| Background and Objective:Worldwide,lung cancer is still the highest incidence and mortality rate.The annual incidence rate of lung cancer is 11.6%of all cancers.The mortality rate of lung cancer is 18.4%of all cancer deaths.According to the data of the National Cancer registered in 2019,there are about 787000 new cases of lung cancer and 631000 deaths every year in China,of which 85%are non-small cell lung cancer(NSCLC)originated from alveolar epithelium and glandular epithelium.Lung adenocarcinoma(LUAD)is the most common type of non-small cell lung cancer.With the development of diagnosis and treatment technology,the prognosis of patients with NSCLC has greatly improved.However,most patients are in the middle and late stage when they are diagnosed because of no obvious symptoms in the early stage of NSCLC.There is no effective treatment for patients with metastatic NSCLC,and the 5-year survival rate is still less than 15%.Therefore,to further elucidated the molecular mechanism of NSCLC,especially in metastasis,and to further explored the novel therapeutic targets are urgently needed for oncology research.Long non-coding RNAs(lncRNAs)are RNA molecules that contain more than 200 nucleotides with little or without translating into a protein.Recent studies demonstrated that lncRN As were associated with various cellular biological processes such as cell development,proliferation,metastasis,differentiation and apoptosis.It was confirmed that lncRNAs were dysregulated in various cancers,including lung cancer.LncRNA LINC00176(LINC00176),a newly identified lncRN A,one of these THOC5 target genes,which could be activated by Myc and expressed at high levels in hepatocellular carcinoma is locates in 10q23.1.Recently,the dysregulation of LINC00176 was also reported in ovarian cancer and esophageal cancer.However,the expression pattern and potential function of most LINC00176 in LUAD remain unknown.Methods and Results:Firstly,online data and QRT-PCR analysis confirmed that LINC00176 is remarkably upregulated in both LUAD samples and cell lines.The Kaplan-Meier plotter database found the overall survival time in LINC00176-high group are lower than that in LINC00176-low group in LUAD,phase Ⅰ LUAD,phase ⅡLUAD,male LUAD,female LUAD,and smoking lung cancer except phase Ⅱ LUAD and non-smoking cancer.Analysis of qRT-PCR revealed that LINC00176 is highly expressed in two LUAD cells.NET1 was successfully silenced by siRNAs compared to its controls.LINC00176 siRNAs,LINC00176 plasmid and their controls took effects in enhancing or weakening LINC00176 levels in LUAD cells.CCK-8 and EdU assays indicated that silencing LINC00176 by its siRNAs leads to a prominent suppression in LUAD cell growth,while overexpressing LINC00176 using LINC00176 plasmid results in a significant promotion in LUAD cells.Colony formation assay confirmed that depletion of LINC00176 decreases cell colony formation,overexpression of LINC00176 increases that.Flow cytometry demonstrated that downregulation of LINC00176 represses cell cycle and induces cell apoptosis,while an overexpressed LINC00176 has a contrary effect.Wound-healing and transwell invasion assays displayed that silencing LINC00176 inhibits cell migration and invasion while an enhanced expression of LINC00176 promotes that effect.QRT-PCR analysis showed that miR-22-3p negatively correlates with LINC00176,while an elevated miR-22-3p can reverse LINC00176 level in A549 and H1650 cells.Luciferase reporter assay further displayed that LINC00176 can bind with miR-22-3p.LINC00176 was upregulated or downregulated via using LINC00176 siRNA or LINC00176 plasmid,respectively.Overexpressing miR22-3p or abating NET1 could reduce LINC00176 levels,while an elevated expression of NET1 further accelerated that.GAPDH was used as a control.Similarly,knockdown of LINC00176 and NET1 enhanced expression level of miR-22-3p,while overexpression of LINC01176 and NET1 had a converse effect.U6 was considered as a loading control.Western blot assay was performed to detect the level of NET1 and signaling pathways downstream targets of RhoA relative genes in each group.Suppression of LINC00176 and NET1 repressed the protein expression of NET1,ROCK,PAK,LIMK and cofilin,as well as the protein phosphorylation of ROCK,PAK,LIMK and cofilin in LUAD A549 cell lines,while overexpressed LINC00176 and NET1 had an opposite effect,and elevating miR-22-3p with transfected miR-22-3p mimics could partly rescue that results.GAPDH was considered as an inteRNA1 control.Proposed illustration depicting molecular mechanism of LINC00176/miR-22-3p/NET1 in regulating cell progression of LUAD cell lines.Secondly,miR-22 is downregulated in human lung cancer.We sought the integrated database for miR-22 expression in normal and cancer tissues using dbDEMC(database of Differentially Expressed MiRNAs in human Cancers)and found that miR-22 was identified to be decreased in human lung cancer compared to normal samples.Quantitative analysis for miR-22 expression standardized against those of U6 using qRT-PCR in normal human lung tissues and in lung tumor tissues.A qRT-PCR assay was performed to confirm the miR-22 level in NSCLC cell lines and their corresponding control,normalized against U6.We performed to analyze NET1 expression in NSCLC tumor and adjacent normal tissues consulted by Oncomine,derived from Hou Lung.NET1 is remarkably elevated in human LUAD comparison to adjacent normal tissues via using western blot assay.NET1 expression in NSCLC cell lines(A549,H1299)and in an immortalized normal lung epithelial cells BEAS-2B as detected by qRT-PCR and western bolting.The related expression between miR-22 and NET1 in 20 paired NSCLC tissues.