LncRNA-HOTAIR Modulates The Proliferation And Apoptosis Of Liver Cancer Cells Through MiR-144-5p/SEMA6A Axis

Background:Liver cancer is the most common cause of death worldwide.Patients are frequently diagnosed late in the disease’s progression and have a poor clinical prognosis.The main treatments for liver cancer include liver transplantation,radiation therapy,chemotherapy and immunotherapy.The pathogenesis of liver cancer is difficult to understand.The key to improving the prognosis of liver cancer is early detection and treatment.Cancer cells have stronger proliferation and anti-apoptosis ability than normal cells,as a result,they can influence disease occurrence and progression by inhibiting cancer cell proliferation and hastening apoptosis..Long non-coding RNA is a new type of non-coding RNA with a length of about 200 nucleotides.It regulates the molecular processes of cancer cells in transcription,translation and epigenetic level,and participates in the invasion,metastasis,drug resistance,proliferation and apoptosis of cancer cells.HOTAIR is a very important carcinogenic lncRNA,which is located between the HOXC11 and HOXC12 gene clusters on chromosome 12q13.13.HOTAIR plays a role in cancer by regulating signal pathways,activating EMT,inhibiting phosphorylation,inactivating autophagy,and affecting the process of proliferation and apoptosis,and has become one of the targets for developing targeted therapies for liver cancer.MicroRNA(miRNA)is a small non-coding endogenous RNA with a length of about 20-24 nucleotides.In most cancers,miRNA controls multiple regulation pathways through post-transcriptional gene silencing.Studies have shown that miRNA can act as a tumor suppressor or oncogene,and the imbalance of its expression level regulates the occurrence and development of cancer.In recent years,research on miRNA in cancer prognosis diagnosis and treatment has reached the level of clinical development,such as miR-34a,miR-122,etc.Semaphorins family exists in vivo.It has been found that Semaphorins contain more than 20 members,and each member has a SEMA domain composed of about 500 amino acids,which may promote or inhibit tumor development through various mechanisms.SEMA6A is involved in many biological processes and plays an important role in the development of nervous system,angiogenesis and tumor cell apoptosis.Phosphatidylinositol 3-kinase/serine/threonine protein kinases signal pathway is one of the most common dysfunctional signal pathways in cancer patients.PI3K/Akt signal pathway is involved in many processes such as cell metastasis,metabolism,proliferation and apoptosis.At present,small-molecule inhibitors targeting the main kinases of PI3K/Akt signaling pathway have become the main trend of cancer targeted therapy.Based on relevant literature and previous results,this study speculated that whether the lncRNA HOTAIR/miR-144-5p/SEMA6A pathway affects the proliferation and apoptosis of liver cancer cells by regulating the activity of PI3K/Akt signaling pathway.Objective:The preliminary experimental results showed that HOTAIR negatively regulated miR-144-5p through PRC2 pathway.Sequencing results showed that SEMA6A was differentially expressed when HOTAIR expression level was increased.By exploring and clarifying the mechanism of HOTAIR/miR-144-5p/SEMA6A pathway regulating the activity of PI3K/Akt pathway and then affecting the proliferation and apoptosis of liver cancer cells,it will play a positive role in the targeted treatment of liver cancer.Methods:1.The total RNA of the cells transfected with the over-expression vector was extracted and sequenced.The results of the sequencing were combined with transcriptomics to analyze the differentially expressed miRNA and mRNA.The HOTAIR-miRNAmRNA interaction network was constructed by GO and KEGG enrichment analysis to determine the network-related molecules.2.Screening differentially expressed genes by transcriptome sequencing and qPCR.3.Western blot and qPCR were used to detect the regulatory relationship between HOTAIR,miR-144-5p and SEMA6A.4.The molecular mechanism of miR-144-5p regulating SEMA6A expression was discovered using a double luciferase reporter gene assay.5.MTT,clone formation and EdU cell proliferation test were used to detect the effect of HOTAIR and miR-144-5p on the proliferation of HepG2 and SNU-387 liver cancer cells.6.By using flow cytometry,Western blot,and immunofluorescence assays,HOTAIR and miR-144-5p on the apoptosis of cells were discovered.7.HOTAIR and miR-144-5p’s effect on PI3K/Akt pathway-related components in cells were discovered using a Western blot test.Results:1.The sequencing results showed that there was a positive correlation between HOTAIR and SEMA6A expression.2.The GEPIA database results showed that when the expression level of SEMA6A was high,the survival rate of patients with liver cancer is decreases;When the expression level is low,the survival rate of patients with liver cancer is increases.3.The results of qPCR and Western blot showed that the expression level of SEMA6A mRNA and protein in HepG2 and SNU-387 cells increased after overexpression of HOTAIR,and decreased after knockdown of HOTAIR.4.The results of qPCR and Western blot showed that the expression level of SEMA6A mRNA and protein decreased after transfection of miR-144-5p analog(mimic),and the expression level of SEMA6A increased after transfection of miR-144-5p inhibitor(inhibitor)5.The results of luciferase experiment showed that miR-144-5p was directly bound to 3’-UTR of SEMA6A.6.The results of MTT,clone formation and EdU cell proliferation experiments showed that the cell proliferation ability decreased after knockdown of HOTAIR and transfection of miR-144-5p analog;After overexpression of HOTAIR and transfection of miR-144-5p inhibitor,the cell proliferation ability increased.7.The results of flow cytometry,Western blot and immunofluorescence showed that the apoptosis rate increased,the expression level of c-Caspase3 and BAX increased,the expression level of Bcl-2 decreased,the ratio of Bax/Bcl-2 increased,the nucleus pyknosis and chromatin agglutination increased after knockdown of HOTAIR and transfection of miR-144-5p analog;After overexpression of HOTAIR and transfection of miR-144-5p inhibitor,the apoptosis rate decreased,the expression level of BAX and c-Caspase3 decreased,the expression level of Bcl-2 increased,the Bax/Bcl-2 ratio decreased,and the nuclear morphology did not change significantly.8.Western blot results showed that the expression of p-PI3K and p-Akt decreased and the ratio of p-PI3K/P13K and p-Akt/Akt decreased after knockdown of HOTAIR and transfection of miR-144-5p analog;After overexpression of HOTAIR and transfection of miR-144-5p inhibitor,the expression of p-PI3K and p-Akt increased,and the ratio of p-PI3K/P13K and p-Akt/Akt increased.Conclusion:1.HOTAIR positively regulates SEMA6A,and miR-144-5p negatively regulates SEMA6A by directly binding with the 3’-UTR terminal of SEMA6A.HOTAIR regulates SEMA6A through miR-144-5p.2.HOTAIR/miR-144-5p/SEMA6A axis affects proliferation and apoptosis of liver cancer cells.3.HOTAIR/miR-144-5p/SEMA6A signal pathway affects the proliferation and apoptosis of liver cancer cells by regulating the activity of PI3K/Akt signal pathway.