Serum MiRNAs As Biomarkers For Early Diagnosis Of Non-small Cell Lung Cancer

BackgroundLung cancer is the highest incidence of morbidity and mortality in the world.According to statistics,half of the patients with lung cancer has metastasized when being diagnosed,that most can not receive radical treatment any more and the median survival time of these patients were only 8-11 months.85%of lung cancer cases were cases of non-small cell lung cancer(NSCLC),and NSCLC is mainly composed of adenocarcinoma and squamous cell carcinoma.Up to now,there is still no accurate and reliable,economical and convenient and less invasive method for the diagnosis of patients with early stage NSCLC.MicroRNA(miRNA)is a class of non-coding single-stranded small RNAs of about 19-24 nucleotides in length,and a large number of literatures confirm that it is associated with progression,metastasis and recurrence.Since miRNA has been detected in the circulatory system for the first time,it has been shown to be stable in serum and may be used as a noninvasive biomarker for early diagnosis of tumor.PurposeWe are trying to find new serum miRNA biomarkers for early diagnosis of NSCLC.Materials and MethodsIn the first part,we selected 6 patients with early-stage NSCLC and 6 healthy volunteers after age and gender matched.First,we extracted the serum total RNA.Second we took advantage of Denmark's Exiqon miRCURY TM LNA Array to screen the differential expressed serum miRNAs in early-stage NSCLC compared to healthy control.Third,we took advantage of bioinformatic analyses to discover serum differential expressed miRNAs in NSCLC.At last,we combined the results above and obtained the potential differential expressed serum miRNAs.In the second Part,we selected 12 patients with early-stage NSCLC and 12 healthy controls after age and gender matched that defined as training set.In the same way,we extracted the serum total RNA first.After that,we took advanced of real-time PCR to preliminarily verified the potential differential expressed serum miRNAs found after first screening in the first part.In the third Part,we selected 60 patients with early-stage NSCLC and 60 healthy controls after age and gender matched that defined as validation set.In the same way,we extracted the serum total RNA first.After that,we took advanced of real-time PCR to further verified the potential differential expressed serum miRNAs found after preliminary verification in the second part.At last,we took advantage of receiver operating characteristic curve to test the ability of the differential expressed serum miRNAs in diagnosed early-stage NSCLC and make sure the potential biomarkers for early diagnosis of NSCLC.ResultsIn the first part,the purity and concentration of the total RNA extracted from the serum of the 12 cases all reached the basic requirements of the follow-up experiment.The probe points of each chip were evenly imaged and no obvious abnormalities were observed.That both the expression level was changed by 2 times or above and p value of less than 0.05 were considered to be meaningful for the results of the microarray.The microarray profiling showed that 122 up-regulated miRNAs and 87 down-regulated miRNAs.For bioinformatic analyses,there were 31 up-regulated miRNAs and 28 down-regulated miRNAs in the serum samples and 172 up-regulated miRNAs and 137 down-regulated miRNAs in the tissue samples.Then we combined the experimental results above and 7 differential expressed serum miRNAs were found,which were miR-185-5p?miR-431-5p?miR-484?miR-492?miR-584-5p?miR-590-3p and miR-631,respectively.In the second part,the average Ct values of serum U6 were 23.57 � 0.23 in the control group and 23.19�0.25 in the experimental group.There was no statistically difference in these 2 groups and it showed that serum U6 was a suitable internal gene.In the training set,compared to normal control,the expression level of serum miR-492 and miR-590-3p were up-regulatd with fold change of 1.88 and 1.71,p value were both less than 0.05;the expression level of serum miR-631 was down-regulatd with fold change of 1.46 and p value was less than 0.05.The Ct of serum miR-584-5p was more than 40 and revealed that serum miR-584-5p may be not a suitable biomarker because of low content.In the third part,using the validation set,compared to normal control,the expression level of serum miR-492 and miR-590-3p were up-regulatd with fold change of 1.65 and 1.69,p value were both less than 0.05;the expression level of serum miR-631 was down-regulatd with fold change of 1.50 and p value was less than 0.05.The area under the ROC curve(AUC)was 0.789.0.792 or 0.711 when using serum miR-492,miR-590-3p or miR-631 as a single index to distinguish healthy control with early-stage NSCLC.Moreover,AUC was 0.828 and 95%of confidence interval was 0.705 to 0.905 when combined these 3 serum miRNAs as a single index,and the sensitivity was 86.7%and the specificity was 71.1%,respectively.ConclusionA panel of 3 serum miRNAs,including miR-492,miR-590-3p and miR-631,could be used as a potential biomarker for the early diagnosis of NSCLC.However,more clinical data,especially large sample,multicenter and prospective trials,are needed.