Study On The Growth Of Esophageal Carcinoma Cells And Expression Of The Related Genes Through Changing Modification Of Methylation And Acetylation

Objective: 1) In order to lay the pre-experimental basis for clinical treatment of esophageal cancer in the future, set up epigenetic reverse modification system in esophageal cancer cell lines, confirm it can produce influnce on cell morphology, cell cycle and cell apoptosis in different experimental groups. 2) As start point in methylation and acetylation modification from the view of epigenetics, observe the changes of characteristic of methylation and acetylation after the effect of the drugs.Further study the results of combination function from methylation and acetylation, preliminary research methylation and acetylation reverse modification in different types genes and combined modification influnceing on single modification accompanied by the incidence of esophageal cancer. 3) Changing genetic modification in genes, the abnormal expression of genes and the occurrence of esophageal cancer are cognate events, detect the change of genes in transcriptionlevel and translation level. Investigate to transform genetic modification in different types of genes will determine expression of genes, which can provide newmolecular detection indexs for early discovery and treatment esophageal cancer. Methods: 1) Use human esophageal squamous cell cancer as research models.Set the concentration and time of drugs action(TSA 0.08μMol/L, 0.4μMol/L, 2μMol/L, 10μMol/L and 50μMol/L. 5-Aza-d C 0.2μMol/L, 1μMol/L, 5μMol/L, 25μMol/L and 125μMol/L, the action time 12h、24h and 48h). Apply CCK-8 detection Kit to draw the groeth curve and calculate cells inhibition rate, then choose the optional concentration and time of drugs.Set the experimental groups, normal control group, TSA group, 5-Aza-d C and combination group and adopt cell morphology, cell cycle and cell apoptosis to verify whether the drugs can play the role before analysing the changing of all indexes in differentgroups. 2) According to the results of previous study in our team(screening of differentially expressed genes in esophageal cancer), now choose PTEN、Survivin、IKKα and IKKβ as target genes and detect them from epigenetics.①Detection of methylation: find 2000 bp base sequence of upstream in target gene’s promoter before using http: //cpgislands.usc.edu/ and http: //www.urogene.org/cgi-bin/methrimer/mehp Rimer. cgi software to observe the CG distribution in sequence and forecast of methylation sites.Select the basesection which is rich in CG island as the subjects through above results and design its methylation primers.Extraction of genomic DNA in experiment groups and decoration it with BSP. Look the modified DNA as the template for PCR amplification and make sequencing of TA clones.Draw the black and white scatter diagram after comparison the sequencing results and the original sequence and calculate methylation frequency, analyse and compare of characteristic of changes. ②Detection of acetylation: extract HDAC coming from sample cell nucleus, setting up standar curve by HDAC activity detection kit operation tips.Use enzyme mark instrument to obtain photometric numerical, calculate the relative content of HDAC by the formula.Each group was done by statistical analysis and compared the changes of characteristics. 3) The level of transcription and translation of results for verify: Real-time PCR Detect the expression of m RNA in target genes of each experimental group.Western Blot inspect the the expression of protein in target genes of each experimental group.Combine thesecond part of the results and make the relativity analysis for them to survey the function of methylation, acetylation and methylation adding acetylation in target genes which will influnce on expression and the relationship of mutual regulation. Results: 1) The results of different cytologic indexes: ①The effective concentration of TSA and 5-Aza-d C on Eca109 cells is 2μMol/Land 5μMol/L.There is a appearance of inhibited growth phenomenon that the drug make the influnce on cells.The TSA effect of the drug is more significant(P<0.05). ②The change of the cells ultrastructure earlier than its morphology change under microscope.Combination therapy for damage cell ultrastructure was more obvious than using TSA alone or 5-Aza-d C. ③The Single or combination action can promote cell apoptosis, but TSA and combined medication of promoting apoptosis show significant effecting(P<0.05). The sensitivity of the different drugs block on KYSE150 cells was weaker than that of Eca109 cell.④The Single or combination action effect on cells cycle and cells were arrested in different time phase in cell cycles. TSA could block Eca109 cells in G0/G1 phase and G2/M phase, block KYSE150 cells in G2/M phase. 5-Aza-d C block cells in S phase, the combination group block the cells in G2/M phase. 