The role of miRNAs in Behçet’s disease

The symptoms of Behçet’s disease (BD), a multisystemic condition with autoimmune and inflammation as hallmarks, include arthritis, recurring oral and vaginal ulcers, skin rashes and lesions, and involvement of the nervous, gastrointestinal, and vascular systems. Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), may be important regulators of inflammation and autoimmune disease. These ncRNAs are essential to the physiological and pathophysiological disease course, and miRNA in particular has received significant attention for its role and function in BD and its potential use as a diagnostic biomarker in recent years. Although promising as therapeutic targets, miRNAs must be studied further to fully comprehend how miRNAs in BD act biologically

MiR-137 Targets Estrogen-Related Receptor Alpha and Impairs the Proliferative and Migratory Capacity of Breast Cancer Cells

ERRα is an orphan nuclear receptor emerging as a novel biomarker of breast cancer. Over-expression of ERRα in breast tumor is considered as a prognostic factor of poor clinical outcome. The mechanisms underlying the dysexpression of this nuclear receptor, however, are poorly understood. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play important roles in tumor initiation and progression. In the present study, we have identified that the expression of ERRα is regulated by miR-137, a potential tumor suppressor microRNA. The bioinformatics search revealed two putative and highly conserved target-sites for miR-137 located within the ERRα 3′UTR at nt 480–486 and nt 596–602 respectively. Luciferase-reporter assay demonstrated that the two predicted target sites were authentically functional. They mediated the repression of reporter gene expression induced by miR-137 in an additive manner. Moreover, ectopic expression of miR-137 down-regulated ERRα expression at both protein level and mRNA level, and the miR-137 induced ERRα-knockdown contributed to the impaired proliferative and migratory capacity of breast cancer cells. Furthermore, transfection with miR-137mimics suppressed at least two downstream target genes of ERRα–CCNE1 and WNT11, which are important effectors of ERRα implicated in tumor proliferation and migration. Taken together, our results establish a role of miR-137 in negatively regulating ERRα expression and breast cancer cell proliferation and migration. They suggest that manipulating the expression level of ERRα by microRNAs has the potential to influence breast cancer progression

ER-α36 mediates cisplatin resistance in breast cancer cells through EGFR/HER-2/ERK signaling pathway

Abstract Background ER-α36, a novel ER-α66 variant, has been demonstrated to promote tamoxifen resistance in breast cancer cells. However, the role and mechanisms of ER-α36 in cisplatin resistance of breast cancer cells remain unclear. This study investigates the expression and role of ER-α36 in cisplatin resistance of breast cancer cells and elucidates its underlying mechanisms. Methods The expression of ER-α36 and the proteins involved in nongenomic estrogen signaling was evaluated by western blot analysis. Cisplatin sensitivity was explored by CCK-8 assay, monolayer colony formation assay and apoptosis assays, respectively. ER-α36 siRNAs/shRNAs and overexpression vector were transfected into cells to down-regulate or up-regulate ER-α36 expression. Loss-and gain-of function assays were performed to investigate the role of ER-α36 in cisplatin sensitivity. The interaction between ER-α36 and EGFR/HER-2 were detected using CoIP. A mouse xenograft model of breast cancer was established to verify the role of ER-α36 in vivo. Results ER-α36 is expressed at higher levels in cisplatin-resistant breast cancer cells compared to cisplatin sensitive cells. Cisplatin induced up-regulation of ER-α36 in a dose-dependent manner in breast cancer cells. Overexpression of ER-α36 leaded to cell resistant to cisplatin and knockdown of ER-α36 in cisplatin-resistant breast cancer cells restored cisplatin sensitivity. The up-regulation of ER-α36 resulted in increased activation of nongenomic estrogen signaling, which was responsible for cisplatin resistance. Disruption of ER-α36-mediated nongenomic estrogen signaling with kinase inhibitors significantly inhibited cisplatin-induced expression of ER-α36 and increased cisplatin sensitivity. The in vivo experiment also confirmed that up-regulation of ER-α36 attenuated cisplatin sensitivity in a mouse xenograft model of breast cancer. Conclusions The results for the first time demonstrated that ER-α36 mediates cisplatin resistance in breast cancer cells through nongenomic estrogen signaling, suggesting that ER-α36 may serve as a novel target for cisplatin resistance and a potential indicator of cisplatin sensitivity in breast cancer treatment

The effect of si-ERRα and miR-137–mediated knockdown of ERRα on the cell proliferation.

<p>Breast cancer cell lines (MCF-7, BT-474, SK-BR-3 and MDA-MB-231) were transfected with 50 nM NC oligos, si-ERRα or miR-137 mimics or treated with transfection reagent alone (mock) and seeded in 96-well plates. Plates were harvested at 1, 2, 3, 4, 5 days after seeding for CCK-8 assay.</p

Identification of two highly conserved miR-137 target sites within the ESRRA 3′UTR.

