High-throughput qRT-PCR validation of blood microRNAs in non-small cell lung cancer.
ABSTRACT: Validation of biomarkers is essential to advance the translational process to clinical application. Although there exists an increasing number of reports on small non-coding RNAs (microRNAs) as minimally-invasive markers from blood, serum or plasma, just a limited number is verified in follow-up studies. We used qRT-PCR to evaluate a known miRNA signature measured from blood that allowed for separation between patients with non-small cell lung cancer (NSCLC), COPD and healthy controls.From the data of our previous microarray studies we selected a panel of 235 miRNAs related to lung cancer and COPD. We observed a high concordance between the AUC values of our initial microarray screening and the qRT-PCR data (correlation of 0.704, p < 10-16). Overall, 90.3% of markers were successfully validated. Among the top markers that were concordant between both studies we found hsa-miR-20b-5p, hsa-miR-20a-5p, hsa-miR-17-5p, and hsa-miR-106a-5p. The qRT-PCR analysis also confirmed that non-small cell lung cancer patients could be accurately differentiated from unaffected controls: a subset of five markers was sufficient to separate NSCLC patients from unaffected controls with accuracy of 94.5% (specificity and sensitivity of 98% and 91%). Beyond differentiation from controls, we also succeeded in separating NSCLC patients from patients with COPD. MiRNAs that were identified as relevant for the separation between lung cancer and COPD by both qRT-PCR and the array-based studies included hsa-miR-26a-5p, hsa-miR-328-3p and hsa-miR-1224-3p. Although for differentiation between NSCLC patients from COPD patients more markers were required, still high accuracy rates were obtained (5 markers: 78.8%; 10 markers: 83.9%; 50 markers: 87.6%).
Project description:Background:Lung cancer is one of the leading diagnosed cancers worldwide, and microRNAs could be used as biomarkers to diagnose lung cancer. hsa-miR-195 has been demonstrated to affect the prognosis of NSCLC (non-small-cell lung cancer) in a previous study. However, the diagnostic value of hsa-miR-195-5p in lung cancer has not been investigated. Methods:To evaluate the ability of hsa-miR-195-5p to diagnose lung cancer, we compared the expression of hsa-miR-195-5p in lung cancer patients, COPD patients, and normal controls. Receiver operating characteristic (ROC) curve analysis was performed to investigate the sensitivity and specificity of hsa-miR-195-5p. Coexpression network and pathway analysis were carried out to explore the mechanism. Results:We found that hsa-miR-195-5p had lower expression in lung cancer and COPD patients than in normal controls, and the AUC was 0.92 for diagnosing lung cancer. hsa-miR-143 correlated with hsa-miR-195-5p, and by combining these two microRNAs, the AUC was 0.97 for diagnosing lung cancer. Conclusions:hsa-miR-195-5p may act as a biomarker that contributes to the diagnosis of lung cancer and the detection of its high-risk population.
Project description:<b>Background: </b>Early diagnosis improves the prognosis for non-small cell lung cancer (NSCLC); therefore, there is a pressing need for effective diagnostic methods for NSCLC. Increasing evidence indicates that serum exosomal micro RNAs (miRNAs) represent promising diagnostic and prognostic markers for multiple cancers. Here, we explored a panel of miRNAs for NSCLC diagnosis and functionally characterized miR-1269a in the pathogenesis of NSCLC.<br><br><b>Methods: </b>First, we analyzed high-throughput data from The Cancer Genome Atlas (TCGA) to identify differentially expressed miRNAs between NSCLC patients and healthy controls. We examined the expression profiles of the identified miRNAs using qRT-PCR.<br><br><b>Results: </b>We found that four micro-RNAs (hsa-miR-9-3p, hsa-miR-205-5p, hsa-miR-210-5p, and hsa-miR-1269a) were more abundant in serum exosomes from NSCLC patients. A logistic regression model validated the diagnostic efficacy of the four-microRNA panel, allowing us to distinguish NSCLC patients from healthy controls with AUCs of 0.915 and 0.878 for the training and validation sets, respectively. Functionally, NSCLC cell proliferation, migration, and invasion were affected by the aberrant expression of hsa-miR-1269a in culture. Reduced expression of miR-1269a resulted in reduced proliferation, migration, and invasion through targeting the forkhead box O1 gene (FOXO1).<br><br><b>Conclusions: </b>Taken together, our study identified a panel of four serum exosomal miRNAs as a potential noninvasive diagnostic biomarker for NSCLC. The interactions between FOXO1 and miR-1269a represent novel potential targets for NSCLC therapy.
