Divergent target recognition by coexpressed 5′-isomiRs of miR-142-3p and selective viral mimicry

Sequence heterogeneity at the ends of mature microRNAs (miRNAs) is well documented, but its effects on miRNA function are largely unexplored. Here we studied the impact of miRNA 5′-heterogeneity, which affects the seed region critical for target recognition. Using the example of miR-142-3p, an emerging regulator of the hematopoietic lineage in vertebrates, we show that naturally coexpressed 5′-variants (5′-isomiRs) can recognize largely distinct sets of binding sites. Despite this, both miR-142-3p isomiRs regulate exclusive and shared targets involved in actin dynamics. Thus, 5′-heterogeneity can substantially broaden and enhance regulation of one pathway. Other 5′-isomiRs, in contrast, recognize largely overlapping sets of binding sites. This is exemplified by two herpesviral 5′-isomiRs that selectively mimic one of the miR-142-3p 5′-isomiRs. We hypothesize that other cellular and viral 5′-isomiRs can similarly be grouped into those with divergent or convergent target repertoires, based on 5′-sequence features. Taken together, our results provide a detailed characterization of target recognition by miR-142-3p and its 5′-isomiR-specific viral mimic. We furthermore demonstrate that miRNA 5′-end variation leads to differential targeting and can thus broaden the target range of miRNAs.     

Curcumin inhibits proteasome activity in triple-negative breast cancer cells through regulating p300/miR-142–3p/PSMB5 axis

BackgroundCurcumin functions as a proteasome inhibitor. However, the molecular mechanisms behind this action need more detailed explanations.PurposeThis study aimed to investigate the inhibitory effect of curcumin on 20S proteasome activity and to elucidate its exact mechanism in triple-negative breast cancer (TNBC) MDA-MB-231 cells.MethodsProteasomal peptidase activities were assayed using synthetic fluorogenic peptide substrates. Knockdown or overexpression of microRNA (miRNA or miR) or protein was used to investigate its functional effect on downstream cellular processes. BrdU (5‑bromo‑2′-deoxyuridine) assay was performed to identify cell proliferation. Western blot and quantitative real-time PCR(qRT-PCR) were carried out to determine protein abundance and miRNA expression, respectively. Correlations between protein expressions, miRNA levels, and proteasome activities were analyzed in TNBC tissues. Xenograft tumor model was performed to observe the in vivo effect of curcumin on 20S proteasome activity.ResultsCurcumin significantly reduced PSMB5 protein levels, accompanied with a reduction in the chymotrypsin-like (CT-l) activity of proteasome 20S core. Loss of PSMB5 markedly inhibited the CT-l activity of 20S proteasome. Furthermore, curcumin treatment significantly elevated miR-142–3p expression. PSMB5 was a direct target of miR-142–3p and its protein levels were negatively regulated by miR-142–3p. Moreover, histone acetyltransferase p300 suppressed miR-142–3p expression. Overexpression of p300 mitigated the promotive effect of curcumin on miR-142–3p expression. The correlations among p300 abundances, miR-142–3p levels, PSMB5 expressions, and the CT-l activities of 20S proteasome were evidenced in TNBC tissues. In addition, loss of p300 and PSMB5 reduced cell proliferation. Inhibition of miR-142–3p significantly attenuated the inhibitory impact of curcumin on cell proliferation. These curcumin-induced changes on p300, miR-142–3p, PSMB5, and 20S proteasome activity were further confirmed in in vivo solid tumor model.ConclusionThese findings demonstrated that curcumin suppressed p300/miR-142–3p/PSMB5 axis leading to the inhibition of the CT-l activity of 20S proteasome. These results provide a novel and alternative explanation for the inhibitory effect of curcumin on proteasome activity and also raised potential therapeutic targets for TNBC treatment.     

