Establishment Of Relative Quantitative PCR Detection Method For Long Non-coding RNA And Preliminary Verification In Non-small Cell Lung Cancer

Objective?s?:Lung cancer is the most common cause of cancer-related deaths all over the world.Non-small cell lung cancer accounts for 85%of all lung cancer cases.Although there have been many positive advances in treatment,the overall survival rate of patients is still low.Most patients already metastasis at the time of diagnosis,and the prognosis is poor.It is urgent need to develop new biomarkers that are non-invasive,with high specific and sensitive,enabling diagnose lung cancer at early stage.More and more study proves that lncRNA participates in the process of the development of non-small cell lung cancer.Based on the previous research,this study selected a series potentially valuable lncRNA by the microarray result,and establish a relatively quantitative PCR detection method.Looking for lncRNA molecules with potential value as new biomarkers for tumor diagnosis,lay the foundation for further mechanism research,and provide new ideas for early diagnosis and prognostic of non-small cell lung cancer.Methods:Based on the results of the previous LncRNA+mRNA microarray,eight candidate lncRNAs with potential value as diagnostic markers were selected.Later,it was found that three of the lncRNA molecules could not be designed with suitable primer.Next,we establish a multiple quantitative PCR detection method to measure the relative expression of 5 lncRNAs in 20 lung cancer tissues and matched adjucent normal tissues,we furtuer analysed the relative expression of 5 lncRNAs from serum in a cohort of 44 lung cancer patients,46 lung benign patients,and 48 healthy controls.Statistical analysis was performed by Mann-Whitney U tests.And the receiver operating characteristic curve?ROC curve?analysis was used to evaluate diagnostic performance of selected IncRNAs in non-small cell lung cancer.Results:Based on the preliminary microarray result,five lncRNAs with high fold change and good consistency were selected as candidate lncRNAs:XLOC₀09677,ENSG00000256564.1,ENSG00 000249859.2?PVT1?,XLOC₀12568,XLOC₀01406.We established multiple real-time quantitative PCR relative quantitative methods,and optimize the experimental conditions.The method was used to measure the expression of 5 IncRNAs in 20 pairs of lung cancer tissues and paired tissues,as well as serum of 44 lung cancer patients,46 lung benign patients,and 48 healthy controls.It was found that compared with adjacent tissues:XLOC₀09677,ENSG00000249859.2?PVT1?,XLOC₀12568,XLOC₀01406 was up-regulated,The result basically consistent with the microarray result,indicating that the microarray results were reliable.Subsequently,we selected 3 lncRNAs:XLOC₀09677,XLOC₀12568,XLOC₀01406.Which were significantly up-regulated?P<0.05?between the lung cancer patients and the healthy controls,but with no statistical difference between the benign lung patients and the healthy controls?P>0.05?to confirm its diagnostic value.The analysis results show that the AUC of XLOC₀09677,XLOC₀12568,and XLOC₀01406 are:0.647?95%CI:0.532-0.762?,0.642?95%CI:0.527-0.756?,0.707?95%CI:0.601-0.813?.The combined detection AUC is:0.747?95%CI:0.527-0.756?,all of three lncRNAs have the potential value to distinguish non-small cell lung cancer patients from healthy people,and the combined detection of three lncRNAs is more valuable for diagnosis.Conclusion?s?:In this study,we found that compared with adjacent tissues:XLOC₀09677,ENSG00000249859.2?PVT1?,XLOC₀12568,XLOC₀01406 were up-regulated;compared with healthy serum controls:XLOC₀09677,XLOC₀12568,XLOC₀01406 were up-regulated in non-small cell lung cancer patients.,all of three lncRNAs have the potential value to distinguish non-small cell lung cancer patients from healthy people.