MicroRNA-421 functions as an oncogenic miRNA in biliary tract cancer through down-regulating farnesoid X receptor expression

MicroRNAs (miRNAs) are involved in the development of most cancers. However, few studies have been conducted to determine their relationship to biliary tract cancer (BTC). Farnesoid X receptor (FXR) has been reported to be a tumor suppressor for hepatocellular carcinoma and breast cancer; but few studies have focused on its correlation with BTC. In this study, we identified miR-421 as a potential regulator of FXR expression. We found that their expression amount was inversely correlated as FXR was aberrantly down-regulated in both primary tumor specimens and cell lines; while miR-421 was significantly up-regulated. Ectopic expression of miR-421 significantly decreased FXR protein concentration in BTC cells and promoted cell proliferation, colony formation and migration in vitro. Furthermore, a decrease in miR-421 expression induced G₀/G₁ cell cycle arrest. In conclusion, our study identified microRNA-421 functions as an oncomiR in BTC by targeting FXR. This finding may provide a novel therapeutic strategy for treatment of biliary tract cancer.     

Hepatocarcinogenesis in FXR-/- mice mimics human HCC progression that operates through HNF1α regulation of FXR expression

Farnesoid X receptor (FXR) (nuclear receptor subfamily 1, group H, member 4) is a member of nuclear hormone receptor superfamily, which plays essential roles in metabolism of bile acids, lipid, and glucose. We previously showed spontaneously hepatocarcinogenesis in aged FXR(-/-) mice, but its relevance to human hepatocellular carcinoma (HCC) is unclear. Here, we report a systematical analysis of hepatocarcinogenesis in FXR(-/-) mice and FXR expression in human liver cancer. In this study, liver tissues obtained from FXR(-/-) and wild-type mice at different ages were compared by microarray gene profiling, histological staining, chemical analysis, and quantitative real-time PCR. Primary hepatic stellate cells and primary hepatocytes isolated from FXR(-/-) and wild-type mice were also analyzed and compared. The results showed that the altered genes in FXR(-/-) livers were mainly related to metabolism, inflammation, and fibrosis, which suggest that hepatocarcinogenesis in FXR(-/-) mice recapitulated the progression of human liver cancer. Indeed, FXR expression in human HCC was down-regulated compared with normal liver tissues. Furthermore, the proinflammatory cytokines, which were up-regulated in human HCC microenvironment, decreased FXR expression by inhibiting the transactivity of hepatic nuclear factor 1α on FXR gene promoter. Our study thereby demonstrates that the down-regulation of FXR has an important role in human hepatocarcinogenesis and FXR(-/-) mice provide a unique animal model for HCC study.     

The miR-545/374a Cluster Encoded in the Ftx lncRNA is Overexpressed in HBV-Related Hepatocellular Carcinoma and Promotes Tumorigenesis and Tumor Progression

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). Previous studies have shown several long noncoding RNAs (lncRNAs) play various roles in HCC progression, but no research has focused on the expression pattern of microRNA clusters encoded in lncRNAs. The Ftx gene encodes a lncRNA which harbors 2 clusters of microRNAs in its introns, the miR-374b/421 cluster and the miR-545/374a cluster. To date, no research has focused on the role of the miR-545/374a and miR-374b/421 clusters in HBV-related HCC. In this study, 66 pairs of HBV-related HCC tissue and matched non-cancerous liver tissue specimens were analyzed for the expression of the Ftx microRNA clusters. Our results showed that the miR-545/374a cluster was upregulated in HBV-HCC tissue and significantly correlated with prognosis-related clinical features, including histological grade, metastasis and tumor capsule. Transfection studies with microRNA mimics and inhibitors revealed that miR-545/374a expression promoted in vitro cell proliferation, cell migration and invasion. The wild-type HBV-genome-containing plasmid or full-length HBx protein encoding plasmid was transfected into the Bel-7402 cell line and observed for their influence on miR-545/374a expression. We found that transfection of the HBV genome or HBx alone resulted in an increase in miR-545/374a expression. Next, by monitoring the expression of sera miR-545/374a before and after surgical tumor excision, we found serum miR-545/374a was tumor-derived and exhibited a sharp decrease 25 days after tumor excision. We also examined the gender-based difference in miR-545/374a expression among HCC patients and utilized microRNA target prediction software to find the targets of miR-545/374a. One of these targets, namely estrogen-related receptor gamma (ESRRG) was inversely correlated with miR-545 expression. In conclusion, the overexpression of miR-545/374a cluster located in the Ftx lncRNA is partially responsible for a poor prognosis, and monitoring sera levels of miR-545/374a may be a useful diagnostic marker for HCC.     