Project description:The diagnosis of non-small cell lung carcinoma (NSCLC) at an early stage, as well as better prediction of outcome remains clinically challenging due to the lack of specific and robust non-invasive markers. The discovery of microRNAs (miRNAs), particularly those found in the bloodstream, has opened up new perspectives for tumor diagnosis and prognosis. The aim of our study was to determine whether expression profiles of specific miRNAs in plasma could accurately discriminate between NSCLC patients and controls, and whether they are able to predict the prognosis of resectable NSCLC patients. We therefore evaluated a series of seventeen NSCLC-related miRNAs by quantitative real-time (qRT)-PCR in plasma from 52 patients with I-IIIA stages NSCLC, 10 patients with chronic obstructive pulmonary disease (COPD) and 20-age, sex and smoking status-matched healthy individuals. We identified an eleven-plasma miRNA panel that could distinguish NSCLC patients from healthy subjects (AUC = 0.879). A six-plasma miRNA panel was able to discriminate between NSCLC patients and COPD patients (AUC = 0.944). Furthermore, we identified a three-miRNA plasma signature (high miR-155-5p, high miR-223-3p, and low miR-126-3p) that significantly associated with a higher risk for progression in adenocarcinoma patients. In addition, a three-miRNA plasma panel (high miR-20a-5p, low miR-152-3p, and low miR-199a-5p) significantly predicted survival of squamous cell carcinoma patients. In conclusion, we identified two plasma miRNA expression profiles that may be useful for predicting the outcome of patients with resectable NSCLC.
Project description:<h4>Background</h4>Two mature microRNAs (miRNAs), hsa-miR-125a-3p and hsa-miR-125a-5p (collectively referred to as hsa-miR-125a-3p/5p), are derived from 3' and 5' ends of pre-miR-125a, respectively. Although impaired regulation of hsa-miR-125a-5p has been observed in some tumors, the role of this miRNA in invasion and metastasis remains unclear, and few studies have examined the function of hsa-miR-125a-3p. In order to characterize the functions of hsa-miR-125a-3p/5p in invasion and metastasis of non-small cell lung cancer (NSCLC), we investigated the relationships between hsa-miR-125a-3p/5p expression and lymph node metastasis in NSCLC tissues. We also explored the impact of expression of these miRNAs on invasive and migratory capabilities of lung cancer cells.<h4>Methods</h4>Expression of hsa-miR-125a-3p/5p in NSCLC tissues was explored using real-time PCR. The relationships between hsa-miR-125a-3p/5p expression and pathological stage or lymph node metastasis were assessed using the Spearman correlation test. For in vitro studies, lung cancer cells were transfected with sense and antisense 2'-O-methyl oligonucleotides for gain-of-function and loss-of-function experiments. Transwell experiments were performed to evaluate cellular migration and invasion.<h4>Results</h4>Expression of hsa-miR-125a-3p/5p was lower in NSCLC tissues than in adjacent normal lung tissues (LAC). Furthermore, the results from the Spearman correlation test showed a negative relationship between hsa-miR-125a-3p expression and pathological stage or lymph node metastasis and an inverse relationship between hsa-miR-125a-5p expression and pathological stage or lymph node metastasis. In vitro gain-of-function experiments indicated that hsa-miR-125a-3p and hsa-miR-125a-5p function in an opposing manner, suppressing or enhancing cell migration and invasion in A549 and SPC-A-1 cell lines, respectively. These opposing functions were further validated by suppression of hsa-miR-125a-3p and hsa-miR-125a-5p expression in loss-of-function experiments.<h4>Conclusion</h4>Hsa-miR-125a-3p and hsa-miR-125a-5p play distinct roles in regulation of invasive and metastatic capabilities of lung cancer cells, consistent with the opposing correlations between the expression of these miRNAs and lymph node metastasis in NSCLC. These results provide new insights into the roles of miR-125a family members in the development of NSCLC.
