| Lung cancer is one of the leading causes of malignancy-related mortality worldwide.In 2015,there were about 4.292 million new cases of cancer in China and 2.814 million cancer deaths.Among them,733,000 cases of lung cancer(17.1%)and 610,000 cases of death(21.1%)have become the main cause of cancer deaths in China.According to pathological classification,lung cancer is divided into adenocarcinoma(LUAD),squamous cell carcinoma(LSCC),small cell lung cancer(SCLC),large cell lung cancer.Lung adenocarcinoma and squamous cell carcinoma account for about 85%,and the 5-year survival rate is less than 20% after surgery and anti-cancer treatments.However,the diagnosis of traditional imaging and pathology is limited in many ways and cannot achieve the purpose of early detection.Therefore,the diagnosis of lung cancer is mostly in the late stage of lung cancer,and the optimal treatment time is missed,coming with the consequence of slow effect,rapid drug resistance and poor prognosis.Thus,accurate and early diagnosis of lung cancer is a critical factor for effective treatment and reducing mortality.A blood-based tumor marker test is an ideal non-invasive way for diagnosing cancer and early detection,it can also be used to monitor the curative effect during follow-up observation.The National Academy of Clinical Biochemistry(NACB)and European Group On Tumor Markers(EGTM)recommended several serum tumor markers for lung cancer,including CEA(carcinoembryonic antigen),proGRP(pro-gastrin-releeasing peptide),SCC(squarmous cell carcinoma antigen)and CYFRA 21-1(cytokeratin 19 fragment)and NSE(neuron specific enolase).NSE and ProGRP are mainly used for the diagnosis and prognosis of SCLC,while CEA,SCC and CYFRA21-1 is used for the diagnosis of NSCLC.However,a lack of sensitivity and specificity has limited their value in detecting lung cancer.For example,ProGRP significantly increased in benign diseases,such as chronic renal failur,and CEA is elevated in various malignancies,and is mainly used for diagnosing gastrointestinal tumors.Besides,detection of biomarkers in blood is convenient and non-invasive.Therefore,the discovery of new lung cancer specific circulating biomarkers with a high sensitivity is critically important.In human genome,more than 80% of the genes are transcribed into non-protein coding RNAs.These non-coding RNAs are classified into long non-coding RNAs(lncRNAs)and small non-coding RNAs(ncRNAs).In a recent study,5,446 lncRNAs were confirmed to be present in the human genome,and the total number of lncRNAs has reached 6,736.Moreover,in the past 20 years,the molecular mechanism of microRNA(miRNAs)has been relatively identified,but the function of lncRNAs remains to be unclear.LncRNA has been found to play an important role in many fields of biology,such as stem cell biology,individual development,X chromosome inactivation,neuroscience and cancer.Overall,however,the functional lncRNAs remains a minority.Recent studies have found that lncRNAs play an important role in different tumors by participating in multiple signaling pathways.Conventional wisdom holds that RNA is not stable in the natural environment without the protection of cells,but the RNA outside cells has been proved to be stable in the circulatory system.RNAs may be protected by vesicles,such as exosomes,from being degraded.Different expressed lncRNAs in lung cancer tissue and cell lines have been reported.But its expression and clinical significance in plasma still lack data support.In order to find a new key lncRNA as a diagnostic tumor marker of lung cancer by screening and validating candidate lncRNAs in plasma sample,this study was carried out as the following two parts.Part I: diagnostic value of plasma PVT1 in lung adenocarcinoma.Objective: 1.To find out the high expressed LncRNAs in plasma of patients with lung adenocarcinoma.2.To understand how it is transported in plasma and its possible sources.3.To clarify its diagnostic value in lung adenocarcinoma.Methods: 30 differentially expressed LncRNAs were selected from lncRNADisease database and reported literature.The plasma of patients with lung adenocarcinoma was set as the experimental group(n=26),and the plasma of healthy volunteers was set as the control group(n=26).The expression of candidate LncRNAs in plasma was detected by qRT-PCR,and LncRNAs with significant difference in lung adenocarcinoma was found by statistical analysis.Validation group included lung adenocarcinoma patients(n=202),healthy volunteers(n=202),and other pulmonary diseases patients(n=198).Sensitivity and specificity were calculated by ROC curve analysis.Statistical analysis showed the correlation between LncRNA and clinical factors.Subsequently,through a series of experiment study the stability of lncRNAs in plasma and its possible origin.Results: PVT1 expression in patients with lung adenocarcinoma was significantly higher than that of healthy volunteers(P<0.001).The ROC curve analysis showed better specificity(78.85%)and sensitivity(78.5%)than tumor biomarkers such as CEA when discriminating non-LUAD patients from LUAD patients.The expression of plasma PVT was associated with distant metastasis of lung adenocarcinoma.By comparing the expression of PVT1 in plasma,exosome and exosome-depleted plasma,we found PVT1 was protected by exosomes in plasma.In addition,(1)plasma PVT1 expression was significantly reduced after surgically resected adenosquamous carcinoma of the lung;(2)The expression of exosomal PVT in culture medium of lung adenocarcinoma cell lines was increased over time;(3)After inhibition of PVT1 expression in A549 cells,the expression of exosomal PVT1 in the culture medium also decreased.All these results indicated that plasma PVT1 was mainly derived from lung adenocarcinoma cells.Conclusion: We found that plasma PVT1 is significantly up-regulated in LUAD patients and serves as a potential biomarker for LUAD.PVT1 expression in plasma is associated with distant metastasis.It is found in the plasma and is mainly in the form of exosome containing substances,and is derived from lung adenocarcinoma cells.In summary,plasma PVT1 is expected to be a plasma marker for dynamic monitoring the progression of lung adenocarcinoma,providing new ideas for the diagnosis and treatment of lung adenocarcinoma.Part II: the diagnostic value of plasma exosomes SOX2-OT in lung squamous cell carcinoma Objective: to identify the expression of 20 lncRNAs which is found by analyzing the results of 5 chip data of lung squamous cell carcinoma tissue in GEO databases and identify their diagnostic value in lung squamous cell carcinoma.Methods: 20 lncRNAs were found to be differentially expressed in lung squamous cell carcinoma tissues using the analysis results of chip data in 5 GEO databases.qRT-PCR was used to detect the expression of the candidate LncRNAs in plasma exosomes,and to determine whether high expression of LncRNA was present.The diagnostic sensitivity and specificity were calculated by ROC curve analysis.Statistical analysis showed the correlation between LncRNAs and clinical features.Results: The expression of lncRNA SOX2-OT and ENSG00000245648 in patients with lung squamous cell carcinoma was significantly increased(P<0.01).Through ROC curve analysis and comparison with markers such as CEA and SCC,it was found that SOX2-OT had a better diagnostic value for lung squamous cell carcinoma.The specificity and sensitivity were 73.17% and 76% respectively,while the sensitivity of ENSG00000245648 was only 44%.Additionaly,it was found that the expression of SOX2-OT in plasma was positively correlated with tumor size,TNM stage and lymph node metastasis.Conclusion: The expression of plasma exosomal SOX2-OT and ENSG00000245648 was significantly increased in patients with lung squamous carcinoma.But only SOX2-OT has a good diagnostic value for lung squamous cell carcinoma.The expression of SOX2-OT in plasma is correlated with tumor size,TNM stage and lymph node metastasis.In summary,the plasma exosomal SOX2-OT is expected to be a potential diagnostic marker for lung squamous cell carcinoma. |
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