| Esophageal carcinoma is an environment-related tumor.It’s supposed that exposure of nitrosamines is one of the risk factors.In recent years,many evidences showed that the occurrence and development of esophageal carcinoma were closely related to the infection of human papillomavirus(HPV).This study used case-control study to examine the prevalence of plasma HPV infection in Huai’an population,and to analyze the relationship between HPV infection and esophageal carcinoma,then to determine the type of major HPV infection in patients with esophageal carcinoma in Huai’an.As target cell line,human esophageal epithelial cell Het-1A was checked primary infection type of HPV.Under combinative exposure both HPV and MNNG,malignant transformation characteristics of Het-1A were observed,in the meantime,the molecular mechanism of miR-218 was investigated.1.HPV infection in people with high risk of esophageal carcinoma in Huai’an(1)According to the 1:1 matched pairs,a case-control sample of 60 pairs was recruited in Huai’an,Jiangsu province.HPV levels in plasma were detected by ELISA technique.The HPV level in plasma in the case group was 1.060(0.436～2.212)nmol/L,it significantly higher than that in the control group 0.756(0.471～1.111)nmol/L(Z=2.643,P=0.008).The correlation between HPV infection rate and the incidence of esophageal carcinoma risk showed that HPV infection significantly increased the risk of esophageal carcinoma(OR=3.273,95%CI:1.181～9.069).(2)Nested PCR was used to amplify the conserved region of HPVL1 in DNA samples from 105 cases of esophageal carcinoma.The results showed that the positive rate of HPV in DNA of cancer tissues samples was 31.4%;Genotyping of HPV gene in HPV positive samples revealed that the HPV 18 subtype infection rate was 24.2%,8 out of 33 HPV infected samples,and other types were not detected.2.Synergistic effects of HPV18 and MNNG on malignant transformation of Het-IA cells(1)Het-1A cell lines with HPV18-e6e7 were established by using lentiviral vector.QRT-PCR and Western Blot were used to detect the expression of HPV18 E6,HPV E7 mRNA and protein in cell lines,and the expression level in transfected group was higher than that in control group.(2)The HPV18 transfection group and the empty vector control group were exposed to 12h,24h,and 48h with MNNG of 0μmol/L,0.625μmol/L,1.25μmol/L,2.5μmol/L,5μmol/L,10μmol/L,20μmol/L,40μmol/L,respectively.Using MTT method to detect cytotoxicity,it was found that the cell inhibition rate increased with the of MNNG exposed dose increasing;the OD value of HVP18 transfection groups exposed MNNG were higher than those of control group.(3)Under concentration of 0μmol/L,0.625μmol/L,2.5μmol/L and 10μmol/L,MNNG could promote the apoptosis of the control group,and the percentage of G1 phase cells increases;S phase in cells with HPV 18 transfection were increased and the apoptosis were inhibited;the cells with HPV18 transfection showed that the proportion of G2 phase cells was increasing with MNNG increasing dosage.(4)The combinative effect of HPV18 and MNNG induced malignant transformation of Het-IA.Results showed that cultured Het-IA of the 25 generation appeared malignant transformation characterics,such as the cells loose,size change,multi directions,etc..The results of ConA agglutination test showed that the ability of cell aggregation was significantly enhanced.The results of plate and soft agar cloning showed that the rate of colony formation was significantly higher than that of the independent exposed group.(5)The results of cell proliferation showed that the 25 generation proliferation were different among several treatment groups(F=7.919,P=0.004);results of multiple comparisons showed that the control group was the lowest(P<0.05),there was no significant difference with other groups(P=0.173).Cell cycle test showed that HPV 18 or MNNG exposure independently resulted in the 25 generations of cells G1-phase increased,and the S-phase cell rate decreased.The combinative exposure of MNNG and HPV18 can significantly increase the percentage of S-phase cells,and there was a significant difference in the rate of apoptosis.The apoptosis rate of the combined HPV18 and MNNG was lower than that of other groups(P<0.05).(6)The cell migration experiments found that cell migration rate had no difference among the groups(F=-0.655,P=0.289);the invasion experiment results show that the number of cell penetrate the membrane in combined MNNG and HPV18 exposed group were higher than that in the independent exposed group and control group(P<0.05).3.Study on the mechanism of miR-218 participating in the malignant transformation of Het-1A cells induced by combined both HPV18 and MNNG(1)The results of case-control study showed that the plasma level of miR-218 expression in case group was lower than that in control group(P<0.05).Association analysis between the plasma level of miR-218 and esophageal carcinoma risk showed that downregulation of miR-218 expression was significantly associated with risk of esophageal carcinoma(OR=0.276,95%CI:0.152～0.501).HPV levels in plasma were negatively correlated with the expression of miR-218(P<0.01).(2)Het-1A cells of malignant transformation induced by combined both HPV18 and MNNG,the miR-218 expression levels of different treatment groups were lower than that of the control group,and the combinative treatment group was the lowest(P<0.05).The expression of ROBO1,CDK6,MMP-2,MMP-9 and Bcl-2 in the combined both HPV18 and MNNG were significantly up-regulated,this suggests that miR-218 may be involved in the carcinogenic process,interfereing with cell cycle,apoptosis and invasion function.The expression of p53 in the combinative effect group was down regulated and the expression of c-Myc was up-regulated,this suggesting that the combined both HPV18 and MNNG may inhibit the regulation of c-Myc transcription by p53 and promote the malignant transformation of cells.Main conclusions as follow:1.HPV infection is associated with an increased risk of esophageal carcinoma in Huai’an,and HPV18 is the major infection type in esophageal carcinoma.2.HPV18 and MNNG can promote malignant transformation of esophageal epithelial cells,inhibit apoptosis and promote cell proliferation and invasion.3.Combinative transformation of HPV 18 and MNNG leads to malignant transformation of esophageal epithelial cells,this process can affect the expression of miR-218 and related functional proteins,it suggests that miR-218 may be involved in the synergistic effect of HPV18 and MNNG on malignant transformation of esophageal epithelial cells. |
PDF Full Text Request