Gga-miR-7b Regulatory Functional Target Genes And Their Roles In The Process Of Tumorigenesis Of Marek's Disease In Chicken

Marek's disease(MD)is an immunosuppressive and lymphocytic neoplastic disease induced by Marek's disease virus(MDV).In recent years,although MD can use commercially available vaccines to protect chickens from MD,it will increase the virulence of MDV with the outbreak of MD,and its pathogenicity will gradually increase.MD is still a serious disease,which has brought huge economic losses.Therefore,there is a need to enhance existing vaccine control measures and to find new alternative strategies.Host micro RNAs(mi RNAs)exert various biological functions at the post-transcriptional regulation level,and they may play a n important regulatory role in the induction of MD tumor formation.We used the self-cultivated B21(resistance)and B19(susceptible)haplotype SPF chickens as models,to construct the differential expression profile of mi RNA in spleen tissue s between B21 and B19 chickens in three key stages of MDV virus infection(5,14 and 25dpi)by using highthroughput sequencing technology.Gga-mi R-7b was found to be diferential expressed significantly between infection vs non infection.Therefore,we conducted in-depth research with gga-mi R-7b as the research goal.(1)Using Targetscan and mi RDB two biological software to predict the target genes of gga-mi R-7b,and the two were combined and combined with GO analysis to screen target genes related to tumor and immunity,and 5 candidate target genes were screened.(VDAC1,KLF4,SATB1,SNCA,TCF12).(2)Real-time quantitative PCR(q RT-PCR)was used to analyze the expression changes of spleen tissues in the three key stages(5,14 and 25 dpi)of B21 and B19 haplotype SPF chickens.By q RT-PCR analysis,we selected VDAC1,KLF4,and SNCA for candidate target gene verification.(3)In vitro validation of the candidate target gene of gga-mi R-7b using the dual luciferase reporter gene system,the results showed that gga-mi R-7b can bind to the 3'UTR region of VDAC1,SNCA m RNA,and inhibit firefly luciferase Activity(p < 0.05).q RT-PCR and Western blot showed that gga-mi R-7b inhibited the m RNA and protein expression of VDAC1 gene(p<0.05).The above results confirmed that VDAC1 is a target gene regulated by gga-mi R-7b.(4)The effects of gga-mi R-7b and VDAC1 on the proliferation and migration of MDCC-MSB1 cells were analyzed by CCK-8 and Transwell chamber experiments.The results showed that gga-mi R-7b inhibited the proliferation and migration of MDCC-MSB1 cells(p<0.05);The results showed that gga-mi R-7b inhibited the proliferation and migration of MDCC-MSB1 cells(p<0.05);After knockdown of VDAC1 by si RNA,proliferation of MDCC-MSB1 cells was inhibited(p<0.05),while migration of MDCC-MSB1 cells was reduced,although not at a significant level.More interestingly,m RNA expression of the MDV tumorigenic gene Meq was also significantly inhibited after knockdown of VDAC1(p<0.05).In summary,this study demonstrates that VDAC1 is a functional target gene regulated by gga-mi R-7b,and that gga-mi R-7b may inhibit proliferation and migration of MDCC-MSB1 cells by regulating VDAC1.gga-mi R-7b and its regulated target gene VDAC1 are likely to be involved in MD tumor formation and MDV infection.