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Objective To investigate the effect of human papillomavirus (HPV) 16 E6 gene on the expression of E-cadherin (E-cad) in cervical cancer cell line SiHa. Methods HPV16 E6 expression system was established in the cervical cancer cell line SiHa by using transient transfection system, MTT method was used to detect SiHa cell proliferation activity, and reverse transcription-polymerase chain reaction (RT-PCR) and western blot method was used respectively to detect E-cad mRNA and protein expression level in cells after HPV16 E6 transfection. Results Compared with the blank control group (non-transfected plasmid) and the vector group (the addition of pcDNA3.1), the cell viability rate of the E6 group (pcDNA3-1-HPV16 E6) was significantly increased (P<0 .05), while there was no significant difference between the vector group and the blank control group (P>0.05). The relative expressions of E-cad mRNA in the E6 group, the vector group and the blank control group were 0.26±0.12, 0.82±0.14, 0.83±0.21 respectively, then the protein relative expressions in the three groups were 0.62±0.02, 1.33±0.04, 1.31±0.05 respectively. The expressions level of E-cad mRNA and protein in E6 group were significantly lower than those in the other two groups (all P<0.05). However, there was no significant difference between the vector group and the blank control group (both P>0.05). Conclusion The instantaneous transfection of HPV16 E6 gene can reduce the expression of E-cad in cervical cancer cells, which is closely related to the occurrence and development of cervical cancer.
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Objective To investigate the effect of human papillomavirus (HPV) 16 E6 gene on the expression of E-cadherin (E-cad) in cervical cancer cell line SiHa. Methods HPV16 E6 expression system was established in the cervical cancer cell line SiHa by using transient transfection system, MTT method was used to detect SiHa cell proliferation activity, and reverse transcription-polymerase chain reaction (RT-PCR) and western blot method was used respectively to detect E-cad mRNA and protein expression level in cells after HPV16 E6 transfection. Results Compared with the blank control group (non-transfected plasmid) and the vector group (the addition of pcDNA3.1), the cell viability rate of the E6 group (pcDNA3-1-HPV16 E6) was significantly increased (P<0 .05), while there was no significant difference between the vector group and the blank control group (P>0.05). The relative expressions of E-cad mRNA in the E6 group, the vector group and the blank control group were 0.26±0.12, 0.82±0.14, 0.83±0.21 respectively, then the protein relative expressions in the three groups were 0.62±0.02, 1.33±0.04, 1.31±0.05 respectively. The expressions level of E-cad mRNA and protein in E6 group were significantly lower than those in the other two groups (all P<0.05). However, there was no significant difference between the vector group and the blank control group (both P>0.05). Conclusion The instantaneous transfection of HPV16 E6 gene can reduce the expression of E-cad in cervical cancer cells, which is closely related to the occurrence and development of cervical cancer.
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Objective To investigate the influence of human papillomavirus (HPV) 16 E6 gene silencing by small interfering RNA (siRNA) on the expression and the promoter hypermethylation status of E-cadherin (E-cad) in cervical cancer SiHa cell line. Methods siRNA which used lentivirus as the vector was used to knock down the HPV16E6 gene in cervical cancer SiHa cell line. The expression levels of HPV16E6 mRNA, E-cad mRNA and protein in siRNA-HPV16E6 SiHa cell line were detected by RT-qPCR and Western blot, respectively. Methylation specific PCR (MSP) method was used to detect the methylation status of E-cad gene (CDH1) promoter in siRNA-HPV16E6 SiHa cell line. Results The E-cad mRNA expression levels in siRNA E6 group, empty vector group and blank control group were 4.755±1.085, 1.224± 0.840, 1.327±1.221, respectively. The protein expression levels were 0.616±0.019, 0.325±0.016, 0.299±0.015, respectively. The expressions of E-cad mRNA and protein in siRNA E6 group were significantly higher than those in the empty vector group and blank control group (F = 21.346, P 0.05). After knocking down HPV16E6 gene, the methylation status of E-cad gene was weakly positive, and the intensity of the amplified products was significantly weaker than that in the empty vector group and blank control group, while the unmethylation amplification was positive. Conclusions Knocking down the HPV16E6 gene increases the expression of E-cad in cervical cancer SiHa cell line, and decreases the level of CDH1 promoter methylation. To a certain extent, it partly reverses the hypermethylation status of CDH1 promoter, and causes E-cad to be re-expressed.
