Serum Exosomal MicroRNA, miR-10b-5p, as a Potential Diagnostic Biomarker for Early-Stage Hepatocellular Carcinoma.
ABSTRACT: Exosomal microRNAs (exo-miRs) have been promising cancer biomarkers. MiRs in hepatocellular carcinoma (HCC) cell-derived exosomes (HEX) were analyzed to identify reliable serum biomarkers for HCC. To detect overexpressed miRs in HEX, extracted exosomal small RNAs from human HCC cell lines and normal hepatocytes were sequenced and analyzed. Clinical significance of the overexpressed miRs in HEX was evaluated using quantitative real-time PCR (qRT-PCR) on serum samples of a validation cohort consisting of 28 healthy individuals, 60 with chronic liver disease, and 90 with HCC. We found 49 significantly overexpressed miRs in HEX compared to a normal hepatocyte. Among them, miR-10b-5p, miR-18a-5p, miR-215-5p, and miR-940 were overexpressed in HCC tissues and also associated with prognosis of HCC in the analysis of a public omics database. qRT-PCR analysis of the four serum exo-miRs in the validation cohort revealed serum exo-miR-10b-5p as a promising biomarker for early-stage HCC with 0.934 area under the curve (AUC) (sensitivity, 90.7%; specificity, 75.0%; cutoff value, 1.8-fold). Overexpression of serum exo-miR-215-5p was found to be significantly associated with poor disease-free survival in patients with HCC. Serum exo-miR-10b-5p is a potential biomarker for early-stage HCC, while serum exo-miR-215-5p can be used as prognostic biomarker for HCC.
Project description:Currently, a reliable serum biomarker for hepatocellular carcinoma (HCC) has not been established, particularly for early-stage HCC (single tumor < 2 cm). We aimed to investigate diagnostic serum exosomal microRNA (exo-miR) panel for early-stage HCC. Driver oncogenic miR (onco-miR) candidates were selected by integrative analysis of miR expression profiles from three different RNA sequencing datasets of human HCC. Expressions of selected onco-miRs in serum exosome were measured using quantitative real-time PCR. Diagnostic performances of serum exo-miRs for HCC were evaluated in the test cohort (N = 24) and validation cohort (N = 144). Serum exo-miR panels were developed using a logistic regression model, and their diagnostic performance was evaluated. Six promising driver onco-miRs, including miR-25-3p, miR-140-3p, miR-423-3p, miR-1269a, miR-4661-5p, and miR-4746-5p, were identified by integrative analysis of three different RNA sequencing datasets. Among the six candidates, four serum exo-miRs (miR-25-3p, miR-1269a, miR-4661-5p, and miR-4746-5p) showed promising performance in the test cohort with area under the receiving operator curve (AUROC) >0.8. In our validation study, serum exo-miR-4661-5p could diagnose HCC in all stages (AUROC = 0.917), even in early stage (AUROC = 0.923), with a greater accuracy than other candidate serum exo-miRs and serum AFP. The panel composed of exo-miR-4661-5p and exo-miR-4746-5p was identified as the most accurate biomarker for early-stage HCC (AUROC = 0.947, 95% confidence interval = 0.889-0.980, sensitivity = 81.8%, and specificity = 91.7%). In conclusion, exo-miR-4661-5p-based serum panel is a promising diagnostic marker for early-stage HCC.
Project description:Exosomal microRNAs (exo-miRs) contribute to cancer metastasis. To identify pro-metastatic circulating exo-miRs in hepatocellular carcinoma (HCC), next-generation sequencing-based plasma exo-miR profiles of 14 patients with HCC (eight non-metastatic and six with metastasis within 1 year of follow-up) were analyzed. Sixty-one miRs were significantly overexpressed among patients with metastatic HCC. Candidate miRs were selected through integrative analyses of two different public expression datasets, GSE67140 and The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA_LIHC). Integrative analyses revealed 3 of 61 miRs (miR-106b-5p, miR-1307-5p, and miR-340-5p) commonly overexpressed both in metastasis and vascular invasion groups, with prognostic implications. Validation was performed using stored blood samples of 150 patients with HCC. Validation analysis showed that circulating exo-miR-1307-5p was significantly overexpressed in the metastasis group (<i>p</i> = 0.04), as well as in the vascular invasion and tumor recurrence groups. Circulating exo-miR-1307-5p expression was significantly correlated with tumor stage progression (<i>p</i> < 0.0001). Downstream signaling pathways of miR-1307 were predicted using TargetScan and Ingenuity Pathway Analysis. On comprehensive bioinformatics analysis, the downstream pathway of miR-1307-5p, promoting epithelial-mesenchymal transition (EMT), showed SEC14L2 and ENG downregulation. Our results show that circulating exo-miR-1307-5p promotes metastasis and helps predict metastasis in HCC, and SEC14L2 and ENG are target tumor suppressor genes of miR-1307 that promote EMT.
