| Objective:hARDl reprsents is an important acetyltransferase witn both the protein of N-terminus and ε protein acetylation activity,it is closely related with cell differentiation, DNA repair and maintain the stability of the genes.Many studies have found that hARD1 can expresse in many types of tumor tissue and other diseases in humans,which has become a research hotspot in recent years, but the research is still less about the colorectal cancer cell factor after the silencing of ARD1 by shRNA.In this study,we constructed the nude mice xenograft model of human colorectal carcinoma SW620 cell after the silencing of ARD1 by shRNA,which detected the expression levels of VEGF, bcl-2 and TNF-a protein in the tumor tissue and analysised the correlation between three protein with colorectal cancer to further explanation of the pathogenesis of colorectal cancer, while providing potential new targets for the clinical treatment of colorectal cancer.Methods:1. Rcovery and cultivation of SW620 colorectal cancer cell strains.2.Construction of hARD1 interference plasmid (pENTRTM/U6-hARDl-shRNA).3. The resuscitation pENTRTM/U6-hARD1-shRNA and pENTRTM/U6-NC-shRNA were transfected into SW620 cells. Screening resistant cells from the culture medium by G418, which is extended culture. cell clones of interference plasmid as experimental group, cell clones of unrelated sequence plasmid as the control group.4. Construction the nude mice xenograft model of colorectal carcinoma:(according to the preliminary experimental results) there was no significant difference between the negative control group and the blank control group in tumor growth, the nude mice were randomly divided into two groups (8 rats in each group, the two groups were: subcutaneous stably expressing pENTRTM/U6-hARDl-shRNA of SW620 cells in the experimental group, subcutaneous stably expressing pENTR TM/U6-NC of SW620 cells as the control group, To observe the growth status of transplanted tmour.5. Using the western blot to examine each group of transplanted tumor tissues’ hARD1 expression.6. Use immunohistochemistry to detect the levels of expression in colorectal cancer cell transplanted tumor tissue s’VEGF, bcl-2 and TNF-a.7. The SPSS 17.0 statistical software package was used for all analyses,measurement data comparing between the two groups using t test or rank sum test, with mean ± standard deviation (x ± s); count data using chi-square test.The level of significant difference is a=0.05. the relationship were analysised between the three proteins by Spearman rank correlation analysis.Results:1. SW620 were cultured successfully and good growth state in vitro.The recombinant plasmid(pENTRTM/U6-hARDl-shRNA) was constructed successfully, G418 selects the stable expression cell lines after it was transfected successfully into human colorectal adeno carcinoma cells-SW620. Inverted fluorescence microscope observation of experimental group and the control group, and most of the cells expressed green fluorescent, indicating that the experimental group the pENTRTM/U6-hARDl-shRNA and control group the pENTR TM/U6-NC were transferred SW620 human colorectal adenocarcinoma cell lines, and enrichment to the stable expression cells.2. Effect of silence hARD1 by shRNA on growth of transplanted tmour from colorectal adenocarcinoma cells in nude mice All of the xenografts have a good growth status in nude mice, Tumor formation rate of 2 groups were 100%, the nude mice were killed by cervical dislocation after 25d, weighting those tumor,we found that it’s volume and weight in the experimental group is significantly less than the other group. the difference was statistically significant (P<0.05).3. Using the western blot to examine each group of transplanted tumor tissues’ hARD1 expression. With GAPDH as the reference protein, using Image J software analysis system conduct quantitative analysis Western strip.The result showed that the two groups of transfected tumor tissues have the expression of hARDl, while the experimental group hARD1 expression was significantly decreased, which showed that the introduction of hARDl-shRNA down regulated the expression of hARDl. The expression of hARD1 in the experimental group compared with the control group has statistically significant (p<0.05).4.The expression of VEGF, Bcl-2 and TNF-a were examined in two groups of colorectal carcinoma xenograft tissue by immunohistochemistry VEGF was expressed mainly in the cytoplasm, the expression of VEGF in the experimental group was significantly lower than the control group, the difference was statistically significant between the two groups (P<0.05). bcl-2 protein was expressed in the nucleus and cytoplasm, the expression rate of of bcl-2 in control group was higher than that in the experimental group, the difference was no statistically significant (P> 0.05). TNF-a was expressed in the cytoplasm, the expression of TNF-a in the experimental group was significantly lower than the control group,the difference was statistically significant(P<0.05).5. The correlation of expression of VEGF, bcl-2 and TNF-a in two groups of colorectal adenocarcinoma xenograft tumor. This study analyzed the correlation of expression of VEGF, bcl-2 and TNF-a in two groups of colorectal adenocarcinoma xenograft tumor by the Spearman rank correlation analysis.The results showed that the expression of between VEGF and bcl-2 was positive correlation (r=0.683, P<0.01); the expression of between VEGF and TNF-a was positive correlation (r=0.775, P<0.01); The expression of bcl-2 and TNF-a was positive correlation with the expression of colorectal cancer cell transplantation tumor (r=0.882, P<0.01)Conclusion:1. The shRNA silence ARD1 can inhibit the growth of transplanted tumor of colorectal adenocarcinoma in nude mice.2. The shRNA silence ARD1 can affect the expression of VEGF and TNF-a,but it dose not affect the expression of bcl-2.3. VEGF, Bcl-2 and TNF-a were closely related with the occurrence of colorectal cancer cell transplantation tumor. the expression of between VEGF and bcl-2 was positive correlation;the expression of between VEGF and TNF-a was positive correlation;the expression of bcl-2 and TNF-a was positive correlation. It provides a potential target for the further study of the clinical treatment of colorectal cancer. |
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