Polypyrimidine tract binding protein (hnRNP I) is possibly a conserved modulator of miRNA-mediated gene regulation

MiRNAs can regulate gene expression through versatile mechanisms that result in increased or decreased expression of the targeted mRNA and it could effect the expression of thousands of protein in a particular cell. An increasing body of evidence suggest that miRNAs action can be modulated by proteins that bind to the same 3'UTRs that are targeted by miRNAs, suggesting that other factors apart from miRNAs and their target sites determine miRNA-modulation of gene expression. We applied an affinity purification protocol using biotinylated let-7 miRNA inhibitor to isolate proteins that are involved in let-7 mediated gene regulation that resulted in an affinity purification of Polypyrimidine Tract Binding protein (PTB). Here we show that PTB interacts with miRNAs and human Argonaute 2 (hAgo2) through RNA as well as identified potential mammalian cellular targets that are co-regulated by PTB and hAgo2. In addition, using genetic approach, we have demonstrated that PTB genetically interacts with Caenorhabditis elegans let-7 indicating a conserved role for PTB in miRNA-mediated gene regulation.     

Targeted Expression of Suicide Gene by Tissue-Specific Promoter and MicroRNA Regulation for Cancer Gene Therapy

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus–thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3’UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM ^+ve/let-7b^{down-regulated}), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM ^−ve/let-7b^up-regulated), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM ^+ve/let-7b^up-regulated) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.     

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.     

let-7 microRNAs induce tamoxifen sensitivity by downregulation of estrogen receptor α signaling in breast cancer

MicroRNAs (miRNAs) play an important regulatory role in breast tumorigenesis. Previously, we found that let-7 miRNAs were downregulated significantly in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. In this study, we further found that endogenous levels of let-7b and let-7i miRNAs are inversely correlated with levels of estrogen receptor (ER)-a36, a new variant of ER-α66, in the FFPE tissue set. Bioinformatic analysis suggested that ER-α36 may be another target of let-7 miRNAs. To test this hypothesis, cotransfection of let-7 mimics or inhibitors together with full-length or a fragment of ER-α36 3'UTR luciferase construct was performed, and we found that let-7b and let-7i mimics suppressed the activity of reporter gene significantly, which was enhanced remarkably by let-7b and let-7i inhibitors. Both mRNA and protein expression of ER-α36 were inhibited by let-7 mimics and enhanced by let-7 inhibitors. Furthermore, ER-α36 mediated nongenomic MAPK and Akt pathways were weakened by let-7b and let-7i mimics in triple negative breast cancer cell line MDA-MB-231. The reverse correlation between let-7 miRNAs and ER-α36 also exists in Tamoxifen (Tam)-resistant MCF7 cell line. Transfection of let-7 mimics to Tam-resistant MCF7 cells downregulated ER-α36 expression and enhanced the sensitivity of MCF7 cells to Tam in estrogen-free medium, which could be restored by overexpression of ER-α36 constructs without 3'UTR. Our results suggested a novel regulatory mechanism of let-7 miRNAs on ER-α36 mediated nongenomic estrogen signal pathways and Tam resistance.     

Role of let-7b/Fzd4 axis in mitochondrial biogenesis through wnt signaling: In neonatal and adult megakaryocytes

BackgroundMegakaryocytes (MKs), a rare population of bone marrow cells, are responsible for the production of platelets. Sick neonates are predisposed to developing thrombocytopenia (platelet count <150 × 109/L) and neonates are affected by several megakaryocyte disorders as compared to adults.HypothesisMicroRNAs (miRNAs) have been shown to crucially involve in the regulation of stem-cell differentiation in normal as well as malignant hematopoiesis, but their role in regulation of biological differences between adult and neonatal megakaryopoiesis is unknown.MethodsTo study this, we cultured human cord blood (CB) and peripheral blood (PB) derived CD34⁺ cells in the presence of thrombopoietin for 14 days and collected cultures expressing >90% CD41⁺ by flow cytometry and studied 88 miRNAs involved in stem cell development and differentiation. miRNA validation studies were performed in Dami cell line.ResultsOut of 88 miRNAs involved in stem cell development, let-7b was the only miRNA down regulated (∼10-fold) in neonates compared to adult-MKs. Let-7b has not been previously described in MKs, however reduced expression of let-7b was found in several human cancers, suggesting that it functions as a tumor suppressor. Our results showed the inhibitory effect of let-7b on wnt signaling pathway by regulating Fzd4 (frizzled family receptor 4) and thereby regulating proliferation as well as differentiation. Let-7b down regulation induced mitochondrial biogenesis and its markers PGC-1α and NRF1 during megakaryocyte development.ConclusionsOur findings for the first time unveil the novel role of let-7b/Fzd4 axis through wnt signaling by regulating mitochondrial biogenesis during megakaryocyte development.     

