The Mechanism Of LINC00222 Regulating Biological Behaviour Of Non-small Cell Lung Cancer Through GSK-3?

Objective: Lung cancer is the most common cause of cancer-related death worldwide,in which non-small cell lung cancer(NSCLC)accounts for about 80-85% of all lung cancer cases.Most NSCLC patients are diagnosed at the advanced stages as they are usually asymptomatic at early stages.The overall 5-year survival rate is still about 16%.Thus,it is important to investigate the molecular mechanisms of NSCLC and identify diagnostic markers for early detection and targeted treatment of NSCLC.The human transcriptome comprises not only protein-coding messenger RNAs(mRNAs),but also a large amount of non–protein-coding transcripts that have structural,regulatory,or unknown functions.The long noncoding RNAs(lncRNAs)are a large class of(>200 nt,with limited protein-coding potential)ncRNAs and have been shown to play vital roles in various aspects of cell biology.Some lncRNAs have been demonstrated to play key roles in cellular development,differentiation,and many other biological processes.In addition,some lncRNAs have been found to be misregulated in various types of human diseases,especially in cancer.However,although over a decade's research has led to considerable progress in understanding lncRNAs,the precise function of most remains unknown.The Wnt/?-catenin signaling pathway has been recognized to play a critical role in regulating oncogenesis and metastasis.The aberrant activation of Wnt signaling was detected in 50% of human NSCLC cell lines and tissues.Accumulated evidence showed that lncRNAs are crucial modulators of Wnt/?-catenin signaling pathway.Glycogen synthase kinase-3?(GSK-3?)is one of key protein in Wnt/?-catenin signaling pathway,which phosphorylates ?-catenin and lead to degradation of ?-catenin.Here we firstly investigate expression level of long intergenic non-protein coding RNA 222(LINC00222)in NSCLC samples and adjacent normal lung tissues,which have been proved downregulared in Hepatoblastoma tissues.Increased expression of LINC00222 suppressed NSCLC cell proliferation,migration and invasion but induced apoptosis.Then we predicted that LINC00222 may interact with GSK-3? from the URL http://pridb.gdcb.iastate.edu/RPISeq/results.php.Then we further verified how LINC00222 affect Wnt/?-catenin signaling pathway through kinase GSK-3?.Therefore,our results provided a new therapeutic target for the treatment of NSCLC.Materials:1.Materails:(1)Non-small cell lung cancer fresh frozen tissue.(2)Non-small cell lung cancer cell line A549,LTEP-a-2.(3)Real-time PCR experiments needed reagent and instrument.(4)Western Blot experiments needed reagent and instrument.(5)Cell culture related reagent and instrument.(6)LINC00222 expression vector: pCDNA-LINC00222.(7)Activity inhibitors of GSK-3?: TWS119.(8)RNA-CHIP related reagents,equipment.2.Method:(1)Real-time PCR method to detect RNA expression level of LINC00222 in fresh frozen non-small cell lung cancer tissue and adjacent normal lung tissues,detect RNA expression level of LINC00222 in cell lines of non-small cell lung cancer and embryonic cell line.(2)Analyze the correlation between LINC00222 expression level and the prognostic indicators to verify if LINC00222 expression is related to the prognosis of non-small cell lung cancer.(3)Build LINC00222 length expression vector pCDNA-LINC00222,transfection non-small cell lung cancer cell line A549,LTEP-a-2.(4)Assay the role of LINC00222 in proliferation and apoptosis with CCK 8,PI method.(5)Assay the role of LINC00222 in migration and invasion of non-small cell lung cancer cell transwell,wound-healing method.(6)Western blot to detect protein phosphorylation level change of GSK-3? after transfection to identify whether GSK-3? activity change.(7)Western Blot to detect protein expression of ?-catenin in cell nucleus to validate if LINC00222 can influence?-catenin phosphorylation and resulting in abnormal nuclear transfer;Join the GSK-3? activity inhibitors(TWS119)to identify whether the expression of ?-catenin callback.(8)RNA-CHIP to identify if LINC00222 can directly interact with GSK-3? protein.Results: 1.LINC00222 was obviously downregulated in 85%(88 /103)of NSCLC samples compared to adjacent normal lung tissues.2.LINC00222 was downregulated in A549 and LTEP-a-2 cells compared to MRC-5 cells.3.Patients in the low LINC00222 expression group had a higher recurrence rate(median DFS: 16 months)and much shorter overall survival(median OS: 22 months)than those in the high LINC00222 expression group(median DFS: 24 months;median OS: 31 months;p = 0.0139 and 0.0189,respectively).4.The expression levels of LINC00222 in NSCLC patients were significantly correlated with tumor size,TNM stage,and lymph node metastasis.5.LINC00222 upregulation inhibits NSCLC cells proliferation but induces apoptosis.6.LINC00222 upregulation inhibits NSCLC cells migration and invasion.7.Overexpression of LINC00222 reduced phosphorylation levels of GSK-3? which meaned it enhanced the activity of GSK-3?.8.The expression of nuclear ?-catenin protein levels was significantly reduced after transfected with pCDNA-LINC00222,the expression of nuclear ?-catenin protein levels was rescued when cotransfected pCDNA-LINC00222 and TWS119.9.LINC00222 was physical bond with GSK-3? protein through RIP.Conclusion: Our data suggested that LINC00222 was aberrantly downregulated in patient NSCLC tissues and LINC00222 could inhibit the proliferation,migration and invasion but promote apoptosis of NSCLC cells,which associated with the Wnt/?-catenin signaling pathway.This study put forward a new research direction for the treatment of non-small-cell lung cancer(NSCLC).