| Objective: DNA methylation occurs in the early stage of tumorigenesis, CpGislands aberrant methylation earlier than the malignant cell proliferation, so genepromoter region hypomethylation or hypermethylation is a highly sensitive tumorbiomarker in early stage of tumorigenesis. Detection of tumor-associated genemethylation status can provide extremely important information in the early diagnosis,effect observation and prognosis judgment of the tumor. Kazak Esophageal cancer (EC)is one of the highest prevalence disease in Xinjiang. Based on there are no systematicearly warning indicator before tumorigenesis, the project intends to screen the digestivetract tumors early related genes, research the different expression of oncogenes and tumorsuppressor genes in EC tissue, then explore their promoter CpG island methylation state,and further study the change of promoter methylation affect on gene expression andbiological functions. All these can provide a theoretical basis in the early diagnosis, theearly warning indicators establishment, as well as tumor treatment and prognosis inXinjiang Kazak’s EC. Methods:1) Detection of the mRNA expression of survivin, cdc42,p16and FHIT in EC: Collected48pairs specimens in the Kazak EC tissues and distalnon-cancerous tissue, extracted the total RNA by Trizol, used reverse transcription obtaincDNA, amplified by PCR technique, then detected the expression of the four genes in ECcancer tissues and distal non-cancerous tissues;2) Detection of the methylation state ofsurvivin, cdc42, p16and FHIT genes CpG island in promoter region in EC: UsingMethprimer software to got survivin, cdc42, p16and FHIT genes CpG islands inpromoter region, and design the methylated primers and normal primers. Modificated thegenomic DNA by sodium bisulfite, and then used methylation-specific PCR (MSP) todetect the survivin and cdc42methylation status, used high-resolution melting curve(HRM)to detect p16and FHIT gene promoter CpG island methylation level. Based onthese, further explore the relationship of the mRNA expression and the methylation status or level;3) Construction of recombinant vector: Using Primer5design primers, thenamplified survivin, cdc42gene promoter CpG island, and one random sequence withoutCpG islands by PCR, divided these sequeces into two groups, one group are methylatedmodification in vitro, another group are not modified. Then connected these sixfragments with the pCAT3-Enhancer vector, constructed the recombinants which containthe methylated or unmethylated CpG island-pCAT3-Enhancer vector. Then therecombinants which contain methylated CpGisland-pCAT3-Enhancer vector andpCAT3-Enhancer empty vector were transformed into E.coli ER1793(Mcr-, Mrr-, whichdefect methylase), the recombinants which contain unmethylatedCpGisland-pCAT3-Enhancer vector and pCAT3-Enhancer empty vector weretransformed into E.coli JM109which can maintain methylation status. Then selectedpositive recombinants, identificated by PCR and sequencing, and detected themethylation degree of the three CpG islands which in methylated CpG island-pCAT3-Enhancer vector recombinants;4) The eight recombinants were transfected into Eca109:The recombinants were transfected into Eca109cell lines respectively, then cultured at37℃for48hours;5) Detection of Chloramphenicol acetyl enzyme (CAT) activity: the cellswere harvested after48hours, then extracted the CAT gene products by disrupt the cell,after label it with the14C chloramphenicol, determined the CAT activity by thin layerchromatography(TLC), analyzed the CAT enzyme activity, determined the impact of genepromoter CpG island methylation on gene expression. Results:1) In the48pairs of ECtissues, survivin，cdc42，p16，FHIT positive expression ratio are87.50%,97.92%,75.00%,83.33%respectively in cancer tissues, positive expression ratio are58.33%,100.00%,77.08%,85.42%respectively in distal non-cancerous tissues. Survivin mRNA expressionlevel and positive ratio in cancer tissues significantly higher than distal non-canceroustissues, the difference has statistically significant (P＜0.05). The positive expression ratioand mRNA expression levels of cdc42, p16, FHIT have no difference between cancertissues and distal non-cancerous tissues(P＞0.05).2) In the48pairs of EC tissues,survivin mRNA expression has a positive correlation with lymph node metastasis, clinicalstage and tumor invasion(r=0.379,0.488,0.393; P=0.017,0.002,0.019), and has nocorrelation with the degree of differentiation(P＞0.05). Cdc42mRNA expression haspositive correlation with the clinical stage and tumor differentiation(r=0.452, r=0.325；P=0.003, P=0.034), and has no correlation with lymph node metastasis and tumorinvasion degree(P＞0.05). P16mRNA expression has no correlation with the clinicalstage, the degree of differentiation, lymph