes HOXD10and protein levels by QPCR and Western blot analysis-line The analysis verified simultaneously high expression of miR-10b on hepatoma cells in vitro cell invasion assay.Results Huh and SSMC-7721HOXD10expression is low, the melting curve impure peaks, HepG2melting curve showed a single peak, high expression levels. PCR analysis of HepG2-HOXD102-AACt value is significantly higher than228.71Huh and SSMC-7721cells. Western Blot protein expression is also elevated in HepG2cells. Detected by high expression of MiR-10b hepatoma cell line HepG2HOXD10not significantly higher at the gene level but the Western blot protein expression was reduced. High expression of miR-10b hepatoma cell HepG invasion.Conclusion HOXD10expression in HepG2cells increased, consider the gene might be the development of liver cancer inhibitor, confirmed the HOXD10subject to the regulation of miR-10b; MiR-10b overexpression and hepatocellular carcinoma (HCC) invasion of closely related can prompt us to promote invasion and metastasis potential target gene silencing induced inhibition of the target genes of the invasion and metastasis by inducing high expression in clinically to inhibit tumor invasion and metastasis of purpose. As well as further study of hepatoma cells in a variety of malignant features, in-depth study of its mechanism of action, and provide experimental basis for a new target for diagnosis and treatment of miR-10b as hepatocellular carcinoma. |
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