| ObjectiveLung cancer is a global health problem that still threatens thousands of lives.In China,the morbidity and mortality of lung cancer ranks first in many years.It poses a great threat to the health and life of the Chinese people and causes a huge social and economic burden.The main histological types of lung cancer include non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).Non-small cell lung cancer accounts for about 85%of the total number of lung cancers.The cause of non-small cell lung cancer is still unclear.In terms of personalized treatment for non-small cell lung cancer,the current research on non-small cell lung cancer has deepened to the gene level.Related molecular markers such as epidermal growth factor receptor,tumor expression protein p53,etc,may directly affect clinicians' choice of treatment options.With the refinement of non-small cell lung cancer treatment programs and the ongoing development of molecular targeted therapies,novel tumor molecular markers related to the diagnosis and prognosis of non-small cell lung cancer still needs to be constantly updated to meet the needs of clinical assessment of the diagnosis and prognosis of NSCLC.LncRNA is more than 200nt in length and lacks protein coding capacity.So far,14,880 human lncRNAs have been identified.LncRNAs are the products of Polymer?(pol ?),most of which have been blocked,polyadenylated and frequently spliced.Transcriptional profiling studies have gradually revealed the role of lncRNAs in various diseases in human.In the field of cancer,lncRNA expression has a significant dysregulation in tumor cells and tissues compared with normal tissues,and is extensively involved in malignant transformation,lineage-specific changes,gene copy number changes,and epigenetic modifications of tumor cells.Such biological processes can regulate cell proliferation,apoptosis,cell cycle progression,cell metabolism,cell invasion and metastasis.Maternally expressed gene 3(MEG3),one of the most important cancer-associated lncRNAs,also known as GTL2,was initially identified on chromosome 12 of the distal mouse.MEG3 is localized on human chromosome 14q32 and is approximately 1.6 kb in length,producing alternative spliced transcripts.Similar to its mouse homologs,the expression and regulation of MEG3 is crucial for embryonic development and growth.The loss of expression of MEG3 has been detected in human pituitary,cervical,urothelial,and prostate cancers,highly suggesting that MEG3 may act as a tumor suppressor gene in various biological processes of tumors.In the study of different types of tumors,it was found that most of MEG3 exert its anti-cancer effect,and its high expression can induce the growth arrest of hepatoma cells,promote the apoptosis of hepatoma cells,and can also inhibit the excessive proliferation of breast cancer cells and reduce their malignancy.In thyroid cancen,it has also been found that the loss of expression of MEG3 is associated with its malignant transformation.In vitro studies have shown that abnormal methylation of MEG3 promoter region was often found in NSCLC cells,which caused the suppression or deletion of MEG3 expression in NSCLC;Overexpression of MEG3 can downregulate NSCLC cell proliferation and induce apoptosis in vitro.As the correlation between the absence of MEG3 expression and tumors is gradually revealed,key cancer-related signaling pathways involved in MEG3 and key cytokines that are regulated are also being elucidated.The abnormal expression of MEG3 could influence p53,MDM2,CDF 15 and other key cytokines,results in the deregulation of biological processes such as proliferation,apoptosis and invasion and metastasis of NSCLC cells.In addition,MEG3 is also considered to be related to the sensitivity of NSCLC to platinum drugs.Studies on clinical tissue specimens have also confirmed that MEG3 expression in NSCLC tissues is reduced or even absent,and its low expression may be a poor prognostic factor in patients with NSCLC.Based on the effect of MEG3 on key genes such as p53 and its down-regulation in NSCLC,the investigators believe that MEG3 may act as a tumor suppressor and affect the proliferation,apoptosis,and invasion of NSCLC cells through p53-related molecular pathways.Biological behavior of MEG3 may affect the prognosis of patients with non-small cell lung cancer.However,up to now,there have not been enough reports of MEG3 down-regulation related to the poor prognosis of patients with NSCLC.The clinical meaning of MEG3,and it's expression in patients with non-small cell lung cancer remains unclear and remains to be further explored.The purpose of this reaserch is to systematically study the relationship between MEG3 and prognosis of patients with non-small cell lung cancer and