Project description:Background:Podocyte migration is actively involved in the process of podocyte loss and proteinuria production, which is closely associated with the development of diabetic nephropathy (DN). Exosomes from adipose-derived stem cells (ADSCs-Exos) effectively inhibit podocyte apoptosis in the treatment of DN. However, how ADSCs-Exos affect the migration of podocytes is obscure. This study is aimed at exploring the regulatory role of ADSCs-Exos on cell migration and the underlying mechanism. Methods:ADSCs-Exo was authenticated by transmission electron microscopy (TEM), western blotting, and flow cytometry. Cell viability and migration ability of podocytes were measured by CCK8 and Transwell assays, respectively. Relative expressions of miRNAs and mRNAs were determined by qRT-PCR. The transmitting between PKH26-labeled exosome and podocytes was evaluated by IF assay. Dual luciferase reporter assay was employed to detect the relationship between miR-215-5p and ZEB2. Results:The exposure to serum from DN patient (hDN-serum) significantly inhibited cell viability of podocytes, but ADSCs-Exo addition notably blunts cytotoxicity induced by the transient stimulus of hDN-serum. Besides, ADSCs-Exo administration powerfully impeded high glucose- (HG-) induced migration and injury of podocyte. With the podocyte dysfunction, several miRNAs presented a significant decline under the treatment of HG including miR-251-5p, miR-879-5p, miR-3066-5p, and miR-7a-5p, all of which were rescued by the addition of ADSCs-Exo. However, only miR-251-5p was a key determinant in the process of ADSCs-Exo-mediated protective role on podocyte damage. The miR-251-5p inhibitor counteracted the improvement from the ADSCs-Exo preparation on HG-induced proliferation inhibition and migration promotion. Additionally, miR-215-5p mimics alone remarkably reversed HG-induced EMT process of podocyte. Mechanistically, we confirmed that ADSCs-Exos mediated the shuttling of miR-215-5p to podocyte, thereby protecting against HG-induced metastasis, possibly through inhibiting the transcription of ZEB2. Conclusion:ADSCs-Exo has the protective effect on HG-evoked EMT progression of podocytes thru a mechanism involving ZEB2. Potentially, the ADSCs-Exo preparation is a useful therapeutic strategy for improving podocyte dysfunction and DN symptoms clinically.
Project description:BACKGROUND The therapeutic potential of endothelial colony-forming cells (ECFCs) may be impaired in an ischemic environment. Direct injection of ECFCs is not an effective method of rescuing the ischemic heart, but exosomes derived from these cells may be a promising therapeutic tool. However, exosomes produced under normoxia and hypoxia may not be identical. Therefore, the purpose of this study was to investigate alterations in the anti-fibrotic effects of hypoxia-treated ECFC-derived exosomes and the underlying mechanism involved. MATERIAL AND METHODS ECFCs were isolated from peripheral blood and exosomes were collected from ECFCs treated with normoxia (nor-exo) or hypoxia (hyp-exo). Effects of exosomes on cardiac fibroblast activation were evaluated in vitro. MicroRNAs (miRNAs) inside the exosomes were extracted and compared using next-generation RNA sequencing. Predicted target mRNAs of miR-10b-5p were validated using a dual-luciferase reporter gene assay method. RESULTS Nor-exo significantly ameliorated cardiac fibroblast activation in vitro. These effects were attenuated in the hyp-exo-treated group. miR-10b-5p was enriched in nor-exo but not in hyp-exo. Dual-luciferase reporter gene assay found that both SMAD-specific E3 ubiquitin protein ligase 1 (Smurf1) and histone deacetylase 4 (HDAC4) mRNAs were inhibited by miR-10b-5p. The expression of neutral sphingomyelinase 2 (N-SMase2) was decreased in hypoxia ECFCs, and this result was consistent with the changes in miR-10b-5p in hyp-exo. CONCLUSIONS Due to a reduction of miR-10b-5p, which targets the fibrotic genes Smurf1 and HDAC4, the anti-fibrotic effects of hyp-exo were abolished.
