MicroRNA-450b-3p inhibits cell growth by targeting phosphoglycerate kinase 1 in hepatocellular carcinoma

Dysregulation of microRNAs frequently contributes to the occurrence and progression of human diseases, including hepatocellular carcinoma (HCC). In this study, the role of miR-450b-3p in HCC was investigated. Gene Expression Omnibus database and HCC specimens were used to evaluate the expression level of miR-450b-3p and the patient's prognosis. Cell functional analyses and tumor xenograft model were used to assess the role of miR-450b-3p in HCC. Bioinformatics was used to predict the downstream target gene of miR-450b-3p, which was verified by dual-luciferase reporter assay. MiR-450b-3p was found to be downregulated in HCC cell lines and tissues, compared with nontransformed immortal hepatic cells and adjacent normal liver tissues, respectively. Lower expression of miR-450b-3p was associated with poor overall survival and disease-free survival in patients with HCC. Ectopic expression of miR-450b-3p inhibited HCC cell viability, colony formation, and cell-cycle progression in vitro, and suppressed the growth of HCC xenograft tumors in vivo. Interestingly, a negative correlation between miR-450b-3p and phosphoglycerate kinase 1 (PGK1) protein was observed among HCC specimens. Additionally, miR-450b-3p inhibited PGK1 expression and phosphorylation of protein kinase B in HCC cell lines. Further experiments confirmed that PGK1 was a direct target of miR-450b-3p. Moreover, restoration of PGK1 abrogated the inhibitory effect of miR-450b-3p on HCC proliferation and cell division. In conclusion, miR-450b-3p is downregulated in human HCC and exerts tumor suppressive effects at least in part by inhibiting PGK1.     

Silencing of long noncoding RNA LEF1-AS1 prevents the progression of hepatocellular carcinoma via the crosstalk with microRNA-136-5p/WNK1

Long noncoding RNAs (lncRNAs) have been recognized as cancer-associated biological molecules, favoring hepatocellular carcinoma (HCC) progression. This study was conducted to elucidate the effects lncRNA lymphoid enhancer-binding Factor 1 antisense RNA (LEF1-AS1) on the pathological development of HCC, along with the crosstalk involving microRNA-136-5p (miR-136-5p) and with-no-K (lysine) kinase 1 (WNK1). The study recruited primary HCC tissues and their corresponding nonneoplastic liver tissues. The gain- and loss-of-function studies were performed in HCC cells HuH-7 and tumor xenografts in nude mice. The dual luciferase reporter gene assay system, RNA pull-down, and radioimmunoprecipitation assays were applied to detect their interactions among lncRNA LEF1-AS1, miR-136-5p, and WNK1. 5-Ethynyl-2′-deoxyuridine staining, scratch test, Transwell assays, and in vitro tube formation assays were conducted to examine HCC cell proliferation, migration, and invasion and HUVEC angiogenesis. HCC tissues and cells contained high lncRNA LEF1-AS1 expression. LncRNA LEF1-AS1 upregulation triggered markedly increased HCC cell proliferation, migration, and invasion and human umbilical vein endothelial cell angiogenesis. In vivo silencing lncRNA LEF1-AS1 resulted in reduced tumor cell vitality and matrix metalloproteinase-9 and the vascular endothelial growth factor expression. Additionally, the role of lncRNA LEF1-AS1 was found to be largely dependent on WNK1. Association of lncRNA LEF1-AS1 with WNK1 blocked the inhibitory effect of miR-136-5p on WNK1, which was confirmed by in vivo experiments. Altogether, our results revealed an important role of lncRNA LEF1-AS1 in regulating the HCC progression by regulating WNK1, providing a potential biomarker for the therapeutic modalities regarding HCC.     

Long noncoding RNA DLGAP1-AS1 promotes cell proliferation in hepatocellular carcinoma via sequestering miR-486-5p

Hepatocellular carcinoma (HCC) is most prevalent tumor in liver and one of the most fatal cancers in the world. Long noncoding RNAs (lncRNAs) have been accepted as important regulators in carcinomas. But there are still many lncRNAs including DLGAP1-AS1 unannotated in HCC. First of all, GEPIA suggested that DLGAP1-AS1 presented high expression in HCC tissue samples relative to the normal tissues. Besides, overexpression of DLGAP1-AS1 was also proved in HCC cell lines. Moreover, DLGAP1-AS1 knockdown efficiently suppressed cell proliferation in HCC. Interestingly, miR-486-5p was predicted and validated to interact with DLGAP1-AS1, while the level of miR-486-5p was significantly increased In HCC after DLGAP1-AS1 knockdown. Moreover, we uncovered that ectopic expression of miR-486-5p induced suppression on HCC cell proliferation and that miR-486-5p inhibition offset the effect of DLGAP1-AS1 silence on HCC cell proliferation and apoptosis. Furthermore, H3F3B was identified as target of miR-486-5p and was therefore positively regulated by DLGAP1-AS1 in HCC. Of note, H3F3B upregulation partly revived the declined cell proliferative capacity in response to DLGAP1-AS1 knockdown. To conclude, DLGAP1-AS1 exerted its oncogenic role in HCC via miR-486-5p/H3F3B axis. Our new findings provided novel theoretical basis for discovery of therapeutic targets of HCC.     

