Reassessing the Abundance of miRNAs in the Human Pancreas and Rodent Cell Lines and Its Implication.

miRNAs are critical for pancreas development and function. However, we found that there are discrepancies regarding pancreatic miRNA abundance in published datasets. To obtain a more relevant profile that is closer to the true profile, we profiled small RNAs from human islets cells, acini, and four rodent pancreatic cell lines routinely used in diabetes and pancreatic research using a bias reduction protocol for small RNA sequencing. In contrast to the previous notion that miR-375-3p is the most abundant pancreatic miRNA, we found that miR-148a-3p and miR-7-5p were also abundant in islets. In silico studies using predicted and validated targets of these three miRNAs revealed that they may work cooperatively in endocrine and exocrine cells. Our results also suggest, compared to the most-studied miR-375, that both miR-148a-3p and miR-7-5p may play more critical roles in the human pancreas. Moreover, according to in silico-predicted targets, we found that miR-375-3p had a much broader target spectrum by targeting the coding sequence and the 5' untranslated region, rather than the conventional 3' untranslated region, suggesting additional unexplored roles of miR-375-3p beyond the pancreas. Our study provides a valuable new resource for studying miRNAs in pancreata.

miRNA-148a Enhances the Treatment Response of Patients with Rectal Cancer to Chemoradiation and Promotes Apoptosis by Directly Targeting c-Met.

Patients with locally advanced rectal cancer (LARC) who achieve a pathological complete response (pCR) to neoadjuvant chemoradiotherapy (NACRT) have an excellent prognosis, but only approximately 30% of patients achieve pCR. Therefore, identifying predictors of pCR is imperative. We employed a microRNA (miRNA) microarray to compare the miRNA profiles of patients with LARC who achieved pCR (pCR group, n = 5) with those who did not (non-pCR group, n = 5). The validation set confirmed that miRNA-148a was overexpressed in the pCR group (n = 11) compared with the non-pCR group (n = 40). Cell proliferation and clonogenic assays revealed that miRNA-148a overexpression radio-sensitized cancer cells and inhibited cellular proliferation, before and after irradiation (p < 0.01). Apoptosis assays demonstrated that miRNA-148a enhanced apoptosis before and after irradiation. Reporter assays revealed that c-Met was the direct target gene of miRNA-148a. An in vivo study indicated that miRNA-148a enhanced the irradiation-induced suppression of xenograft tumor growth (p < 0.01). miRNA-148a may be a biomarker of pCR following NACRT and can promote apoptosis and inhibit proliferation in CRC cells by directly targeting c-Met in vitro and enhancing tumor response to irradiation in vivo.

Inverse relationship between the level of miRNA 148a-3p and both TGF-ß1 and FIB-4 in hepatocellular carcinoma.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a major health burden globally. Dysregulation of miRNA 148a-3p is engaged in carcinogenesis. TGF-ß is a profibrogenic cytokine. This study assesses the expression level of miRNA 148a-3p and its relationship with serum TGF-ß1 and fibrosis index based on four factors (FIB-4) in Egyptian patients with HCV-associated HCC. SUBJECTS: and Methods: The study included 72 HCC patients with HCV, 48 HCV cirrhotic patients, and 47 healthy controls. Serum TGF-ß1 was assessed by ELISA and the expression of miRNA 148a-3p was measured by RT-PCR. RESULTS: Patients with HCC had lower plasma miRNA 148a-3p, higher serum TGF-ß1, and higher FIB-4 levels than patients with cirrhosis and controls. miRNA 148a-3p discriminated HCC either from control (AUC: 0.997, 95.83% sensitivity, 85.11% specificity) or from cirrhosis (AUC: 0.943, 91.67% sensitivity, 81.25% specificity). Moreover, it distinguished metastatic from nonmetastatic patients (AUC: 0.800, 88.89% sensitivity, 60.0% specificity). The decreased miRNA 148a-3p and the increased TGF-ß1 levels were related to distant metastasis, multinodular lesions, advanced TNM stage, and BCLC score (C). A negative correlation between miRNA 148a-3p and each of FIB-4 and TGF-ß1 was detected. The decreased miRNA 148a-3p was associated with poor overall survival and poor progression-free survival. CONCLUSION: An inverse relationship between miRNA 148a-3p and both TGF-ß1 and FIB-4 was observed, which could be involved in HCC pathogenesis. Moreover, this miRNA is a potential diagnostic and prognostic biomarker for HCC.

miRNA-148a and miRNA-30c expressions as potential biomarkers in breast cancer patients.

