Inhibition Of HaCaT Cell Proliferation By Let-7a-5p And Detection Of SNP Associated With Psoriasis

Research objective:Through differential gene expression analysis,let-7a-5p was associated with the pathogenesis of psoriasis.Predict the target of let-7a-5p,explore the effect of let-7a-5p on HaCaT cell proliferation and detect and analyze the SNP associated with psoriasis through in vitro experiments and as SNP detection.Research method:（1）Screening candidate miRNAs related to psoriasis.Download psoriasis related expression profiles and transcriptome data in the GEO database of NCBI.The differentially expressed genes were analyzed by DEseq2,the screening condition is FDR<0.1,|log2FC|>1;（2）The candidate has-let-7a-5p corresponding targets are predicted based on miRDB.The target genes were obtained by gene enrichenment analysis,and the expression of target genes in patients and healthy controls were compared;（3）To investigate the effect of let-7a-5p on the proliferation of HaCaT cells in vitro;（4）SNP variation of let-7 binding site was detected.Identify candidate miRNA related SNP sites.Blood samples from patients with psoriasis and control group in Northeast China were collected for DNA extraction,PCR amplification and DNA sequencing.The genotype distribution of SNPs between the two populations was counted and chi square test（Graphpad prism 6.0,χ 2,P < 0.05 means statistically significant）.Research results:（1）Through differential gene expression analysis and screening,it was found that let-7a-5p was most significantly down regulated,which may be the miRNA that plays a major role in let-7 family.We obtained 133 downregulated miRNAs and 99 upregulated miRNAs associated with psoriasis.Among the down regulated miRNAs,six genes of let-7 family（hsa-let-7c-5p,hsa-let-7e-3p,hsa-let-7a-5p,hsa-let-7b-5p,hsalet-7d-5p and hsa-let-7g-5p）were significantly down regulated,and has-let-7a-5p was the most significantly down regulated.（2）The predicted targets of let-7a-5p may be CCL7,MAPK6 and MAPK8,but because the expression of CCL7 gene in psoriasis and control group is small,it is regarded as no difference.（3）Let-7a-5p can significantly inhibit the proliferation of HaCaT cells in vitro.HaCaT cell proliferation was induced,30 nmol / L let-7a-5p simulant was transiently transfected.At the same time,the random sequence of miRNA molecules transfected with 30 nmol /Lwas used as a negative control.The results showed that the proliferation of HaCaT cells was significantly inhibited in the experimental group transfected with let-7a-5p simulant.（4）The SNP associated with let-7a-5p is rs848（A / C）on IL-13 gene,and the C allele will increase the risk of psoriasis.It was found that the SNP locus related to let-7 family was rs848 locus located at the 3 ’UTR end of the untranslated region of IL-13 gene.After DNA amplification and sequencing,the genotype statistics of rs848 locus in each sample showed that there was a significant difference in allele frequency between psoriasis patients and healthy controls（P < 0.05）.CC >AA.Similarly,there was significant difference in genotype frequency between the two groups（P < 0.05）,that is,C >A.Research conclusion:The down-regulation of let-7 was the highest in patients with psoriasis;Let-7a-5p can significantly inhibit the proliferation of HaCaT cells in vitro,and its targets may be MAPK6 and MAPK8;The associated SNP of let-7a-5p is rs848（A / C）on IL-13 gene,and C allele increases the risk of psoriasis.