The Preliminary Study On The Regulation Of Sln And Sank1 Gene Expression And Its Mechanism By Palmitate

Palmitic acid(PA)is a saturated fatty acid found in everyday life.It converts it into free fatty acids in the blood when the body ingests too much palmitic acid.It will affect the synthesis of proteins and cause more metabolic diseases.Some studies have found that excessive fatty acids can affect Ca2+ transport in cells and have a certain influence on the activity of sarcoplasmic reticulum(endoplasmic reticulum)Ca2+ ATPase(SERCA).In skeletal muscle,Sln and s Ank1 are important regulatory factors of SERCA1,and play a key role in the coupling of body heat production and muscle contraction.It needs to be further studied that the relationship between the intake of excess fatty acids and Sln and s Ank1 which are the main regulatory factors of SERCA1.And the mechanism between them are not clear.This study first explored whether PA activates the unfolded protein response(UPR)in endoplasmic reticulum stress and whether PA regulates the expression of the target genes Sln and s Ank1.Next,we investigated whether the three UPR pathways(PERK,IRE1,and ATF6 pathways)were involved in PA regulation of Sln and s Ank1 gene expression.For the study of the PERK and IRE1 pathways,pathway inhibitors and transfection of interfering segments were mainly used to inhibit and block the pathway.and q RT-PCR was used to check the resistance of the two pathways at the m RNA level and using western blot at the protein level.In this case,the expression of Sln and s Ank1 genes was detected under successful blocking conditions.For the ATF6 pathway,the CRISPR/Cas9 technique is mainly used to construct Cas9-ATF6single-target and double-target knockout cell lines.The knockout cell line is used to detect the expression of the target gene.Thus,it was initially determined whether the three pathways were involved in the expression of palmitate-induced musclin gene.The experimental results showed that when the PERK pathway was blocked,the downregulation of Sln gene expression by PA was significantly attenuated,but there was no significant effect on the down-regulation of s Ank1 gene.However,PA down-regulated the expression of s Ank1 gene significantly when the IRE1 pathway was blocked.A study of the ATF6 pathway knockout cell line found that the effect of PA on the Sln and s Ank1 genes in the ATF6 knockout cell line was approximately the same as that in the ordinary cell line.Based on the above results,it can be initially confirmed that the PERK pathway participates in the regulation of PA expression in the Sln gene,the IRE1 pathway participates in the PA regulation of s Ank1 gene expression,and theATF6 has no effect on PA regulation of the expression of the two genes.This study provided new evidences for the effect of excessive palmitic acid on the body and provided new ideas for the next study of Ca2+ homeostasis and metabolism balance.