Project description:Studies suggest that microRNAs that regulate expression of matrix metalloproteinase (MMP)-9 may be involved in hemorrhagic transformation (HT) after cardioembolic stroke, so we examined whether such microRNAs could predict HT in acute cardioembolic stroke patients. Blood samples were prospectively collected from patients who later experienced HT (n = 29) or did not (n = 29), and the samples were assayed for eight microRNAs identified as related to MMP-9 based on three microRNA databases. Expression levels of these microRNAs were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in 28 of the 58 patients, 14 of whom suffered HT and 14 of whom did not. Four differentially expressed miRNAs were identified: hsa-miR-21-5p, hsa-miR-206, hsa-miR-491-5p, and hsa-miR-3123. Subsequent qRT-PCR analysis of these four miRNAs across all 58 patients showed that levels of miR-21-5p, miR-206, and miR-3123 were significantly higher in patients with HT than in those without HT, while expression of miR-491-5p was similar between the two groups. The area under the receiver operating characteristic curve for predicting HT was 0.677 (95% CI 0.535-0.818) for miR-21-5p, 0.687 (95% CI 0.543-0.830) for miR-206, and 0.661 (95% CI 0.512-0.810) for miR-3123. Our results suggest that these three microRNAs may be prognostic markers for HT after cardioembolic stroke, which should be verified by future studies with large samples.
Project description:Recently, increasing studies of miRNA expression profiling has confirmed that miRNA plays an essential role in non-small cell lung cancer (NSCLC). However, inconsistent or discrepant results exist in these researches. In present study, we performed an integrative analysis of 32 miRNA profiling studies compared the differentially expressed miRNA between NSCLC tissue and non-cancerous lung tissue to identify candidate miRNAs associated with NSCLC. 7 upregulated and 10 downregulated miRNAs were identified as miRNA integrated-signature using Robust Rank Aggregation (RRA) method. qRT-PCR demonstrated that miR-21-5p, miR-210, miR-205-5p, miR-182-5p, miR-31-5p, miR-183-5p and miR-96-5p were up-regulated, whereas miR-126-3p, miR-30a-5p, miR-451a, miR-143-3p and miR-30d-5p were down-regulated more than 2 folds in the NSCLC, which was further validated in Tumor Cancer Genome Atlas (TCGA) database. Receiver operating characteristic (ROC) curve analysis confirmed that 9 miRNAs had good predictive performance (AUC > 0.9). Cox regression analysis revealed that miR-21-5p (hazard ratio [HR]: 1.616, 95% CI: 1.114-2.342, p = 0.011) and miR-30d-5p (HR: 0.578, 95% CI: 0.400-0.835, p = 0.003) were independent prognostic factors in NSCLC for overall survival. The accumulative effects of the two miRNAs on the prognosis of NSCLC were further estimated. The results showed that patients with two positive markers had a worse prognosis than those with one or none positive marker. In conclusion, this study contributes to the comprehension of the role of miRNAs in NSCLC and provides a basis for further clinical application.
Project description:Non-small cell lung cancer (NSCLC) accounts for more than 85% of lung cancer cases which cause most of cancer-related deaths globally. However, the results vary largely in different studies due to different platforms and sample sizes. Here, we aim to identify the key miRNAs in the carcinogenesis of NSCLC that might be potential biomarkers for this cancer.Meta-analysis was performed on miRNA profile using seven datasets of NSCLC studies. Furthermore, we predicted and investigated the functions of genes regulated by key miRNAs.Eleven key miRNAs were identified, including 2 significantly upregulated ones (hsa-miR-21-5p and hsa-miR-233-3p) and 9 downregulated ones (hsa-miR-126-3p, hsa-miR-133a-3p, hsa-miR-140-5p, hsa-miR-143-5p, hsa-miR-145-5p, hsa-miR-30a-5p, hsa-miR-30d-3p, hsa-miR-328-3pn, and hsa-miR-451). The functional enrichment analysis revealed that both up- and downregulated miRNAs were proportionally associated with regulation of transcription from RNA polymerase II promoter. According to transcription factor analysis, there were 65 (43.9%) transcription factors influenced by both up- and downregulated miRNAs.In this study, 11 meta-signature miRNAs, as well as their target genes and transcription factors, were found to play significant role in carcinogenesis of NSCLC. These target genes identified in our study may be profitable to diagnosis and prognostic prediction of NSCLC as biomarkers.
