| Background:Lung cancer has become one of the leading causes of cancer-related deaths worldwide.Non-small cell lung cancer(NSCLC)accounts for approximately 85%of all lung cancers,among which,lung adenocarcinoma(LUAD)is the most common subtype of NSCLC.Although advances have been made in chemotherapy and molecular targeted therapy,the overall 5-year survival rate for patients remains low.Therefore,the search for diagnostic markers and effective therapeutic targets for LUAD is crucial.Variations or abnormal functions of individual genetic factors have been identified as the direct cause of cancer at the molecular level.Gene mutations,mutation sites,copy number changes in different cancers and changes in the function of essential genes in cancer-induced signaling pathways have become the focus of research on tumorigenesis and therapeutic mechanisms.As messengers for genes to perform their biological functions,there are complex interactions among different types of RNAs,and their role in regulating gene expression and biological functions in tumorigenesis is receiving increasing attention.Protein-coding mRNAs and non-coding RNAs,such as long non-coding(lnc)RNA(a non-coding RNA>200bp nucleotide in length)and micro(mi)RNA(a tiny RNA containing 18-25 nucleotides),influence lung cancer development by regulating mRNAs,i.e.lncRNAs can bind to miRNA loci that regulate the expression of genes encoding proteins bind to and prevent them from regulating the translation of target mRNAs.The FXYD protein family contains seven members that play a role in a variety of cancers.The FXYD domain-containing ion transport regulator-6 is an ion channel-associated transmembrane protein.Dysregulation of FXYD6 contributes to the development of several cancers,but its role in the pathogenesis of LUAD is unclear.Analysis of large-scale sequencing data showed that FXYD6 is closely related to the prognosis of LUAD.OIP5-AS1 is a conserved lncRNA that plays an important role in the progression of NSCLC,but its oncogenic or oncogenic role is still controversial.On the basis of this,database search and comparison revealed that FXYD6 is the target gene of miR-4735-3p,and the expression of miR-4735-3p may be regulated by the LncRNA OIP5-AS1,they together constitute an RNA interplay network for the regulation of FXYD6 expression,and their regulatory role on the malignancy of LUAD needs to be further elucidated.Objective:In this study,a retrospective clinical cohort and a series of ex vivo experiments were conducted to investigate whether LncRNA OIP5-AS1 affects FXYD6 expression in LUAD by acting on miR-4735-3p,and thus plays a role in LUAD proliferation and migration.Method:1.Bioinformatics analysis:Differential analysis of normal and tumor groups was performed by GEO’s own R language analysis tool GEO2R to obtain differentially expressed mRNAs.Publicly available cancer transcriptome data were obtained from UALCAN to verify the expression of differential genes in the TCGA database and survival curves.The upstream miRNAs of target genes were predicted by TargetScan.ENCORI was used to predict the regulatory lncRNAs of miRNAs.2.Clinical experiments:54 clinical samples were collected,of which 27 cases were corresponding non-tumor normal tissues as the control group and 27 cases were LUAD tissues as the LUAD group.The expression of FXYD6 in fresh clinical tissues was detected through Western blot.Based on RT-PCR,the expression of LncRNA OIP5-AS1,miR-4735-3p was detected in fresh clinical tissues.3.Cellular experiments:normal lung epithelial cell lines and LUAD cell lines were used to verify the expression differences of FXYD6,LncRNA OIP5-AS1,miR-4735-3p in different cell lines based on western blot and RT-PCR;gene intervention was performed by lentiviral infection of cells,which were divided into enhanced group,silenced group and enhanced cotransfection group and silenced cotransfected group.The effects of LncRNA OIP5-AS1,miR-4735-3p and FXYD6 on LUAD proliferation and migration were verified by MTT,cloning assay,Transwell and Wound Healing;whether LncRNA OIP5-AS1 regulates the transcriptional expression of miR-4735-3p was verified by dual luciferase reporter gene;whether miR-4735-3p regulates the transcriptional expression of FXYD6.4.Animal experiments:PC9 cell line was selected,and the intervention was performed by lentiviral transfection of cells,and the effect of intervention of LncRNA OIP5-AS1,miR-4735-3p and FXYD6 on LUAD tumorigenesis was verified in vivo by tumorigenic assay in tumor-bearing mice.Results:1.Compared with corresponding non-tumor normal and normal tissues and cell lines,FXYD6 showed low expression,miR-4735-3p showed high expression and OIP5-AS1 showed low expression in LUAD tissues and lung adenocarcinoma cell lines,and the differences were statistically significant;2.Compared with the control group,the overexpression of FXYD6 in lung cancer cells inhibited the proliferation and invasion of LUAD,and the difference was statistically significant;3.LncRNA OIP5-AS1 can promote FXYD6 protein expression and inhibit proliferation,invasion and migration of lung adenocarcinoma through adsorption of miR-4735-3p.Conclusions:1.FXYD6 is lowly expressed in LUAD tissues and cell lines;FXYD6 will inhibit proliferation and migration of LUAD.Therefore,FXYD6 may be an oncogene in LUAD,as well as a new potential biomarker and therapeutic target for lung cancer.2.miR-4735-3p is an upstream regulatory gene of FXYD6,it acts as a tumor promoter in LUAD and promotes proliferation and migration of LUAD by targeting and inhibiting FXYD6;LncRNA OIP5-AS1 regulates FXYD6 protein expression through adsorption of miR-4735-3p and affects the proliferation and migration of lung adenocarcinoma.lncRNA OIP5-AS1/miR-4735-3p/FXYD6 may be a diagnostic or therapeutic target for LUAD. |
