HNRNPA2B1 Up-Regulates MiR-93-5p Through M~6A Mechanism To Promote The Invasion And Migration Of Liver Cancer Cells

Background:Primary liver cancer is one of the most common cancers in the world(especially in my country).According to the latest statistics of the global cancer field published by the American Cancer Society,from 2019 to 2020,the incidence of liver cancer ranks sixth in the world,causing death It ranks third in the world and has seriously threatened human health.There are three pathological types of primary liver cancer.Among them,hepatocellular carcinoma(HCC)accounts for about 90%of all primary liver cancer cases.Compared with other types of tumors,HCC has more complex intratumoral heterogeneity,which greatly reduces the efficacy of HCC targeted therapy.Therefore,studying the molecular mechanism of the occurrence and development of HCC is essential for the development of new treatment strategies for HCC.Research materials and methods:Sequencing data of liver cancer/para-cancerous tissues were obtained and analyzed through the TCGA database.The m~6A barcode reader HNRNPA2B1,which was highly expressed in liver cancer,was screened out,and its expression level was analyzed with the clinical characteristics of liver cancer patients in the TCGA database.QRT-PCR and Western Blots detect the expression of HNRNPA2B1 in normal liver cells and HCC cell lines;predict the m~6A site of pri-miR-93 by SRAMP,and use RIP experiment to detect the binding of HNRNPA2B1 and pri-miR-93 in HCC cell lines,and silence HNRNPA2B1 Then QRT-PCR was used to detect the expression of pri-miR-93 and miR-93-5p,and the RIP experiment was used to detect its effect on the binding of pri-miR-93 and DGCR8;HNRNPA2B1 was silenced in the HCC cell line through the scratch test and Transwell Experiment to study its effect on HCC cell migration and invasion;after silencing or over-expressing miR-93-5p in HCC cell lines,scratch experiment and Transwell experiment to study its effect on HCC cell migration and invasion;use loss/gain of function The method used to verify the function of HNRNPA2B1’s hypothesis that miR-93-5p promotes HCC migration and invasion;Western Blots experiment to silence HNRNPA2B1/silence or overexpress miR-93-5p to verify the effect of HNRNPA2B1/miR-93-5p on the EMT pathway;By Western Blots experiment to silence HNRNPA2B1/silence or overexpression of miR-93-5p to verify the effect of HNRNPA2B1/miR-93-5p on downstream KPNA2 expression;through CHIP experiment overexpression of miR-93-5p to confirm miR-93-5p to KPNA2 The effect of enrichment with H3K27ac.Research result:Sequencing data of liver cancer/para-cancerous tissues were obtained from the TCGA database and analyzed,and 20 m~6A-related protein genes abnormally expressed in liver cancer were obtained.Among them,8 were up-regulated in liver cancer tissues,and 12 were down-regulated in liver cancer tissues.Combined with relevant literature and clinical characteristics,we selected HNRNPA2B1 for follow-up research.In in vitro experiments,QRT-PCR and Western Blots experiments proved that the expression of HNRNPA2B1 in HCC cell lines SMMC-7721 and Hep G2was higher than that of normal liver cell line LO2;SRAMP predicted the presence of m~6A site in pri-miR-93;in HCC cells The RIP experiment in the line proved that HNRNPA2B1 directly binds to pri-miR-93.After silencing HNRNPA2B1 in the HCC cell line,QRT-PCR and RIP experiments proved that HNRNPA2B1 can promote the maturation of pri-miR-93 and up-regulate the expression of miR-93-5p;After silencing HNRNPA2B1 in the HCC cell line,the scratch test and the Transwell test proved that the migration and invasion ability of HCC cells decreased after silencing HNRNPA2B1;the scratch test and the Transwell test after silencing or overexpressing miR-93-5p in the HCC cell line Prove that the ability of HCC cells to migrate and invade after silencing or overexpression of miR-93-5p decreases or increases;use the loss/gain method to verify that HNRNPA2B1 promotes HCC migration and invasion through miR-93-5p;silence HNRNPA2B1/silence through Western Blots experiment Or overexpress miR-93-5p to verify that HNRNPA2B1/miR-93-5p activates the EMT pathway;use Western Blots to silence HNRNPA2B1/silence or overexpress miR-93-5p to verify that HNRNPA2B1/miR-93-5p positively regulates downstream KPNA2 Expression;CHIP experiment overexpression of miR-93-5p verified that miR-93-5p can be used as an enhancer regulator to positively regulate KPNA2.Analysis conclusion:1.HNRNPA2B1 is highly expressed in hepatocellular carcinoma and can be used as an m~6A reader to identify the m~6A site of pri-miR-93 in hepatocellular carcinoma and promote its maturation.2.HNRNPA2B1 activates the EMT pathway of HCC through miR-93-5p,thereby promoting the migration and invasion of hepatocellular carcinoma cells.3.HNRNPA2B1 up-regulates the expression of miR-93-5p,and miR-93-5p may act as an enhancer regulator of KPNA2 to promote its expression.