Low expression of miR-186-5p regulates cell apoptosis by targeting toll-like receptor 3 in high glucose–induced cardiomyocytes

To investigate the effect and mechanism of microRNA-186-5p (miR-186-5p) on the apoptosis in high glucose (HG)–treated cardiomyocytes. Diabetic cardiomyopathy model was established in cardiomyocytes by stimulating with HG. The expressions of miR-186-5p and toll-like receptor 3 (TLR3) were detected by quantitative polymerase chain reaction or Western blot analysis, respectively. Apoptosis was detected in HG-treated cardiomyocytes by flow cytometry and Western blot analysis. The interaction between miR-186-5p and TLR3 was explored by bioinformatics analysis and luciferase activity assay. Results showed that miR-186-5p expression was downregulated in HG-treated cardiomyocytes and its overexpression reversed HG-induced apoptosis and cleaved caspase-3 protein expression. Moreover, TLR3 was indicated as a target of miR-186-5p and regulated by miR-186-5p. Knockdown of TLR3 suppressed HG-induced apoptosis and cleaved caspase-3 protein expression. Besides, restoration of TLR3 ablated the effect of miR-186-5p on cell apoptosis. Collectively, miR-186-5p attenuated HG-induced apoptosis by regulating TLR3 in cardiomyocytes, providing novel biomarker for treatment of diabetic cardiomyopathy.     

Assignment of rat Jun family genes to Chromosome 19 (Junb), Chromosome 5q31–33 (Jun), and Chromosome 16 (Jund)

By means of somatic cell hybrids segregating rat chromosomes, we determined the chromosome localization of three rat genes of the Jun family: Jumb (Chr 19), Jun (=c-Jun) (Chr 5) and Jund (Chr 16). The Jun gene was also localized to the 5q31–33 region by fluorescence in situ hybridization. These rat gene assignments reveal two new homologies with mouse and human chromosomes, and provide a new example of synteny conserved in the human and a rodent species (the mouse), but split between the two rodent species.     

miR-605-5p promotes invasion and proliferation by targeting TNFAIP3 in non–small-cell lung cancer

Lung cancer is an significant cause of death worldwide, and non–small-cell lung cancer (NSCLC) is the most common type of lung cancer. MicroRNAs (miRNAs) have been identified to play key roles in NSCLC development. Recently, it has been reported that miR-605-5p is a cancer-related miRNA in several types of tumors. In this study, we study the role of miR-605-5p in NSCLC cells. We find that miR-605-5p is upregulated in NSCLC cells. Overexpression of miR-605-5p significantly promotes lung cancer invasion and migration in H460 and H1299 cells. Besides this, miR-605-5p also promotes lung cancer cell carcinoma proliferation and metastasis in vivo. However, downregulation of miR-605-5p inhibits cell invasion and migration by inhibiting lung cancer cell carcinoma proliferation and metastasis. In addition, the luciferase report assay identifies 3′-untranslated region tumor necrosis factor α-induced protein 3 (TNFAIP3) as a target of miR-605-5p. Silencing of TNFAIP3 promotes invasion and proliferation in lung cancer. In addition, the knockdown of TNFAIP3 restores the significant decrease in invasion and proliferation in miR-605-5p-inhibitor–transfected lung cancer cells. In conclusion, miR-605-5p promotes invasion and proliferation by targeting TNFAIP3 in NSCLC, and may provide possible biomarkers for NSCLC therapy.     

Promoter hypermethylation of tumour suppressor genes (p14/ARF and p16/INK4a): case–control study in North Indian population

The epigenetic modifications have been reported to be key factors in breast carcinogenesis. In the current study, it has been tried to determine the methylation status of two tumour suppressor genes p14/ARF and p16/INK4a in 150 breast cancer patients as well as 150 controls by using MSP-PCR. There was, highly significant difference in methylation of p14/ARF and p16/INK4a (P = 0.000) between patients and controls. Methylation of both the genes together significantly increased the risk of breast cancer by 12.31 folds. The present study concludes that hypermethylation of p14/ARF and p16/INK4a promoters demonstrate significant association with the risk of breast cancer, hence indicating these as important tumour suppressor genes involved in the pathogenesis of breast cancer in North Indian population (i.e. Punjab, Haryana, Uttar Pradesh, Himachal Pradesh as well as Union Territory of Chandigarh).     

circ-016910 sponges miR-574-5p to regulate cell physiology and milk synthesis via MAPK and PI3K/AKT–mTOR pathways in GMECs

