| Background:Lung cancer is the leading cause of cancer-associated mortality,with～1.35 million new cases every year worldwide.Between 75 and 80%of lung cancer cases are non-small cell lung cancer(NSCLC),which has an overall 5-year survival rate of only 10%.Despite novel methods targeting early diagnosis and recent advancements in treatments,the prognosis and survival rate of NSCLC patients remains relatively poor.Between 40 and 50%of patients will eventually succumb to relapse or metastatic disease after curative resection.However,few reliable prognostic biomarkers are available in clinical practice,particularly in patients who have undergone curative surgical resection.Therefore,there is a requirement to identify novel prognostic biomarkers that could aid prognosis prediction and optimize the treatment of NSCLC patients.MicroRNAs(miRNAs/miRs)are a class of small(19-24 nucleotides in length),highly conserved non-coding RNAs that bind to the 3’-untranslated regions of target mRNAs and suppress their translation to proteins.It has become increasingly evident that different miRNAs can play either oncogenic or tumor-suppressive roles in a wide variety of pathways,depending on the target genes or the cellular context.miR-200c is a member of the miR-200 family,which consists of five members(miR-200a,miR-200b and miR-429 comprise cluster 1,which is located on chromosome 1 p36;and miR-200c and miR-141 comprise cluster 2,which is located on chromosome 12p]3).Recent investigations have shown that members of the miR-200 family could promote or repress different cancer types via various pathways.However,studies investigating the association between the expression level of miR-200c and the clinical outcome in resected NSCLC patients are few in number and have returned contradictory results.The specific role of miR-200c in NSCLC has,therefore,not yet been elucidated.In the present study,we perform the following two parts research:(1)The expression of miR-200c was examined in 110 clinical NSCLC samples and the association between miR-200c expression and variable clinicopathological features and patient prognosis was analyzed.(2)The target genes of miR-200c were then identified and verified by bioinformatics software,dual-luciferase reporter assay.And then explore the regulatory effects and mechanism of miR-200c via target genes in A549 cells.Part 1:The correlation between microRNA-200c and prognosis in non-small cell lung cancerObjective:To detect the expression levels of miR-200c in patients diagnosed non-small cell lung cancer,and investigate the correlation of miR-200c to the clinicopathologic features and prognosis.Methods:A total of 110 tumor samples were collected from patients who had been pathologically diagnosed with primary NSCLC and who underwent complete tumor resection(lobectomy or pneumonectomy)with regional lymph node dissection at the Department of Thoracic Surgery,Qilu Hospital Shandong University,between January and December 2008.Collect the clinicopathological characteristics and the follow-up data of these 110 patients.Detect the expression levels of miR-200c using stem-loop RT-PCR.All statistical analyses were performed using SPSS 18.0 software.The associations between clinical variables and miR-200c expression were analyzed using the Pearson X2 test.Survival curves were plotted using the Kaplan-Meier method and assessed with a log-rank test to identify significant differences,with mortality due to lung cancer as the end point.Multivariate Cox regression was used to perform multivariate survival analysis(5-year disease-free survival and 5-year overall survival).P<0.05 was considered to indicate statistical significance.Results:miR-200c levels were quantified by performing stem-loop RT-PCR on 110 NSCLC specimens and 43 normal lung tissues.qPCR confirmed that,compared with their corresponding normal tissues,the NSCLC specimens exhibited upregulated miR-200c expression.Higher miR-200c expression was significantly associated with positive lymph node metastasis(P<0.05);there was no statistical significance in the associations between miR-200c expression and other clinicopathological variables(P>0.05).Of the 110 NSCLC patients examined in this study,tumor relapse developed in 89(80.9%)within the follow-up period:Local recurrence occurred in 22 patients,distant metastasis in 46 patients,and local recurrence and distant metastasis in 21 patients.Univariate analysis demonstrated that high miR-200c expression(15.2 vs 25.0%,P=0.004),positive lymph node metastasis(9.3 vs 28.6%,P<0.001)and advanced TNM stage(0 vs 15.6 vs 30.4%for stage Ⅲ,Ⅱ,and Ⅰ,respectively;P=0.011)significantly predicted decreased 5-year disease-free survival rates.Of the 110 NSCLC patients,74(67.3%)succumbed to cancer-associated causes within 5 years of surgery,and the 5-year overall survival was 32.7%.Univariate analysis demonstrated that high miR-200c expression(21.2 vs 50.0%,P<0.001),positive lymph node metastasis(13.0 vs 51.8%)and advanced TNM stage(0 vs 24.4 vs 54.3%for stage Ⅲ,Ⅱ and Ⅰ,respectively;P=0.002)significantly predicted poor 5-year overall survival rates.The results of multivariate Cox regression analysis showed that TNM stage(both P=0.000)and miR-200c expression(P=0.030 and P=0.006)retained significance as independent prognostic factors for unfavorable 5-year disease-free survival and poor 5-year overall survival rates,respectively.Conclusions:MiR-200c overexpression is common in non-small cell lung cancer,and significantly associated with positive lymph node metastasis and poor survival.The present results suggest that miR-200c may have clinical potential as an effective predictor to identify individuals with poor prognosis.Part 2:The target genes and the regulatory mechanism of miRNA-200c in non-small cell lung cancerObjective:To identify and verify the target genes of miR-200c in non-small cell lung cancer,and explore the regulatory effects and mechanism of miR-200c via target genes in A549 cells.Methods:The target genes of miR-200c were identified by bioinformatics software(TargetScan;microRNA.org;PicTar).And then verify the target genes by the dual-luciferase reporter assay in HEK-293 cells.Detect the expression levels of the mRNAs of target genes in non-small cell lung cancer by RT-PCR.Make the expression of miR-200c upregulation and downregulation respectively in A549 cells,and then detect the expression levels of the target genes proteins by Western blot,mRNAs by RT-PCR.And analyze the regulatory mechanism of miR-200c in non-small cell lung cancer.Results:We identify three potential target genes of miR-200c by bioinformatics software,ATRX,HFE and DLC1.And these three target genes were verified by the dual-luciferase reporter assay in HEK-293 cells successfully.The mRNAs expression of ATRX,HFE and DLC1 showed significantly downregulation in non-small cell lung cancer samples,which substantiated the regulatory effects of miR-200 on these three target genes.The overexpression of miR-200c in A549 cells resulted in significantly downregulation of ATRX,HFE and DLC1 proteins,and downregulation of ATRX and HFE mRNAs,but not DLC1 mRNA.The lower expression of miR-200c in A549 cells resulted in significantly upregulation of ATRX,HFE and DLC1 proteins,and upregulation of ATRX and HFE mRNAs,but not DLC1 mRNA.The above results indicated that miR-200c regulate ATRX and HFE at mRNA level,and regulate DLC1 at protein level.Conclusions:ATRX,HFE and DLC1 are the direct target genes of miRNA-200c in non-small cell lung cancer.MiRNA-200c could regulate ATRX and HFE at mRNA level,and regulate DLC1 at protein level in A549 cells. |
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