Let-7b contributes to hepatocellular cancer progression through Wnt/β-catenin signaling

Elevated evidences show that microRNAs (miRNAs) play vital roles in tumor progression regulation. However, the functional role of let-7b in hepatocellular carcinoma (HCC) is still largely unknown. In this study, we try to investigate the biological activity of let-7b in human HCC cells and try to find the potential regulatory signaling pathway. Our results indicate that let- 7b was remarkably down-regulated in human HCC tissues by qRT-PCR. In addition, let-7b overexpression decreased the expression of β-catenin and c-Myc, while upregulated E-cadherin expression in HCC cells which was verified by quantitative real-time PCR (qRT-PCR) and western blotting. Furthermore, Wnt/β-catenin was involved in let-7b biological activity which was revealed by luciferase assay. Moreover, Wnt/β-catenin signaling inhibitor blocks HCC cell proliferation which is as the same pattern as let-7b overexpression inhibits in HCC cells proliferation. In conclusion, down-regulated let-7b promotes HCC cell proliferation through Wnt/β-catenin signaling in HCC cells. These results suggested that appropriate manipulation of let-7b might be a new treatment of human HCC in the future.     

Identification and expression analysis of miRNAs during batch culture of HEK-293 cells

MicroRNAs (miRNAs) are small non-coding RNAs of about 20–24 nucleotides in length. They regulate gene expression negatively and have been implicated in a wide variety of biological processes. To identify potential miRNAs that may influence the growth and proliferation of mammalian cells cultured in bioreactors, we applied miRNA microarray expression profiling technology to batch cultures of HEK293 cells in protein free media. In our study, we identified miRNAs that were differentially expressed during the exponential and stationary phases, 13 of these showed distinct up regulation trends while 1 exhibited down regulation. These miRNAs have been implicated in cellular differentiation, growth arrest and apoptosis. Specifically, miR-16 and let-7b are potentially useful in the enhancement of bioreactor cell cultures.     

Impact of preovulatory estradiol concentrations on conceptus development and uterine gene expression

The objectives of this study included: (1) identify the expression of miRNAs specific to bovine cumulus-oocyte complexes (COCs) during late oogenesis, (2) characterize the expression of candidate miRNAs as well as some miRNA processing genes, and (3) computationally identify and characterize the expression of target mRNAs for candidate miRNAs. Small RNAs in the 16-27 bp range were isolated from pooled COCs aspirated from 1- to 10-mm follicles of beef cattle ovaries and used to construct a cDNA library. A total 1798 putative miRNA sequences from the cDNA library of small RNA were compared to known miRNAs. Sixty-four miRNA clusters matched previously reported sequences in the miRBase database and 5 miRNA clusters had not been reported. TaqMan miRNA assays were used to confirm the expression of let-7b, let-7i, and miR-106a from independent collections of COCs. Real-time PCR assays were used to characterize expression of miRNA processing genes and target mRNAs (MYC and WEE1A) for the candidate miRNAs from independent collections of COCs. Expression data were analyzed using general linear model procedures for analysis of variance. The expression of let-7b and let-7i were not different between the cellular populations from various sized follicles. However, miR-106a expression was greater (P<0.01) in oocytes compared with COCs and granulosa cells. Furthermore, all the miRNA processing genes have greater expression (P<0.001) in oocytes compared with COCs and granulosa cells. The expression of potential target mRNAs for let-7 and let-7i (i.e., MYC), and miR-106a (i.e., WEE1A) were decreased (P<0.05) in oocytes compared with COCs and granulosa cells. These results demonstrate specific miRNAs within bovine COCs during late oogenesis and provide some evidence that miRNAs may play a role regulating maternal mRNAs in bovine oocytes.     

Systematic gene regulation involving miRNAs during neuronal differentiation of mouse P19 embryonic carcinoma cell

MicroRNAs (miRNAs) are small noncoding RNA and play an essential role in gene regulation. In this study, we investigated regulation of gene expression during neuronal differentiation of mouse P19 embryonic carcinoma cells and described a systematic pathway of gene regulation involving miRNAs. In the pathway, downregulation of Lin28 involved in blocking the let-7 maturation and upregulation of let-7 occur following induction of the differentiation, thereby triggering suppression of the downstream High Mobility Group A2 (Hmga2) gene expression via activation of gene silencing mediated by let-7. Our data further suggest that miR-9, as well as miR-125b, participate in the reduction of the Lin28 expression. The gene regulation involving miRNAs likely contributes to a rapid and programmed change in gene expression in neuronal differentiation of P19 cells.