Incremental proofs demonstrate that miRNAs, the essential regulators of gene expression, are implicated in various biological procedures, including mammary development and milk synthesis. Here, the role of miR-574-5p in milk synthesis, apoptosis, and proliferation of goat mammary epithelial cells (GMECs) are explored without precedent, and the molecular mechanisms for the impacts are elucidated. Small RNA libraries were constructed using GMECs transfected with miR-574-5p mimics and negative control followed by sequencing via Solexa technology. Overall, 332 genes were distinguishingly expressed entre two libraries, with 74 genes upregulated and 258 genes downregulated. This approach revealed mitogen-activated protein kinase kinase kinase 9 (MAP3K9), an upstream activator of MAPK signaling, as a differentially expressed unigene. miR-574-5p targeted seed sequences of the MAP3K9 3′-untranslated region and suppressed its messenger RNA (mRNA) and protein levels, correspondingly. GMECs with miR-574-5p overexpression and MAP3K9 inhibition showed increased cell apoptosis and decreased cell proliferation resulting from sustained suppression of MAPK pathways, while MAP3K9 elevation manifested the opposite results. miR-574-5p repressed the phosphorylation of members of protein kinase B (AKT)–mammalian target of rapamycin pathway via downregulating MAP3K9 and AKT3, resulting in reducing the secretion of β-casein and triglycerides in GMECs. Finally, according to the constructed circular RNA (circRNA) libraries and bioinformatics prediction approach, we selected circ-016910 and found it acted as a sponge for miR-574-5p and blocked its relevant behaviors to undertake biological effects in GMECs. The circRNA–miRNA–mRNA network facilitates further probes on the function of miR-574-5p in mammary development and milk synthesis.     

TGF-β2-induced circ-PRDM5 regulates migration,invasion, and EMT through the miR-92b-3p/COL1A2 pathway in human lens epithelial cells

Journal of Molecular Histology - CircRNA circ-PRDM5 (PR/SET domain 5) (circ-PRDM5) is overexpressed in age-related cataracts. Nevertheless, the biological role of circ-PRDM5 in posterior capsule...     

Aldose reductase regulates miR-200a-3p/141-3p to coordinate Keap1–Nrf2, Tgfβ1/2, and Zeb1/2 signaling in renal mesangial cells and the renal cortex of diabetic mice

Aberrant regulation in oxidative stress, fibrogenesis, and the epithelial–mesenchymal transition (EMT) in renal cells under hyperglycemic conditions contributes significantly to the onset and progression of diabetic nephropathy. The mechanisms underlying these hyperglycemia-induced dysregulations, however, have not been clearly elucidated. Herein, we report that aldose reductase is capable of regulating the expression of miR-200a-3p/141-3p negatively in renal mesangial cells. MiR-200a-3p/141-3p, in turn, act to target Keap1, Tgfβ2, fibronectin, and Zeb2 directly and regulate Tgfβ1 and Nrf2 indirectly under high-glucose conditions, resulting in profound dysregulations in Keap1–Nrf2, Tgfβ1/2, and Zeb1/2 signaling. In vivo in streptozotocin-induced diabetic mice, we found that aldose reductase deficiency caused significant elevations in miR-200a-3p/141-3p in the renal cortex, which were accompanied by a significant downregulation of Keap1, Tgfβ1/2, and fibronectin but significant upregulation of Nrf2. Moreover, in vivo administration of inhibitors of miR-200a-3p in diabetic animals significantly exacerbated cortical and glomerular fibrogenesis and increased urinary albumin excretion, tightly linking dysregulated miR-200a-3p with the development of diabetic nephropathy. Collectively, our results reveal a novel mechanism whereby hyperglycemia induces aldose reductase to regulate renal expression of miR-200a-3p/141-3p to coordinately control hyperglycemia-induced renal oxidative stress, fibrogenesis, and the EMT. Our novel findings also suggest that inhibition of aldose reductase and in vivo renal cortical restoration of miR-200a-3p/141-3p or their combination are very promising avenues for the development of therapeutic strategies or drugs against diabetic nephropathy.     

Effects of miR-202-5p silencing PIK3CA gene expression on proliferation,invasion, and epithelial–mesenchymal transition of cervical cancer SiHa cells through inhibiting PI3K/Akt/mTOR signaling pathway activation

Molecular and Cellular Biochemistry - To explore the mechanism of miR-202-5p targeting the expression of PIK3CA and mediating the activation of PI3K/Akt/mTOR signaling pathway on the proliferation,...     

