Abstract.Evidence is rapidly accumulating that long non-coding RNAs (lncRNAs) are involved in human tumorigenesis and are dysregulated in multiple cancers, including hepatocellular carcinoma (HCC). lncRNAs can regulate essential pathways that contribute to tumor initiation and progression with tissue specificity, which suggests that lncRNAs may be valuable biomarkers and therapeutic targets. HOX transcript antisense intergenic RNA (HOTAIR) has previously been demonstrated to be an oncogene and a negative prognostic factor in a variety of cancers; however, the factors that contribute to the upregulation of HOTAIR and the interaction between HOTAIR and microRNAs (miRNAs or miRs) are largely unknown. In the present study, the expression levels of HOTAIR, forkhead box C1 (FOXC1) and miRNA-1 were examined in 50 matched pairs of HCC and HCC cells. The effects of HOTAIR on HCC cell proliferation were tested using trypan blue exclusion assay. The effect of HOTAIR on HCC growth in vivo was determined in a (nu/nu) mouse model. A computational screening of HOTAIR promoter was conducted to search for transcription factor-binding sites. FOXC1 binding to the promoter region of HOTAIR was confirmed using a chromatin immunoprecipitation assay. A search for miRNAs that had complementary base paring with HOTAIR was performed utilizing an online software program.The interaction between miR-1 and HOTAIR was examined using a luciferase reporter assay. Gain and loss of function approaches were used to determine the changes of HOTAIR or miR-1 expression. The relative levels of FOXC1 and HOTAIR expression in HCC tissues and HepG2 cells were significantly higher than those in normal liver LO2 cells and adjacent carcinoma tissues; the relative expression of miR-1 exhibited the opposite pattern. Overexpression of HOTAIR promoted HCC cell proliferation and progression of tumor xenografts. The present authors have demonstrated that FOXC1 binds to the upstream region of HOTAIR in HCC cells and that FOXC1 activates lncRNA HOTAIR expression in HCC HepG2 cells, which suggests that HOTAIR harbors a miRNA-1 binding site. The present data revealed that this binding site is vital for the regulation of miRNA-1 by HOTAIR. Furthermore, HOTAIR negatively regulated the expression of miRNA-1 in HepG2 cells. Additionally, the present study demonstrated that the oncogenic activity of HOTAIR is in part based on the negative regulation of miR-1. Taken together, these results suggest that HOTAIR is a FOXC1-activated driver of malignancy, which acts in part through the repression of miR-1. IntroductionHepatocellular carcinoma (HCC) is the third leading cause of cancer-associated mortalities, with nearly 600,000 mortalities occurring worldwide each year (1). Although resection is considered a potentially curative treatment for HCC patients, the 1-year post-operative survival rate is only 30-40% (2). Thus, it is necessary to improve our understanding of the disease-causing mechanisms and to identify specific biomarkers for HCC progression to aid in the predictio...

No abstract

Mesenchymal stem cells (MSCs) are able to differentiate into hepatocytes, promote the regeneration of hepatic cells and inhibit the progression of hepatic fibrosis. Transforming growth factor (TGF)-β1 is one of the key factors in the development of liver fibrosis, which also promotes extracellular matrix (ECM) formation. Drosophila mothers against decapentaplegic 7 (Smad7) is an essential negative regulator in the TGF-β1/Smad signaling pathway. In the present study, bone mesenchymal stem cells (BMSCs) were isolated from rat bone marrow and transfected with lentiviral vectors carrying the Smad7 gene. Smad7-enhanced green fluorescent protein (EGFP)-BMSCs stably expressing Smad7 were subsequently co-cultured with hepatic stellate cells (HSCs) for 48 h. Smad7 and TGF-β1 levels in the culture medium were detected using ELISA, and the levels of collagen (Col) I, Col III, laminin (LN) and hyaluronic acid (HA) were measured using immunoassays. The early apoptosis rates of HSCs were determined via flow cytometry. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to evaluate the mRNA and protein expression profiles, respectively. The results indicated that Smad7-EGFP-BMSCs stably expressing Smad7 were successfully constructed. Upon co-culturing with rat Smad7-EGFP-BMSCs, the early apoptotic rate of HSCs was significantly increased (P<0.05). Levels of Smad7 in the culture medium were also significantly increased (P<0.05), whereas the levels of TGF-β1, Col I, Col III, LN and HA were significantly decreased (P<0.05). Furthermore, the mRNA and protein levels of Smad7 and matrix metalloproteinase 1 were significantly increased (P<0.05), whereas those of TGF-β1, α-SMA, Smad2, smad3, TGF-β receptor I, Col I, tissue inhibitors of metalloproteinase-1 and Col III were significantly decreased. The results of the present study suggest that rat BMSCs overexpressing Smad7 may inhibit the fibrosis of HSCs by regulating the TGF-β1/Smad signaling pathway. This provides a novel insight into future treatments for liver fibrosis.

Background Bone mesenchymal stem cells (MSCs) can promote liver regeneration and inhibit inflammation and hepatic fibrosis. MSCs also can serve as a vehicle for gene therapy. Smad7 is an essential negative regulatory gene in the TGF-β1/Smad signalling pathway. Activation of TGF-β1/Smad signalling accelerates liver inflammation and fibrosis; we therefore hypothesized that MSCs overexpressing the Smad7 gene might be a new cell therapy approach for treating liver fibrosis via the inhibition of TGF-β1/Smad signalling. Methods MSCs were isolated from 6-week-old Wistar rats and transduced with the Smad7 gene using a lentivirus vector. Liver cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl4) for 8 weeks. The rats with established liver cirrhosis were treated with Smad7-MSCs by direct injection of cells into the main lobes of the liver. The expression of Smad7, Smad2/3 and fibrosis biomarkers or extracellular matrix proteins and histopathological change were assessed by quantitative PCR, ELISA and Western blotting and staining. Results The mRNA and protein level of Smad7 in the recipient liver and serum were increased after treating with Smad-MSCs for 7 and 21 days (P < 0.001). The serum levels of collagen I and III and collagenase I and III were significantly (P < 0.001) reduced after the treatment with Smad7-MSCs. The mRNA levels of TGF-β1, TGFBR1, α-SMA, TIMP-1, laminin and hyaluronic acid were decreased (P < 0.001), while MMP-1 increased (P < 0.001). The liver fibrosis score and liver function were significantly alleviated after the cell therapy. Conclusions The findings suggest that the MSC therapy with Smad7-MSCs is effective in the treatment of liver fibrosis in the CCl4-induced liver cirrhosis model. Inhibition of TGF-β1 signalling pathway by enhancement of Smad-7 expression could be a feasible cell therapy approach to mitigate liver cirrhosis.