<p>A. Schematic representation of the ERRα (ESRRA) mRNA with two putative sites (A and B) targeted by miR-137. B. Sequence alignment of predicted miR-137 target sites located within ESRRA 3′UTR showing high conservation among different species. The sequence of miR-137 target sites in ESRRA 3′UTR is shown in underlined. C. Luciferase reporter assay to verify activity of miR-137 upon the consensus miR-137 target site. HepG2 cells were transfected with Empty reporter plasmids, luciferase constructs containing perfect match miR-137 target site (miR-137 target) or mismatch miR-137 target site (△miR-137 target) and either miR-137 mimcs or NC oligos. Luciferase activity was determined 24 hr after transfection. Relative luciferase expression (firefly normalized to Renilla) values are the ratio of miR-137-treated reporter vector compared with the same NC oligos-treated reporter vector. Data are representative of at least three independent experiments. Error bars: SD. ***: P<0.0001. D. Luciferase reporter assay to evaluate the interaction between miR-137 and 3′-UTR of ESRRA. HepG2 cells were transfected with luciferase constructs containing wild-type (WT 3′UTR) or deletion mutated ESRRA 3′UTR (mutant A, mutant B and mutant C) and either miR-137 mimcs or NC oligos. Luciferase activity was determined 24 hr after transfection. Relative luciferase expression (firefly normalized to Renilla) values are the ratio of miR-137-treated reporter vector compared with the same NC oligos-treated reporter vector. Data are representative of three independent experiments. Error bars: SD. *: p<0.05, ***: P<0.0001.</p

MiR-137 and ERRα levels in normal breast epithelial and breast cancer cell lines.

<p>A. Western-blot analysis for ERRα protein level in normal breast epithelial cell line (MCF-10A) and five breast cancer cell lines. β-actin was used as the loading control. B. qRT-PCR analysis for miR-137 expression level. The miR-137 expression was normalized to RNU6B-small nuclear RNA. Data are representative of three independent experiments performed in triplicate. Error bars: SD.</p

MiR-137 influences the migratory capacity of MDA-MB-231 cells partly through ERRα-WNT11 signaling pathway.

<p>A. re-expression of ERRα (without 3′-UTR) in MDA-MB-231 cells restored the impaired migratory capacity induced by miR-137. MDA-MB-231 cells were co-transfected with indicated RNA oligonucleotides (50 nM) and plasmids (1 µg), and serum starved for 12 hr, followed by assessment of cell invasion and viability. Error bars: SD; *: p<0.05; **: P<0.01; ***: P<0.0001. B. re-expression of ERRα (without 3′-UTR) in MDA-MB-231 cells reversed the decrease of WNT11 expression induced by miR-137. MDA-MB-231 cells were co-transfected with indicated RNA oligonucleotides (50 nM) and plasmids (1 µg). 48 hr after transfection, protein and mRNA levels of WNT11 and ERRα were assayed using western bolt and qRT-PCR respectively. WNT11 or ERRα mRNA expression was normalized to β-actin mRNA expression. The relative level of WNT11 or ERRα determined using the 2-^△△CT method. Data are representative of three independent experiments performed in duplicate. Error bars: SD; **: P<0.01; ***: P<0.0001.</p

Ectopic transfection of miR-137 regulates the endogenous ERRα expression level.

<p>A. Western blot analysis for ERRα protein level and qRT-PCR analysis for ERRα mRNA level in SK-BR-3 cells 48 hr after transfection regent treatment (mock) or transfection with indicated RNA oligonucleotides (50 nM). B. Western blot analysis for ERRα protein level and qRT-PCR analysis for ERRα mRNA level in SK-BR-3 cells 48 hr after transfection regent treatment (mock) or cotransfection with equal amount of indicated RNA oligonucleotides. ERRα mRNA expression was normalized to β-actin mRNA expression. The relative level of ERRα expression determined using the 2-^△△CT method. Data are representative of three independent experiments performed in triplicate. Error bars: SD; *: p<0.05; ***: P<0.0001.</p

Primers used for PCR.

<p>The recognition sites of restriction endonuclease are underlined.</p

The expression of ERRα downstream target gene CCNE1 is regulated by miR-137.

<p>A. Western blot analysis for ERRα and CylinE1 protein level and qRT-PCR analysis for CCNE1 mRNA level in SK-BR-3 cells 48 hr after DMSO or XCT-790 treatment. B. Western blot analysis for CylinE1 protein level and qRT-PCR analysis for CCNE1 mRNA level in SK-BR-3 cells 48 hr after transfection regent treatment (mock) or transfection with NC oligos or si-ERRα. C. Western blot analysis for CylinE1 protein level and qRT-PCR analysis for CCNE1 mRNA level in SK-BR-3 48 hr after transfection regent treatment (mock) or co-transfection with equal amount of indicated RNA oligonucleotides. CCNE1 mRNA expression was normalized to β-actin mRNA expression. The relative level of CCNE1 mRNA was determined using the 2-^△△CT method. Data are representative of three independent experiments performed in triplicate. Error bars: SD; *: p<0.05; **: P<0.01; ***: P<0.0001.</p