BACKGROUND: Breast cancer is an extensively identified malignant tumor and is a prime cause of cancer mortalities in females. It has been shown that alteration of miRNAs expression (up or down regulation) can affect the initiation and progression of many malignancies. We aimed to evaluate the role of circulating miRNA-148a and miRNA-30c in female patients with breast cancer and estimate their usage as potential biomarkers in the diagnosis, prognosis and survival of breast cancer. METHODS: This study included 75 breast cancer female patients.They were compared with 55 apparently healthy female subjects. miRNAs expression analysis was assessed via real-time PCR. RESULTS: To discriminate breast cancer patients from controls, miR-30c showed the best performance at a cut off value of ≤20.6 (AUC = 0.998, 97.33% sensitivity, 96.36% specificity, p < 0.001), followed by miR-148a (AUC = 0.995, 94.67% sensitivity, 90.91% specificity, p < 0.001 at a cut off value of ≤0.1), CA 15-3 (AUC = 0.930, 88.0% sensitivity, 81.82% specificity, p < 0.001 at a cut off value of >21.3), and finally CEA (AUC = 0.751, 70.67% sensitivity, 63.64% specificity, p < 0.001 at a cut off value of >2.5). CONCLUSION: miRNA-148a and miRNA-30c expressions were down regulated in female patients with breast cancer and might be considered as potential blood biomarkers. Both also might have rule in disease treatment and selection of therapeutic targets. Future studies are needed to improve their role in predicting response to treatment and prognosis.

Molecular Mechanism of Mir-148A regulating cell adhesion molecule1 gene methylation by acting on DNA methyltransferase 1 in ovarian cancer / 中华内分泌外科杂志

Objective:To investigate the effects of cell adhesion molecule 1 (CADM1) promoter methylation in ovarian cancer on gene transcription and protein expression levels, and the regulation mechanism of mirNA-148A on CADM1 methylation levels.Methods:A total of 86 patients with ovarian cancer who received surgical treatment in the Affiliated Hospital of Hangzhou Normal University from Jun. 2018 to Jun. 2020 were selected as study subjects. The methylation level of CADM1 gene CpG island in ovarian cancer tissues and adjacent tissues was quantitatively detected. Quantitative real-time polymerase chain reaction was used to detect the mRNA and mirNA-148a expressions of CADM1 gene. The CADM1 gene and DNMT1 protein levels were detected by Western blot. Human ovarian cancer SKOV3 cells were treated with different concentrations of methyltransferase inhibitors (5-Azacytidine, 5-aza) , and CADM1 mRNA expression was detected 72 h later. Human ovarian cancer cell lines SKOV3 were transfected with mir-335-5p mimic, inhibitor and negative control respectively. Then mir-335-5p expression level and CADM1 gene methylation level were detected after transfection.Results:The methylation level of CADM1-1 island in ovarian cancer tissues was 2.89%±0.82%, significantly higher than that of paracancerous normal tissues 1.86%±0.68% ( t=4.936, P<0.001) , and that of CADM1-2 island in ovarian cancer tissues was 3.12%±0.93%, significantly higher than that of paracancerous normal tissues (2.27%±0.69%, t=5.114, P<0.001) . Pearson correlation analysis showed that the methylation level of CADM1-1 island and CADM1-2 island in ovarian cancer tissues was significantly negatively correlated with the relative mRNA expression (r was -0.615 and -0.582, respectively, and both P<0.001) , and with the protein expression level of CADM1 (r was -0.521 and -0.612, respectively, and both P<0.001) . The relative expression level of mirNA-148a in ovarian cancer tissues was 1.53±0.42, significantly lower than that in paracancer tissues (2.59±0.73, t=6.113, P<0.001) . After treatment with different concentrations of 5-AZA, mRNA expression levels of CADM1 gene in SKOV3 cells were significantly higher in the low concentration group and the high concentration group than in the control group (both P<0.05) , and mRNA expression levels in the high concentration group were significantly higher than in the low concentration group ( P<0.05) . After mirNA-148A transfected SKOV3 cells, the relative expression levels of mirNA-148a in the mimic group were significantly increased, while those of inhibitor group were significantly decreased ( P<0.001) . The DNMT1 expression level and CADM1 gene methylation level of mimic group were significantly decreased, while those of inhibitor group were significantly increased (P<0.001) . Conclusion:In ovarian cancer, miRNA-148a can regulate the DNA methylation level of CADM1 gene by acting on the downstream target protein DNMT1, thus affecting the mRNA and protein expression levels of CDM1 gene and participating in the pathogenesis of ovarian cancer.