βactin and U6 acted as inteRNA1 controls separately.TargetScan and miRDB bioinformatics analysis revealed that NET1 is predicted to be a target of miR-22.Predicted miR-22 target sequences in the wildtype and mutant 3’UTR regions of NET1.The Luciferase activity assay illustrated that overexpression of miR-22 could decrease the intensity of the fluorescence in both A549 and H1299 cells transfected with the NET1 3’UTR WT vector,while was invalid with the NET1 3’UTR MUT vector.The expression of NET1 in both mRNA and protein in NSCLC cell lines was significantly reduced following upregulation of miR-22,which was determined by qRT-PCR and western blotting.Quantitative reverse transcription polymerase chain reactions(qRT-PCRs)showed that miR-22 mimics and inhibitors successfully take effect in both A549 and H1299 cells.CCK-8 assay indicated that miR-22 mimics decreased cell’s vitality,while miR-22 inhibitors increased that.Similarly,EdU staining confirmed that miRNA-22 mimics resulted in a reduced cell’s proliferation,while miR-22 inhibitors increased that effect.miR-22 mimics attenuated NSCLC cell migration in A549 and H1299 cells.Western blot was used to analyze EMT andβ-actin was considered as a loading control.Induction of cell apoptosis confirmed by flow cytometry.Apoptosis associated protein was analyzed using western blot,and β-actin was used as a control.The expression level of miR-22 and its target gene NET1 were evaluated by using qRT-PCR and western blot,respectively.Overexpression of NET1 counteracted the inhibition of the cell vitality and growth capacity using CCK-8 and EdU assays.The suppression of migration of A549 and H1299 cells could be reversed by NET1 vector using wound healing assay.Analogously,NET1 vector neutralized the pro-apoptotic effect of miR-22 via using western blotting analyses.Thirdly,TGCA and oncomine database showed that NET1 is remarkably upregulated in LUAD samples.QRT-PCR and western bolt assays further confirmed that NET1 was identified to be increased in human lung cancer compared to normal samples.Transfection of small interfering RNA successfully took effects in decreasing NET-1 levels in A549 cells.The transfection of either NET-1 si-01 or NET-1 si-02 led to efficient knockdown of NET-1 as detected by both qRT-PCR and western bolt.Analysis of EdU-positive cells and CCK-8 confirms thatNET-1 siRNA decreases A549 cell proliferation and viability.Up-regulation of NET-1 attenuates migration of A549 cells.The healing ability of A549 cells in NET1 siRNA group was significantly weaker than that of A549 cells in control group at 24 and 48 h after wound scratched.The expression of migration suppressor gene E-cadherin increased,and the expression of migration promoting genes vimentin and F-actin decreased.NET1 may mediate proliferation and migration of A549 cells through regulating RhoA.Knockdown of NET1 was shown to inhibit activation of RhoA.Transfection of either small interfering RNA for NET-1 decreased A549 cell proliferation and migration,whereas co-transfection of NET-1 siRNA and C3 further weaken A549 cell proliferation and migration in comparison with the control group.Conclusions:Firstly,LINC00176 facilitated human LUAD progression via sponging miR-22-3p,and then made an upregulation of NET1 to promote tumor growth.Mechanistically,to our knowledge,we have elucidated,for the first time,that the LINC001 76/miR-22/NET1 axis regulates the property of two LUAD cells though activity of RHO/ROCK and PAK/LIMK pathway.Our studies evaluated the roles and mechanism of a new LINC00176/miR-22/NET1 axis in the progression of LUAD,which might contribute to treatment in NSCLC.Secondly,we demonstrated that the lack of miR-22 is a common event in patients with NSCLC and may serve as a tumor-suppressor by endogenously and negatively regulating NET1.To the best of our knowledge,we have identified,for the first time,that the miR-22/NET1 axis regulates the proliferation,migration and apoptosis of NSCLC cells.These findings may provide a new evidence for better understanding the functional roles of miR-22 in the pathogenesis of NSCLC and developing new therapeutic strategy for NSCLC.Thirdly,we have shown that NET1 plays fundamental roles in A549 cell progression by regulating cancer cell migration and proliferation.Furthermore,we also have demonstrated that NET 1 is a key player in activation of RhoA and the subsequent proliferation and migration of lung cancer cells.Suppression of NET1 was considered as effective at reducing cell migration as treatment with the RhoA inhibitor,C3 exoenzyme,highlighting its importance in this setting.As NET1 is important to the growth and migration,we therefore propose that NET1 is an ideal potential therapeutic target in lung carcinoma. |
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