Screening And Mechanism Validation Of Human Adipose-Derived MiRNA Targeting Liver MST1

PurposesNon-alcoholic fatty liver disease（NAFLD）is a common cause of chronic liver disease and liver-related disease incidence and mortality.It has become an important cause of chronic liver disease in developed countries such as Europe and the United States and in rich areas of China.The prevalence rate has exceeded 15%.For the treatment of NAFLD,there are still no specific targeted drugs,so it is imperative to find new targets.Through literature study and accumulation of previous work of the research group,we realized that circulating miRNAs derived from adipose tissue may mediate liver lipid metabolism and affect the occurrence and development of NAFLD by mediating the expression of MST1 gene in liver tissue.The discussion and verification of this hypothesis will provide new experimental evidence for the metabolic linkage between liver and adipose tissue,and provide new solutions and means for the treatment and diagnosis of NAFLD.Method1.References"Adipose-derived circulating miRNAs regulate gene expression in other tissues"in the Supplementary Table 1-human adipose tissue-derived circulating miRNA database to obtain circulating information of human adipose tissue miRNA data;then using TargetScan,miRDB database screening to obtain possible target humans miRNAs of MST13’UTR;Finally,nine miRNAs were obtained by taking the intersection of Venn diagrams,ie,screening for adipose-derived miRNAs that may target the human MST1 3’UTR region.MicroRNAs with significant MST1 targeting were screened by Western blotting and dual luciferase reporter assays.2.Design and synthesis of hsa-miR-448 and hsa-miR-518a-3p primers,using human genomic DNA as a template,using PrimeSTAR?Max DNA Polymerase high-fidelity enzyme to amplify the target genes hsa-miR-448 and hsa-miR-518a-3p full length.The CD810A-1-EGFP vector and the target fragment hsa-miR-448 and hsa-miR-518a-3p genes were digested with restriction endonucleases BamH I and Sal I,and purified by nucleic acid electrophoresis using a gel recovery kit.The target band was rapidly ligated with the DNA Ligation Kit Ver.2.1 ligase in a water bath at 16℃for 30 minutes to rapidly link the linearized vector to the target fragment.The ligation product was transformed into Stbl3competent cells,cultured on LB solid medium for 12 hours-16 hours overnight,and the appropriate monoclonal was selected and dissolved in micro-sterilized ddH₂O,and subjected to rapid PCR identification.The successfully identified monoclonal clones were cultured overnight in LB liquid medium for 12 hours-16 hours.The plasmid was extracted using the plasmid extraction kit,and the recombinant plasmid was identified by double enzyme digestion.The correct colony clones were used for sequencing and sequencing.The clones were successfully constructed CD810A-1-EGFP-miR448 and D810A-1-EGFP-miR518a-3p recombinant plasmids,and were subjected to a toxoid-free preparation.3.HepG2 cells were plated in 6-well plates at about 1.2×10⁶ cells per well.After incubation for 12h,HepG2 cells were incubated with 0μM,125μM,250μM palmitic acid（PA）for 12 hours or 24 hours,respectively.The cell culture model of non-alcoholic fatty liver disease（NAFLD）was established in vitro.The expression of MST1 was determined by oil red O staining,intracellular triglyceride（TG）content and Western blotting.The recombinant plasmids CD810A-1-EGFP-miR448,CD810A-1-EGFP-miR518a-3p and the control group were transfected into HepG2 cells,and 48 hours after transfection,they were induced by PA for 24 hours.Oil red O staining was used to detect the accumulation of lipid droplets in cells.The content of intracellular triglyceride was determined by triglyceride detection kit.The expression levels of pMST1/MST1 protein and its downstream lipid metabolism related proteins pSREBP/SREBP and pAMPK/AMPK were detected by Western blotting.4.Blood samples,liver tissue,and adipose tissue from the NAFLD patient group and the NAFLD control group were collected from the clinic.The lipid accumulation in liver cells was determined by B-ultrasound examination,blood biochemical examination and HE staining of liver tissue sections.Among them,13 cases were enrolled in the NAFLD disease group and 10 cases were in the NAFLD negative control group.qPCR was used to detect the expression level of MST1 gene in blood samples,liver tissues and adipose tissue,and the difference in expression of hsa-miR-448 and hsa-miR-518a-3p.Result1.Successful screening of hsa-miRNA targeting the liver MST1 geneFrom the Supplementary Table 4-of the literature"Adipose-derived circulating miRNAs regulate gene expression in other tissues",a total of 231 hsa-miRNAs down-regulated in patients with lipodystrophy can be obtained;A total of 15 hsa-miRNAs targeting the human MST1-3’UTR region were predicted by TargetScan,miRbase and other databases,and the intersection of the two was used to screen for adipose-derived hsa-miRNAs that may target the human MST1-3’UTR region.A total of nine.The expression of MST1 protein in each group was detected by Western blotting.The protein expression levels of miR-448 and miR-518a-3p were significantly decreased in the candidate 9 hsa-miRNAs.The dual luciferase reporter assay detects the expression of luciferase activity in the 3’UTR region of