A Novel Set Of DNA Methylation Markers Of Bladder Cancer And Apoptosis Mechanism Induced By PA-MSHA And Procaine

Bladder cancer is the most common solid tumors in male, which ranked fifth. In thefemale the incidence of bladder cancer ranks seventh worldwide, new cases of bladdercancer are 386,300 cases,150, 200 cases of death each year. The epigenetic variation isanother major factor in addition to in gene mutation. Some studies have shown thatmethylation of tumor suppressor gene as a major epigenetic change can be used as markerfor a bladder cancer diagnosis, treatment and prognosis targets. In this study, we performedthe screening of tumor suppressor gene methylation status in 1 DNA from 112 casesof clinical bladder cancer tumor tissue samples with urine samples and peripheral bloodusing Methylation-Specific PCR (MSP), and successfully established a novel set of DNAMethylation Markers including DAPK,ITGA4,RARβgenes by establishing the methylationscreening system, display a certain application prospects. We also investigated themechanism of bladder tumor apoptosis induced by treatment with procaine and PA-MSHA.1 Methylation status of tumor suppress gene in tumor tissues of BTCC1.1 Methylation status of tumor suppressor genes in bladder cancerWe first investigated methylation status of tumor suppressor genes p14ARF, RUNX3,DAPK, E-cad, ITGA4, RARβ, HPP1 and APAF1 in tumor tissue of BTCC by MSP. Theresults showed that: methylation positive rate ofP14ARF was 26.7%; methylation-positiverate of RUNX3 was 56.2%; methylation-positive rate of DAPK was 29.4%; methylation-positive rate of E-cad was 11.6%; methylation-positive rate of ITGA4 was 75.0 %;methylation-positive rate of RARβwas 91.0%; methylation -positive rate of HPP1 was48.2%; methylation-positive rate of APAF1 was 90.1%; Dispite to the ITGA4 gene, the otherseven gene was methylation status in normal bladder and glandular cystitis tissue and there isno difference (p>0.05), methylation -positive rate of ITGA4 gene in normal bladdertissue and cystitis glandularis tissue have statistical difference (p=0.003) ;In addition to ITGA4 and E-cad gene (p =0.081, p=0.214), methylation-positive rate insix other tumor suppressor genes in bladder cancer group compared with the healthycontrol and cystitis glandularis group were statistically significant withχ~2test (p <0.05),the results show that these six kinds of tumor suppressor genes can be diagnosed as bladder cancer methylation candidate markers;1.2 The relationship between methylation status of tumor suppressor gene andgender in Bladder cancer patientsMethylation-positive of P14ARF in male patients with bladder cancer was 30 cases,negative for 65 cases, 15 cases in 17 cases of female patients were negative for methylation,2 cases were methylation; positive rate between male patients and female patients wasstatistically significant; DAPK methylation-positive male patients were 21 cases, 74 caseswere negative; 4 cases were negative for methylation; In 17 female patients, 12 cases weremethylation-positive cases;5 cases were negative, between male patients and femalepatients positive rate was statistically significant (p<0.001);1.3 The relationship between methylation status of tumor suppressor gene andpatients ageNo correlation were found between the methylation status rate of eight kinds of tumorsuppressor gene in patients and patient's age, showed that methylation of tumor suppressorgene can occur at any age in patients with bladder cancer;1.4 The relationship analysis of tumor suppressor gene methylation status in bladdercancer patients and pathological gradeWe have obtained the basic pathological data of each patients with bladder cancer,methylation status of tumor suppressor gene and histological grade were classified, wefound: the gene expression of RUNX3, DAPK, ITGA4, RARβ, APAF1 were higher in PhaseI stage; Low expression of P14ARF, E-cad, HPP1 gene; between them have a significantdifference (p <0.05). With the progress in the tumor stage and tumor growth, expressionlevels of DAPK gene was reduced, in stageⅠandⅡexpression levels of DAPK weresignificant differences (p =0.001);1.5 The relationship analysis of methylation status of tumor suppressor gene with thedegree of infiltration in bladder cancerInfiltration is an important indicator to determine the prognosis of bladder cancer, wefound that methylation-positive rate of E-cad and RUNX3 gene in non-invasive group andinvasive group have difference (p <0.01); there was no difference between the rest of tumorsuppressor gene in non-invasive and invasive group. In non-invasive bladder cancerpatients, the