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Objective To explore the interaction between folate and the expression of HPV16 E6/E7 mRNA in the progression of cervix carcinogenesis.Methods Subjects were selected from the participants who were diagnosed pathologically,including 64 patients with cervical squamous cell carcinoma (SCC),55 patients with low-grade cervical intraepithelial neoplasm (CIN1),55 patients with high-grade cervical intraepithelial neoplasm (CIN2 +) and 80 with normal cervix (NC).The levels of serum folate and RBC folate were detected by microbiological assay,and the expression levels of HPV16 E6/E7 mRNA were measured,using the real-time polymerase chain reaction (real-time PCR).Data was analyzed by methods as chi-square test,analysis of variance (ANOVA),Welch test,Kruskal-Wallis H test and ordinal logistic regression.Spearman correlation was tested using the SPSS statistical software (version 16.0) while the interaction effects were evaluated by additive model.Results There was a positive correlation seen between the serum folate and RBC folate (r=0.41,P＜0.001).The levels of serum folate and RBC folate decreased gradually along with the severity of cervical lesions (x2=32.71,P＜0.001;x2=16.32,P＜0.001).The expression levels of HPV 16 E6/E7 mRNA increased gradually with the severity of cervical lesions (x2 =30.11,P＜ 0.001;x2 =38.99,P＜0.001).There was a negative correlation between the levels of RBC folate,expression levels of HPV16 E6 (E6:r=-0.14,P=0.009) and HPV16 E7 mRNA (E7:r=-0.21,P=0.001),respectively.Both RBC folate deficiency and HPV16 E6/E7 mRNA high expression showed additive interaction in CIN 1,CIN2 + and SCC.Conclusion Folate deficiency and high expression of HPV16 E6/E7 mRNA might increase the risk of cervical cancer and cervix precancerous lesions,and having a synergistic action in the progression of cervix carcinogenesis.
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Objective To explore the interaction between folate and the expression of HPV16 E6/E7 mRNA in the progression of cervix carcinogenesis.Methods Subjects were selected from the participants who were diagnosed pathologically,including 64 patients with cervical squamous cell carcinoma (SCC),55 patients with low-grade cervical intraepithelial neoplasm (CIN1),55 patients with high-grade cervical intraepithelial neoplasm (CIN2 +) and 80 with normal cervix (NC).The levels of serum folate and RBC folate were detected by microbiological assay,and the expression levels of HPV16 E6/E7 mRNA were measured,using the real-time polymerase chain reaction (real-time PCR).Data was analyzed by methods as chi-square test,analysis of variance (ANOVA),Welch test,Kruskal-Wallis H test and ordinal logistic regression.Spearman correlation was tested using the SPSS statistical software (version 16.0) while the interaction effects were evaluated by additive model.Results There was a positive correlation seen between the serum folate and RBC folate (r=0.41,P＜0.001).The levels of serum folate and RBC folate decreased gradually along with the severity of cervical lesions (x2=32.71,P＜0.001;x2=16.32,P＜0.001).The expression levels of HPV 16 E6/E7 mRNA increased gradually with the severity of cervical lesions (x2 =30.11,P＜ 0.001;x2 =38.99,P＜0.001).There was a negative correlation between the levels of RBC folate,expression levels of HPV16 E6 (E6:r=-0.14,P=0.009) and HPV16 E7 mRNA (E7:r=-0.21,P=0.001),respectively.Both RBC folate deficiency and HPV16 E6/E7 mRNA high expression showed additive interaction in CIN 1,CIN2 + and SCC.Conclusion Folate deficiency and high expression of HPV16 E6/E7 mRNA might increase the risk of cervical cancer and cervix precancerous lesions,and having a synergistic action in the progression of cervix carcinogenesis.
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Objective To investigate the effects of human papilloma virus 16 E6 (HPV16 E6) on endoplasmic reticulum (ER) stress-autophagic response in the cervical cancer C33A cells.Methods Polymerase chain reaction was used for detecting the integration of HPV DNA.The eukaryotic expression vector of HPV16 E6 was constructed and transfected via lipofectamine into C33A cells.Experimental cells were classified into 3 groups:pcDNA3.1--HPV16 E6 group,pcDNA 3.1-group and C33A group.Western blot was used to measure expression of protein of HPV16 E6,Beclin 1,LC3 Ⅱ,IRE1,PERK and ATF6 in transfected cells.Results here was no HPV DNA integration in C33A cells that were confirmed as the intervention cells.Eukaryotic expression vector pcDNA3.1--HPV16 E6 was constructed successfully.The eukaryotic expression vector pcDNA3.1--HPV16 E6 significantly improved the expression of protein of HPV 16 E6 in C33A cells.The protein expression of Beclin 1,LC3 Ⅱ,IRE1,PERK and ATF6 were significantly improved after transfection with vector pcDNA3.1 +-HPV16 E6 (P ＜ 0.05).Furthermore,LC3 Ⅱ protein level was reduced by treatment with ER stress inhibitor.Conclusion HPV16 E6 can improve autophagy through the ER stress pathway,and this response may play an important role in the process of HPV16 E6 inducing cervical cancer,providing one of the new strategies for gene therapy of cervical carcinoma.