The Mechanism Of MicroRNA-375 Target ATG14 To Restrain Autophagy And Sensitize Hepatocellular Carcinoma Cells To Sorafenib

Objective: Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer death,ranking fourth among all malignant tumors in China.For HCC patients,current treatment is limited and cannot improve their survival rate.Sorafenib is a multi-kinase inhibitor that affects both cell surface tyrosine kinase receptors and intracellular serine/threonine kinases.Many factors can lead to drug resistance in liver cancer.One important factor is autophagy activation.In recent years,a large number of studies have shown that autophagy is closely related to tumors,and autophagy can regulate tumor formation,proliferation,metastasis,and energy metabolism.Studies have shown that autophagy can inhibit tumor growth,proliferation,and metastasis.At the same time,it can help maintain the balance of tumor cell product synthesis and metabolism.In addition,autophagy-related genes(ATGs)plays a dual role in the occurrence and development of a variety of cancers.Micro RNAs are a class of endogenously expressed,short non-coding RNAs,which post-transcriptionally regulate gene expression.It can affect many biological processes,such as cell development,infection,immunity and carcinogenesis.Micro RNAs are involved in various stages of autophagy,including the phagophore induction,nucleation and expansion,as well as the maturation of autolysosome and autophagosome,and play a regulatory role.In this article,we demonstrated that mi R-375 sensitizes hepatocellular carcinoma cells to sorafenib by blocking sorafenib-induced autophagy.We also provide evidence that a key autophagic protein,ATG14,which is a direct autophagy-related target of mi R-375.So we come to the conclusion that mi R-375-ATG14 is heavily involved in the development of sorafenib resistance in HCC.Methods:1.Determine the relationship between sorafenib and autophagy using bioinformatics methods such as GEO database and KEGG analysis.2.The expressions of LC3 and P62 in Huh7 treated with sorafenib were detected by western blot.The the level of autophagy under sorafenib treatment were detected by immunofluorescence in HCC.Transmission electron microscopy was used to observe the autophagosomes of HCC treated with sorafenib.3.After blocking autophagy in HCC to do the following experiment: MTT assay,Colony formation and Flow cytometric analysis of apoptosis.4.Use TCGA database,GEO database,KEGG analysis and other bioinformatics methods to determine the research object was mi R-375.Using plastid transfection technology and real-time PCR was used to detect transfection efficiency.5.Huh7 were transfected with mi R-375 mimics.The subjects were divided into 4 groups: control group,sorafenib treatment group,mi R-375 mimics group,and sorafenib treatment with transfection mi R-375 mimics group.Western blot was used to detect the expression of LC3 and P62.Immunofluorescence was used to observe the expression of LC3 content.Transmission electron microscopy was used to observe the status of autophagosomes.6.Bioinformatics methods were used to screen the target of mi R-375,and real-time PCR was used to detect the expression of the screened indicators in HCC cells.The luciferase reporter was used to verify whether mi R-375 was directly targeted binding to ATG14.After co-transfection of mi R-375 mimics and pc DNA3.1-ATG14,the cell viability status of the transfection group and the control group was detected by MTT assay under the treatment of sorafenib,and the colony formation was used to detect the change of cell proliferation level.Results: 1.Sorafenib treatment group was positive in autophagy gene enrichment.2.Western blot showed the increase of LC3-II / LC3-I ratio and the decrease of P62 expression in HCC treated with sorafenib.3.Immunofluorescence observation showed that LC3 green fluorescence increased in HCC cells treated with sorafenib.4.The autophagosomes was observed by transmission electron microscope after the treatment of sorafenib.5.The number of HCC cells treated with BAF-A1 and transfected with SiULK was decreased by MTT assay,Colony formation and Flow cytometric analysis respectively(P< 0.01).6.Mi R-375 had the lowest expression in Huh7 treated with sorafenib,so the research object was determined to be mi R-375.8.Western blot results showed that the expression of LC3-II / LC3-I ratio in sorafenib-treated and transfected with mi R-375 mimics group was lower.9.Immunofluorescence showed that the content of green fluorescent spots in LC3 in sorafenib-treated and transfected mi R-375 mimics group was relatively low,and the number of sorafenib-treated groups was decreased.10.The number of autophagosomes in the sorafenib-treated and transfected mi R-375 mimics group decreased.At the same time,cell proliferation experiments(MTT),clone formation and flow cytometry detection of apoptosis experiments observed that the number of cells treated with sorafenib with transfection mi R-375 mimics was decreased(P<0.01).11.Bioinformatics methods were used to screen the target of mi R-375,and real-time PCR was used to detect the expression of the screened indicators in hepatocellular carcinoma cells.ATG14 was found to be the most obvious.12.Luciferase reporter assay showed binding affinity between mi R-375 and 3′-UTR of ATG14 m RNA(P<0.05).13.Western blot results showed that ATG14 protein levels were lower in the sorafenib-treated and transfected mi R-375 mimics group compared to the control group.14.Rescue experiments showed that the cell viability of cotransfection of mi R-375 mimics and pc DNA3.1-ATG14 was increased by MTT assay.The colony formation experiment showed that the cell proliferation level was increased.Western blot results showed that the expression of apoptotic proteins was reduced.Finally,using the bioinformatics website,it was found that mi R-375 was negatively correlated with ATG14.Conclusion: 1.There was a functional correlation between autophagy and sorafenib in GEO liver cancer clinical tissue samples.In addition,sorafenib can promote the increase of autophagy expression in HCC cells.After inhibiting autophagy,sensitized HCC cells to sorafenib.2.The research object was identified as mi R-375.Mi R-375 inhibits sorafenib-induced autophagy and can increase the sensitivity of HCC cells to sorafenib.3.mi R-375 regulates autophagy by targeting ATG14,which affects the sensitivity of HCC cells to sorafenib.