LncRNA NEAT1 Sponges MiRNA-148a-3p to Suppress Choroidal Neovascularization and M2 macrophage polarization.

PURPOSE: Long noncoding RNAs (lncRNAs) have emerged as essential regulators in many biological processes; however, little is known about the role of lncRNAs in choroidal neovascularization (CNV). The aim of this study was to investigate the role of lncRNA NEAT1 in CNV formation, and assessed whether inhibition of lncRNA NEAT1 could suppress M2-type macrophage polarization and CNV. METHODS: The expression profiles of lncRNAs in a CNV mice model were accessed via microarray analysis. The role of lncRNA NEAT1 on macrophage polarization was assessed both in vitro and vivo. The interaction between lncRNA NEAT1, miR-148a-3p, and PTEN was assessed using a dual-luciferase reporter assay and RNA immunoprecipitation assay. Additionally, to evaluate the role of lncRNA NEAT1 on CNV development, eyes of mice in the mice CNV model were examined by Fluorescein Angiography (FA) and choroidal flatmounts on days 3 and 7 after intravitreal injection. RESULTS: The results revealed that 128 lncRNAs were significantly altered in the RPE-choroid-sclera complexes of CNV mice (P < 0.05, fold change > 2.0). Additionally, lncRNA NEAT1 increased in CNV formation and M2 macrophage polarization. LncRNA NEAT1 sponging miRNA-148a-3p targeting PTEN can modulate M2 macrophage polarization in mice CNV models as well as in bone marrow-derived macrophages cultured in vitro. Inhibition of lncRNA NEAT1 can suppress M2 macrophage both in vitro and vivo. Moreover, the intravitreal injection of a lncRNA NEAT1 Smart Silencer can inhibit CNV leakage and neovascularization. CONCLUSION: LncRNA NEAT1 via miRNA-148a-3p targeting PTEN plays a significant role in M2 macrophage polarization, while the inhibition of lncRNA NEAT1 can suppress choroidal neovascularization by inhibiting M2 macrophage polarization.

A Novel System to Discriminate HLA-C mir148a Binding Site by Allele-Specific Quantitative PC R.

The levels of expression of the HLA-class I molecules are critical for modulating T/NK lymphocytes effector functions. Among HLA molecules, HLA-C, the most recent developed form of class I antigens, is subjected to multiple post transcriptional level of regulation that affect its cell surface expression.We describe a new method of allele-specific real-time PCR that monitor the integrity/disruption of the binding site of the microRNA Hsa-miR-148a, a key factor associated to the levels of HLA-C expression in the Caucasian populations.

Milk exosomal miRNAs: potential drivers of AMPK-to-mTORC1 switching in ß-cell de-differentiation of type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) steadily increases in prevalence since the 1950's, the period of widespread distribution of refrigerated pasteurized cow's milk. Whereas breastfeeding protects against the development of T2DM in later life, accumulating epidemiological evidence underlines the role of cow's milk consumption in T2DM. Recent studies in rodent models demonstrate that during the breastfeeding period pancreatic ß-cells are metabolically immature and preferentially proliferate by activation of mechanistic target of rapamycin complex 1 (mTORC1) and suppression of AMP-activated protein kinase (AMPK). Weaning determines a metabolic switch of ß-cells from a proliferating, immature phenotype with low insulin secretion to a differentiated mature phenotype with glucose-stimulated insulin secretion, less proliferation, reduced mTORC1- but increased AMPK activity. Translational evidence presented in this perspective implies for the first time that termination of milk miRNA transfer is the driver of this metabolic switch. miRNA-148a is a key inhibitor of AMPK and phosphatase and tensin homolog, crucial suppressors of mTORC1. ß-Cells of diabetic patients return to the postnatal phenotype with high mTORC1 and low AMPK activity, explained by continuous transfer of bovine milk miRNAs to the human milk consumer. Bovine milk miRNA-148a apparently promotes ß-cell de-differentiation to the immature mTORC1-high/AMPK-low phenotype with functional impairments in insulin secretion, increased mTORC1-driven endoplasmic reticulum stress, reduced autophagy and early ß-cell apoptosis. In contrast to pasteurized cow's milk, milk's miRNAs are inactivated by bacterial fermentation, boiling and ultra-heat treatment and are missing in current infant formula. Persistent milk miRNA signaling adds a new perspective to the pathogenesis of T2DM and explains the protective role of breastfeeding but the diabetogenic effect of continued milk miRNA signaling by persistent consumption of pasteurized cow's milk.

miR-148a suppresses cell invasion and migration in gastric cancer by targeting DNA methyltransferase 1.