Project description:Dysregulation of microRNAs (miRNAs) has been associated with malignant behavior in a variety of cancers. Our previous study demonstrated that miRNA expression profiles are predictors for patients with advanced non-small cell lung cancer (NSCLC). We also showed that miRNAs are involved in small-cell lung cancer metastasis. Here, we used qRT-PCR to re-analyze our previous microarray results using serum samples from 75 patients with NSCLC. Surprisingly, we found that miR-574-5p and miR-874 were overexpressed in patients with metastatic advanced NSCLC but not in patients with non-metastatic advanced NSCLC. Additionally, miR-574-5p expression was correlated between matched serum and tissue samples from 68 patients. However, these 2 miRNAs are not prognostic factors for NSCLC. Transwell and wound-healing assays showed that miR-574-5p promotes the migration and invasion of NSCLC cells. Furthermore, miR-574-5p enhanced the tyrosine phosphorylation of ?-catenin by repressing PTPRU expression in vitro. In conclusion, this study explored the expression of miR-574-5p in clinical samples and its molecular mechanisms in the metastasis of advanced NSCLC.
Project description:Objective. Here, we aim to investigate the microRNA (miR) profiling in human gastric cancer (GC). Methods. Tumoral and matched peritumoral gastric specimens were collected from 12 GC patients who underwent routine surgery. A high-throughput miR sequencing method was applied to detect the aberrantly expressed miRs in a subset of 6 paired samples. The stem-loop quantitative real-time polymerase chain reaction (qRT-PCR) assay was subsequently performed to confirm the sequencing results in the remaining 6 paired samples. The profiling results were also validated in vitro in three human GC cell lines (BGC-823, MGC-803, and GTL-16) and a normal gastric epithelial cell line (GES-1). Results. The miR sequencing approach detected 5 differentially expressed miRs, hsa-miR-132-3p, hsa-miR-155-5p, hsa-miR-19b-3p, hsa-miR-204-5p, and hsa-miR-30a-3p, which were significantly downmodulated between the tumoral and peritumoral GC tissues. Most of the results were further confirmed by qRT-PCR, while no change was observed for hsa-miR-30a-3p. The in vitro finding also agreed with the results of both miR sequencing and qRT-PCR for hsa-miR-204-5p, hsa-miR-155-5p, and hsa-miR-132-3p. Conclusion. Together, our findings may serve to identify new molecular alterations as well as to enrich the miR profiling in human GC.
Project description:Increasing evidence supports that microRNA (miRNA)-mediated gene regulation plays a significant functional role in cancer progression. To investigate the expression and clinical significance of ITGB1 in non small cell lung cancer (NSCLC), the expression levels of ITGB1 in NSCLC tissues and human normal lung tissues were analyzed in silico using genes microarray, KEGG pathway and hierarchical clustering analysis followed by validation with quantitative RT-PCR. Our results showed that ITGB1 was upregulated in NSCLC tissues when compared with normal lung tissues. Survival analysis based on the qRT-PCR data established that ITGB1 expression was attentively related to the prognosis of NSCLC, and patients with higher ITGB1 expression had shorter overall survival (OS). Moreover, ITGB1 was confirmed to be a direct target of miR-493-5p. Furthermore, concomitant high expression of ITGB1 and low expression of miR-493-5p correlated with a shorter median OS and PFS in NSCLC patients. In conclusion, our results provide the first evidence that ITGB1 is a direct target of miR-493-5p suggesting that ITGB1 and miR-493-5p may have potential prognostic value and may be useful as tumor biomarkers for the diagnosis of NSCLC patients.