human MST1 and also found that miR-448 and miR-518a-3p significantly attenuated luciferase activity in the 3’UTR region of human MST1.2.Successful construction of hsa-miR-448 and hsa-miR-518a-3p overexpression vectorsFirst,we modified the newly purchased CD810A-1 plasmid and added EGFP green fluorescent label to facilitate the evaluation of transfection efficiency and tracking of expression vectors.The constructed recombinant plasmid CD810A-1-EGFP was transferred into competent cell Stbl3 for amplification,by PCR identification of bacterial water and double restriction enzyme digestion of recombinant plasmid,and transfection of 293T with recombinant vector CD810A-1-EGFP.Fluorescence collection of the cells indicated that the CD810A-1-EGFP recombinant vector was successfully engineered.The constructed recombinant plasmids CD810A-1-EGFP-miR448 and CD810A-1-EGFP-miR518a-3p were transfected into competent cells Stbl3 for amplification,identified by PCR for bacterial water and double enzyme digestion of recombinant plasmids.Fluorescence collection of recombinant vector CD810A-1-EGFP-miR448,CD810A-1-EGFP-miR518a-3p transfected293T cells indicated that the CD810A-1-EGFP recombinant vector was successfully transformed.3.PA induces HepG2 cells to construct NAFLD cell modelHepG2 cells were plated in 6-well plates at about 1.2×10⁶ cells per well.After incubation for 12 hours,HepG2 cells were incubated with 0μM,125μM,250μM PA for 12 hours and24 hours respectively.The oil red O staining showed that 250μM PA induced intracellular cells in 24 hours group.The lipid droplet content was significantly higher than the other groups.The content of intracellular triglyceride was measured by triglyceride assay kit,and the total protein concentration obtained by BCA method was used to correct the intracellular triglyceride content.It was observed that the intracellular triglyceride content of the 24 hours group induced by 250μM PA was obvious.Compared with other groups,the expression of MST1 protein in cells was also significantly lower than other groups.4.Overexpression of hsa-miR-448 and hsa-miR-518a-3p promotes the development of NAFLDHepG2 cells were plated in 6-well plates at about 1.2×10⁶ cells per well.After 12 hours of culture,100 nM miRNA control,hsa-miR-448 and hsa-miR-518a-3p were transfected into HepG2 cells,respectively,and transfected for 48 hours.After induction with 250μM PA,the HepG2 cells were induced for 24 hours,and the oil droplets in the PA-induced group were significantly increased compared with the PA-inducible group.The expression of MST1 and the expression of lipid metabolism pathway-related regulatory molecules in PA-induced and PA-inducible cells were detected by Western blotting.The data showed that the expression levels of MST1,AMPK and pSREBP were significantly down-regulated in the PA-induced group compared with the PA-inducible group.SREBP protein expression levels were significantly upregulated.The content of TG in the cells induced by intracellular PA was also significantly increased by the determination of triglyceride content.The above results demonstrated that there was significant liver lipid accumulation in the PA-induced group,and the NAFLD model of HepG2 cells was successfully constructed.At the same time,the hsa-miR-448 and hsa-miR-518a-3p intervention experiments were carried out by oil red O staining.The results showed that the lipid droplets in the hsa-miR-448 and hsa-miR-518a-3p intervention groups were higher than those in the control group.There is an increasing trend.The expression levels of MST1,AMPK and pSREBP in hsa-miR-448 and hsa-miR-518a-3p groups were significantly lower than those in the control group.The expression level of SREBP protein was significantly higher than that in the control group.The intracellular TG content increased significantly,indicating that at the cellular level,hsa-miR-448 and hsa-miR-518a-3p are important for regulating liver lipid metabolism by regulating the expression of human liver MST1 gene.5.There was significant difference in expression between hsa-miR-448 and hsa-miR-518a-3p in the NAFLD patient group and the NAFLD control groupThe various miRNAs secreted by adipose tissue mainly act on the liver tissue through blood circulation,so we collected the serum,adipose tissue and liver tissue of the same patient clinically.HE staining of liver tissue was the main basis for the diagnosis of NAFLD.The clinical B-ultrasound and blood student examination were used to determine 13 samples in the NAFLD group and 11 samples in the NAFLD control group.The expressions of hsa-miR-448 and hsa-miR-518a-3p in serum,adipose tissue and liver tissues were detected by qPCR,and the hsa-miR of the above tissues or organs was found in the NAFLD group compared with the control group.The expression of hsa-miR-448 and hsa-miR-518a-3p was significantly increased,and the expression level of liver MST1 gene was significantly decreased.It is indicated that at the level of human clinical samples,adipose tissue-derived hsa-miR-448 and hsa-miR-518a-3p are important for regulating liver lipid metabolism by regulating the expression of human liver MST1 gene.The results are consistent with cellular levels.ConclusionIt was confirmed that adipose-derived hsa-miR-448 and hsa-miR-518a-3p affect liver lipid metabolism by acting on liver MST1.