LncRNA HOTAIR Affects The Invasion And Migration Of Liver Cancer Cells Through The MiR-144-5p/FRK Axis

Background:Liver cancer is the most common malignant tumor in the world and the second leading cause of cancer-related death worldwide.Liver cancer accounts for 90%of primary liver cancer,which is highly malignant and has poor prognosis.At present,the potential molecular mechanism of liver cancer has not been discovered,so surgery,radiotherapy and chemotherapy are still the main treatment methods for hepatocellular carcinoma.However,hepatocellular carcinoma cells are highly invasive and prone to metastasis,which leads to poor prognosis and low survival rate.Non-coding RNA can be involved in tumorigenesis,invasion and metastasis,metabolism,apoptosis and drug resistance.Non-coding RNA can be divided into two types according to its length:small non-coding RNA（<200nt）and long non-coding RNA（lncRNA>200nt）.HOTAIR is a lncRNA with a length of 2158 bp,which is located on the twelfth chromosome.HOTAIR plays a role in various diseases such as tumor,diabetes,heart disease and so on.HOTAIR is highly expressed in a variety of cancers and is now considered as an oncogene.The high expression of HOTAIR in tumors can promote tumor invasion and metastasis,lead to tumor recurrence,affect the prognosis of patients,and shorten the survival time of patients.Therefore,HOTAIR has been regarded as a new tumor marker for hepatocellular carcinoma.In addition,HOTAIR can also regulate a variety of microRNAs.MicroRNA（miRNA）is a 19-25 nt endogenous small non-coding RNA with a highly conserved structure that can directly bind to the 3’-end non-coding region（3’UTR）of messenger RNA（mRNA）to regulate transcription Gene expression levels afterward,thereby inhibiting the transformation of proteins in various biological and pathological processes.In addition,miRNAs have been confirmed to play an important role in a variety of tumors,acting as tumor-promoting or tumor-suppressing genes,involved in the regulation of tumorigenesis and tumor cell proliferation,differentiation,invasion and metastasis,metabolism and apoptosis.Fyn family related kinase（FRK）is a non-receptor tyrosine kinase with tissue specificity.FRK plays an inhibitory role in brain cancer and breast cancer,and promotes cancer in lung cancer,pancreatic cancer and liver cancer.FRK is a multifunctional signal transduction molecule,which can participate in the regulation of multiple signal pathways and is an important regulatory protein of tumor.Aims:It has been proved by studies that HOTAIR can promote the invasion and migration of hepatoma cells.Sequencing results show that the expression of miR-1445p decreases and the expression of FRK increases when HOTAIR is overexpressed.However,the molecular mechanism of HOTAIR regulating miR-144-5p is not clear,and whether HOTAIR regulates FRK through miR-144-5p is not known.By studying whether HOTAIR regulates FRK through miR-144-5p,and the mechanism of HOTAIR regulating miR-144-5p and miR-144-5p regulating FRK,the new molecular mechanism of HOTAIR promoting invasion and migration of hepatocellular carcinoma cells was clarified,which provided a new idea for preventing invasion and migration of hepatocellular carcinoma and a new target for treating hepatocellular carcinoma.Methods:1.We used RNA-sequencing（RNA-Seq）-based SmallRNA-omics and transcriptomic analysis combined with bioinformatics prediction websites and TCGA,GEO,GTExpert data mining websites,to link lncRNA-miRNA interactions with miRNA target gene mRNA interactions Combined,the HOTAIR-miRNA-mRNA regulatory network was established,and the regulatory role of the HOTAIR-miRNA-mRNA regulatory network in HCC was systematically studied.2 The expressions of HOTAIR and FRK in hepatocellular carcinoma were detected by q-PCR.The effects of HOTAIR and miR-144-5p on the invasive ability of HepG2 and SNU387 cells were detected by Transwell assay.4 The effects of HOTAIR and miR-144-5p on lateral migration of HepG2 and SNU387 cells were detected by scratch test.5 The effect of HOTAIR on the expression of miR-144-5p and FRK was detected by qPCR.6 The effect of HOTAIR on the expression of FRK and EMT related genes was detected by Western Blot test.7 Through q-PCR and CHIP assay,it was proved that HOTAIR indirectly regulated the expression of miR-144-5p through PRC2 pathway.8 It was proved by double luciferase reporter gene experiment that miR-144-5p directly regulated the expression of FRK.Results:1 Through the analysis of sequencing results,it was found that the expression of miR144-5p was down-regulated and the expression of FRK was up-regulated when HOTAIR was over-expressed.2 Through q-PCR assay,compared with normal liver tissues,the expression levels of HOTAIR and FRK in hepatocellular carcinoma tissues were increased.3 Overexpression of HOTAIR and miR-144-5p decreased,while FRK RNA and protein increased.When HOTAIR was knocked down,the expression of miR-144-5p increased,and the expression of FRK RNA and protein decreased.The expression levels of RNA and protein of FRK were decreased after transfection of miR-144-5p mimics;The expression level of RNA and protein of FRK transfected with miR-144-5p inhibitor increased.5 After transfection of sh-HOTAIR and miR-144-5p mimics,HepG2 and SNU-387 cells showed weaker ability of invasion and lateral migration.Western Blot assay showed that E-cadherin protein expression increased,while N-cadherin,Vimentin,MMP2,MMP9 protein expression decreased.HepG2 and SNU-387 cells were transfected with p-HOTAIR and mir-144-5p inhibitor,respectively.The expression level of E-cadherin protein was decreased by Western Blot assay,while the expression level of N-cadherin,Vimentin,MMP2 and MMP9 protein was increased.The expression of miR-144-5p was up-regulated by q-PCR.Transfection of miR-1445p mimics,Western Blot detection of EZH2 protein expression level decreased.Transfection of miR-144-5p inhibitor and Western Blot showed that the expression of EZH2 protein increased.8 CHIP assay detected the direct binding of EZH2 and H3K27ME3 to miR-144-5p TSS region.9 double luciferase reporter gene test showed that miR-144-5p directly bound to FRK.Experimental conclusions:1 HOTAIR can act as a molecular scaffold and negatively regulate the expression of miR-144-5p through PRC2 pathway.2 HOTAIR can positively regulate the expression of FRK,miR-144-5p can bind to 3’UTR of FRK and negatively regulate the expression of FRK.3 HOTAIR can promote the invasion and migration of hepatocellular carcinoma through EMT pathway,and miR-144-5p can inhibit the invasion and migration of hepatocellular carcinoma through EMT pathway.4 HOTAIR/miR-144-5p/FRK axis can regulate the invasion and migration of hepatocellular carcinoma.