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

Targeting the <i>MIR34C-5p</i>-ATG4B-autophagy axis enhances the sensitivity of cervical cancer cells to pirarubicin

<p>Pirarubicin (THP) is a newer generation anthracycline anticancer drug. In the clinic, THP and THP-based combination therapies have been demonstrated to be effective against various tumors without severe side effects. However, previous clinical studies have shown that most patients with cervical cancer are not sensitive to THP treatment, and the associated mechanisms are not clear. Consistent with the clinical study, we confirmed that cervical cancer cells were resistant to THP in vitro and in vivo. Our data demonstrated that THP induced a protective macroautophagy/autophagy response in cervical cancer cells, and suppression of this autophagy dramatically enhanced the cytotoxicity of THP. By scanning the mRNA level change of autophagy-related genes, we found that the upregulation of <i>ATG4B</i> (autophagy-related 4B cysteine peptidase) plays an important role in THP-induced autophagy. Moreover, THP increased the mRNA level of <i>ATG4B</i> in cervical cancer cells by promoting mRNA stability without influencing its transcription. Furthermore, THP triggered a downregulation of <i>MIR34C-5p</i>, which was associated with the upregulation of <i>ATG4B</i> and autophagy induction. Overexpression of <i>MIR34C-5p</i> significantly decreased the level of ATG4B and attenuated autophagy, accompanied by enhanced cell death and apoptosis in THP-treated cervical cancer cells. These results for the first time reveal the presence of a <i>MIR34C-5p</i>-ATG4B-autophagy signaling axis in THP-treated cervical cancer cells in vitro and in vivo, and the axis, at least partially, accounts for the THP nonsensitivity in cervical cancer patients. This study may provide a new insight for improving the chemotherapeutic effect of THP, which may be beneficial to the further clinical application of THP in cervical cancer treatment.</p

AU4S: A novel synthetic peptide to measure the activity of ATG4 in living cells

<div><p>ATG4 plays a key role in autophagy induction, but the methods for monitoring ATG4 activity in living cells are limited. Here we designed a novel fluorescent peptide named AU4S for noninvasive detection of ATG4 activity in living cells, which consists of the cell-penetrating peptide (CPP), ATG4-recognized sequence “GTFG,” and the fluorophore FITC. Additionally, an ATG4-resistant peptide AG4R was used as a control. CPP can help AU4S or AG4R to penetrate cell membrane efficiently. AU4S but not AG4R can be recognized and cleaved by ATG4, leading to the change of fluorescence intensity. Therefore, the difference between AU4S- and AG4R-measured fluorescence values in the same sample, defined as “F-D value,” can reflect ATG4 activity. By detecting the F-D values, we found that ATG4 activity paralleled LC3B-II levels in rapamycin-treated cells, but neither paralleled LC3B-II levels in starved cells nor presented a correlation with LC3B-II accumulation in WBCs from healthy donors or leukemia patients. However, when DTT was added to the system, ATG4 activity not only paralleled LC3B-II levels in starved cells in the presence or absence of autophagy inhibitors, but also presented a positive correlation with LC3B-II accumulation in WBCs from leukemia patients (<i>R²</i> = 0.5288). In conclusion, this study provides a convenient, rapid, and quantitative method to monitor ATG4 activity in living cells, which may be beneficial to basic and clinical research on autophagy.</p></div

Additional file 1: Figure S1. of Upregulation of microRNA-122 by farnesoid X receptor suppresses the growth of hepatocellular carcinoma cells

Activation of FXR upregulates miR-122 expression in HCC cells. HepG2, Huh7, PLC and SMMC-7721 cells were separately treated with GW4064 (5 μM) or vehicle DMSO for 24 h, and then the expression of pri-miR-122 (A) and mature miR-122 (B) was examined by qRT-PCR. **P < 0.01 vs vehicle. Table S1. The primer sets for qRT-PCR. Table S2. The primer sets for PCR amplification of different fragments of human miR-122 promoter region. (DOC 82 kb