miR-421 promotes apoptosis and suppresses metastasis of osteosarcoma cells via targeting LTBP2

Increasing evidence has confirmed that microRNAs (miRs) are involved in tumor development and progression. A previous study reported that miR-421 could serve as a diagnostic marker in patients with osteosarcoma (OS). The present study explored the potential roles of miR-421 in the regulation of cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition of OS cells. Our results showed that miR-421 was upregulated in OS tissues and cell lines (MG63, U2OS, HOS, and Saos-2) compared with the corresponding adjacent tissues or human osteoblast cells hFOB1.19, while the latent transforming growth factor β-binding protein 2 (LTBP2) expression was reduced. In MG63 and U2OS cells, CCK8 assay displayed that cell proliferation was repressed by the miR-421 inhibitor, conversely increased by miR-421 mimics. Inhibition of miR-421 promoted cell apoptosis rate, caspase 3 activity, cleaved-caspase 3 (c-caspase 3) expression, and Bax/Bcl-2 ratio, restoration of miR-421 showed the opposite functions. Suppression of miR-421 blocked migration and invasion, whereas miR-421 overexpression promoted the migration and invasion of MG63 and U2OS cells. In addition, real-time polymerase chain reaction and Western blot analysis revealed that miR-421 negatively regulated E-cadherin expression, and positively regulated the expression of N-cadherin and vimentin. The luciferase reporter assay determined that miR-421 could target LTBP2-3′-UTR, and LTBP2 expression was regulated negatively by miR-421 both in mRNA and protein levels. Depletion of LTBP2 partly abolished the biological functions of miR-421 inhibitor in OS. In conclusion, miR-421 plays an oncogenic role in OS via targeting LTBP2, suggesting that miR-421 may be a potential therapeutic target against OS.     

MiR-199a Regulates Cell Proliferation and Survival by Targeting FZD7

A growing amount of evidence indicates that miRNAs are important regulators of multiple cellular processes and, when expressed aberrantly in different types of cancer such as hepatocellular carcinoma (HCC), play significant roles in tumorigenesis and progression. Aberrant expression of miR-199a-5p (also called miR-199a) was found to contribute to carcinogenesis in different types of cancer, including HCC. However, the precise molecular mechanism is not yet fully understood. The present study showed that miR-199a is frequently down-regulated in HCC tissues and cells. Importantly, lower expression of miR-199a was significantly correlated with the malignant potential and poor prognosis of HCC, and restoration of miR-199a in HCC cells led to inhibition of the cell proliferation and cell cycle in vitro and in vivo. Furthermore, Frizzled type 7 receptor (FZD7), the most important Wnt receptor involved in cancer development and progression, was identified as a functional target of miR-199a. In addition, these findings were further strengthened by results showing that expression of FZD7 was inversely correlated with miR-199a in both HCC tissues and cells and that over-expression of miR-199a could significantly down-regulate the expression of genes downstream of FZD7, including β-catenin, Jun, Cyclin D1 and Myc. In conclusion, these findings not only help us to better elucidate the molecular mechanisms of hepatocarcinogenesis from a fresh perspective but also provide a new theoretical basis to further investigate miR-199a as a potential biomarker and a promising approach for HCC treatment.     

A transcriptional target of androgen receptor,miR-421 regulates proliferation and metabolism of prostate cancer cells

Prostate cancer is one of the most common malignancies, and microRNAs have been recognized to be involved in tumorigenesis of various kinds of cancer including prostate cancer (PCa). Androgen receptor (AR) plays a core role in prostate cancer progression and is responsible for regulation of numerous downstream targets including microRNAs. This study identified an AR-repressed microRNA, miR-421, in prostate cancer. Expression of miR-421 was significantly suppressed by androgen treatment, and correlated to AR expression in different prostate cancer cell lines. Furthermore, androgen-activated AR could directly bind to androgen responsive element (ARE) of miR-421, as predicted by bioinformatics resources and demonstrated by ChIP and luciferase reporter assays. In addition, over-expression of miR-421 markedly supressed cell viability, delayed cell cycle, reduced glycolysis and inhibited migration in prostate cancer cells. According to the result of miR-421 target genes searching, we focused on 4 genes NRAS, PRAME, CUL4B and PFKFB2 based on their involvement in cell proliferation, cell cycle progression and metabolism. The expression of these 4 downstream targets were significantly repressed by miR-421, and the binding sites were verified by luciferase assay. Additionally, we explored the expression of miR-421 and its target genes in human prostate cancer tissues, both in shared microarray data and in our own cohort. Significant differential expression and inverse correlation were found in PCa patients.     