Downregulation of circDYNC1H1 exhibits inhibitor effect on cell proliferation and migration in hepatocellular carcinoma through miR-140-5p

Circular RNAs have been found to be aberrantly expressed in tumors and their significance in tumorigenesis has been focused on. The role of circDYNC1H1 in hepatocellular carcinoma (HCC) pathogenesis and its relationship with miR-140-5p were explored. The expression of circDYNC1H1, miR-140-5p, and SULT2B1 in HCC tissues and cells was measured, and Pearson's analysis was used to analyze their expression correlation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Transwell assays were performed to determine cell proliferation and migration. Binding between circDYNC1H1 and miR-140-5p was evaluated with RNA pull-down assay. A luciferase reporter assay was conducted to assess the interaction between circDYNC1H1 and miR-140-5p and between miR-140-5p and SULT2B1. circDYNC1H1 was highly expressed in HCC tissues (n = 20), and it was negatively associated with the expression of miR-140-5p but positively correlated with SULT2B1 messenger RNA expression. circDYNC1H1 was upregulated in cell lines of HCC. Interference of circDYNC1H1 suppressed cell proliferation and migration of HCC. circDYNC1H1 acted as a sponge of miR-140-5p. miR-140-5p controlled SULT2B1 expression by targeting its 3′-untranslated region. circDYNC1H1 enhanced SULT2B1 expression via sponging miR-140-5p. Downregulation of circDYNC1H1 disturbed cell proliferation and migration of HCC through miR-140-5p/SULT2B1 pathway. Silencing of circDYNC1H1 delayed tumor growth in HCC mouse model. Acting like a sponge of miR-140-5p, silenced circDYNC1H1 downregulated SULT2B1 to restrain HCC cell proliferation and migration, which is adverse to HCC growth and progression.     

Effects of microRNA-30a on migration,invasion and prognosis of hepatocellular carcinoma

The role of microRNA-30a (miR-30a) deregulation in tumor progression and its downstream signaling pathways remain unknown. Here we confirmed significant downregulation of miR-30a in hepatocellular carcinoma (HCC) tissues and cell lines compared with non-tumor counterparts. MiR-30a downregulation was significantly associated with worse disease-free survival (DFS) of HCC patients. Gain- and loss-of-function studies revealed that downregulation of miR-30a facilitated tumor cell migration, invasion and epithelial–mesenchymal transition (EMT). We identified SNAI1 as a direct target of miR-30a and demonstrated miR-30a as a novel regulator of EMT by targeting SNAI1, indicating its potential therapeutic value for reducing invasion and metastasis of HCC.     

lncRNA A1BG-AS1 suppresses proliferation and invasion of hepatocellular carcinoma cells by targeting miR-216a-5p

Extensive evidence indicate that long noncoding RNAs (lncRNAs) regulates the tumorigenesis and progression of hepatocellular carcinoma (HCC). However, the expression and biological function of lncRNA A1BG antisense RNA 1 (A1BG-AS1) were poorly known in HCC. Here, we found the underexpression of A1BG-AS1 in HCC via analysis of The Cancer Genome Atlas database. Further analyses confirmed that A1BG-AS1 expression in HCC was markedly lower than that in noncancerous tissues based on our HCC cohort. Clinical association analysis revealed that low A1BG-AS1 expression correlated with poor prognostic features, such as microvascular invasion, high tumor grade, and advanced tumor stage. Follow-up data indicated that low A1BG-AS1 level evidently correlated with poor clinical outcomes of HCC patients. Moreover, forced expression of A1BG-AS1 repressed proliferation, migration, and invasion of HCC cells in vitro. Conversely, A1BG-AS1 knockdown promoted these malignant behaviors in HepG2 cells. Mechanistically, A1BG-AS1 functioned as a competing endogenous RNA by directly sponging miR-216a-5p in HCC cells. Notably, miR-216a-5p restoration rescued A1BG-AS1 attenuated proliferation, migration and invasion of HCCLM3 cells. A1BG-AS1 positively regulated the levels of phosphatase and tensin homolog and SMAD family member 7, which were reduced by miR-216a-5p in HCC cells. Altogether, we conclude that A1BG-AS1 exerts a tumor suppressive role in HCC progression.     

