Functional Study On Candidate Gene Tmem176a Associated With Lipid Metabolism In Mice

For many years,researchers in China and abroad have been committed to finding potential genes involved in the high-fat response in the liver to further enrich the gene family of lipid metabolism.However,lipid metabolism is a complex trait,and its phenotype is subject to genetic action on the one hand,and environmental factors on the other hand.Therefore,in this study,we explored lipid metabolism in mice from both genetic and environmental aspects.First,in this study,Tmem176a was used as a candidate gene for lipid metabolism,and the cell model was preliminarily selected and constructed on the basis of mouse liver cancer Hepa1-6and Hepa1c1c7 cells.The lowest lethal concentrations of Hepa1-6 and Hepa1c1c7 mouse hepatoma cell lines were obtained by puromycin resistance screening.sh RNA-Tmem176a(designated as Tmem176a^KD)and sg RNA-Tmem176a(designated as Tmem176a^KO)were respectively transfected into HEK293T cells for lentivirus packaging.The collected virus liquid was infected with Hepa1-6and Hepa1c1c7 cells,and the knocked-down and knock-out positive stably transfected cells were obtained after four days of puromycin treatment and screening.The knock-down efficiency of Tmem176a^KD detected by QPCR in Hepa1-6 and Hepa1c1c7 mouse liver cancer cells was 0.64 and0.23,respectively.Next,that knock-out efficiency of Tmem176a^KD in Hepa1-6 cell was detected by T7EI enzyme.Finally,QPCR was used to detect the expression levels of genes responsible for lipid metabolism after knock-down and knock-out of Tmem176a.In this study,Tmem176a^KD-Hepa1c1c7was finally identified as a subsequent experimental cell model.Second,to facilitate the identification of whether the candidate gene Tmem176a is involved in lipid metabolism?We performed preliminary verification on the function of lipid metabolism candidate gene Tmem176a.First,a lipid metabolism positive gene Srebf2 and a negative gene Hlx were introduced,and QPCR detected the expression levels of lipid metabolism genes after the positive genes Srebf2(si-Srebf2)and the negative gene Hlx(si-Hlx)were knocked down,respectively.Then the expression levels of lipid metabolism genes after Tmem176a^KD were compared with those of si-Srebf2 and si-Hlx,and visualized mapping was performed by R language.The results showed that:First,the expression pattern of lipid metabolism gene after Tmem176a^KDwas more similar to that of si-Srebf2 gene.Second,among the genes related to TG synthesis,Fasn,Dgat1,Elovl 6,and Scd1 were significantly down-regulated after Tmem176a^KD,while among the genes related to TG decomposition,Atgl,Hsl,and Lipc were significantly up-regulated.Hmgcs1,Sqle,and Acat1 were all significantly up-regulated in genes related to CHOL synthesis,while Srebf1 was significantly up-regulated in genes related to CHOL decomposition.Third,after Tmem176a^KD,the relative m RNA expression level of the gene Sar1a related to chyle microparticle transport changed by about 0.49 times,and the protein expression level was significantly decreased.At the same time,over-expression of Sar1a resulted in a 158-fold change in m RNA relative to the control group and an approximately 2.8-fold increase in expression of the lipid metabolism candidate gene Tmem176a.Finally,cell biochemical level detection showed that the lipid droplet area was further reduced after Tmem176a was knocked down by oil red O staining,suggesting that the lipid level was down-regulated.LDL-C,TG and CHOL contents also decreased to different degrees.Conclusion:The results of cell biochemical tests such as QPCR,Western Blot,and oil red O staining further confirmed that the lipid metabolism candidate gene Tmem176a was involved in the lipid metabolism of mouse hepatoma Hepa1c1c7 cells.Finally,in this study,the Tmem176a^KD-Hepa 1c1c7 cell model was constructed by introducing environmental factors combined with Tmem176a^KD.The results showed that the differences between the experimental group and the control group were more significant at the molecular and cellular levels.First,the tolerance threshold of cells to rapamycin was found through experiment,and the optimal concentration of rapamycin was determined to be 1n M.Then Hepa1c1c7 cells were treated with the combination of Tmem176a^KD and rapamycin.The results of cell biochemical levels such as QPCR,Western Blot,and oil red O staining showed that the intracellular lipid levels were significantly decreased after the combination of rapamycin and Tmem176a ^KD(Rapa+Tmem176a^KD),with statistical significance.In summary,the cell model for lipid metabolism candidate genes constructed in this study was successful.The method has reference value for subsequent lipid metabolism gene mining.