its mechanism.Section ?:The clinical and pathological parameters in GEO database were selected and analyzed,it was found that the low expression of LncRNA-MEG3 in non-small cell lung cancer indicated poor prognosis.Section ?:Basic experiments to verify the expression of MEG3 in non-small cell lung cancer and its biological behavior,so as to further demonstrate and analyze its anti-oncogene mechanism;Section ?:Review of association between lncRNA-MEG3 and tumor.Through the MEG3 above-mentioned from clinical data,basic research,research progress in three section,the study intends to investigate the relationship between the expression of human maternally expressed gene 3(MEG3)and prognosis in non-small cell lung cancer,in order to find a potential target for cancer diagnosis,treatment and prognosis.Section I:Down-regulation of long non-coding RNA MEG3 indicates an Unfavor-able prognosis in non-small cell lung cancer:Evidence from the GEO database.MethodsTo investigate the association of MEG3 with the prognosis of NSCLC patients,seven datasets with 1144 patients were obtained from Gene Expression Omnibus(GEO)database(Affymetrix U133 Plus 2.0 platform).The correlation between MEG3 expression and age,smoking index,gender,histology,staging,smoking status,ethnicity and adjuvant therapy was tested using SPSS 22.0 software using chi-square test.Kaplan-Meier survival analysis was used to explore the association between MEG3 expression and overall/progression-free survival.Univariate Cox proportional hazards regression analysis was performed on clinical and pathological data affecting patients' OS and PFS.Multivariate regression analysis was performed on the P<0.05 variables in a univariate Cox regression analysis.Subgroups included age,gender,histology,stage,ethnicity,smoking status and adjuvant chemotherapy;What's more,we did meta-analysis with all data in the seven datasets to further analyze summarized impact of MEG3 expression on survival.Random effect model was applied to estimate pooled HR(hazard ration).Forest plots were presented with HR and 95%confidence interval(95%CI).And sensitivity analysis was done to evaluate the influence of each dataset to the summarized result.We did Egger's tests in order to evaluate publication bias.P values were two-sided and it was regarded as statistically significant with value<0.05.Meta-analysis was performed with STATA 12.0.The correlation between MEG3 and OS/PFS in NSCLC patients was analyzed in detail in each database.ResultsDifferential expression of MEG3 in GEO database was related to stage(GSE31210OS and GSE31210PFS),histology(GSE29013OS and GSE29013PFS)and gender(GSE29013PFS).Further analysis of the correlation between MEG3 and OS/PFS in patients,we found that the GSE3141OS(p = 0.039),GSE30219OS(p =0.008)GSE30219PFS(p= 0.048)were correlated with the expression level of MEG3.The low expression of MEG3 was associated with OS shortening in GSE31210 dataset patients(p = 0.050).In GSE31410S,GSE30219OS,GSE31210OS,GSE377450S,GSE50081OS,GSE29013PFS,GSE30219RFS,GSE31210PFS and GSE37745PFS,the 5-year survival rate of NSCLC patients in MEG3 high expression group was significantly higher than that in MEG3 low expression group.MEG3 was an independent prognostic factor for PFS in patients with GSE30219(HR 0.666,95%Cl 0.458-0.969,p = 0.033).The differential expression of MEG3 in lung adenocarcinoma,squamous cell carcinoma and large cell lung cancer tissues were compared with that in non-tumor tissues in the oncomine database,the result showed that MEG3 was low expressed in NSCLC patients(median = 4346.0,p<0.001).The summarized result suggested that low MEG3 expression was a poor prognostic factor in NSCLC(HR = 0.77,95%CI 0.63-0.95).Specifically,the association between low MEG3 expression and poor prognosis was markedly significant in younger patients(?60 years old)(HR0.602,95%CI 0.417-0.867,P= 0.007).ConclusionMEG3 expression is affected by clinical staging,histology,and gender of patients.Non-small-cell lung cancer patients often exhibit suppression or absence of MEG3 expression,and the reduction in the overall survival or progression-free survival of patients is closely related to the down-regulation of MEG3;Especially in young patients(? 