Project description:Noninvasive biomarkers for early pancreatic ductal adenocarcinoma (PDAC) diagnosis and disease risk stratification are greatly needed. We conducted a nested case-control study within the Prospective Investigation into Cancer and Nutrition (EPIC) cohort to evaluate prediagnostic microRNAs (miRs) as biomarkers of subsequent PDAC risk. A panel of eight miRs (miR-10a, -10b, -21-3p, -21-5p, -30c, -106b, -155 and -212) based on previous evidence from our group was evaluated in 225 microscopically confirmed PDAC cases and 225 controls matched on center, sex, fasting status and age/date/time of blood collection. MiR levels in prediagnostic plasma samples were determined by quantitative RT-PCR. Logistic regression was used to model levels and PDAC risk, adjusting for covariates and to estimate area under the receiver operating characteristic curves (AUC). Plasma miR-10b, -21-5p, -30c and -106b levels were significantly higher in cases diagnosed within 2 years of blood collection compared to matched controls (all p-values <0.04). Based on adjusted logistic regression models, levels for six miRs (miR-10a, -10b, -21-5p, -30c, -155 and -212) overall, and for four miRs (-10a, -10b, -21-5p and -30c) at shorter follow-up time between blood collection and diagnosis (?5 yr, ?2 yr), were statistically significantly associated with risk. A score based on the panel showed a linear dose-response trend with risk (p-value?=?0.0006). For shorter follow-up (?5 yr), AUC for the score was 0.73, and for individual miRs ranged from 0.73 (miR-212) to 0.79 (miR-21-5p).
Project description:BACKGROUND:Prostate cancer is one of the most common and socially significant cancers among men. The aim of our study was to reveal changes in miRNA expression profiles associated with lymphatic dissemination in prostate cancer and to identify the most prominent miRNAs as potential prognostic markers for future studies. METHODS:High-throughput miRNA sequencing was performed for 44 prostate cancer specimens taken from Russian patients, with and without lymphatic dissemination (N1 - 20 samples; N0 - 24 samples). RESULTS:We found at least 18 microRNAs with differential expression between N0 and N1 sample groups: miR-182-5p, miR-183-5p, miR-96-5p, miR-25-3p, miR-93-5p, miR-7-5p, miR-615-3p, miR-10b, miR-1248 (N1-miRs; elevated expression in N1 cohort; p?<?0.05); miR-1271-5p, miR-184, miR-222-3p, miR-221-5p, miR-221-3p, miR-455-3p, miR-143-5p, miR-181c-3p and miR-455-5p (N0-miRs; elevated expression in N0; p?<?0.05). The expression levels of N1-miRs were highly correlated between each other (the same is applied for N0-miRs) and the expression levels of N0-miRs and N1-miRs were anti-correlated. The tumor samples can be divided into two groups depending on the expression ratio between N0-miRs and N1-miRs. CONCLUSIONS:We found the miRNA expression signature associated with lymphatic dissemination, in particular on the Russian patient cohort. Many of these miRNAs are well-known players in either oncogenic transformation or tumor suppression. Further experimental studies with extended sampling are required to validate these results.
Project description:BACKGROUND:Alpha-fetoprotein (AFP) has received extensive attention in the differential diagnosis of hepatocellular carcinoma (HCC), especially for AFP-negative HCC (AFP-NHCC). The current study aimed to explore the value of targeted regulation of LHPP expression-related microRNAs (miRs) and protein induced by vitamin K deficiency or antagonist-II (PIVKA-II) in the differential diagnosis of AFP-NHCC. METHODS:A retrospective study was conducted on a testing set-including 214 AFP-NHCC patients, 200 cirrhosis, and 210 controls, and a validation set-including 140 AFP-NHCC patients, 134 cirrhosis, and 128 controls recruited from The First Affiliated Hospital of Hunan Normal University. Serum miRs were examined using quantitative real-time PCR method. Serum PIVKA-II was measured by enzyme-linked immunosorbent assay. RESULTS:Compared with adjacent tissues, LHPP protein levels in cancer tissues were significantly decreased (P < .05). Predictive software and dual-luciferase reporter assays showed that miR-363-5p and miR-765 can target LHPP expression. Serum miR-363-5p, miR-765, and PIVKA-II levels were significantly higher in AFP-HCC patients than in cirrhosis and controls. A logistic regression model combining miR-363-5p, miR-765, and PIVKA-II was performed. This model presented a high discriminating value (AUC: 0.930, sensitivity/specificity: 79.4%/95.4%) than any single indicator. In the validation set, this model still showed a high discriminating value (AUC: 0.936, sensitivity/specificity: 83.6%/94.7%). CONCLUSION:Current model combining serum miR-363-5p, miR-765, and PIVKA-II has potential significance for diagnosis of AFP-NHCC.
