Prediction And Mechanism Of Recurrence Related LncRNAs After Surgery In Early Lung Adenocarcinoma

Objectives: Patients with early-stage lung adenocarcinoma(LUAD)are still at risk of recurrence within five years after surgery.Although histological subtypes and driver genes significantly affect LUAD recurrence,the early recurrence mechanism of some patients is still unknown.This study aimed to explore the functional long non-coding RNA(lnc RNA)that may exist in early recurrent LUAD patients and construct a prediction model based on recurrence-related lnc RNAs(RRLnc RNAs).We then confirmed the biological functions of each RRLnc RNA through a series of cell experiments,screened the key lnc RNA AC022613.1,and analyzed its potential mechanism to provide potential molecular targets for the prognosis evaluation and treatment of early-stage LUAD.Methods:(1)We retrospectively collected intraoperative samples of 97 stage I lung cancer radical surgery patients and followed up for one year to obtain the lnc RNA expression library through transcriptome sequencing of samples from patients who developed recurrence.We then screened differentially expressed lnc RNAs(DElnc RNAs)from the library and analyzed them using public database information.Then,we used single-factor and multi-factor Cox regression analysis to construct an integrated prognostic model based on RRLnc RNAs in the form of a nomogram and validated the predictive performance of the model.(2)We used RT-q PCR to determine the expression levels of AC022784.1,AC022613.1,and AC092718.4 in clinical samples of LUAD and several commonly used LUAD cell lines.We knocked down AC022784.1,AC022613.1,and AC092718.4 in various LUAD cell lines,then used CCK-8,Brd U,and Transwell experiments to observe the effects of RRLnc RNA knockdown on LUAD proliferation,DNA synthesis,and cell migration.We used a combination of tools such as GENCODE,Annolnc2,Coding Potential Calculator(CPC),and lnclocator to analyze the molecular characteristics,chromosomal location,secondary structure,coding potential,and subcellular localization of AC022784.1,AC022613.1,and AC092718.4,and performed GSEA pathway enrichment and GSVA tumor-associated immune infiltration cell correlation analysis on differentially expressed genes of AC022784.1,AC022613.1,and AC092718.4.(3)We constructed knockdown and overexpression AC022613.1 H1299 cell lines and used a DNA demethylating agent,5-Aza,to reverse the effect of AC022613.1knockdown to explore the role of hypomethylation in AC022613.1 activation.We then used FISH and dual luciferase reporter gene experiments to verify the subcellular localization and interaction targets of AC022613.1.We used RNA interference to knock down downstream m RNA CCDC85 B,CDCP1,and SLC16A3(MCT4)in H1299 cells with knocked down AC022613.1 and used CCK-8,Transwell,clone formation,PCR,flow cytometry,ELISA,WB,and other techniques to detect phenotypic features and key molecular changes.We then used Agomi R-184 transfection to perform a functional recovery(rescue)experiment on the AC022613.1-mi R-184-SLC16A3 axis in H1299 cells that overexpressed AC022613.1.Results:(1)Among 97 stage I LUAD patients who underwent radical surgery,there were 3 cases of recurrence within 1 year.After sequencing the lnc RNAs of the corresponding surgical samples,1498 up-regulated lnc RNAs were found.Among the top 20 Lnc RNAs with the highest fold change(FC),early LUAD recurrence-related lnc RNAs(RRLnc RNAs)were selected by combining TCGA clinical information:AC022784.1,AC022613.1,and AC092718.4.TCGA clinical information revealed that the expression levels of RRLnc RNAs were related to disease-specific survival time(DSS).Pan-cancer analysis revealed significant differential expression of RRLnc RNAs in multiple tumors,and the expression level of RRLnc RNAs in LUAD tissues was significantly higher than that in normal tissues.The higher the clinical and pathological TNM stage,the higher the expression level of RRLnc RNAs.Using AC022784.1,AC022613.1,and AC092718.4 to predict the overall survival(OS)of LUAD patients showed good predictive ability.By integrating RRLnc RNAs,a highly effective prognostic model for LUAD was constructed(AUC=0.967).(2)AC022784.1,AC022613.1,and AC092718.4 were highly expressed in clinical samples and several commonly used LUAD cell lines.Knocking down AC022784.1,AC022613.1,and AC092718.4 in various LUAD cell lines could inhibit cell proliferation,DNA synthesis,and cell migration.Bioinformatic analysis of RRLnc RNAs revealed that AC022613.1 was the most likely to affect LUAD characteristics through the ce RNA mechanism.GSEA pathway enrichment and GSVA tumor-related immune infiltration cell correlation analysis suggested that AC022613.1and AC092718.4 were associated with multiple tumor-related pathways and tumor immune cell infiltration.(3)Knocking down AC022613.1 promoted apoptosis of H1299 cells,decreases the proportion of cells in G2 and S phase,increases the proportion of cells in G0/G1 phase,and inhibits EMT of cells.The high expression of AC022613.1 in LUAD is mediated by low methylation levels of the gene promoter.FISH confirmed that it is mainly distributed in the cytoplasm.Knocking down AC022613.1 inhibits the expression of SLC16A3 by targeting mi R-184 to weaken the expression of EMT-related proteins,HIF-1α,and Kras,and also reduces H1299 cell proliferation,anti-apoptosis,cell cycle,and cell migration.Overexpression of AC022613.1 and the use of Agomi R-184 can restore the promoting effects of AC022613.1 on SLC16A3,EMT-related proteins,HIF-1α,and lactate efflux,as well as its effects on cell proliferation,anti-apoptosis,cell cycle,and cell migration.The subcutaneous tumor formation experiment in nude mice confirmed that overexpression of AC022613.1 can inhibit mi R-184,promote the expression of SLC16A3 and HIF-1α,and promote tumor growth and lactate metabolism in vivo.Conclusions: Early postoperative RRlnc RNAs in LUAD include AC022784.1,AC022613.1,and AC092718.4.RRlnc RNAs promote malignant behavior in LUAD cell lines,and AC022613.1 promotes the malignant behavior of LUAD through mi R-184/SLC16A3 axis.