miR­425­5p promotes cell proliferation, migration and invasion by directly targeting FOXD3 in hepatocellular carcinoma cells.

MicroRNAs (miRs) are important regulators of the tumorigenesis and metastasis of various cancers. In the present study, the roles and underlying mechanisms of miR­425­5p in the development of hepatocellular carcinoma (HCC) were investigated. RT­qPCR analysis revealed that miR­425­5p was upregulated in HCC tissues and cell lines. A functional study in vitro using MTT assays, colony formation and Transwell assays demonstrated that overexpression of miR­425­5p promoted the proliferation, migration, and invasion of HCC cells, prevented cell apoptosis and accelerates the epithelial­mesenchymal transition process, whereas miR­425­5p knockdown induced opposing effects. A further mechanistic study revealed that forkhead box D3 (FOXD3) was a direct target of miR­425­5p, and gain­ and loss­of­function of FOXD3 studies demonstrated that FOXD3 suppressed HCC cell proliferation, migration, and invasion. Furthermore, rescue experiments revealed that overexpression of FOXD3 counteracted the positive effects of miR­425­5p on HCC malignant behaviors. Collectively, the present results demonstrated that miR­425­5p promoted HCC cell proliferation, migration, and invasion by suppressing FOXD3 expression, potentially providing a novel target for the treatment of HCC.

PM2.5 induces liver fibrosis via triggering ROS-mediated mitophagy.

BACKGROUND: The increasing epidemic of fine particulate matter (PM2.5) is a serious threat to human health. It induces the occurrence of liver fibrosis, but its molecular mechanism is not yet clear. The molecular mechanisms of PM2.5 inducing liver fibrosis were investigated in this study. METHODS: The cell viability of LX-2 cells and primary hepatic stellate cells (HSCs) was detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In vitro enzyme-linked immune sorbent assay (ELISA) kits were used to detect the concentrations of antioxidant enzymes and reactive oxygen species (ROS). The mitochondrial transmembrane potential (MTP) was determined by JC-1 dye. Knockdown of Parkin was carried out by Parkin-specific siRNA transfection. Relative mRNA and protein expressions were evaluated by qRT-PCR, Western blotting, and immunofluorescence analysis. RESULTS: PM2.5 activated LX-2 cells and primary HSCs, inducing the liver fibrosis along with down-regulation of the gelatinases MMP-2, and up-regulation of myofibroblast markers collagen type I and α-SMA. The levels of ROS and reactive nitrogen species (RNS), as well as the lipid peroxidation marker malondialdehyde (MDA) were significantly up-regulated in LX-2 cells and primary HSCs treated with PM2.5. Also, the enzymatic antioxidants levels were disturbed by PM2.5. Furthermore, PM2.5 decreased the MTP, releasing cytochrome c from the mitochondria to the cytosol. The dynamics of mitochondria were regulated by PM2.5 via facilitating mitochondrial fission. The excess ROS induced by PM2.5 triggered the mitophagy by activating PINK1/Parkin pathway, and inhibition of mitophagy induced by PM2.5 diminished the liver fibrosis. CONCLUSION: PM2.5 may induce mitophagy via activating PINK1/Parking signal pathway by increasing ROS, thereby activating HSCs and causing liver fibrosis.

MiR-410 Acts as a Tumor Suppressor in Estrogen Receptor-Positive Breast Cancer Cells by Directly Targeting ERLIN2 via the ERS Pathway.

BACKGROUND/AIMS: Endoplasmic reticulum lipid raft-associated 2 (ERLIN2) is reported to be overexpressed in human breast cancer cells and plays an important role in cell proliferation. MicroRNAs (miRNAs) act as post-transcriptional regulators of gene expression and are involved in the development of multiple malignancies, including breast cancer. However, the molecular mechanism of the aberrant ERLIN2 expression in human breast cancer remains poorly understood. METHODS: MiR-410 expression level was analyzed using Real-time PCR, and ERLIN2 expression was analyzed using Western blot, Real-time PCR and immunohistochemical staining. The effect of miR-410 on ERLIN2 3'UTR intensity was performed using a luciferase assay. Cell proliferation was analyzed using CCK-8 and colony formation assay, together with an Annexin V-PE/7-AAD kit for cell apoptosis assay. Cell migration and invasion was detected using a Transwell migration and invasion assay. Methylation specific PCR was used to examine whether miR-410 promoter was demethylated. RESULTS: In this study, we validated that ERLIN2 was a direct target of miR-410 and miR-410 suppressed ERLIN2 expression at the post-transcriptional level. Importantly, the regulation of ERLIN2 by miR-410 was estrogen receptor (ER) dependent. Functional studies demonstrated that miR-410 inhibited breast cancer cell proliferation, migration and invasion, but promoted cell apoptosis. However, inhibition of miR-410 resulted in opposite effects. A xenograft nude mouse model further confirmed that miR-410 suppressed breast tumor growth. In addition, miR-410 modulated the expression levels of epithelial-mesenchymal transition (EMT)-related genes. ERLIN2 knockdown suppressed cell proliferation, migration and invasion, as well as EMT. ERLIN2 overexpression can restore the cell proliferation, migration and invasion that were inhibited by miR-410. Furthermore, our data demonstrated that miR-410 inhibition suppressed the expression of endoplasmic reticulum-stress (ERS)-related genes, while ERLIN2 knockdown abrogated the effects of miR-410 inhibitor. Finally, we showed that miR-410 was downregulated in human ER-positive breast cancer tissues, inversely correlated with ERLIN2. We further demonstrated the downregulation of miR-410 in breast cancer might be due to the hypermethylation of its promoter. CONCLUSIONS: Our study indicates that miR-410 suppresses cell growth, migration and invasion by directly downregulating ERLIN2 in ER positive breast cancer, acting as a tumor suppressor. Our study also suggests that miR-410 may serve as a potential therapeutic target for patients with ER positive breast cancer.

A negative feedback loop between miR-200b and the nuclear factor-κB pathway via IKBKB/IKK-ß in breast cancer cells.

MicroRNAs (miRNAs) act as important post-transcriptional regulators of gene expression in diverse signalling pathways. However, the relationship between miR-200b and the nuclear factor-κB (NF-κB) signalling pathway remains poorly understood in breast cancer cells. In the current study, we show that IKBKB is a direct target of miR-200b, and that miR-200b downregulates IKBKB expression via directly binding to its 3'-UTR. miR-200b inhibits IκBα phosphorylation, nuclear p50/p65 expression, NF-κB-binding activity, and the translocation of p65 to the nucleus. In addition, miR-200b also suppresses tumour necrosis factor (TNF)-α-induced NF-κB activation and the expression of NF-κB target genes. Importantly, IKBKB overexpression attenuates the inhibitory roles of miR-200b in NF-κB expression, NF-κB-binding activity, and the nuclear translocation of p65. We also show that NF-κB p65 knockdown reduces the binding of NF-κB to the miR-200b promoter and miR-200b promoter activity. Furthermore, p65 knockdown or inhibition of IκBα phosphorylation suppresses miR-200b expression. Finally, functional studies show that IKBKB overexpression can restore the cell growth and migration that are suppressed by miR-200b. In conclusion, our results demonstrate that miR-200b, a transcriptional target of NF-κB, suppresses breast cancer cell growth and migration, and NF-κB activation, through downregulation of IKBKB, indicating that miR-200b has potential as a therapeutic target in breast cancer patients.