Expression Of MiR-374a And Preliminary Study On Its Function And Molecular Mechanisms In NSCLC

Lung cancer is one of the deadliest types of cancer as demonstrated by the poor survival and high relapse rates after surgery.Non-small cell lung cancer(NSCLC)is the most commonly diagnosed type of lung cancer and accounts for approximately 85%of all cases and the 5-year survival rate is less than 15%.Poor survival of lung cancer patients is largely due to the lack of reliable early diagnostic markers,often resulting in diagnosis of tumors at advanced stages.Thus,further elucidation of the molecular mechanisms during NSCLC is urgently required.MicroRNAs(miRNAs)are small(18-22 nucleotides)noncoding RNAs that were first discovered in Caenorhabditis elegans.miRNAs regulate a wide range of biological processes,including development,cell differentiation,proliferation and apoptosis.Accumulating evidence has shown that microRNAs(miRNAs)play a pivotal role in NSCLC pathogenesis,which has provided new insights into targeted therapy of this disease.Several studies have shown that miRNAs are frequently dysregulated in cancers and can modulate both oncogenes and tumor suppressor genes.The studies suggest that miR-374a functions as an oncogene during cancer progression.In lung cancer,miR-374a was first reported to be upregulated in primary small cell lung cancer compared to normal lung.Furthermore,functional assays revealed that miR-374a acts as an oncogene by directly targeting Wnt5a to regulate proliferation,gefitinib-induced apoptosis,EMT,migration,and invasion of NSCLC in vitro and in vivo.Interestingly,low miR-374a expression in early-stage NSCLC was associated with poor patient survival,which suggested that miR-374a may also serve a tumor-suppressive role in NSCLC.Nevertheless,to date no studies have attempted to address the dual functions and molecular mechanisms of miR-3 74a in NSCLC.In this study,to know the exact function of miR-374a in NSCLC,in vitro gain-of-function analyses were performed using lentivirus or mimics overexpression in A549,SPCA-1 and H1975.Subsequently,A549,SPCA-1 and H1975 cell proliferation was measured by MTT assay,colony formation assay,cell cycle analysis and Edu assay.Transwell and Boyden Chamber assays were used to examine the effects of miR-3 74a overexpression on migration and invasion.Iuciferase reporter assays was performed to confirm that miR-374a could directly target the 3'UTR of CCND1 in A549 cells and the 3'UTR of PTEN in SPCA-1 and H1975 cells.So the expression patterns of miR-374a were examined in NSCLC by in situ hybridization,the result show a correlation between miR-374a and CCND1 in early-stage NSCLC and a correlation between miR-374a and PTEN in advanced-stage NSCLC.Results show:1.miR-374a suppresses A549 but promotes SPCA-1 and H1975 cell proliferation as well as cell cycle transition in vitro and in vivo,2.To understand the biological effects of miR-3 74a deregulation in human NSCLC cells,in vitro gain-of-function analyses were performed using lentivirus or mimics overexpression in A549,SPCA-1 and H1975 cell lines.More than 10-fold increase in miR-374 expression was observed in miR-3 74a lentivirus or mimics treated NSCLC cells compared with the control group by RT-qPCR(with P<0.01;P<0.001).To further explore its biological role in NSCLC,miR-374a inhibitors were transfected into Lv-miR-374a-A549 cells,Lv-miR-374a-SPCA-1 and Lv-miR-374a-H1975 cells,and expression levels of miR-374a detected by RT-qPCR.Subsequently,NSCLC cell proliferation was measured in vitro.Compared with negative controls,we found that ectopic miR-374a inhibited A549 cell growth and G1 to S cell cycle transition by MTT assay,colony formation assay,cell cycle analysis and Edu assay.Interestingly,the opposite results were observed in SPCA-1 and H1975 cells.We also observed that miR-374a overexpression markedly suppressed in vivo tumorigenesis of A549 cells yet significantly promoted SPCA-1 and H1975 tumor growth.These above mentioned results indicated that miR-374a could exert