| Objective: To explore the reason of the difference of MUS81 expression occurs in EBV-positive gastric carcinoma(EBVa GC)and EBV-negative gastric carcinoma(EBVn GC).To further explore the role and mechanism of MUS81(MUS81structure-specific endonuclease subunit)in the development of EBVa GC.Methods:(1)qRT-PCR(Real time quantitative PCR,real time qPCR)was used to explore the difference of transcriptional level of MUS81 and EME2(essential meiotic structure-specific endonuclease subunit 2)in EBVa GC and EBVn GC.(2)Western-blot was used to explore the difference of protein level in EBVa GC and EBVn GC cells.(3)SGC7901 cell lines stably transfected with EBV latent membrane protein 1(LMP1),latent membrane protein 2A(LMP2A),and EBV-encoded small RNA(EBER)were constructed to detect the effects of exogenous LMP1,LMP2 A and EBER expression on MUS81 expression.(4)Immunohistochemistry(IHC)was used to detect the difference of MUS81 protein level in EBVa GC and EBVn GC tissues.(5)The methylation status of MUS81 gene promoter region in EBV-positive and negative gastric cancer cell lines was detected by bisulfite genomic sequencing(BGS),and the effects of demethylation reagent5-Aza-Cd R on the expression of MUS81 in BGC823 and SGC7901 cell lines were observed.(6)Effects of si RNA targeting MUS81 and EME2 on cell cycle and apoptosis.Results:(1)The results of qRT-PCR showed that the transcriptional levels of MUS81 gene,EME2 gene,EME1 gene and SLX4 gene in EBVn GC cell lines were significantly higher than those in EBVa GC cell lines.The transcriptional level of SLX1 gene in EBVa GC cell lines was significantly higher than that in EBVn GC cell lines and no difference of EME1.(2)The protein expression level of MUS81 gene in EBVn GC cell lines was significantly higher than that in EBVa GC cell lines.There was no significantly difference of EME2 protein expression level in EBVa GC cell lines and EBVn GC cell lines.(3)There was no significantly difference between transcriptional level of SGC7901-LMP1,SGC7901-EBER and SGC7901-NC cell lines by q RT-PCR.The transcriptional level in SGC7901-LMP2 A cell line was higher than that in SGC7901-NC cell line.There was no significantly difference in protein level between SGC7901-EBER,SGC7901-LMP2 A cell lines and that of SGC7901-NC cell line.(4)The results of q RT-PCR showed that the transcription level of SGC7901-LMP1 increased after treated with 5-Aza-2'-deoxycytidine.Accordingly,the results of Western-blot showed that the protein level of SGC7901-LMP1 cell line did not change after treated with5-Aza-2-deoxycytidine.(5)The methylation level in promoter region of MUS81 gene in EBV negative gastric cancer cell line was significantly higher than that in EBV positive gastric cancer cell line.Western blot results showed that the expression of MUS81 protein in EBV-negative gastric cancer cells treated with 5-aza-2'-deoxycytidine did not change significantly.(6)The protein expression level of MUS81 gene was significantly lower in EBVa GC tissues than in EBVn GC tissues.(7)The expression of EME2 was down-regulated by making MUS81 protein silience.Compared with the control group,the cells had G2/M arrest,but the apoptotic rate had no significant difference.Conclusion:(1)EBV can inhibit the expression of MUS81 in gastric cancer cell line,and methylation of MUS81 promoter may not be involved in the regulation of MUS81 protein expression.Exogenous LMP1,LMP2 A and EBER expression had no significant effect on MUS81 expression.(2)In EBV associated gastric cancer,the expression of MUS81-EME2 complex was down-regulated and synergistic.(3)Silience of MUS81 and EME2 expression could block SGC7901 cell line in G2/M phase,while interference of EME2 could promote SGC7901 cell apoptosis,and interference of MUS81 had no significant effect on SGC7901 cell apoptosis. |
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