Elevated TFPI is a prognostic factor in hepatocellular carcinoma: Putative role of miR-7-5p and miR-1236-3p.

BACKGROUND: Primary liver cancer is the third leading cause of cancer related deaths worldwide, and the disease is associated with high incidence rate of thrombosis. Studies indicate that Tissue Factor Pathway Inhibitor (TFPI) plays a role in cancer development. We aimed to study its expression, clinical role and regulation by micro RNAs (miRNAs) in hepatocellular carcinoma (HCC). METHODS: Publically available datasets were used for clinical analysis of TFPI and miRNAs expression by web analysis tools. miRNA mimics targeting TFPIα 3'untranslated region (UTR) were selected from target prediction programs and verified by luciferase reporter assay. In vitro effects of miRNAs overexpression in HCC cell lines on TFPI expression and cell proliferation and apoptosis were analysed. RESULTS: TFPI expression was significantly increased in HCC tumours compared to normal tissue. Low TFPI tumour expression was associated with better survival probability. Four candidate miRNAs were selected from the target prediction programs. miR-7-5p and miR-1236-3p were validated in HepG2 and Huh7 cells to reduce TFPI mRNA and protein levels following overexpression. Furthermore, miR-7-5p and miR-1236-3p reduced TFPIα-3'UTR-controlled luciferase activity. The two validated miRNAs inhibited proliferation of HepG2 cells, and had clinical significance in HCC. CONCLUSIONS: TFPI was increased in HCC tumours compared to normal tissue and high TFPI expression was associated with an unfavorable outcome in HCC patients. miR-7-5p and miR-1236-3p were identified as novel regulators of TFPI in vitro.

The role of microRNA-27a/b and microRNA-494 in estrogen-mediated downregulation of tissue factor pathway inhibitor α.

UNLABELLED: Essentials Estrogens are known to influence the expression of microRNAs in breast cancer cells. We looked at microRNAs in estrogenic regulation of tissue factor pathway inhibitor α (TFPIα). Estrogen upregulated microRNA-27a/b and microRNA-494 through the estrogen receptor α. MicroRNA-27a/b and microRNA-494 are partly involved in estrogenic downregulation of TFPIα. SUMMARY: Background Tissue factor pathway inhibitor (TFPI) has been linked to breast cancer pathogenesis. We have recently reported TFPI mRNA levels to be downregulated by estrogens in a breast cancer cell line (MCF7) through the estrogen receptor α (ERα). Accumulating evidence also indicates that activation of ERα signaling by estrogens may modulate the expression of target genes indirectly through microRNAs (miRNAs). Objectives To examine if miRNAs are involved in the estrogenic downregulation of TFPIα. Methods Computational analysis of the TFPI 3'-untranslated region (UTR) identified potential binding sites for miR-19a/b, miR-27a/b, miR-494, and miR-24. Transient overexpression or inhibition of the respective miRNAs was achieved by transfection of miRNA mimics or inhibitors. Direct targeting of TFPI 3'-UTR by miR-27a/b and miR-494 was determined by luciferase reporter assay in HEK293T cells. Effects of 17α-ethinylestradiol (EE2) and fulvestrant on relative miR-27a/b, miR-494, and TFPI mRNA levels in MCF7 cells were determined by qRT-PCR and secreted TFPIα protein by ELISA. Transient knockdown of ERα was achieved by siRNA transfection. Results EE2 treatment lead to a significant increase in miR-19a, miR-27a/b, miR-494, and miR-24 mRNA levels in MCF7 cells through ERα. miR-27a/b and miR-494 mimics lead to reduced TFPI mRNA and protein levels. Luciferase assay showed direct targeting of miR-27a/b and miR-494 on TFPI mRNA. Impaired estrogen-mediated downregulation of TFPI mRNA was detected in anti-miR-27a/b and anti-miR-494 transfected cells. Conclusions Our results provide evidence that miR-27a/b and miR-494 regulate TFPIα expression and suggest a possible role of these miRNAs in the estrogen-mediated downregulation of TFPIα.

Effect of hypoxia on tissue factor pathway inhibitor expression in breast cancer.

UNLABELLED: ESSENTIALS: A hypoxic microenvironment is a common feature of tumors that may influence activation of coagulation. MCF-7 and SK-BR-3 breast cancer cells and breast cancer tissue samples were used. The results showed transcriptional repression of tissue factor pathway inhibitor expression in hypoxia. Hypoxia-inducible factor 1α may be a target for the therapy of cancer-related coagulation and thrombosis. BACKGROUND: Activation of coagulation is a common finding in patients with cancer, and is associated with an increased risk of venous thrombosis. As a hypoxic microenvironment is a common feature of solid tumors, we investigated the role of hypoxia in the regulation of tissue factor (TF) pathway inhibitor (TFPI) expression in breast cancer. OBJECTIVES: To explore the transcriptional regulation of TFPI by hypoxia-inducible factor (HIF)-1α in breast cancer cells and their correlation in breast cancer tissues. METHODS AND RESULTS: MCF-7 and SK-BR-3 breast cancer cells were cultured in 1% oxygen or treated with cobalt chloride (CoCl2 ) to mimic hypoxia. Time-dependent and dose-dependent downregulation of TFPI mRNA (quantitative RT-PCR) and of free TFPI protein (ELISA) were observed in hypoxia. Western blotting showed parallel increases in the levels of HIF-1α protein and TF. HIF-1α inhibitor abolished or attenuated the hypoxia-induced downregulation of TFPI. Luciferase reporter assay showed that both hypoxia and HIF-1α overexpression caused strong repression of TFPI promoter activity. Subsequent chromatin immunoprecipitation and mutagenesis analysis demonstrated a functional hypoxia response element within the TFPI promoter, located at -1065 to -1060 relative to the transcriptional start point. In breast cancer tissue samples, gene expression analyses showed a positive correlation between the mRNA expression of TFPI and that of HIF-1α. CONCLUSIONS: This study demonstrates that HIF-1α is involved in the transcriptional regulation of the TFPI gene, and suggests that a hypoxic microenvironment inside a breast tumor may induce a procoagulant state in breast cancer patients.