divergent effects in different NSCLC cells.miR-374a respectively inhibits or promotes A549 or SPCA-1 and H1975 cell migration,invasion,and metastasis in vitro and in vivo To examine the effects of miR-374a overexpression on migration and invasion,Lv-miR-374a cells were cultured with or without inhibitors.Compared with LEV control cells,we found that miR-374a significantly reduced cell migration and invasion in transwell and Boyden Chamber assays.The addition of inhibitors could rescue this effect in A549 cells.Inversely,we observed overexpressed miR-374a significantly promoted cell migration and invasion in SPCA-1 and H1975 cells.Since in vitro experiments revealed that miR-374a expression was associated with metastatic phenotypes,we next asked whether this effect was present in vivo.A549,SPCA-1 and H1975 cells expressing miR-374a stably were transplanted under the liver capsule of mice,4 weeks later the tissues were harvested.Strikingly,mice bearing SPCA-1/miR-374a and H1975/miR-374a tumors developed prominent lung metastases,whereas no visible metastases were found in mice transplanted with A549/miR-374a cells.Histological analyses further revealed that miR-3 74a could promote metastasis of SPCA-1 and H1975 cells but inhibit A549 metastasis.Together,these data indicate that miR-374a plays a pivotal role in NSCLC metastasis in vitro and in vivo but its function is unique in different NSCLC cell lines.2.miR-374a directly targets the CCND1 3'UTR in A549 and the PTEN 3'UTR in SPCA-1 and H1975 cells.To delineate the mechanism by which miR-374a inhibited or promoted cell proliferation,invasion and migration in NSCLC cells,we used TargetScan(http://www.targetscan.org/)and PicTar(http://pictar.mdc-berlin.de/)bioinformatics algorithms.We found that CCND1 and PTEN were predicted to be direct targets of miR-374a.Western blot analysis revealed that miR-374a mimics downregulated CCND1 expression in A549 and PTEN in SPCA-1 and H1975 cells respectively.Conversely,transfection of miR-374a inhibitors into these 3 lines resulted in upregulation of CCND1 or PTEN.No significant change in PTEN protein was observed for A549-miR-374a cells but CCND1 was upregulated in SPCA-1-miR-374a and H1975-miR-374a cells.luciferase reporter assays was performed to confirm that miR-374a could directly target the 3'UTR of CCND1 and PTEN in NSCLC cells.We found that miR-374a could directly target the CCND13'UTR but not the PTEN 3'UTR in A549 cells.In SPCA-1 and H1975 cells,miR-374a directly targeted the PTEN 3'UTR but not the CCND1 3'UTR.Immnohistochemistry of xenografts derived from A549-miR-374a,SPCA-1-miR-374a or H1975-miR-374a cells revealed a drastic reduction in CCND1 and PTEN expression,respectively.Taken together,these data suggest that miR-3 74a directly suppresses CCND1 in A549 cells but PTEN in SPCA-1 and H1975 cells.levels of p-PI3K,p-AKT,c-Myc,CDK4 and N-cadherin were significantly reduced while E-cadherin and P21 were increased after siRNA-mediated suppression of CCND1 in A549 cells.Downregulation of PTEN in SPCA-1 and H1975 cells increased the expression of invasion-associated genes including ?-catenin,TCF4 and N-cadherin,as well as elevated oncogenic cell cycle regulator c-Myc but reduced P21.These results indicate that miR-374a can mediate CCND1 and PTEN and their downstream factors..Ectopic expression of CCND1 or PTEN mitigates miR-374a-mediated effects on proliferation,invasion and migration in NSCLC cells3.To explore whether miR-374a targeting of CCND1 or PTEN was responsible for its effects on proliferation,migration and invasion,we utilized an expression construct that encodes the entire CCND1 and PTEN coding sequence but lacks the 3'-UTR.These functional studies demonstrated that CCND1 or PTEN overexpression respectively inhibited or promoted proliferation,migration and invasion.Ectopic expression of CCND1 or PTEN partially rescued miR-374a-mediated effects on growth,migration and invasion in A549 or SPCA-1 and H1975 cells,respectively.This suggested that the effects of miR-374a overexpression in NSCLC cells specifically,depend on CCND1 or PTEN suppression.miR-374a modulates expression of cell cycle-associated and EMT-related genes by inactivating PI3K/Akt pathway or activating ?