| Objectives Hepatocellular carcinoma(HCC)is one of the most common malignancies in the world.It is highly invasive and has a poor prognosis,with a5-year survival rate of only 5%.The recurrence rate of 5 years after early radical surgical resection is still as high as 70%.It is particularly important to clarify the molecular mechanism of HCC invasion and metastasis.Long non-coding RNA(Lnc RNA)is a class of non-coding RNA whose length is more than 200 nucleotides.At present,many scholars have found that various Lnc RNAs are involved in the occurrence and development of human cancer,and the study of the relevant mechanism of Lnc RNAs provides new targets and new ideas for cancer treatment.Epidermal growth factor receptor protein tyrosine kinase Substrate 15(EPS15)and Lnc RNA Epidermal growth factor receptor protein tyrosine kinase Substrate 15-Antisense(EPS15-AS)are menbers of epidermal growth receptors Factor receptor protein(EGFR)family,Lnc RNA EPS15-AS is the antisense Lnc RNA of EPS15.he purpose of this project is to investigate the role of Lnc RNA EPS15-AS in regulating the malignant biological behaviors of HCC by targeting EPS15 and its mechanism,so as to improve the diagnosis and treatment efficiency of HCC,further predict the survival of patients,,and developing new targeted drugs.Methods1.To analyze the expression of EPS15 and Lnc RNA EPS15-AS1 in hepatocellular carcinoma and normal tissues by bioinformatics database,to analyze the correlation between EPS15 and Lnc RNA EPS15-AS1 and clinical features and prognosis of HCC,and to analyze the possible pathways of EPS15 and Lnc RNA EPS15-AS on HCC.2.To analyze the expression of Lnc RNA EPS15-AS1 and EPS15 in clinical hepatocellular carcinoma tissues and paracancer tissues of 27 cases,and their correlation with clinicopathological features and prognosis of patients with HCC.3.PCR was used to detect the expression level of Lnc RNA EPS15-AS1 in liver cancer cells(Hep G2,Hep3 B,SMMC7721,Bel7404,PLC/PRF/5).The stable knockdown cell lines(SMMC7721-EPS15-AS1-KD,PLC/PRF/5-EPS15-AS1-KD)and the stable overexpressed cell lines(Hep3B-EPS15AS1-OE,Bel7404-EPS15AS1-OE)were established.The expression of Lnc RNA EPS15-AS1 and EPS15 m RNA were detected by PCR.Western blot was used to detect EPS15 and epithelial-mesenchymal Transition(EMT)related proteins.CCK-8assay,flow cytometry,Transwell assay,Matrigel-Transwell assay and rhodamine labeled peptide fluorescence labeling were used to detect the proliferation,apoptosis,migration,invasion and cytocytotic morphology of the cells.4.EPS15-overexpressed hepatoma cell lines(7404-EPS15-AS1-OEEPS15-OE,Hep3B-EPS15-AS1-OE-EPS15-OE)were established after stably transfected lentiviral vector and packed with overexpressed Lnc RNA EPS15-AS1 recombinant plasmid.Compared with the effects of EPS15-AS1 overexpression(7404-EPS15-AS1-OE,Hep3B-EPS15-AS1-OE)on proliferation,apoptosis,migration,invasion,cytoskeleton morphology and EMT-related protein levels of hepatocellular carcinoma cells.5.7404-EPS15-AS1-OE and 7404-EPS15-AS1-OE-EPS15-OE cells were inoculated subcutaneous into BALB/ C nude mice,with 8 cells in each group.The size of the tumor was observed.Six weeks later,the tumor tissue was collected and the degree of malignancy was observed by HE staining.Immunohistochemical staining of Ki67,e-cadherin,ZO-1,N-cadherin,and Vimentin proteins in tumors.6.Western Blot was used to detect the expression of EPS15/EGFR/PI3K/AKT/m TOR signaling pathway after Lnc RNA EPS15-AS1 overexpression.Results1.EPS15 was abnormally high in liver cancer tissues,while Lnc RNA EPS15-AS was significantly low in liver cancer tissues.The expression of Lnc RNA EPS15-AS1 was correlated with the expression of EPS15;Abnormal expression of EPS15 and Lnc RNA EPS15-As is associated with survival and prognosis of HCC.EPS15 and lnc RNA EPS15-AS act on liver cancer through a variety of pathways.2.The expression of Lnc RNA EPS15-AS1 in HCC cells was lower than that in normal cells;Silencing Lnc RNA EPS15-AS1 can promote proliferation,migration and invasion of HCC cells and inhibit apoptosis of HCC cells,while overexpressing Lnc RNA EPS15-AS1 can inhibit proliferation,migration and invasion of HCC cells and promote apoptosis of HCC cells.Silencing or overexpressing Lnc RNA EPS15-AS1 can affect the expression of EMT-related proteins and cytoskeletal morphology of HCC cells.3.This study demonstrated that Lnc RNA EPS15-As could regulate the expression of EPS15 in HCC from the level of RNA and protein.In vivo and in vitro experiments demonstrated that Lnc RNA EPS15-AS targets EPS15 to regulate the malignant biological behavior of hepatocellular carcinoma through EPS15/EGFR/PI3K/AKT/m TOR signaling pathway.Conclusions1.Lnc RNA EPS15-AS may play an inhibitory role in the development of HCC.2.Lnc RNA EPS15-AS regulates the expression of EPS15 in hepatocellular carcinoma.3.Lnc RNA EPS15-AS targets EPS15 and regulates malignant biological properties of hepatocellular carcinoma. |
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