Gastric cancer (GC) is the fourth most common malignant tumor globally. The highest incidence of GC is found in Eastern Asia, particularly in China. It is therefore imperative to further elucidate the molecular pathogenesis of GC in order to identify new biomarkers and targets for effective therapy. In the present study, we determined whether miR-148a was aberrantly downregulated in gastric cancer tissues and significantly correlated with aggressive clinicopathological characteristics in the MGC-803, HGC-27 and GES-1 cell lines using reverse transcription-quantitative PCR and western blot analysis. The cell lines were obtained from 60 patients who presented at our hospital between September 2010 and July 2015. The results showed that, miR-148a was aberrantly downregulated in GC tissues and its expression was relatively lower in the MGC-803 and HGC-27 GC cell lines than in the normal gastric epithelial cell line, GES-1. The clinicopathological analysis revealed that a decrease of miR-148a was significantly correlated with lymph-node metastasis (P<0.01) and tumor node metastasis (TNM) stage (P<0.05). The transwell assay showed that the re-expression of miR-148a significantly reduced cell migratory and invasive abilities in vitro (P<0.01). The luciferase assay confirmed that, DNA methyltransferase 1 (DNMT1) was a direct and functional target of miR-148a. The miR-148a inhibitor increased the expression of DNMT1 in HGC-27 cells and the re-expression of miR-148a reduced the expression of DNMT1 in MGC-803 cells as confirmed by western blot analysis. Furthermore, we found that the re-expression of DNMT1 reversed the inhibition of cell migration and invasion induced by miR-148a. Taken together, we demonstrated that miR-148a suppresses cell invasion and migration in gastric cancer by regulating DNMT1 expression. The miR-148a/DNMT1 axis may therefore be a new potential target for GC therapy.

Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo.

In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory.

MicroRNAs: Milk's epigenetic regulators.

Our perception of milk has changed from a "simple food" to a highly sophisticated maternal-neonatal nutrient and communication system orchestrating early programming of the infant. Milk miRNAs delivered by exosomes and milk fat globules derived from mammary gland epithelial cells play a key role in this process. Exosomes resist the harsh intestinal environment, are taken up by intestinal cells via endocytosis, and reach the systemic circulation of the milk recipient. The most abundant miRNA found in exosomes and milk fat globules of human and cow's milk, miRNA-148a, attenuates the expression of DNA methyltransferase 1, which is critically involved in epigenetic regulation. Another important miRNA of milk, miRNA-125b, targets p53, the guardian of the genome, and its diverse transcriptional network. The deficiency of exosomal miRNAs in infant formula and the persistent uptake of milk miRNAs after the nursing period via consumption of cow's milk are two epigenetic aberrations that may induce adverse long-term effects on human health.

miRNA-148a regulates the expression of the estrogen receptor through DNMT1-mediated DNA methylation in breast cancer cells.

Breast cancer remains the most prevalent cancer among women worldwide. The expression of estrogen receptor-α (ER-α) is an important marker for prognosis. ER-α status may be positive or negative in breast cancer cells, although the cause of negative or positive status is not yet fully characterized. In the present study, the expression of ER-α and miRNA-148a was assessed in two breast cancer cell lines, HCC1937 and MCF7. An association between ER-α and miRNA-148a expression was identified. It was then demonstrated that DNA methyltransferase 1 (DNMT1) is a target of miRNA-148a, which may suppress the expression of ER-α via DNA methylation. Finally, an miRNA-148a mimic or inhibitor was transfected into MCF7 cells; the miRNA-148a mimic increased ER-α expression whereas the miRNA-148a inhibitor decreased ER-α expression. In conclusion, it was identified that miRNA-148a regulates ER-α expression through DNMT1-mediated DNA methylation in breast cancer cells. This may represent a potential miRNA-based strategy to modulate the expression of ER-α and provide a novel perspective for investigating the role of miRNAs in treating breast cancer.

Milk's Role as an Epigenetic Regulator in Health and Disease.

