| Objective:1. Detection of the Kiss-1gene methylation in normal tissue, colorectaladenomas, colorectal carcinoma and tissue adjacent to cancer, and research the role incolorectal carcinoma development.2. Detection of the expression of Kiss-1gene in colorectal carcinoma, andresearch the relationship between the expression of Kiss-1gene and clinicalpathological features.3. Detection of the Kiss-1gene methylation in colorectal carcinoma, andresearch the relationship between the Kiss-1gene methylation and clinicalpathological features.Methods:1. The Kiss-1gene promotor methylation were detected respectively bymethylation-specific PCR (MSP) in normal tissue, colorectal adenomas, colorectalcarcinoma and tissue adjacent to cancer.2. The mRNA expression of the Kiss-1gene were detected by real-time PCR incolorectal carcinoma tissue.3. The metastine expression of Kiss-1gene were detected by western-blot incolorectal carcinoma tissue.Results:1. The positive rate of Kiss-1promoter hyper-methylation was82.19%(60/73)in colorectal carcinoma tissue,30.01%(22/73) in tissue adjacent to cancer,21.7%(5/23)in colorectal adenomas and6.3%(9/142) in normal tissue. The rate of Kiss-1promoter methylation was significantly higher in colorectal carcinoma tissue than innon-carcinoma tissue (P<0.05).2. The positive expression rate of Kiss-1gene methylation in poorlydifferentiated of the colorectal carcinoma tissue was significantly higher than that in well differentiated group (90.5%vs60%, P<0.05), it was higher in T3+T4stagegroup than that in the T1+T2stage group (98.1%vs35.0%, P<0.05), and it washigher in lymph nodes metastasis group than that in lymph nodes negative group(93.3%vs74.4%, P<0.05), that in distant metastasis group was higher in non-distantmetastasis group (100%vs87.3%, P<0.05); It was independent of the patient’sgender, age, tumor location and tumor diameter (P>0.05).3. In the cancer tissue, the Kiss-1gene mRNA expression of Kiss-1methylation-positive group was lower than that in negative group (0.240±0.036vs1.138±0.136, P <0.05); The Kiss-1gene mRNA expression was negative correlationwith tumor differentiation, depth of invasion, lymph node metastasis and distantmetastasis (P<0.05), but it was independent of the patient’s gender, age, tumorlocation and tumor diameter (P>0.05).4. To detect the Kiss-1gene metastine expression of the cancer tissue, it is lowerin the Kiss-1gene methylation-positive group than in negative group (0.388±0.032VS0.945±0.050, P<0.05). The Kiss-1gene metastin expression was negative correlationwith depth of invasion, lymph node metastasis and distant metastasis (P<0.05), but itwas independent of the patient’s gender, age, tumor location, tumor differentiation andtumor diameter (P>0.05).Conclusions:1. The Kiss-1gene promoter methylation is one of the common ways ofepigenetic modification and may be closely related to the characteristics of metastasis,invasion and differentiation in colorectal carcinoma.2. Kiss-1gene promoter hyper-methylation may induce to the Kiss-1geneexpression decreased in colorectal cancer tissues.3. Kiss-1gene methylation may be used as a reference indicator of the risk ofcolorectal cancer metastasis and prognosis. Objective:1. Analysis the relationships between the Kiss-1gene promoter methylation andthe Kiss-1gene expression in different human colorectal carcinoma cell lines;research the effect of the Kiss-1gene promoter methylation on the invasion andmigration ability of colorectal carcinoma cell.2. Research the effect of5-aza-2’-deoxycytidine (5-Aza-CdR) on the Kiss-1genemethylation and the Kiss-1gene expression of colorectal carcinoma cells HCT116.3. Research the effect of5-Aza-CdR on the biological characteristics such asinvasion and metastasis of colorectal cancer cells HCT116, and explore if thebiological characteristics of colorectal cancer would be changed by reversal of theKiss-1gene methylation.Methods:1. The Kiss-1gene promotor methylation and the Kiss-1gene mRNA andmetastin expression were detected respectively by methylation-specific PCR（MSP）,realtime-PCR and western-blot in the colorectal carcinoma cells of HCT116, SW480,SW1116and VOLO. And the invasion and migration ability of colorectal carcinomacells were detected by transwell.2. Human colorectal carcinoma cell HCT116were treated with0umol/L、0.1umol/L、1umol/L、5umol/L and10umol/L5-Aza-CdR, and the The Kiss-1genepromotor methylation and the Kiss-1gene mRNA and metastin expression weredetected affter24hour、3day and5day.3.The Cell Counting Kit-8were used to detect cell growth and transwell assaywere used to detect the migration and movement capacity.Results:1．