The Effect Of RNA Editing Enzymes In Induced Differentiation Of Colorectal Carcinoma Cells

Colorectal carcinoma is one of the most common malignant tumors among human beings.In the world,it is the fourth in the common malignant tumors. It is the second main reason of cancer causing death in western countries. The annually morbidity is about 1,023,000 cases , 529,000 men died with colorectal carcinoma annually. The tendency of morbidity is increasing, among the humen beings that over 40 years old ,the morbidity is obviously increasing , adds one times annually, it displays a persistent exponential growth.It is a result of multi-procedure,polygene heterogeny or activated from the normal colorectal enterocyte to colorectal cancer cells,the pathogenetic related gene of colorectal cancer transformed in different component element in the course of carcinogensis,it included that the activation of oncogene,inactivation of anti-oncogene,the germ line mutateon and methylation of mismatch repair gene.With the development of molecular biology and genetic technique, gene treatment had gained some results,but it is not satisfactory in the total consequence. The human being gradually realized that it cann't completely suppressed the growth of cancer with tradition technique.at present,it is not possible that the gene treatment was clinically applicated in wide-bound.It demonstrated that:malignant tumor is the result of cell proliferation and disdifferentiation,although it is tumor,but it has a ability that it controls normal growth and differentiation.Therefore,with the certain factor influence,tumor cells may change,and alter toward normal cells,which is induced-differentiation to tumor cells,and it has been the new hot field of colorectal carcinoma research.RNA editing is a biotic basic-phenomenon,and a physiological course which retains normal metabolic activity.It is one of important mechanism which creates biomolecule versatility and complexity.The development of some disease concerned with RNA editing dysfunction. The relation of RNA editing dysfunction and tumor were more and more payed close attention by researchers.At present,it has been indicated that RNA editing enzymes may educe important effect.Purpose: To investigate the expression of RNA editing in colorectal carcinoma tissue and cells ,and significance in proliferation and differentiation, discuss the effect and mechanism of phenylacetate induced differentiation.Methods: Use MTT colorimetry to investigate the inhibitory action of phenylacetate to the proliferation of colorectal carcinoma cell HCT-8. Changes of HCT-8 cell cycle phases with different concentration and action time of phenylacetate were detected quantificationaly by floating cytometry. The morphological changes of HCT-8 were detected by phase contrast microscope. The expression of ADAR mRNA in HCT-8 and colorectal carcinoma specimen with and without phenylacetate were detected by RT-PCR.Results: (1),Use MTT colorimetry to investigate the inhibitory action of phenylacetate to the proliferation of colorectal carcinoma cell HCT-8. The proliferation inhibitory rate were that:24 hours:4.61%,11.72%,41.09%,65.82%,88.40%; 48 hours:7.55%,27.00%,58.36%,77.02%,88.68%; 72 hours:22.65%,41.36%,89.07%,93.32%,94.45%.The results indicated that the inhibitory action depended on both action time and dosage. (2),Use floating cytometry to detect changes of HCT-8 cell cycle phases with different concentration and action time of phenylacetate such as 24h and 72h. The results indicated that after using of phenylacetate, the proliferation of HCT-8 cells were inhibited in phase G0/G1, abundant cells accumulated in the end stage of phase S, DNA synthesis could not be accomplished entirely. (3),The morphological changes of colorectal carcinoma cell HCT-8 without and with different concentration of phenylacetate for 24h,72h were detected by phase contrast microscope. The results indicated that phenylacetate restrained the proliferation of HCT-8, elevated the differentiation stage,and made tumor cells transform to normal cells. (4),The expression of RNA editing enzymes mRNA in HCT-8 cells,human colorectal carcinoma specimen and tissue beyond carcinoma were detected by RT-PCR.The results indicated that ADAR1 had not visible expression, however ADAR2 had a positive expression in HCT-8 cells,human colorectal carcinoma specimen and tissue beyond carcinoma. (5),The expression changes of ADAR2mRNA in HCT-8 cells after use of phenylacetate with different concentration and action times were detected by RT-PCR. The gray scale rate of without and with PA in 1.0mM24h,1.0mM72h, 2.0mM24h,2.0mM72h were 1.668±0.060, 1.141±0.030,1.058±0.036, 1.075±0.034,0.894±0.026. The results indicated that the expression of ADAR2 gradually weakened with the increasing of phenylacetate on concentration and action times.Conclusions: (1),The inhibitory action of phenylacetate to the cell HCT-8 depended on both action time and dosage. (2),Through the detection of cell cycle, it indicated that the regulation of cell proliferation and differentiation of HCT-8 may through the effect on HCT-8 cells RNA functional expression, rather than in-specific inhibition to RNA expression. The action point may be phase G1. (3),High-expression of ADAR2mRNA may related to the occurrence and evolution of colorectal carcinoma. (4),ADAR2 mRNA played a vital role of regulation in the process of colorectal carcinoma cells growth and differentiation. Phenylacetate could regulate the proliferation and differentiation of colorectal carcinoma cells through the regulation of ADAR2mRNA expression.