| BackgroundLung cancer is the most frequently diagnosed cancer and remains the number one cancer-related cause of death in China.The majority(-80%)of lung cancer cases are non-small cell lung cancer(NSCLC).Even though we have made great progress in the therapies including surgery,targeted therapy and radiotherapy,the overall 5-year survival rate of the disease is less than 15%.Thus,it remains a priority of exploring the early detection and treatment targets for NSCLC.Long noncoding RNAs(IncRNAs)are a class of transcribed RNA molecules that have a length>200 nucleotides that may not encode proteins.Accumulating studies have demonstrated that IncRNAs are emerging as key regulators involved in a broad range of biological processes and complex diseases,especially in cancers.A growing number of researches have shown that IncRNAs could regulate the tumorigenesis and development of lung cancer,which indicates that IncRNAs may serve as novel therapeutic targets in lung cancer.Long noncoding RNA 00312(linc00312),also called NAG7,is a lincRNA located at 3p25.3.It was first found in nasopharyngeal carcinoma and was reported to be negatively correlated with tumor size but positively correlated with lymph node metastasis in nasopharyngeal carcinoma.In addition,a recent study showed that linc003 12 could inhibit bladder cancer cell invasion and metastasis through mediating miR-197-3p.The integrative analysis of two NSCLC microarray datasets(GSE19188 and GSE18842)showed that linc00312 was one of the most down-regulated 1ncRNAs in NSCLC tumors,which indicates that linc00312 may be associated with tumorigenesis in NSCLC.In this regard,the further exploring of the function of linc00312 in NSCLC is essential.In the present study,we present for the first time a detailed analysis of linc00312 in NSCLC.We aim to explore the effect and mechanism of linc00312 on proliferation,invasion and migration in vivo and in vitro,finally to provide the theoretical basis for NSCLC carcinogenesis.Methods and Materials1.Real-time quantitative PCR(qRT-PCR)was performed to detect the relative expression of linc00312 in paired NSCLC tissues and analyze its expression level and clinicopathological characteristics.QRT-PCR was performed to detect the relative expression level of linc00312 in the plasma of NSCLC patients,other pulmonary diseases(OPD)patients and healthy volunteers.The prediction values of linc003 12 in NSCLC patients was analyzed by ROC curve.2.We use loss or gain of function approaches to investigate the biological functions of linc00312 in NSCLC.MTT assay,colony formation assay,5-Ethynyl-2,-deoxyuridine(EdU)assay,flow-Cytometric analysis,terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)and xenograft mouse model were used to detect the effect of linc00312 on proliferation and apoptosis.The impact of linc00312 on cell migration and invasion were furthermore observed in transwell experiment without or with matrigel matrix respectively.3.QRT-PCR was performed to detect the relative expression level of linc00312 in NSCLC cells after the cells were transfected with siRNAs-HDAC1/3,siRNA-EZH2,siRNAs-SUZ12 or plasmid of HOXA5 or after cells were treated by pan-HDACs inhibitor.ChIP-qPCR were used to detect the effect of HOXA5 on linc00312.In addition,rescue experiments to investigate whether linc00312 is involved in the HOXA5-induced decrease in NSCLC cell proliferation,invasion and metastasis.4.Bioinformatics analysis,qRT-PCR,RIP and western blot assays were used to investigate the underlying targets of linc00312 in NSCLC tissues and cells.MTT assay and transwell experiment were performed to detect the effect of miR-197-3P on NSCLC cell proliferation,invasion and metastasis.Rescue experiments were used to investigate whether miR-197-3p was involved in the linc00312-induced decrease in NSCLC cell proliferation,invasion and metastasis.Results1.Quantified the expression of linc00312 in 100 paired clinical NSCLC tissues and matched non-tumor tissues showed that it was down-regulated(4.56-fold change)in clinical NSCLC tissues and decreased linc00312 expression in NSCLC was associated with larger and the later stage tumors(P<0.05).Moreover,linc00312 was down-regulated in the plasma of NSCLC patients compared with in that of healthy volunteers or other pulmonary diseases patients.We analyzed the prediction values of linc00312 in the plasma of NSCLC patients by ROC curve.The cutoff value was 8.418(ACT)and the AUC is 0.6743(95%confidence interval:0.5648 to 0.7838,P=0.0042).2.The ability of cell proliferation,invasion and metastasis was decreased and cell apoptosis was increased in linc00312 overexpressed PC9 and SPC-A1 cells,which were contrast in siRNAs-linc00312 treated A549 cells.Linc00312 overexpression treatment dramatically decreased tumor growth,which was demonstrated bysignificantly reduced tumor size and weight,with lower ki-67 staining and higher tunel staining.3.Inhibiting of HDACs,EZH2 and SUZ12 could not upregulate the expression of linc00312.HOXA5 mRNA expression level was positively correlated with linc00312 in these normal lung tissues and NSCLC cancer tissues.In addition,increasing HOXA5 expression resulted in the up-regulation of linc00312.Furthermore,ChIP experiments results showed that HOXA5 could bind with the promoters of linc00312.SPC-A1 cell were cotransfected with SL-LINC00312 and overexpression plasmid pcDNA3.1-HOXA5.This cotransfection could partially rescue pcDNA3.1-HOXA5-impaired proliferation,migration and invasion in SPC-A1 cells.4.Linc00312 could increase the protein level of p53,p21,BAX and E-cadherin,while decrease the protein level of N-cadherin and MMP9.Linc00312 expression level was negatively correlated with miR-197-3 p in these normal lung tissues and N SCLC cancer tissues.Up-regulation of linc00312 induced decrease of miR-197-3 P,while increase in down-regulating of linc00312.The expression level of miR-197-3p in NSCLC was high.In addition,inhibition of miR-197-3p resulted the decrease of proliferation,migration and invasion of NSCLC cells.The cotransfection of miR-197-3p inhibitor and SL-LINC00312 could partially rescue SL-LINC00312-increased proliferation,migration and invasion in SPC-A1 cells.Conclusion1.We proved that linc00312 was down-regulated in N SCLC cancer tissues.We firstly detected the lower expression level of linc00312 in the plasma of NSCLC patients and the sensitivity of linc00312 to distinguish non-cancerous patients from NSCLC patients is high,which indicates the down-regulated linc00312 may serve as a biomarker of detection of NSCLC.2.We firstly proved that the anti-tumor role of linc00312 in NSCLC.It is demonstrated that the overexpression of linc00312 resulted in the decrease of the proliferation,invasion and migration and increase of apoptosis of NSCLC cells.3.We firstly found that HOXA5-linc00312-miR-197-3p axis could regulate the proliferation,invasion and migration of NSCLC cells.In addition,linc00312 increased the apoptosis via upregulating the protein level of the p53,p21 and BAX.Furthermore,we demonstrated that linc00312 could affect the epithelial-mesenchymal transition(EMT).This provides a new theoretical foundation for elucidating the molecular mechanism of NSCLC. |
PDF Full Text Request