LncRNA MKRN3-AS1 Promotes Metastasis And Autophagy Of Hepatocellular Carcinoma By Sponging Multiple MiRNAs

Background:Hepatocellular carcinoma(HCC),a common malignant tumor originating from the liver.And high metastasis rate is the main reason for poor prognosis of HCC patients.In malignant cells,autophagy usually promotes tumor development by providing means for cell survival under stress conditions.In recent years,a large number of long non-coding RNAs(lncRNAs)have been proven to be involved in regulating the occurrence and development of various cancers such as HCC.Our group’s previous transcriptome sequencing data discovered lncRNA MKRN3-AS1,which is significantly overexpressed in HCC.Its clinical significance,biological function and regulatory mechanism in HCC are still unclear.Objects:(1)To verify the expression difference of MKRN3-AS1 in HCC and adjacent tissues and analyze its clinical relevance;(2)To explore the effect and molecular mechanism of MKRN3-AS1 on invasion and migration of HCC cells;(3)To explore the effect and molecular mechanism of MKRN3-AS1 on autophagy of HCC cells.Methods:(1)The abnormal expression of MKRN3-AS1 in HCC tissues was verified by large-sample Real-time Quantitative PCR(qRT-PCR)combined with TCGA database.The correlation between MKRN3-AS1 expression level and clinical pathological indicators of HCC patients was analyzed by Kaplan-Meier method,Cox regression and chi-square test.(2)The effects of MKRN3-AS1 on invasion and migration of HCC cells and epithelial-mesenchymal transition were explored by Transwell assay,cell scratch assay and protein immunoblotting assay(Western blot).NCG mice were used to construct a lung metastasis model of HCC to evaluate the effect of MKRN3-AS1 on HCC metastasis in vivo.(3)Fluorescence in situ hybridization assay was used to explore the subcellular localization of MKRN3-AS1.Bioinformatics tools such as lncRNASNP2,Lnc Base and miRDB were used to predict miRNAs potentially binding to MKRN3-AS1,and their expression levels in HCC cells and tissues were verified by qRT-PCR for correlation with MKRN3-AS1.Luciferase reporter assay was used to verify whether MKRN3-AS1 interacts with miR-601,miR-4487,miR-6881-3p.(4)The effect of MKRN3-AS1/miR-601/ZEB1 axis on the invasion and migration of hepatocellular carcinoma cells was explored by replenishing miR-601,together with phenotype experiment,Luciferase reporting experiment and Western blot.In addition,transmission electron microscopy,immunofluorescence experiments,and replenishing miR-4487 and miR-6881-3p were used to explore the effects of the MKRN3-AS1/miR-4487/ULK1 axis and the MKRN3-AS1/miR-6881-3p/ATG5-ATG7 axis on autophagy of liver cancer cells.Results:(1)MKRN3-AS1 is significantly upregulated in HCC tissues and patients with high expression of this lncRNA have a poor prognosis.The expression of MKRN3-AS1 is significantly correlated with the tumor capsule and alpha-fetoprotein in patients,and its expression level is an independent risk factor for assessing the overall survival of HCC patients.(2)MKRN3-AS1 promotes invasion,migration and autophagy of HCC cells and promotes lung metastasis of HCC in vivo.(3)MKRN3-AS1 is mainly located in the cytoplasm and as micro RNA "sponge" to downregulate of miR-601,miR-4487,and miR-6881-3p in HCC cells and tissues.(4)The MKRN3-AS1/miR-601/ZEB1 axis regulates invasion and migration of HCC cells;the MKRN3-AS1/miR-4487/ULK1 axis and the MKRN3-AS1/miR-6881-3p/ATG5-ATG7 axis regulate autophagy in HCC cells and tissues.Conclusion:MKRN3-AS1 is significantly upregulated in HCC tissues and is mainly located in the cytoplasm.It regulates tumor metastasis through the miR-601/ZEB1 axis and cell autophagy through the miR-4487/ULK1 and miR-6881-3p/ATG5-ATG7 axes.This gene is expected to be a new biomarker for prognosis and a new target for treatment of HCC.