Association Between The SNP In The Coding Region Of SNORD105 And Hepatocellular Carcinoma Susceptibility

Objective:In Chinese populations,the correlation of a single nucleotide polymorphism(SNP)rs2305789 in the coding region of SNORD105 with HCC susceptibility and the underlying mechanism of this polymorphism in hepatocellular carcinoma were evaluated by using molecular genetics and other methods.Methods:(1)Screening polymorphisms in the coding region of SNORD105 using bioinformatics method,and selecting rs2305789,which has a high known frequency,as candidate.(2)The genotyping results of a single nucleotide polymorphism(rs2305789)in 712 HCC cases and 801 healthy controls was assayed by pyrosequencing.(3)Logistic regression analysis was used to detect the relationship between different genotypes of rs2305789 and the risk of HCC.(4)To identify whether different genotypes of rs2305789 has a regulatory role in the expression levels of SNORD105 and PPAN(the host gene of SNORD105),we explored the expression of SNORD105 and PPAN in HCC tissues from patients by Real-time PCR.(5)To validate whether rs2305789 polymorphism has an effect on SNORDI05 expression,we constructed two SNORD105 vectors containing A or G allele,respectively,and transfected them into Huh7 and HepG2 cell lines.Real-time PCR analysis was used to further verify the correlation between different subtypes and the expressions of SNORD105 and PPAN.(6)CCK8 method and wound-healing migration assay were performed to evaluate the effect of overexpression of SNORD105 on the viability and motility.Results:(1)The analysis of pyrosequencing results documented that the genotype distributions of the rs2305789 polymorphism in the normal control group had no deviation from Hardy-Weinberg equilibrium(P>0.05),and the genotypic frequencies of rs2305789 were 0.34(AA),0.46(AG),0.20(GG)in control group,respectively.(2)After being adjusted for age,gender and other major risk factors,Logistic regression analysis indicated that the susceptibility of GG genotype to HCC was significantly lower than AA genotype(OR=0.61,95%CI:0.45-0.83,P<0.001).(3)In Real-time PCR assay,the transcript level of SNORD105 with GG genotype was significantly downregulated in HCC tissues compared with that in samples with AA genotype.As expected,the results of PPAN showed a similar trend.(4)Further validation showed that the SNORD105 and PPAN expression level in the SNORD105 vector containing G allele transfected group was significantly lower than that in the SNORD105 vector containing A allele transfected group.The results in HepG2 cell line showed a similar trend.(5)The CCK8 assay demonstrated that the cell viability of HepG2 in the SNORD105pDNA transfected group increased significantly.And in the Huh7 cell lines,it showed a similar trend,and more significant than that in HepG2 cell line.(6)The results of wound-healing migration assays showed that the overexpressed SNORD105 increased the motility of both HepG2 and Huh7 cell lines.Conclusion:(1)In Chinese populations,the SNP rs2305789 in the coding region of SNORD105 was highly relevant to the risk of HCC.(2)Different genotypes of rs2305789 has a regulatory role in the transcript levels of SNORD105 and PPAN.(3)The overexpressed SNORD105 increased the viability and motility of both HepG2 and Huh7 cell lines.(4)rs2305789 may play a vital part in development of HCC through affecting the transcript of SNORD105 and PPAN.