The Kiss-1gene methylation was positive in the colorectal carcinoma cells of HCT116、SW116and SW480, but the cell of LoVo is negative. The mRNA andmetastin expression of Kiss-1gene, the results show: LoVo> SW480> SW1116>HCT116(p<0.05). The proliferation speed of SW480、VoLo were lower than thespeed of HCT116(p<0.05), and invasion capacity of the SW1116、SW480、VoLo werelower than the capacity of HCT116(p<0.05), the movement of the SW480、VoLowere also lower than the HCT116(p<0.05).2. Each dose group in the5-Aza-CdR intervention HCT116cells24h,3days and5days, Kiss-1gene mRNA expression levels were significantly increased to thecontrol group (P<0.05), and show as time-dependent manner. Kiss-1mRNAexpression level of5umol/L and10umol/L groups were significantly higher than thecontrol group after24hours of5-Aza-CdR intervented (P<0.05), and each groups butthe exception of0.1umol/L group were significantly higher than the control groupafter3days intervented (P<0.05); Five days later, each groups were significantlyhigher than the control group (P<0.05), but there was no significant increase between10umol/L group and5umol/L group (P>0.05).3. After different doses of5-Aza-CdR intervented HCT116cell5days, eachgroup of the metastin expression levels were significantly higher than the controlgroup (P<0.05), but no significantly different between10umol/L group and5umol/Lgroup (P>0.05). With5umol/L of5-Aza-CdR intervented HCT116cell, comparedwith the control group, after24h,3days and5days the Kiss-1gene metastinexpression level was significantly increased (P<0.05); It was gradually increased withtime and show as time-dependent manner.5. Each dose group compared with the control group,10umol/L group of24hand3days intervented, the ability of HCT116cell proliferation was significantlyinhibited (P<0.05); After5days, each group were significantly reduced, and withextension of the dose increased, the inhibition rate increased (P<0.05), show asdose-dependent manner.6. After24hours、3days and5days, the same dose group5-Aza-CdRintervention HCT116cells’ invasion capacity was significantly decreased with thecontrol group (P<0.05), and show as the time-dependent manner. After24hours, the cell invasion capacity of the10umol/L group was significantly decreased with thecontrol group (P<0.05); After3days and5days, four doses of cell invasion abilitywas significantly decreased with which in the control group (P<0.05), and show asdose-dependent manner.7. After24hours、3days and5days, each dose group of5-Aza-CdR interventionof HCT116cells’ migration ability were significantly decreased with the controlgroup (P<0.05), and show as the time-dependent manner. After24hours5-Aza-CdRintervention in HCT116cells, the cell migration capacity of10umol/L group wassignificantly decreased with the control group (P<0.05); After3day and5days, thecell migration of each doses group were significantly decreased with the control group(P<0.05), and show as dose-dependent manner.Conclusion:1. Kiss-1gene promoter hyper-methylation may induce to the Kiss-1geneexpression decreased in colorectal carcinoma cells, and may associate with theinvasion and movement capacity of colorectal carcinoma cells.2. The Kiss-1gene promoter hyper-methylation were reversed by5-Aza-CdR incolorectal carcinoma cell HCT116, and the expression of Kiss-1were elevated by5-Aza-CdR， and the proliferation speed、 invasion and movement capacity ofcolorectal carcinoma cell HCT116were declined by5-Aza-CdR. |
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