node metastasis and tumor invasion degree(P ＞0.05). FHIT mRNA expression positive correlated with clinical stage (r=0.338,P=0.035), but has no correlation with the degree of differentiation, lymph nodemetastasis and invasion degree(P＞0.05).3) In20paired cases of EC,70%of survivingene promoter CpG islands are heterozygous methylated in the cancer tissues,75%of thesurvivin gene promoter region CpG islands are heterozygous methylated in distalnon-cancerous tissues, there are no significant difference between the cancerous tissuesand distal non-cancerous tissues in methylation status(P＞0.05). Four cases of highmethylation in cancer tissues, which corresponding to survivin mRNA expression levelswere higher than distal non-cancerous tissues, four cases of high methylation in distalnon-cancerous tissues, which corresponding to negative mRNA expression.4) In20paired cases of EC,90%of cdc42promoter CpG islands show methylation status incancer tissues,80%of the cdc42promoter CpG islands show the methylation status indistal non-cancerous tissues, there were no significant difference in the cancer and distalnon-cancerous tissues methylation status(P＞0.05). Compare to the high expression ofcdc42mRNA, cdc42promoter CpG island methylation status has no relation to cdc42mRNA levels in cancer tissues and distal non-cancerous tissues.5) In20paired cases ofEC,100%p16promoter CpG islands show methylated in cancer tissues and distalnon-cancerous tissues. The methylation level has correlation with clinical stage (P＜0.05),but has no correlation with lymph node metastasis, tumor invasion tissues anddifferentiation level(P＞0.05). Compare to the expression of its mRNA, there were norelation between high methylation level and expression level.6) In20paired cases of EC,35%FHIT promoter CpG islands show methylated in cancer tissues and30%in distalnon-cancerous tissues. The methylation level has no correlation with clinical stage, lymphnode metastasis, tumor invasion tissues and differentiation level(P＞0.05). Compare tothe expression of its mRNA, perhaps there are little effect of low methylation level onexpression level.7) The reporter gene CAT enzyme activity in methylated null vector wassignificantly lower than non-methylated null vector after transfected into EC cell line.8)The CAT activity were both decrease to zero both in methylated and unmethlatedRandom region-pCAT3-Enhancer recombinants after transfected into EC cell line.9)TheCAT activity were both decrease to zero after methylated and unmethylated survivinpromoter region CpG island-pCAT3-Enhancer recombinant transfected into EC cell line.10) The CAT activity in methylated cdc42promoter region CpG island-pCAT3-Enhancerrecombinant was significantly lower than unmethylated recombinants after transfectedinto EC cell line. Conclusion:1) High expression of survivin suggests it was involved in the apoptosis and cell cycle regulation in the occurrence and development of EC.4casesin distal non-cancerous tissues have negative expression corresponding to promotermethylated, which means methylated promoter has the ability of regulation on theexpression. The CAT activity were both decrease to zero after methylated andunmethylated survivin promoter region CpG island-pCAT3-Enhancer recombinanttransfected into EC cell line. All these notes survivin promoter methylation in cancertissue has no relation to the gene expression, which may be involved in other mechanismin EC development.2) Cdc42high expression and high positive ratio both in cancer anddistal non-cancerous tissues tips the expression is not a mechanism in EC development.Cdc42promoter CpG islands are both methylated in cancer and distal non-canceroustissues, which has a clue that cdc42promoter region CpG island methylation has norelation to the high gene expression. Methylated CpG island of cdc42, which in EC cellline Eca109, can reduces the expression of the downstream reporter gene CAT activity,rather than the unmethyled can maintain CAT high activity. This tips cdc42promoterCpG island methylation has the ability to regulate the expression in Eca109. But in KazakEC, its expression was not affected by the promoter region methylation, which suggestedthere are may have more regulatory mechanisms involved in gene expression in thedevelopment of EC.3) P16mRNA in cancer tissues and distal non-cancerous tissues arehighly expressed. P16promoter region CpG island methylation ratio is100%. These notep16expression has no relation to the development of EC.4) FHIT mRNA in cancertissues and distal non-cancerous tissues are highly expressed, this means FHIT are notparticipates in the occurrence of the tumor. The low level and ratio of FHIT promoterCpG island methylation perhaps has no effect on the gene expression. |
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