MiR-200c-3p Regulates DUSP1/MAPK Pathway In The Nonalcoholic Fatty Liver After Sleeve Gastrectomy

Objective:Obesity is closely related to nonalcoholic fatty liver disease(NAFLD).Weight loss is the only verified effective method to treat NAFLD at present,and metabolic surgery induces the rapid and durable remission of obesity.However,the effect of metabolic surgery on NAFLD is still controversial,and the underlying mechanism is still unclear.The expression levels of hsa-miR-200c-3p and dual-specificity protein phosphatase 1(DUSP1)were tested in human serums.The effects of sleeve gastrectomy(SG)on body weight,alanine aminotransferase(ALT),aspartate aminotransferase(AST),total cholesterol(TC),three acyl glycerin triacylglycerol(TG),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),mmu-miR-200c-3p,DUSP1?phospho-extracellular regulated protein kinases(p-ERK1/2)?phospho-c-Jun N-terminal kinases(p-JNK),and phospho-p38 mitogen-activated protein kinases(p-p38)were studied in obese C57BL/6J mice with NAFLD.Ribonucleic acid interference(RNAi)technology and luciferase reporter gene assay were used in the cellular and molecular experiments to verify the targeting interaction relationship between hsa-miR-200c-3p and DUSP1.The study was designed to provide a deeper theoretical basis for the improvement of NAFLD following the SG procedure,and search a new marker or drug tar get for the diagnosis and treatments of NAFLD.Methods:In the first part,the serums of healthy adults and those underwent LSG complicated with NAFLD were harvested.The clinical and biochemical characters such as BMI,ALT,AST,TG,TC,HDL-C,LDL-C,and liver color ultrasound were recorded at preoperation,the first month,the third month,and the sixth month outpatient visits after laparoscopic sleeve gastrectomy(LSG).The dynamic expression changes of hsa-miR-200c-3p and DUSP1 were determined with reverse transcription-polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA)at the follow-up time points.In the second part,at the cellular and molecular level,we used hsa-miR-200c-3p mimic,hsa-miR-200c-3p inhibitor,and H₂O₂to treat Hep G2 cells,then observed the changes of DUSP1 followed by the changes of miR-200c-3p with RT-PCR and Western Blot methods.The target genes of miR-200c-3p were predicted with online databases.Luciferase assay was used to verify the expression level of DUSP1 at the protein level.In the third part,C57BL/6J mice were fed with high-fat diets(HFD)to establish NAFLD animal models.After 12 weeks of modeling,one mouse was randomly selected from the control group(CON)and the NAFLD group,respectively.The liver tissue sections were treated with hematoxylin-eosin staining(HE)and oil red O staining.After the successful modeling was verified,the NAFLD mice were randomly divided into the NAFLD+SHAM group,NAFLD+SG group,and NAFLD+food restriction(FR)group.Food intake and body weight of mice in each group were measured continuously for six weeks after the operation,and mice were sacrificed six weeks after surgery.The expression level of miR-200c?DUSP1 m RNA?DUSP1?p-ERK1/2?p-JNK and p-p38 in the liver of each group were detected with RT-PCR and Western Blot methods.Meanwhile,the blood of the angular vein was harvested to examine the levels of ALT,AST,TC,TG,HDL-C,and LDL-C.The morphological changes of liver cells were observed through the HE and oil red O staining.Results:In the first part,1.No severe complications occurred during the perioperative and follow-up periods of the clinical cases in this study.According to the liver ultrasound assessments of NAFLD,one patient remitted at the 1st month after the operation,four patients remitted at the 3rd month after the operation,one patient remitted at the 6th month after the operation,and one patient showed no significant changes during the follow-up period;2.The expression levels of DUSP1 in obese patients with NAFLD who received LSG surgery were significantly lower than that in healthy adults without