Project description:Circulating miRNAs (microRNAs) are emerging as promising biomarkers for several pathological conditions, and the aim of this study was to investigate the feasibility of using serum miRNAs as biomarkers for liver pathologies. Real-time qPCR (quantitative PCR)-based TaqMan MicroRNA arrays were first employed to profile miRNAs in serum pools from patients with HCC (hepatocellular carcinoma) or LC (liver cirrhosis) and from healthy controls. Five miRNAs (i.e. miR-885-5p, miR-574-3p, miR-224, miR-215 and miR-146a) that were up-regulated in the HCC and LC serum pools were selected and further quantified using real-time qPCR in patients with HCC, LC, CHB (chronic hepatitis B) or GC (gastric cancer) and in normal controls. The present study revealed that more than 110 miRNA species in the serum samples and wide distribution ranges of serum miRNAs were observed. The levels of miR-885-5p were significantly higher in sera from patients with HCC, LC and CHB than in healthy controls or GC patients. miR-885-5p yielded an AUC [the area under the ROC (receiver operating characteristic) curve] of 0.904 [95% CI (confidence interval), 0.837-0.951, P<0.0001) with 90.53% sensitivity and 79.17% specificity in discriminating liver pathologies from healthy controls, using a cut off value of 1.06 (normalized). No correlations between increased miR-885-5p and liver function parameters [AFP (?-fetoprotein), ALT (alanine aminotransferase), AST (aspartate aminotransferase) and GGT (?-glutamyl transpeptidase)] were observed in patients with liver pathologies. In summary, miR-885-5p is significantly elevated in the sera of patients with liver pathologies, and our data suggest that serum miRNAs could serve as novel complementary biomarkers for the detection and assessment of liver pathologies.
Project description:Study on the role of microRNAs (miRs) as regulators of gene expression through posttranscriptional gene silencing is currently gaining much interest,due to their wide involvement in different physiological processes. Understanding mammary gland development, lactation, and neoplasia in relation to miRs is essential. miR expression profiling of the mammary gland from different species in various developmental stages shows their role as critical regulators of development. miRs such as miR-126, miR-150, and miR-145 have been shown to be involved in lipid metabolism during lactation. In addition, lactogenic hormones influence miR expression as evidenced by overexpression of miR-148a in cow mammary epithelial cells, leading to enhanced lactation. Similarly, the miR-29 family modulates lactation-related gene expression by regulating DNA methylation of their promoters. Besides their role in development, lactation and involution, miRs are responsible for breast cancer development. Perturbed estrogen (E2) signaling is one of the major causes of breast cancer. Increased E2 levels cause altered expression of ER?, and ER?-miR cross-talk promotes tumour progression. miRs, such as miR-206, miR-34a, miR-17-5p, and miR-125 a/b are found to be tumour suppressors; whereas miR-21, miR-10B, and miR-155 are oncogenes. Oncogenic miRs like miR-21, miR-221, and miR-210 are overexpressed in triple negative breast cancer cases which can be diagnostic biomarker for this subtype of cancer. This review focuses on the recent findings concerning the role of miRs in developmental stages of the mammary gland (mainly lactation and involution stages) and their involvement in breast cancer progression. Further studies in this area will help us to understand the molecular details of mammary gland biology, as well as miRs that could be therapeutic targets of breast cancer.
Project description:MicroRNA-219-5p (miR-219-5p) is a key post-transcriptional regulator of gene expression that is known to regulate cancer progression, but its role in the context of hepatocellular carcinoma (HCC) remains to be fully elucidated. Herein, it was found that this miRNA functions as a tumor suppressor. Specifically, significant decreases in miR-219-5p expression were detected in HCC cells and patient serum samples relative to that found in the serum of 15 healthy people, and it was concluded that miR-219-5p overexpression was sufficient to impair HCC cell proliferation <i>in vitro</i> and <i>vivo</i> and migration <i>in vitro</i>. At the mechanistic level, it was found that miR-219-5p was able to suppress the expression of NEK6 (never in mitosis gene a-related kinase 6), thereby resulting in dysregulated ?-catenin/c-Myc-regulated gene expression. When NEK6 was overexpressed in HCC cells, this was sufficient to reverse the inhibitory impact of miR-219-5p on HCC cell proliferation both <i>in vitro</i> and <i>vivo</i> and metastasis <i>in vitro</i>. Bioinformatics analyses were also conducted, and both miR-219-5p and Nek6 were linked to disease progression in HCC patients with advanced disease. More importantly, the serum specimen data showed that reduced perioperative plasma miR-219-5p correlated significantly with increased risk of early recurrence after curative hepatectomy, whereas it was opposed to NEK6. Together, these findings highlight miR-219-5p as a potentially valuable diagnostic biomarker that can potentially be leveraged to improve clinical outcomes in HCC patients.