The Function And Molecular Mechanism Investigation Of SBF2-AS1 Promoting Lung Adenocarcinoma Malignant Progression

Background:Lung cancer is the leading cause of cancer death worldwide.During the past decades,the pathological constitution of lung cancer has gradually changed and lung adenocarcinoma(LUAD)has becoming a most prevalent subtype accounting for approximately 70% of total lung cancer,especially in Eastern Asia.Screening new LUAD specific theraputic targets has been the current hotspot in lung cancer fields.At present,the study of transcripology has become hotspot in the field of cancer research.Over 75% of human transcriptome products are long non-coding RNA(lnc RNA)which are larger than 200 nt.In the past 5 years,there have been many important research papers published about the regulation of oncogenesis,proliferation,invasion and metastasis of cancer.Therefore,further exploration of new molecular targets of lung adenocarcinoma from the perspective of lnc RNA will help to reveal the new mechanism of the occurrence and development of lung adenocarcinoma,and provide new clues for the diagnosis and treatment of lung cancer.Methods:We first analyzed LUAD transcriptome data from The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)database by weighted gene network co-expression analysis(WGCNA).We discovered that lnc RNA SBF2-AS1 was upregulated in early LUAD.It indicated that the Lnc RNA SBF2-AS1 may closely relate to the malignant progression of early lung adenocarcinoma.We further verify the clinical characteristics of lnc RNA SBF2-AS1 and its correlation with the prognosis by TCGA data.Then cell proliferation and cycle experiments,a subcutaneous tumor bearing model in nude mice and a human tumor xenograft(PDTX)model were constructed.These in vivo and in vitro experiments showed the possible biological functions of lnc RNA SBF2-AS1.RNA immune coprecipitation(RIP),biotin-based pulldown(biotin-pulldown),dual-luciferase reporter gene system was used to study the molecular mechanism of lnc RNA SBF2-AS1 of biological function.Results:1 Through the analysis of LUAD transcriptome data from TCGA and GEO database by WGCNA.Five early LUAD-specific lnc RNAs were filtered out and only SBF2-AS1 was upregulated in LUAD.High expression of SBF2-AS1 indicates poor survival of LUAD,especially the early stage LUAD,but not lung squamous cell carcinoma.2 Lnc RNA SBF2-AS1 is closely related to the prognosis of LUAD.By gene set enrichment analysis(GSEA)of lung cancer samples,it is found that SBF2-AS1 expression is negatively correlated with prognosis.SBF2-AS1 expression was then analyzed online using microarray data of 1145 lung cancer patients,showed that high SBF2-AS1 expression is associated with poor overall survival in LUAD,but not with lung squamous cell carcinoma(LUSC).This relationship is more obvious in early LUAD.The same results are also obtained in the further validation of TCGA data.These results suggest that SBF2-AS1 may be a biomarker for poor prognosis.3 SBF2-AS1 can promote the proliferation of LUAD cells in vivo and in vitro.We have designed specific small interfering RNA(si RNA)and construct the over-expression plasmid of SBF2-AS1.After interfering with lung cancer cell line A549,the cell cycle related genes changed most obviously,suggesting that SBF2-AS1 may be related to cell cycle and proliferation,and exert biological function through regulation of cell cycle and proliferation.After the interference of SBF2-AS1 in the lung cancer cell lines,the cell cycle was blocked at G1 phase.Cyclin D1 and P21 are important cell cycle related proteins related to G1 phase.After interfering and overexpressing SBF2-AS1,Cyclin D1 and P21 expression level has changed significantly,further confirming that SBF2-AS1 may be related to cell cycle.Further in vitro experiments showed that interference with SBF2-AS1 could significantly inhibit the proliferation of LUAD cells,while overexpression of SBF2-AS1 results in the opposite.By constructing the PDTX model,we found that the growth rate of adenocarcinoma of the lung was significantly inhibited after silence of SBF2-AS1.4 SBF2-AS1 regulates transcription factor E2F1 through endogenous competitive RNA(ce RNA)mechanism.Through nuclear separation experiment,SBF2-AS1 is mainly distributed in cytoplasm,suggesting that SBF2-AS1 may regulate LUAD cells at post-transcriptional level.The ce RNA mechanism is one of the important mechanisms for lnc RNA to play the post-transcriptional level of regulation.We examined the SBF2-AS1 sequence and found 4 micro RNAs binding site by Target Scan and Miranda mi RNA prediction programs.RNA immunoprecipitation(RIP)assay demonstrated SBF2-AS1 could bind with Ago2 protein.Biotin pull-down experiment suggested that mi R-338-3p and mi R-362-3p could bind to SBF2-AS1.In addition,overexpression of mi R-338-3p/362-3p did not alter SBF2-AS1 expression level.After inhibition of SBF2-AS1,E2F1 was obviously downregulated among all the candidate genes.The biotin-pull-down experiment and the dual-luciferase reporter gene confirmed mi R-338-3p/362-3p could target E2F1.Mi R-338-3p/362-3p can reduce the expression of E2F1 at the m RNA and protein level.Mi R-338-3p/362-3p mimics partly reverse the effects of SBF2-AS1.5 Animal model verifies that SBF2-AS1 can be a new target for LUAD treatment SBF2-AS1 plays a regulatory role in the proliferation of LUAD cells by adsorbing mi R-338-3p/362-3p.If SBF2-AS1 binding sites of mi R-338-3p/362-3p are mutated,SBF2-AS1 is no longer promote the proliferation of LUAD cells.Immunohistochemistry(IHC)further confirmed that E2F1 was the target gene of SBF2-AS1.Conclusion:Our study demonstrates SBF2-AS1,an early stage specific lnc RNA,promotes LUAD tumorigenesis by sponging mi R-338-3p and mi R-362-3p and increasing E2F1 expression.The SBF2-AS1-mi R-338-3p/362-3p-E2F1 regulatory axis may serve as prognostic marker and potential therapeutic targets for LUAD.