Impact On Human Hepatocellular Carcinoma And Cirrhosis Cell Strain Xenografts In Nude Mice Growth Gene Expression By RNA Interference Dlk1

Hepatocellular carcinoma (HCC) is the most common malignant tumors, the incidence of occult, mostly advanced when diagnosed, early diagnosis and early treatment of clinical treatment is extremely important. Molecular mechanisms of liver cancer has not yet been understood. dlkl gene as in recent years, newly discovered genes associated with liver cancer has aroused widespread concern. However, dlkl gene in the development of liver cancer in the role is not clear. Therefore, we use the expression of hepatocellular carcinoma and cirrhosis in two cell lines and inhibition studies dlkl gene expression after the two tumor cell lines grown in influence, so as to explore the role of development dlkl gene occur in liver cancer.Chapter I Construction of shRNA-dlkl plasmid expression vectorObjective:To construct shRNA expression vectors of human dlkl gene.Methods:Four groups of oligonucleotides for the template-dlkl gene sequence, a plasmid ligated with a vector after annealing pGPU6/GFP/Neo vector, which was then restrictive enzyme digested and sequenced.Results:DNA sequencing analysis showed that RNA interference targeting dlkl plasmid vector was constructed successfully.Conclusion:We successfully constructed shRNA expression plasmid pGPU6/GFP/Neo-shRNA-dlkl against recombinant human dlkl gene. Chapter II RNA interference studies of human hepatocellular carcinoma (QGY-7703) and cancer cirrhosis (QSG-7701) cell lines dlkl gene silencing effectObjective:The interference plasmid human liver cancer and cirrhosis cancer cell lines, observing its dlkl gene silencing effect. Interference filter out the best results and the establishment of a plasmid stably transfected cell lines.Methods:1. Fluorescence microscopy of green fluorescent protein.2. Interference plasmids were transfected liver cancer and cirrhosis cancer cell lines. Using real-time quantitative PCR and Western blot analysis to detect dlkl mRNA and protein expression.3. Interfere with the efficiency of the highest expression plasmid used to create stably transfected cell lines. Meanwhile expression plasmids used as a negative control NC stably transfected cell lines. After G418selection, the two cell lines were established two stably transfected cell lines, called QGY-7703-522, QGY-7703-NC and QSG-7701-522, QSG-7701-NC.4. Using real-time quantitative PCR and Western Blot to detect changes in the expression of stably transfected cell lines and protein dlkl mRNA again to confirm the effect of stably transfected cell lines interfere with gene expression.Results:1. After interference plasmid hepatocellular cirrhosis and carcinoma cell lines, real-time PCR analysis showed that, shRNA-522, shRNA-1052, shRNA-1171and shRNA-1233interference plasmid expression of the target gene mRNA are two kinds of cells inhibited, in both cell interference plasmid shRNA-522inhibition are the most obvious. Inhibition was69%in QGY-7703cells, the inhibition was72%in QSG-7701cells. Compared with the negative control group, the inhibition rate was significantly (P<0.05); Western blot method to detect the protein expression levels in cells display, shRNA-522, shRNA-1052, shRNA-1171and shRNA-1233interference of the two plasmids kinds of cells inhibited the expression of target genes, which interfere with plasmid shRNA-522inhibition of the most significant inhibition rate QGY-7703cells was77%in the QSG-7701cells was80%, and the negative control group and blank compared inhibition rate (P<0.05). Visible shRNA-522interference suppression of gene expression plasmid for dlkl most obvious. 2. Each cell lines were screened by expressing shRNA-522and shRNA-NC plasmid stably transfected cell lines, named QGY-7703-522, QGY-7703-NC, QSG-7701-522, QSG-7701-NC.3. Real-time PCR and Western blot detected again on stable cell lines showed that cell lines stably transfected with the negative control group and compared in QGY-7703cells interfere with the efficiency of83%, in QSG-7701cells interfere with efficiency of86%.Conclusion:1. shRNA-522can inhibit the expression plasmid interference in human hepatoma cell lines and cancer cirrhosis dlkl mRNA and protein from the.2. successfully established stably transfected cell lines. Chapter Ⅲ Impact on the biological behavior of hepatocellular carcinoma strains (QGY-7703) and cancer cirrhosis (QSG-7701) cells, expression of RNA interference dlklObjective:To study dlklshRNA cirrhosis of the liver and cancer cell lines in vitro proliferation, apoptosis, migration and invasion.Methods:Successfully constructed stably transfected cell lines QGY-7703-522, QSG-7701-522as the research object to QGY-7703-NC, QSG-7701-NC and non-transfected cells were used as control. CCK8Law draw cell growth curves growth differences, changes in cell scratch assay, cell migration assay, Transwell chamber detected in vitro invasiveness.Results:QGY-7703cell lines not transfected cells, no significant difference between the indicators of negative control group cells. Cells compared with the control group, experimental group QGY-7703-522cell growth was slow, the third day from the growth rate was lower than the other two groups, more apparent to the logarithmic growth phase. Cloning efficiency QGY-7703-522cells was significantly lower than the control group (P<0.05). Cells QGY-7703-522cells through Matrigel membrane was significantly lower than the control group (P<0.05). Growth rate QSG-7701cell lines and experimental cloning efficiency of cells with the negative control group and blank control group were lower (P<0.05), no difference (P negative control group and blank control group>0.05). Flow cytometry showed that the cell cycle: QGY-7703-522cell line G0/G1phase cells increased, there are differences (p<0.05) groups compared with QGY-7703-BLANK group and QGY-7703-NC; QGY-7703-BLANK group and QGY-7703-NC group compared to P>0.05; S and G2/M phase ratio lower (P<0.05), week ending QGY-7703-BLANK group and QGY-7703-NC group cells the above changes (P>0.05). QSG-7701-522cell line G0/G1phase cells increased, compared with the QSG-7701-BLANK group and QSG-7701-NC group differences p<0.05; QSG-7701-BLANK group and QSG-7701-NC compared to the group, no significant difference (p<0.05), S and G2/M phase ratio lower (P<0.05), change (P above appears QSG-7701-BLANK group and QSG-7701-NC cells the end of week>0.05). Conclusion:shRNA-522stably transfected plasmid interference in vitro growth and inhibit migration and invasion ability of liver cancer and cirrhosis of the liver cancer cell lines week. Chapter IV Experimental study of dlkl gene silencing by RNA interference in human hepatoma (QGY-7703) and cancer cirrhosis (QSG-7701) cell lines in nude miceObjective:To establish human hepatocellular carcinoma in nude mice with liver cirrhosis and cancer cell lines growing tumor model to study the effect of gene silencing dlkl two cell lines in nude mice.Methods:The experimental groups, liver cancer cell lines:QGY-7703-BLANK, QGY7703-NC, QSY-7703-522, cirrhosis cancer cell lines:QSG-7701-BLANK, QSG-7701-NC, QSG-7701-522. Nude mice were inoculated subcutaneously implanted tumor growth was observed, the measurement of the volume of the tumor tissue and tumor formation in nude mice weighed see every5days. Six weeks after mice were sacrificed and tumor weight measured by immunohistochemical detection of tumor tissue dlkl protein.Results:The rate of tumor growth in two cell lines were slow in its control group (P <0.05), when the end of the experiment experimental tumor tissue and tumor weight and volume of the corresponding control group were significantly lower (P<0.05), immunohistochemistry confirmed the experimental group DLK1protein expression in tumor tissue and the corresponding control group comparison of the two cell lines were lower (p<0.05).Conclusion:Silence dlkl can inhibit liver cancer and cirrhosis of the liver cell carcinoma week tumor growth.