The Mechanism Of MiR-425 Regulates Lipophagy Via SIRT1 To Promote Sorafenib Resistance In Hepatocellular Carcinoma Cells

Objective:Hepatocellular carcinoma(HCC)is the third most common cancer associated with cancer-related death worldwide.The incidence of hepatitis B virus infection is highest,representing approximately 85%of China's liver cancers.At present,the treatment of liver cancer mainly involves surgery,radiotherapy and chemotherapy.Although sorafenib has been established as an effective drug for advanced HCC,renal cell carcinoma and thyroid cancer,the number of HCC patients exhibiting a complete response to sorafenib is very small.In view of the emerging crisis of sorafenib resistance in hepatocellular carcinoma,further research on drug resistance is urgently needed.Autophagy is a cellular metabolic process in which cells decompose their own components through lysosomes or vacuoles to maintain normal physiological activities and homeostasis.In the process of tumour development,autophagy can provide tumour cells with growth requirements by degrading organelles and proteins.Altering the autophagy level in tumour cells to treat tumours has become a new idea to treat tumours.Recently,a type of selective autophagy called lipophagy that selectively recognizes and degrades lipids has been identified.It plays an important role in regulating lipid metabolism and maintaining intracellular lipid homeostasis.Lipophagy is directly or indirectly regulated by genes,enzymes,transcription regulators and other factors Noncoding RNA(nc RNA)has recently become an important regulator in the signalling pathway related to drug resistance in hepatoma cells.Therefore,pharmacological targeting of these nc RNAs may represent a novel strategy for reversing drug resistance.As an important protein involved in the nutrition-sensing pathway juxtaposed with rapamycin protein and AMP-activated kinase in mammals,silent information regulator 2homologue 1(SIRT1)directly induces autophagy by deacetylating ATG5,ATG7 and LC3.SIRT1 can also deacetylate FOXO to regulate the expression of autophagy regulatory molecules and modulate lipophagy.However,the role of nc RNAs in regulating lipophagy in drug resistance has not been reported.In this study,we aimed to explore the mechanism by which miR-425 regulates lipophagy and its effect on sorafenib resistance.Methods:(1)GEO and TCGA datasets were obtained by R,and difference analysis was performed by LIMMA package,and survival analysis of miR-425 was performed by survive package.(2)The knockdown cell lines of miR-425 were constructed,and the expression of the cell lines was detected after transfection by PCR assay.The drug resistance of miR-425 knockdown to sorafenib was detected by CCK8 assay.(3)Western blot assay was performed on miR-425 knockdown cell lines to detect the expression of LC3.(4)LC3 protein expression under si-miR-425 and si-NC conditions,LC3expression and LD_Sexpression after combined use of BAF were detected by immunofluorescence assay,and drug resistance to sorafenib was detected by CCK8 assay.(5)The binding targets of miR-425 and SIRT1 were analyzed by bioinformatics.(6)The binding of miR-425 to SIRT1 was detected by dual luciferase assay.Results:1.(1)Bioinformatics screening of miRNA indicated that miR-425 may be involved in the process of sorafenib resistance,and is closely related to the poor survival rate and prognosis of patients;(2)PCR results showed that miR-425 was highly expressed in hepatoma cells,and successfully constructed hepatoma cell lines with low expression of miR-425;(3)The results of Transwell assay showed that si-miR-425inhibited the invasion and metastasis of HCC cells;(4).CCK8 assay showed that miR-425 knockout could improve the sensitivity of hepatoma cells to sorafenib;2.(1)The results showed that miR-425 knockdown could increase the expression of LC3?;(2)Immunofluorescence assay was used to detect the changes of lipid droplets after miR-425knockdown,and quantitative analysis showed that the expression of LC3?increased and the LD_Sdecreased;(3)CCK8 experiment showed that BAF could reverse the process of lipophagy and drug resistance;3.(1)Starbase website analyzed the correlation and binding sites of miR-425 and SIRT1;(2)PCR experiment verified the expression of SIRT1 in miR-425 overexpression/knockdown hepatoma cells,which showed that SIRT1 was highly expressed in miR-425 knockdown hepatoma cells;(3)Luciferase reporter gene results showed that miR-425 could bind to SIRT1;(4)Western blot analysis showed that miR-425 could bind to SIRT1 The results of bolt and immunofluorescence showed that SIRT1 knockdown could reverse the expression of LC3 promoted by si-miR-425;Conclusion:1.miR-425 is a potential gene to regulate sorafenib resistance of hepatoma cells;2.High expression of miR-425 indicates poor prognosis;3.miR-425 promotes metastasis and invasion of hepatoma cells;4.Knockdown of miR-425 weakens sorafenib resistance of hepatoma cells;5.Knockdown of miR-425 enhances lipophagy of Hep G2cells;6.Bioinformatics prediction and luciferase experiments prove that miR-425 can bind to SIRT1;7.miR-425 regulates lipophagy through SIRT1.In conclusion,miR-425can regulate the lipophagy process of hepatoma cells through SIRT1,thus mediating the resistance to sorafenib.