60 years old),the relationship between low expression of MEG3 and poor prognosis is very significant.MEG3 can be a new prognostic factor for patients with non-small cell lung cancer.In view of the weakness of retrospective study,further evidence from prospective study is necessary.Further research is needed to elucidate the mechanism of MEG3.Sectionll The expression of MEG3 in non-small cell lung cancer and its biological behaviorWe used GEO database in our previous study to confirm that MEG3 was down-regulated in NSCLC and was associated with poor prognosis,but the mechanism is unknown.MethodsRelying on the database of tumor organizations of Shandong Provincial Hospital,we randomly selected 40 patients(NSCLC)from the Provincial Hospital of Shandong Province who had undergone lung cancer resection and preserved frozen tissues from 2011 to 2012 through the data management system,and obtained their frozen tumor tissues,and matched normal tissues.The human lung adenocarcinoma cell line A549 was purchased from the American Type Culture Collection(ATCC);The expression of MEG3 in lung cancer and normal tissues were explored by real-time quantitative PCR.MEG3 was overexpressed by plasmid transfection;MEG3 was knocked down by siRNA inA549 cell lines;we used SRB assay for cell proliferation,Annexin V-FITC/PI assay for apoptosis,Ethidium bromide(PI)assay for cell cycle,Scratch test for migration,and Western blotting for some EMT indicators.As result,its biological behaviors were detected.Results1.By detecting the relative expression of MEG3 in lung cancer tissues and matched normal tissues,the expression of MEG3 in most of the patients was lower than that in normal tissues;And the expression of MEG3 in tumor tissues was significantly lower than that in normal tissues(p<0.0001);2.Detection of proliferation of lung cancer cells by SRB assay confirmed that MEG3 overexpression can significantly inhibit the proliferation of A549 cells,while knockout of MEG3 can promote the proliferation of A549 cells to a certain extent;3.Overexpression of MEG3 induced significant G0/G1 phase cell arrest(p = 0.02),but no significant effect on G2/M phase;4.Scratch experiments confirmed that MEG3 overexpression can inhibit cell migration significantly;5.Real-time quantitative PCR revealed that CDH1 was upregulated after MEG3 upregulation,but significant downregulation of CDH2 and VIM was observed in interstitial cells.SNAI1 and SNAI2 were significantly downregulated as transcription factors of epithelial-mesenchymal transition;TGFB1 was significantly downregulated.However,the changes of VEGFA,VEGFB and KDR were not obvious,and MMP3 and MMP9 also showed a certain degree of down-regulation.Western showed that when MEG3 was knocked out,PDGFRA and PDGFRB were significantlyupregulated,and EMT-related parameters such as Snail1,Snail2,CDH2 and KDR15 were significantly up-regulated,further confirming the important role of MEG3 in EMT.ConclusionMEG3 is down-regulated in most NSCLC patients than normal tissues by real-time quantitative PCR.Basic experiments confirmed that MEG3 can affect the biological behaviors of NSCLC such as proliferation,apoptosis,cycle and migration,epithelial mesenchymal transition.1.The detection of MEG3 expression in tumors and matched normal tissues by real-time quantitative PCR confirmed the existence of MEG3 expression difference in NSCLC tumors and normal tissues,and its expression in tumors was significantly lower than that in normal tissues;2.MEG3 can affect the cycle of NSCLC cells and cause cycle inhibition.3.MEG3 can promote the apoptosis of tumor cells;4.MEG3 plays an important role in NSCLC epithelial mesenchymal transition and angiogenesis.Section? Review of association between LncRNA-MEG3 and tumorIn this section,we reviewed the recent research progress of lncRNA-MEG3,including the function of lncRNAs,the regulatory mechanism of MEG3,MEG3 and cancer,MEG3 and non-small cell lung cancer,the prospect,application value and possibility of cancer therapy.So as to further verify our previous clinical research and experimental research results.Conclusion:lncRNA has multiple functions and complex mechanism,which needs further study.MEG3 is closely related to the clinicopathological results of various cancers.The deletion or decrease of MEG3 expression is very common in human cancer.However,MEG3 is an RNA-based tumor suppressor,which is involved in the pathogenesis and progression of tumor,and its activity is mediated by TP53.It's differential expression between normal and different levels of cancer makes MEG3 a biomarker for assessing the stage and prognosis of cancer.SummaryThrough our research on GEO clinicopathological parameters,basic research and review of domestic and foreign research progress on lncRNA-MEG3,we can draw a final conclusion:low expression of lncRNA-MEG3 in non-small cell lung cancer indicates poor prognosis,MEG3 is a tumor suppressor gene,which can be used as a potential target for evaluation of prognosis;At the same time,with the progress of real-time quantitative PCR and other modern scientific research technology,low cost,MEG3 can be widely used in clinical,provide guidance for clinical diagnosis,treatment and evaluation of prognosis. |
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