MicroRNA-99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma

In our in-depth analysis carried out by the Illumina Solexa massive parallel signature sequencing, microRNA-99a (miR-99a) was found to be the sixth abundant microRNA in the miRNome of normal human liver but was markedly down-regulated in hepatocellular carcinoma (HCC). Compelling evidence has suggested the important roles of microRNAs in HCC development. However, the biological function of miR-99a deregulation in HCC remains unknown. In this study, we found that miR-99a was remarkably decreased in HCC tissues and cell lines. Importantly, lower miR-99a expression in HCC tissues significantly correlated with shorter survival of HCC patients, and miR-99a was identified to be an independent predictor for the prognosis of HCC patients. Furthermore, restoration of miR-99a dramatically suppressed HCC cell growth in vitro by inducing the G(1) phase cell cycle arrest. Intratumoral injection of cholesterol-conjugated miR-99a mimics significantly inhibited tumor growth and reduced the α-fetoprotein level in HCC-bearing nude mice. Insulin-like growth factor 1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) were further characterized as the direct targets of miR-99a. Furthermore, protein levels of IGF-1R and mTOR were found to be inversely correlated with miR-99a expression in HCC tissues. miR-99a mimics inhibited IGF-1R and mTOR pathways and subsequently suppressed expression of cell cycle-related proteins, including cyclin D1 in HCC cells. Conclusively, miR-99a expression was frequently down-regulated in HCC tissues and correlates with the prognosis of HCC patients, thus proposing miR-99a as a prospective prognosis predictor of HCC. miR-99a suppresses HCC growth by inducing cell cycle arrest, suggesting miR-99a as potential tumor suppressor for HCC therapeutics.     

MicroRNA-145 suppresses hepatocellular carcinoma by targeting IRS1 and its downstream Akt signaling

Accumulating evidences have proved that dysregulation of microRNAs (miRNAs) is involved in cancer initiation and progression. In this study, we showed that miRNA-145 level was significantly decreased in hepatocellular cancer (HCC) tissues and cell lines, and its low expression was inversely associated with the abundance of insulin receptor substrate 1 (IRS1), a key mediator in oncogenic insulin-like growth factor (IGF) signaling. We verified IRS1 as a direct target of miR-145 using Western blotting and luciferase reporter assay. Further, the restoration of miR-145 in HCC cell lines suppressed cancer cell growth, owing to down-regulated IRS1 expression and its downstream Akt/FOXO1 signaling. Our results demonstrated that miR-145 could inhibit HCC through targeting IRS1 and its downstream signaling, implicating the loss of miR-145 regulation may be a potential molecular mechanism causing aberrant oncogenic signaling in HCC.     

Long noncoding RNA HOXD-AS1 induces epithelial-mesenchymal transition in breast cancer by acting as a competing endogenous RNA of miR-421

Breast cancer (BCa) is the most common malignant tumor in females. Long noncoding RNAs (lncRNAs) are deregulated in many types of human cancers, including BCa. The purpose of the present study was to examine the expression profile and biological role of HOXD cluster antisense RNA 1 (HOXD-AS1) in BCa. Our results revealed that HOXD-AS1 was upregulated in BCa tissues and cell lines, and high HOXD-AS1 expression was correlated with aggressive clinicopathological characteristics of BCa patients. Further gain-of-function and loss-of-function analysis showed that HOXD-AS1 overexpression promoted, whereas HOXD-AS1 knockdown inhibited BCa cell proliferation, cell cycle progression, migration, and invasion, indicating that HOXD-AS1 may function as a novel oncogene in BCa. Mechanistically, HOXD-AS1 could activate epithelial-mesenchymal transition (EMT) in BCa cells. We further proved that HOXD-AS1 might serve as a competing endogenous RNA of miR-421 in BCa cells, and miR-421 was downregulated and negatively correlated with HOXD-AS1 expression in BCa tissues. Besides, we confirmed that SOX4, a master regulator of EMT, was a direct target gene of miR-421. Further, rescue experiments suggested that miR-421 overexpression partly abrogated the oncogenic role of HOXD-AS1 in BCa cells. Therefore, we shed light on that HOXD-AS1/miR-421/SOX4 axis may be considered as a novel therapeutic target for the treatment of BCa patients.     