2)Detection of methylation and acetylation: ①The methylation of tumor suppressor PTEN in the promoter region show high frequency. The methylation frequency show decreasing after using the 5-Aza-d C(P<0.01), combined to TSA of the methylation frequency show decreasing significantly(P < 0.01). Oncogene Survivin promoter region show low frequency of methylation. The methylation frequency did not significantly decrease after using drug intervention. The methylation frequency in promoter region of tumor metastasis gene IKK alpha and IKK beta was higher than Survivin, but less than PTEN.The methylation frequency decreased after using the 5-Aza-d C(P < 0.05), combined to TSA for demethylation frequency decreased significantly(P<0.01). ②The concentration of HDAC in different esophageal cancer was inconsistent. After the treatment of TSA can effectively reduce the content of HDAC in the nucleus. Combining with 5-Aza-d C does not appear to significantly reduce the HDAC concentration. 3) The effect on genes expression because of changing the epigenetic modification: ①Anti-oncogenes PTEN: The expression of m RNA and protein in different esophageal carcinoma is almost consistent. TSA and 5-Aza-d C is helpful to increase the expression of PTEN in esophageal carcinoma, and the combined effect of the two drugs increase the expression of genes significantly(P < 0.01). Correlation analysis showed that the methylation changes was negatively correlated with m RNA(P<0.01), same result to the protein expression(not statistically significant); the acetylation changes was negatively correlated with m RNA(not statistically significant), same result to the protein expression(P < 0.05). ② Oncogene Survivin: the expression of m RNA in different esophageal carcinoma is consistent, protein expression in the Eca109 was higher than KYSE150(P<0.05). TSA or 5-Aza-d C can respectively inhibit the high expression of Survivin in esophageal carcinoma, the combined effect of the two drugs can further inhibited its expression. Correlation analysis showed that the change of methylation and acetylation was positively related to the expression of Survivin, in which aberrant methylation and m RNA expression change is consistent(P<0.05), the rest had no statistical significance. ③Tumor metastasis gene IKKα/IKKβ: Expression of m RNA for IKKα/IKKβ in different esophageal carcinoma is consistent. Expression of IKKα protein was higher in KYSE150, the expression of IKKβ protein was higher in Eca109. TSA or 5-Aza-d C can respectively inhibit the high expression of IKKα/IKKβ in the esophageal carcinoma and the combined effect of the two drugs can further inhibit the expression(P<0.01), Correlation analysis showed that it was negatively for acetylation and the expression of IKKα(P<0.05), methylation changesand the expression of IKKβ has positive correlation trend(notstatistically significant). Conclusions: 1) There are differences between TSA and 5-Aza-d C in esophageal cancer cell growth inhibition that the effects of alone and combined treatment on the growth of esophageal cancer cells are different, which indicate that TSA led increased acetylation and 5-Aza-d C dominated lower methylation and their combined inhibit the growth of esophageal cancer cells. This provides reference for guiding clinical medication treatment in the future through epigenetic theory. 2) Represented by PTEN, Survivin and IKKα/IKKβ belongs to tumor suppressor genes, oncogenes and tumor metastasis suppressor gene, which Methylation level carcinoma are different in esophageal squamous cell. This study showed that Methylation level of the tumor suppressor gene PTEN greater than tumor metastasis suppressor gene IKKα/IKKβ greater than oncogene Survivin. In the three gene, 5-Aza-d C can effectively generate demethylation, but TSA can not significantly change the methylation status in the promoter region, TSA helps 5-Aza-d C demethylation enhancement because of combined drug therapy. 3) In different types of esophageal squamous cancer cells, the modification degree for acetylation of inconsistent reflected in different concentration of HDAC. TSA can effectively reduce the content of HDAC in cells, combined with demethylation does not increase the acetylation produced by TSA. 4) The modified result for changes epigenetic in esophageal squamous carcinoma cells leading in target genes of m RNA and protein levels are expression or inhibition. The detail is a tumor suppressor gene PTEN for lower methylation, higher acetylation increase its expression. The epigenetic modification of cancer gene Survivin after drug action is not obvious, but in the end the gene expression changes, may be regulated by the action of PTEN. Lower methylation and acetylation of IKKα can promotes the expression of the gene. Lower methylation, higher acetylation of IKKβ can promotes the expression of the gene. As the point of changing methylation and acetylation may be more involved in the regulation of PTEN and IKKα/IKKβ expression, but not for the Survivin expression.