NAFLD(p<0.01),the expression levels of DUSP1 showed an uptrend with no significant difference at the1st-month post-operation(p>0.05),the expression levels of DUSP1 were up-regulated at the 3rd-month post-operation(p<0.05),the difference was more evident at the 6th-month post-operation(p<0.05).3.Before the surgery,the expression of hsa-miR-200c-3p was significantly up-regulated in obese NAFLD patients who received LSG surgery compared with healthy adults without NAFLD(p<0.001),and the expression level of hsa-miR-200c-3p showed a downward trend with no statistical significant difference at the 1st-month post-operation(p>0.05).The expression level of hsa-miR-200c-3p was down-regulated at the 3rd-month post-operation(p<0.01),the difference was more obvious at the 6th-month post-operation(p<0.01).In the second part,we found that DUSP1 was a possible regulatory target gene of miR-200c-3p with bioinformatics analysis.We found that the expression levels of DUSP1 and DUSP1 m RNA were significantly decreased in the hsa-miR-200c-3p mimic group in the Hep G2 cell line(p<0.05),while the expression levels of DUSP1 and DUSP1 m RNA in the hsa-miR-200c-3p inhibitors group showed an increasing trend(p<0.01).The expression levels of DUSP1 and DUSP1 m RNA in the hsa-miR-200c-3p mimic and inhibitor group were significantly decreased after treatment with 100u M H₂O₂(p<0.001).The expression level of hsa-miR-200c-3p showed an increasing trend(p<0.01),which proved that hsa-miR-200c-3p might target and regulate the expression of DUSP1.Further luciferase assay confirmed that DUSP1 was a direct target of miR-200c-3p.In the third part,1.There was no death during the modeling of NAFLD;2.The mortality of SG was 14%in the NAFLD+SG group,and no mice died in the other groups;3.The preoperative body weight of mice in the NAFLD+SG group,the NAFLD+SHAM group,and the NAFLD+FR group were significantly higher than that in the CON group(p<0.001),in which the successful modeling was proved.At the 6th week postoperation,the bodyweight of the NAFLD+SG group was lower than the NAFLD+FR group and NAFLD+SHAM group(p<0.01);4.The serum levels of TG,TC,ALT,AST were significantly increased in the NAFLD+SHAM group compared with the CON group(p<0.001),HDL-C was significantly decreased(p<0.001).Compared with the NAFLD+SHAM group and NAFLD+FR group,the serum level of TG,TC,ALT,and AST in the NAFLD+SG group were significantly decreased(p<0.001),HDL-C was significantly increased(p<0.01).5.At the 6th week after surgery,little vesicular steatosis was observed in the NAFLD+SG group by HE staining,and lamellar steatosis and fibrosis were observed in the NAFLD+SHAM group.Inflammatory cell infiltration in the central vein and blood sinusoid were observed in the NAFLD+FR group,and no steatosis was observed in the CON group.6.The background was lighter in the NAFLD+SG group with red oil staining,while the NAFLD+SHAM group presented a wide range of red fat droplets,and the blue nuclei were challenging to be stained.In the NAFLD+FR group,obvious pointlike necrosis and fatty and eosinophilic changes of liver cells were still observed,which were slightly less serious than those in the NAFLD+SHAM group but not as obvious as those in the NAFLD+SG group.In the CON group,only a few intracellular fat droplets were positive;7.At the 6th week postoperatively,the expression level of mmu-miR-200c-3p in the liver tissue of the NAFLD+SG group was decreased compared with that of the NAFLD+SHAM group and NAFLD+FR group(p<0.001);8.At the 6th week after surgery,the expression level of DUSP1 in liver tissue of the NAFLD+SG group was increased compared with that of the NAFLD+SHAM group and the NAFLD+FR group(p<0.001).Conclusions:A novel mechanism was identified in which miR-200c-3p regulates the MAPK-dependent signals that were linked to the promotion of hepatosteatosis via DUSP1.The findings suggested that miR-200c-3p should be further explored as a potential target for the treatments of NAFLD.