MicroRNA200a enhances antitumor effects in combination with doxorubicin in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is often treated with doxorubicin. MicroRNAs have been shown to have important regulatory roles in cancer and serve as a target in chemoresistance. In this study, we investigated the effects of specific microRNA-200a (miR-200a) on HCC tumor cell growth and effect of doxorubicin-mediated cytotoxicity. Our results show miR-200a is downregulated in human HCC and HCC tumor cell lines. Increasing miR-200a expression inhibited HCC growth and synergized with the antitumor effects of doxorubicin. Inhibiting endogenous miR-200a promoted tumor growth and chemotherapeutic resistance. Increasing miR-200a expression inhibited tumor metabolism (ATP production, mitochondrial respiration, glycolysis), while inhibition of endogenous miR-200a reversed these effects. MiR-200a expression also increased autophagy and synergized with doxorubicin-mediated cytotoxicity. This study identifies a novel role of miR-200a in potentiating doxorubicin-mediated therapeutic effects in HCC.     

Upregulation of microRNA-372 associates with tumor progression and prognosis in hepatocellular carcinoma

MicroRNA-372 (miR-372) has been demonstrated to play a crucial role in cellular proliferation and apoptosis of cancer cells. However, its effects in hepatocellular carcinoma (HCC) have not been explored. The aim of this study was to investigate the clinical significance of miR-372 in human HCC. Quantitative RT-PCR was performed to detect miR-372 expression in HCC clinical samples and cell lines. Then, Kaplan–Meier and Cox proportional regression analyses were performed to determine the association of miR-372 expression with survival of HCC patients. Moreover, the effects of miR-372 on tumorigenicity of HCC cell lines were evaluated by in vitro assays. miR-372 expression in HCC tissues was significantly higher than in the corresponding normal adjacent liver tissues (P < 0.001). There was a correlation between miR-372 upregulation and advanced TNM stage of HCC patients (P = 0.02). In addition, HCC patients with higher miR-372 expression had significantly poorer recurrence-free survival (P = 0.006) and overall survival (P = 0.001). Multivariate analysis revealed that high miR-372 expression was an independent predictor of poor prognosis (for recurrence-free survival: Hazard Ratio [HR] 6.826, P = 0.01; for overall survival: HR 9.533, P = 0.008). Moreover, in vitro assays demonstrated that the ectopic expression of miR-372 may significantly promote the cellular proliferation, invasion, and migration of HCC cell lines. Our findings showed that miR-372 may serve as a potent prognostic marker for tumor recurrence and survival of HCC patients. Furthermore, miR-372 has been identified as a promoter for tumorigenicity of HCC cells, suggesting that it might be a prospective therapeutic target for HCC.     

Retracted: MicroRNA-34a inhibit hepatocellular carcinoma progression by repressing hexokinase-1

Hepatocellular carcinoma (HCC) is known as a frequent type of primary cancer in the liver, and it is the third-most common cause of cancer-related death all over the world. However, the molecular mechanism in the progression of HCC is still unclear. The current study was designed to investigate the expression and function of microRNA-34a (miR-34a) in HCC. In HCC tissues and cells, the expression levels of miR-34a were analyzed by quantitative real-time polymerase chain reaction. The association between the level of miR-34a and hexokinase (HK)-1 was also investigated via luciferase reporter assay. Cell viability and proliferation were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry. To assess whether miR-34a can limit tumor growth in vivo, animal models and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were used for examining the role of miR-34a on the development of HCC and cell apoptosis. The expression level of miR-34a was reduced in HCC samples and cells. The expression of miR-34a was associated with the viability and proliferation capacity of HCC cells, and miR-34a could inhibit HCC cells proliferation by inhibiting HK1. In the mouse model of HCC, volumes and weight of the tumors were significantly decreased by transfection with miR-34a mimic compared with the control group. Furthermore, miR-34a mimics could induce apoptosis in a greater proportion of cells compared with the control group. Taken together, the data may provide some novel insights into the molecular mechanism of miR-34a and HK1 in the progression of HCC. Thus, miR-34a/HK1 axis might be a novel promising therapeutic target for treating HCC.     