-catenin signaling4.To explore the molecular mechanisms by which miR-374a mediates its effects through CCND1 or PTEN,we examined protein levels of several cell-cycle and invasion associated genes in Lv-miR-374a cells transfected with ectopic CCND1 or Lv-miR-374a inhibitor-treated cells after PTEN overexpression.As shown in Figures 4E,we observed that miR-374a not only suppressed levels of phos-PI3K(Tyr458)and phos-Akt(Ser473),but also significantly decreased the expression of cell cycle-related genes including CCND1,c-Myc and CDK4 in A549 cells.Mesenchymal marker N-cadherin was also downregulated while E-cadherin expression was increased.Increased the expression of ?-catenin,TCF4,ZEB2 and N-cadherin,CCND1 and c-Myc but decreased P21 in SPCA-1 and H1975 cells overexpressed miR-374a.Both suppressing miR-374a with specific inhibitors and overexpressing CCND1 or PTEN can play reverse effects.Taken together,these data suggest that in A549 cells,miR-374a suppresses cell growth and metastasis by inactivating the PI3K/Akt pathway thus modulating its downstream cell cycle and EMT-related genes.However,in SPCA-1 and H1975 cells,miR-374a exerts its effects by activating ?-catenin signaling,consistent with a previous report of miR-374a function in breast cancer.5.PCR analysis indicated that miR-374a expression levels were significantly increased in NSCLC samples compared to PT tissues.In situ hybridization assay confirmed expression of miR-374a was highly expressed in 72.2%(114/158)of NSCLC samples,we observed that the expression level of miR-374a was positively correlated with lymph node status(N classification;NO-N1 versus N2-N3)(P =0.040).Subsequently,we found that NSCLC patients with low miR-374a expression had longer survival compared with patients with high miR-374a levels(P = 0.037).Interestingly,we found that low expression of miR-374a in early-stage NSCLC is associated with poor patient survival(P = 0.046),which was consistent with a previous report of miR-374a in NSCLC.Furthermore,in patients with advanced NSCLC,low levels of miR-374a correlated with longer survival(P = 0.000).These results provide support that miR-374a plays distinct roles at different stages of NSCLC tumorigenesis.We next asked whether there was an association between the expression level of miR-374a and CCND1 in NSCLC tissues.Unfortunately,a significant association didn't be found between miR-374a and CCND1 expression in the whole NSCLC cohort(P = 0.1550)and advanced NSCLC specimens(Stage ?-?and ?)(P-0.8720).However,81%of samples with low CCND1 expression exhibited high levels of miR-374a(22/27 cases),whereas 35%of samples with high CCND1 expression had low expression of miR-374a(19/54 cases)in early stage of NSCLC specimens(Stage ?-?)(P = 0.0164).More interestingly,80%of samples with low PTEN expression exhibited high levels of miR-374a(39/49 cases),whereas 36%of samples with high PTEN expression had low expression of miR-374a(10/28 cases)in advanced NSCLC specimens(Stage ?-? and ?)(P = 0.0117).There was no correlation between miR-374a and PTEN levels in our whole NSCLC cohort(P =0.3920)nor only early stage of NSCLC specimens(Stage ?-?)(P = 0.0922).In early stage NSCLC,patients with high miR-374a expression together low CCND1 expression groups had significantly longer survival times(P<0.001).This effect was not observed for patients with advanced stage NSCLC(P = 0.475)nor the entire NSCLC cohort(P = 0.088).Patients with low miR-374a expression together with high PTEN expression had the longest survival times among advanced stage patients(P<0.001)as well was the entire NSCLC cohort(P = 0.005).This effect was not found for early stage NSCLC patients(P = 0.916).The study concluded that miR-374a plays a different role in different non-small cell lung cancer cells,and plays a different role in different stages of non-small cell lung cancer.