The Effect Of ShRNA Silence HARD1 Gene On Growth Of Transplanted Tmour And Expression Of Apoptosis-related Proteins From Colorectal Adenocarcinoma In NudeMice

Objective:N acetyl transferase has important biological functions in eukaryotes, the gene of human arrest defective 1 encoding the N-terminal-alpha-acetyl transfer enzyme protein lOis the catalytic subunit of NatA. Human arrest defective 1 is widely involved in the biological process of the organism, and it can transfer acetyl coA acetyl group (-CO-CH3) to the N end of the nascent polypeptide. Human arrest defective 1 plays an important role in the regulation of cell apoptosis, which is closely related to the occurrence and development of colorectal cancer.. This study was to investigate the effect of ShRNA silencing ARD1 gene on the growth and apoptosis related protein expression of colorectal carcinoma in nude mice. And to provide a new treatment concept and treatment measures for diagnosis and treatment of colorectal cancer.Method:1. Recovery and cultivation of SW620 cell line in colorectal adenocarcinoma.2. (pENTR/U6-hARD1 interference plasmid-shRNA) hARDl construction.3. The successful construction of the plasmid pENTR/U6-hARDl-shRNA and plasmid pENTR/U6-NC were transfected into SW620 cells with liposome. Cell lines with stable over expression in the culture medium were screened by G418 (Geneticin, GA). Cell clonal integration plasmid integration interference plasmid then screened the empty cell clones, and untransfected cells were used as experimental group, negative control group and blank control group; to observe the change of cell morphology.4. Mice were randomly divided into 3 groups,12 rats in each group were selected, nude mice armpit of forelimb lateral subcutaneous injection of central point, three groups were respectively, (the pentr it /U6-hARDl-shRNA) subcutaneous injection of a stable expression of siRNA plasmid cells as the experimental group, pentr it/U6-NC SW620 subcutaneous injection of a stable expression vector plasmid cells as negative control group, subcutaneous injection of non transfected SW620 cells as the blank control group carefully kept and always observe three groups of tumor growth.5. The expression of P53 and mRNA in and Bel-2 in three groups were detected by RT-PCR.6. Using Western blotting (Western blot) to detect hARDl stable over expression of hARD1 three expression in colorectal cancer group, the different protein expression level with band and GAPDH band gray reference intensity ratio quantitative representation.7. The expression of apoptosis related protein P53 and Bcl-2 in three groups of colorectal cancer cells was detected by immunocytochemistry. SP method for immunohistochemical staining. With a semi quantitative method to determine the results of random in each sample were randomly selected 3 different HPF (each 100 cells), the calculation of p53 positive cells and bcl-2 expression in tumor cells number. The calculation of p53 and Bcl-2 protein positive cells. Positive result:Bcl-2 positive cytoplasm appeared Brown particles in the nucleus of p53, brown yellow particles for positive expression, reference Mathew grading standards:0; no expression of <50% positive cells or lighter staining is one point; more than 50% positive is two points.0～1 points recorded as negative (a),2 points positive(+).8. The proliferation of colorectal cancer cells in three groups were detected by four methyl groups (MTT).9. Application of spss 19.0 software for statistical analysis of the data, the data of non-normal distribution or homogeneity of variance, with the median (four percentile interval) said that the data of normal distribution and homogeneity of variance to mean± standard deviation. Paired samples of the two groups were paired t test or Wilcoxon test, between the two groups of independent samples using U Mann.Whitney test, P<0.05 for the difference was statistically significant.Result:1. SW620 cell lines were cultured in vitro and grew well.2. HARD1 overexpression plasmid was successfully constructed, and the stable cell lines were transfected into each group by G418.3.(1) As time went by, the tumor volume of the three groups increased gradually. In this study, three groups of 24 nude mice, in addition to the blank group, there was a dead tumor, the tumor formation rate was 95.83%. Continuous observation for three weeks after the cervical dislocation in nude mice were sacrificed and carefully isolated subcutaneous transplantation tumor, measured and recorded the tumor weight and volume (prior to each injection use vernier caliper to measure the tumor the long diameter and short diameter, and the tumor body quality. separation of subcutaneous transplantation tumor, measure and record the tumor weight and volume (before each injection use vernier caliper to measure the longest diameter of the tumor and the shortest path, and the tumor weight. Results:this experimental group body weight and tumor volume was significantly lower than that in negative control group and blank control group, experimental group and negative control group, experimental group and blank control group in nude mice transplanted tumor body weight and tumor volume were significantly different, with statistical significance (P<0.05), negative control group and blank control group in nude mice transplanted tumor body weight and tumor volume difference is not obvious and no statistical significance (P>0.05).(2) No death in nude mice two weeks before, eighteenth days control group one cases died,due to huge and serious with tumor related cachexia.4. RT-PCR. In the three groups of nude mice transplanted tumor tissue P53, mRNA Bcl-2 expression analysis, Relative expression level was obtained using 2-△△ct CT method, provisions of the blank control group was 1.00, analysis of three groups of nude mice transplanted tumor tissue p53 and bcl-2 mRNA expression, the expression level of p53 expression in the experimental group was significantly higher than that of negative control group and blank control group; and Bcl-2 protein expression was significantly lower than the negative control group and blank control group.5. Blot Western method was used to detect the expression of hARD1 protein in three groups of colorectal cancer tissues. The relative expression of hARD1 protein in three groups of nude mice transplanted tumor cells was analyzed by gray scale value analysis: The expression of hARD1 in the three groups was higher than that in the negative control group and blank control group.6. The positive expression of p53 and Bcl-2 in the three groups of xenograft tumor tissue rate in the experimental group were 87.5%(7/8),37.5%(3/8); the negative control group were 37.5%(3/8) and 50.0%(4/8); blank control group were 28.57%(2/7), 85.71%(6/7). P53 positive expression rate of the experimental group and the negative control, experimental group and blank control group, the difference was statistically significant (P<0.05), negative control group and blank control group p53 positive expression rate difference is not obvious (P>0.05). Bcl-2 positive expression rate in the experimental group and negative control group, negative control group and blank control group, the difference was not statistically significant (P>0.05); experimental group and blank control group, the difference was statistically significant (P<0.05).7. Four methyl thiazolyl tetrazolium (MTT) was detected in three groups of colorectal cancer cell proliferation. The method principle is activity of succinate dehydrogenase in mitochondria to exogenous MTT reduction for water insoluble blue purple crystal formazan (formazan for) and deposition in cells, the cells die without this feature. MTT can indirectly reflect the number of living cells. The OD value of 48h was detected in three tumor cells were removed from the nude mice after, calculate the inhibition rate. The result is:the prevalence of inhibition of the experimental group were higher than those in negative control group and blank control group (P<0.05)Conclusion:1. This study confirmed that the shRNA interference technology in the silent ARD1 gene expression levels of p53 in colorectal adenocarcinoma was significantly increased, Bcl-2 expression was often inhibited; In the protein level is to promote the expression of Bcl-2, while the impact on the expression of P53 is not obvious.2.The experimental verification of shRNA silencing ARD1 gene can suppress the subcutaneous colorectal carcinoma xenograft in nude mice; expression of tumor cells in nude mice transplanted colorectal adenocarcinoma in hARD1 mRNA at the gene level.3. The expression of apoptosis related proteins could be regulated in different degrees after shRNA silence ARD1 by this study., which can inhibit colorectal cancer cell growth and promote cell apoptosis of colorectal adenocarcinoma.