Detection of frequent BglII polymorphism by polymerase chain reaction and TaqI restriction fragment length polymorphism for 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase at the human HSDβ3 locus (1p11–p13)

Summary Analysis of amplified polymerase chain reaction products of 575 bp from the fourth exon of the human type I 3-hydroxysteroid dehydrogenase/⁵-⁴ isomerase gene at locus HSD3 1p11–p13, reveals a frequent two-allele polymorphism at codon Leu³³⁸ due to a silent substitution of T by C, thus creating a BglII site leading to 371- and 204-bp fragments. Southern blot analysis of BglII-digested DNA from 57 individuals using a genomic probe detects two allelic fragments of 5.3kb and 0.77 kb, respectively, while two allelic fragments of 3.7 kb and 3.4 kb are obtained in TaqI digests with multiple constant bands, as also observed with BglII digests.     

Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4(n–3), to eicosapentaenoic acid, 20:5(n–3), in a cell line from the turbot, Scophthalmus maximus

The TF cell line, derived from a top predatory, carnivorous marine teleost, the turbot (Scophthalmus maximus), is known to have a limited conversion of C₁₈ to C₂₀ polyunsaturated fatty acids (PUFA). To illuminate the underlying processes, we studied the conversions of stearidonic acid, 18:4(n–3), and its elongation product, 20:4(n–3), in TF cells and also in a cell line, AS, derived from Atlantic salmon (Salmo salar), by adding unlabelled (25 μM), U-¹⁴C (1 μM) or deuterated (d5; 25 μM) fatty acids. Stearidonic acid, 18:4(n–3), was metabolised to 20:5(n–3) in both cells lines, but more so in AS than in TF cells. Δ5 desaturation was more active in TF cells than in AS cells, whereas C₁₈ to C₂₀ elongation was much reduced in TF as compared to AS cells. Only small amounts of docosahexaenoic acid (22:6(n–3)) were produced by both cell lines, although there was significant production of 22:5(n–3) in both cultures, especially when 20:4(n–3) was supplemented. We conclude that limited elongation of C₁₈ to C₂₀ fatty acids rather than limited fatty acyl Δ5 desaturation accounts for the limited rate of conversion of 18:3(n–3) to 20:5(n–3) in the turbot cell line, as compared to the Atlantic salmon cell line. The results can account for the known differences in conversions of C₁₈ to C₂₀ PUFA by the turbot and the Atlantic salmon in vivo.     

Apparent relative retention of the phosphatidylethanolamine molecular species 18:0–20:5(n−3), 16:0–22:6(n−3) and the sum 16:0–20:4(n−6) plus 16:0–20:3(n−9) in the liver microsomes of pig on an essential fatty acid deficient diet

Attempts at a better understanding of the cell membrane organization and functioning need to assess the physical properties which partly depend (i) on the positional distribution of the fatty acids in the membrane phospholipids (PLs) and (ii) on the way by which the PL molecular species are affected by exogenous fatty acids. To do that, the effects of essential (polyunsaturated) fatty acid (EFA) deficiency and enrichment were studied in the liver microsomes of piglets feeding on either an EFA-deficient diet or an EFA-enriched diet containing hydrogenated coconut oil or a mixture of soya + corn oils, respectively. After derivatization, the diacylated forms of choline and ethanolamine PLs were analyzed using a combination of Chromatographic techniques and fast-atom bombardmentmass spectrometry. The dinitrobenzoyl-diacylglycerol derivatives corresponding to the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were identified. It appears that three factors brought about a marked apparent relative retention: the nature of (i) the base of the polar head, (ii) fatty acids at the sn−1 position and (iii) fatty acids at the sn−2 position. The highest apparent relative retentions were displayed by the 18:0–20:5(n−3)-PE and 16:0–22:6(n−3)-PE. It is noteworthy that the behavior of 20:3 n−9 — which is synthesized during the EFA-deficient diet by the same bioconversion system as 20:4 n−6 — was very similar to that of 20:4 n−6 during the formation of PC and PE molecular species and that the molecular species of PE containing 20:4(n−6) and 20:3(n−9), gathered together as metabolical homologues, were also apparently retained, particularly in association with 16:0. Present observations are consistent with some others showing retention or preferential distribution of EFA in PE and suggest that specific acyltransferase(s), ethanolamine phosphotransferase and methyltransferase would be mainly involved for PE and PC formation in liver endoplasmic reticulum. Fast-atom bombardment-mass spectrometry of intact phospholipids enables us to show that there is no very long chain dipolyunsaturated phospholipid in liver endoplasmic reticulum.