Correlation of MicroRNA-16, MicroRNA-21 and MicroRNA-101 Expression with Cyclooxygenase-2 Expression and Angiogenic Factors in Cirrhotic and Noncirrhotic Human Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression.

Methods

Tissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue.

Results

COX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma.

Conclusions

In human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels.     

MicroRNA-363-mediated downregulation of S1PR1 suppresses the proliferation of hepatocellular carcinoma cells

S1PR1 plays a crucial role in promoting proliferation of hepatocellular carcinoma (HCC). Over expression of S1PR1 is observed in HCC cell lines. The mechanisms underlying the aberrant expression of S1PR1 are not known well. MircroRNAs are important regulators of gene expression and disproportionate microRNAs can result in dysregulation of oncogenes in cancer cells. In this study, we found that miR-363, a potential tumor suppressor microRNA, downregulated the expression of S1PR1 and inhibited the proliferation of HCC cells. Bioinformatic analysis predicted a putative binding site of miR-363 within the 3′-UTR of S1PR1 mRNA. Luciferase reporter assay showed that miR-363 directly targeted the 3′-UTR of S1PR1 mRNA. Transfection of miR-363 mimics suppressed S1PR1 expression in HCC cells, followed by the repression of the activation of ERK and STAT3. Moreover, we found that the expression of downstream genes of ERK and STAT3, including PDGF-A, PDGF-B, MCL-1 and Bcl-xL, was suppressed after miR-363 transfection. Taken together, the present study demonstrated that miR-363 was a negative regulator of S1PR1 expression in HCC cells and inhibited cell proliferation, suggesting that the miR-363/S1PR1 pathway might be a novel target for the treatment of HCC.     

A Feedback Inhibition between miRNA-127 and TGFβ/c-Jun Cascade in HCC Cell Migration via MMP13

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is increasing in frequency in the U.S. The major reason for the low postoperative survival rate of HCC is widespread intrahepatic metastasis or invasion, and activation of TGFβ signaling is associated with the invasive phenotype. This study aims at determining the novel function of miR-127 in modulating HCC migration. Overexpression of miR-127 inhibits HCC cell migration, invasion and tumor growth in nude mice. MiR-127 directly represses matrix metalloproteinase 13 (MMP13) 3′UTR activity and protein expression, and diminishes MMP13/TGFβ-induced HCC migration. In turn, TGFβ decreases miR-127 expression by enhancing c-Jun-mediated inhibition of miR-127 promoter activity. In contrast, p53 transactivates miR-127 promoter and induces miR-127 expression, which is antagonized by c-Jun. The inhibition of miR-127 by c-Jun is through TGFβ-mediated ERK and JNK pathways. The lower miR-127 expression shows a negative correlation with the higher MMP13 expression in a subset of human HCC specimens. This is the first report elucidating a feedback regulation between miR-127 and the TGFβ/c-Jun cascade in HCC migration via MMP13 that involves a crosstalk between the oncogene c-Jun and tumor suppressor p53.     

Circular RNA hsa_circRNA_103809 promoted hepatocellular carcinoma development by regulating miR-377-3p/FGFR1/ERK axis

In the last decade, circular RNAs (circRNAs) emerge as important regulators in multiple biological processes. Lately, it is reported hsa_circRNA_103809 could play vital parts in several types of cancers. Based on the analysis of GEO data (GSE97332), hsa_circRNA_103809 was found to be dysregulated in hepatocellular carcinoma (HCC). However, the biological function and underlying regulatory mechanisms of hsa_circRNA_103809 in HCC remain unclear. Our results suggested that hsa_circRNA_103809 was overexpressed in HCC patients, and hsa_circRNA_103809 knockdown remarkably inhibited the proliferation, cycle progression, and migration of HCC cells. The investigations of molecular showed that hsa_circRNA_103809 could elevate the protein expression of a miR-377-3p target, fibroblast growth factor receptor 1 (FGFR1), through interacting with miR-377-3p and decreasing its expression level. Additionally, in vivo assays revealed hsa_circRNA_103809 short hairpin RNA served as a tumor suppressor through downregulating FGFR1 in HCC. This study systematically investigated novel regulatory signaling of hsa_circRNA_103809/miR-377-3p/FGFR1 axis, providing insights into hepatocellular carcinoma treatment from bench to clinic.