It is the intention of this review to characterize milk's role as an epigenetic regulator in health and disease. Based on translational research, we identify milk as a major epigenetic modulator of gene expression of the milk recipient. Milk is presented as an epigenetic "doping system" of mammalian development. Milk exosome-derived micro-ribonucleic acids (miRNAs) that target DNA methyltransferases are implicated to play the key role in the upregulation of developmental genes such as FTO, INS, and IGF1. In contrast to miRNA-deficient infant formula, breastfeeding via physiological miRNA transfer provides the appropriate signals for adequate epigenetic programming of the newborn infant. Whereas breastfeeding is restricted to the lactation period, continued consumption of cow's milk results in persistent epigenetic upregulation of genes critically involved in the development of diseases of civilization such as diabesity, neurodegeneration, and cancer. We hypothesize that the same miRNAs that epigenetically increase lactation, upregulate gene expression of the milk recipient via milk-derived miRNAs. It is of critical concern that persistent consumption of pasteurized cow's milk contaminates the human food chain with bovine miRNAs, that are identical to their human analogs. Commercial interest to enhance dairy lactation performance may further increase the epigenetic miRNA burden for the milk consumer.

Down-regulation of miRNA-148a and miRNA-625-3p in colorectal cancer is associated with tumor budding.

BACKGROUND: MiRNAs are often deregulated in colorectal cancer and might function as tumor suppressors or as oncogenes. They participate in controlling key signaling pathways involved in proliferation, invasion and apoptosis and may serve as prognostic and predictive markers. In this study we aimed to evaluate the role of miRNA-148a and miRNA-625-3p in metastatic colorectal cancer. METHODS: Fifty-four patients with a first-time diagnosed CRC receiving FOLFOX ± Bevacizumab were involved in the study. Tumor samples underwent routine pathology examination including evaluation for tumor budding and KRAS. MiRNA-148a and miRNA-625-3p expression analysis was done by RT-PCR. Associations between expression of both miRNAs and clinico-pathological factors, treatment outcomes and survival were analyzed. RESULTS: Both miRNA-148a and miRNA-625-3p were down-regulated in the tumors compared to normal colonic mucosa. Significantly lower expression of both miRNAs was noticed in tumors with budding phenomenon compared to tumors without it (median values of miRNA-148a were 0.314 and 0.753 respectively, p = 0.011, and 0.404 and 0.620 respectively for miRNA-625-3p, p = 0.036). Significantly lower expression of miRNA-625-3p was detected in rectal tumors, compared to tumors in the colon (median 0.390 and 0.665 respectively, p = 0.037). Progression free survival was significantly lower in patients with high miRNA-148a expression (6 and 9 months respectively, p = 0.033), but there were no significant differences in PFS for miRNA-625-3p and in overall survival for both miRNAs. CONCLUSIONS: There was a significant relationship between low miRNA-148a and miRNA-625-3p expression and tumor budding, which is thought to represent epithelial-mesenchymal transition. Both studied miRNAs may be associated with a more aggressive phenotype and could be the potential prognostic and predictive biomarkers in CRC. Further investigation is needed to confirm miRNAs involvement in EMT, and their prognostic and predictive value.

Expression of miRNA-148a in Bladder Carcinoma Tissues and Its Bioinformatics Analysis / 现代检验医学杂志

Objective To explore the role of miRNA-148a in bladder tumorous development and progression.Methods Ex-pression of miRNA-148a was assessed in 35 bladder carcinoma tissues and 16 non-carcinoma tissues by fluorescence quanti-tative real time PCR,and correlation with clinical features was evaluated.Target genes and transcription factors of miRNA-148a were predicted using bioinformatic analysis,then TF-miRNA-148a-target genes network diagram was built and the tar-get genes was analyzed of gene ontology enrichment and KEGG pathway.Results Expression of miRNA-148a was lower in bladder carcinoma tissues than in non-carcinoma tissues(0.000 8±0.000 2 vs 0.002 1±0.000 5)(t=2.46,P 0.05).268 target genes of miRNA-148a were predicted by three softwares at the same time,60 transcription factors were predicted and the binding sites with combination scroes above 80 was 657.The target genes of miRNA-148a was enriched in many biological processes,such as neuron differentiation,generation of neurons,neuron projec-tion development,cytoplasmic mRNA processing body,cytoplasm(P <0.001).They also participated in p53 signaling path-way,proteoglycans in cancer-homo sapiens,pathways in cancer,prostate cancer,protein processing in endoplasmic reticulum, focal adhesion and so on(P <0.05).According to TF-miRNA-148a-target genes network diagram,miRNA-148a was regula-ted by SP1,ESR1,AP1,MYC and BRCA1,genes of IGF1,P27kip1 ,NCOA1,PTEN,SERPINE1 might be regulated by miR-NA-148a.Conclusion miRNA-148a which was significantly down-regulation in bladder carcinoma tissues may be participate in bladder tumorous development and progression,bioinformatics analysis provides some ideas for further research.