ITGB1-DT/ARNTL2 Axis As A Potential Mechanism Regulates The Development Of Lung Adenocarcinoma

Background: Lung cancer is one of the most deadly malignancies affecting human health.The incidence of lung adenocarcinoma(LUAD)has increased dramatically in recent decades,accounting for nearly 40% of all lung cancers.Increasing evidence points to the importance of the competitive endogenous RNA(ceRNA)intrinsic mechanism in several human cancers.However,the behavioural characteristics of the ceRNA network in lung adenocarcinoma require further investigation.SLC2A1(Solute Carrier Family 2 Member 1)is a major glucose transporter protein in the mammalian blood-brain barrier,located mainly at the cell membrane and cell surface,and also serves as a receptor for human T-cell leukaemia virus(HTLV)types I and II.It has also been shown to have aberrant regulatory functions in tumours and a close relationship between cellular immunity and glucose metabolism.In addition,SLC2A1 has been shown to promote tumor progression and induce the malignant phenotype of lung adenocarcinoma cells.Therefore,we collected a large amount of microarray sequencing and clinical data from the TCGA database,and screened some genes with similar phenotypes based on the expression of SLC2A1 for in-depth analysis and study.We hypothesized that genes with similar phenotypes to SLC2A1 might have the same function and mechanism to induce the development of lung adenocarcinoma.Methods: Based on the expression profile of the microarray,all the genes were classified into three main categories of differentially expressed genes(lnc RNA,miRNA and m RNA).In addition,several specific key genes were obtained by a series of statistical methods such as survival prognosis analysis and correlation analysis to construct the interaction network in combination with clinical data.The mechanism of interaction network of lnc RNA-miRNA-m RNA has been a hot topic of research in recent years,and several studies have demonstrated its key role in various tumors.We identified three potentially interacting genes,ITGB1-DT,miR-30b-3p and ARNTL2 via COX regression,GO,KEGG,gene set enrichment analysis(GSEA),immune infiltration and other bioinformatics analyses,as well as deeper mechanisms through corresponding experiments such as luciferase reporter assay,Western Blot,q RT-PCR,CCK-8 and animal models,and finally determined that the interaction of these three genes induced persistent lung adenocarcinoma progression.Results: Bioinformatics analysis showed that the expression of ITGB1-DT and ARNTL2 was also abnormally elevated in tumor samples with abnormally elevated SLC2A1,while the expression of miR-30b-3p was significantly decreased.This means that ITGB1-DT and ARNTL2 may have similar functions as SLC2A1,while miR-30b-3p may have the opposite effect.And it also suggests that there may be a mutual regulatory relationship between the three genes.In addition,the abnormal upregulation of ITGB1-DT and ARNTL2 can induce poor prognosis of lung adenocarcinoma like SLC2A1,while the outcome of patients with high expression of miR-30b-3p is better.Further experiments found that ITGB1-DT and ARNTL2 were significantly up-regulated in a variety of lung adenocarcinoma cell lines,while miR-30b-3p was significantly down-regulated in lung adenocarcinoma cell lines(compared to human normal bronchial epithelial cell lines),indicating that these three key genes do play a key role in lung adenocarcinoma.Next,the expression of ITGB1-DT and miR-30b-3p were changed by using small interfering and lentivirus.And we found that the down-regulation of ITGB1-DT could induce the down-regulation of ARNTL2 and the up-regulation of miR-30b-3p.Then,downregulation of ITGB1-DT and ARNTL2 expression was observed in cells transfected with miR-30b-3p mimic.The luciferase reporter assay further confirmed our hypothesis that ITGB1-DT can competitively bind to miR-30b-3p to regulate the expression of ARNTL2.The results of cell function experiments showed that the up-regulation of ITGB1-DT could promote the proliferation,invasion and migration of lung adenocarcinoma cells,while miR-30b-3p could reverse the effect of ITGB1-DT on the malignant phenotype of H1299 cells.The results of GSEA showed that ITGB1-DT was positively correlated with TGF-β signaling pathway.The western blot results showed that the protein expression levels of ARNTL2 and TGF-β1 also decreased after down-regulating ITGB1-DT.It was also found that down-regulation of miR-30b-3p expression could also reverse the ITGB1-DT-mediated TGF-β signaling pathway activation.In addition,A subcutaneous tumor model in nude mice was observed that compared with the control group,the formation of subcutaneous tumors in nude mice with knockdown of ITGB1-DT expression was slower,and the volume of the final tumor mass was smaller.Meanwhile,the protein expression of ARNTL2 and TGF-β1 was observed to be decreased in ITGB1-DT knockdown tumor tissues.Conclusions: In summary,our study found that the up-regulation of ITGB1-DT can competitively bind to miR-30b-3p to regulate the expression of ARNTL2.At the same time,the up-regulation of ARNTL2 expression can induce the activation of TGF-β signaling pathway,thus promoting the proliferation and metastasis of lung adenocarcinoma cells,leading to the occurrence and development of the disease.