methylation-positive rate of RUNX3, ITGA4, RARβ, APAF1 was significantlyhigher than the other four groups of genes, with statistical significance (p <0.01);1.6 The sensitivity of MSP : We have proved that 1 ng of methylated DNA as a PCR template could get a significantPCR product, the electrophoretic bands were very significant, this results shows thatthe MSP can be used as a method of early diagnosis and screening.2 Analysis of methylation status of tumor suppressor gene of DNA fromurinary sediment cells in bladder cancer patientsThe results via MSP to analyze the methylation status of the urine sediment cellsshowed that: methylation-positive rate of p14ARF was 20.5%; methylation-positiverate of RUNX3 was 56.25%; methylation-positive rate of DAPK was 26.7%; methylation-positive rate of E-cad was 1.7% ; methylation-positive rate of ITGA4 was 69.6%;methylation-positive rate of RARβwas 87.5%; methylation-positive rate of HPP1 was27.6%; methylation-positive rate of APAF1 was 77.6%; detection rate of E-cad genein Glandular cystitis and bladder cancer patients is extremely low, suggesting that the genemarker is not suitable as a urine sample test, MSP analysis of urine sediment cells isconsistent with the results via bladder cancer tissue;3 Analysis of methylation status of tumor suppressor gene of circulatingDNA in peripheral blood of bladder cancer3.1 Comparision of DNA yield in peripheral blood of patients between blood donorsand patient with bladder cancerWe compared plasma circulating DNA yield of the healthy blood donors, glandularcystitis patients and bladder cancer patients. The assessment results showed:plasma circulating DNA yield is not significantly different between healthy blood donors andpatients with cystitis glandularis, however the extraction plasma circulating DNA frombladder cancer patients was significantly higher than that of healthy donors and patientswith glandular cystitis, with statistical significance;3.2 The comparision of DNA yield by several plasma DNA extraction methodsWe performed experiment of extraction of circulating DNA that used by ZR GENOMICDNA II kit from ZYMO Corporation, phenol-chloroform method, and Wizard GenomicDNA Purification Kit produced by Promega Corporation, respectively.The results are asfollows: DNA yield by ZR GENOMIC DNAⅡkit were higher than that other method,meanwhile with less time, production of circulating DNA can be increased by 30%, theentire operating time is less than 20 min;3.3 Methylation status of tumor suppressor gene in peripheral blood circulating DNAin patients with bladder cancer We investigated peripheral blood circulating DNA methylation-positive rate ofRUNX3, ITGA4, RARβ, APAF1 gene by MSP, the results are as follows: methylation-positive rate of these four genes is consistent with the results by tumor tissue and urinesediment cells, however, the positive rate was significantly lower than the tumor tissue andurine sediment cells from the same patients including RUNX3(19.6%), ITGA4 (29.4%),RARβ(44.6%), APAF1 (30.3%), respectively;4 The mechanism of inhibiting proliferation induced by PA-MSHA andProcaine on T24 and 5637 bladder cancer cells4.1 PA-MSHA and Procaine could inhibit T24 and 5637 cell proliferation and induceapoptosisBy CCK-8 assay and fluorescence staining with Hoechst 33258 we confirmed that thePA-MSHA (4 mmol/L) and Procaine (4×1011/L) inhibited T24 and 5637 bladder cancercell proliferation in a dose-effect and can induce cell apoptosis with typical punctuatednuclear morphology;4.2 Procaine could inhibit the APAF1 gene methylation status of T24 or 5637 cellsThe results by real-time MSP showed that the APAF1 gene methylation index (MI)treatment with procaine in T24 and 5637 was from 11.2%±0.26% to 6.4%±0.34%,however, methylation status of tumor suppressor gene in T24 or 5637 cell does not havea change when cells was treatment with PA-MSHA for 72 h;4.3 PA-MSHA and Procaine induced apoptosis induced by caspase-3/9 and sFASLup-regulation and activation,and inhibition of MMP-9 expression in T24 and 5637 cellSpectrophotometry was used to detect the expression of caspase-3/9 protein expressiontreatment with or without the PA-MSHA and Procaine, respectively. The results showedthat: caspase-3 and caspase-9 as the key effector protein in apoptosis signaling pathwaycould be activation with Procaine and PA-MSHA in T24 cells; however, procaine does notinduced activation of caspase-3 and caspase-9 in 5637 cells, the activation of caspase-9protein induced by PA-MSHA. We also proved that PA-MSHA and Procaine couldup-regulation expression of sFASL, inhibition of MMP-9 expression in T24 and 5637 cell.