MicroRNA-7 arrests cell cycle in G1 phase by directly targeting CCNE1 in human hepatocellular carcinoma cells

Growing evidence has demonstrated that the aberrant expression of miRNA is a hallmark of malignancies, indicating the important roles of miRNA in the development and progression of cancer. MiR-7 is considered as a tumor suppressor miRNA in multiple types of cancer. However, the role of miR-7 in human hepatocellular carcinoma (HCC) and its underlying mechanism remain elusive. In this study, we found that overexpression of miR-7 arrested cell cycle at G1 to S transition in HCC. By combinational use of bioinformatic prediction, reporter assay, quantitative real-time PCR (qRT-PCR) and Western blot, we confirmed that CCNE1, an important mediator in G1/S transition is one of new direct target genes of miR-7. Further studies revealed that silencing of CCNE1 recapitulated the effects of miR-7 overexpression, whereas enforced expression of CCNE1 reversed the suppressive effects of miR-7 in cell cycle regulation. Finally, analysis of qRT-PCR showed a reciprocal relationship between miR-7 and CCNE1 in clinical cancer tissues and multiple types of tumor cell lines. These findings indicate that miR-7 exerts tumor-suppressive effects in hepatocarcinogenesis through the suppression of oncogene CCNE1 expression and suggest a therapeutic application of miR-7 in HCC.     

Over-Expression of miR-106b Promotes Cell Migration and Metastasis in Hepatocellular Carcinoma by Activating Epithelial-Mesenchymal Transition Process

Hepatocellular carcinoma (HCC) is one the the most fatal cancers worldwide. The poor prognosis of HCC is mainly due to the developement of distance metastasis. To investigate the mechanism of metastasis in HCC, an orthotopic HCC metastasis animal model was established. Two sets of primary liver tumor cell lines and corresponding lung metastasis cell lines were generated. In vitro functional analysis demonstrated that the metastatic cell line had higher invasion and migration ability when compared with the primary liver tumor cell line. These cell lines were subjected to microRNA (miRNAs) microarray analysis to identify differentially expressed miRNAs which were associated with the developement of metastasis in vivo. Fifteen human miRNAs, including miR-106b, were differentially expressed in 2 metastatic cell lines compared with the primary tumor cell lines. The clinical significance of miR-106b in 99 HCC clinical samples was studied. The results demonstrated that miR-106b was over-expressed in HCC tumor tissue compared with adjacent non-tumor tissue (p = 0.0005), and overexpression of miR-106b was signficantly correlated with higher tumor grade (p = 0.018). Further functional studies demonstrated that miR-106b could promote cell migration and stress fiber formation by over-expressing RhoGTPases, RhoA and RhoC. In vivo functional studies also showed that over-expression of miR-106b promoted HCC metastasis. These effects were related to the activation of the epithelial-mesenchymal transition (EMT) process. Our results suggested that miR-106b expression contributed to HCC metastasis by activating the EMT process promoting cell migration in vitro and metastasis in vivo.     

miR-130b Promotes CD133(+) liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear protein 1

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%-13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133? counterparts, CD133(+) cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133(+) cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133(+) tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133(+) and CD133? cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133(+) TICs. Functional studies on miR-130b lentiviral-transduced CD133? cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133(+) TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133? cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133(+) liver TICs, in part, via silencing TP53INP1.     

The competing endogenous circular RNA ADAMTS14 suppressed hepatocellular carcinoma progression through regulating microRNA-572/regulator of calcineurin 1

Emerging evidence have discovered that circular RNAs (circRNAs) may serve as diagnostic or tumor promising biomarkers. This study aimed to investigate how circular RNA ADAMTS14 (circADAMTS14) regulates microRNA-572/ regulator of calcineurin 1(miR-572/ RCAN1) in hepatocellular carcinoma (HCC). The expression profiles of circRNA/microRNA (mRNA) between HCC tissues and paired adjacent tissues were analyzed via microarray analysis. The expressions of circADAMTS14, miR-572, and RCAN1 were measured by real-time polymerase chain reaction (PCR). The protein expression level of RCAN1 in HCC cells was detected by western blot. The viability and apoptosis levels of HCC cell lines were measured by the cell counting Kit-8 (CCK-8) assay and fluorescence-activated cell sorter. The invasiveness and migration of cells were detected based on the transwell and wound-healing assay, respectively. The dual-luciferase reporter assays were used to reveal circADAMTS14 and RCAN1 as a potential target of miR-572, which was predicted by TargetScan and miRBase. The effect of circADAMTS14 on HCC cells was demonstrated by tumor formation in nude mice in vivo. CircADAMTS14 and RCAN1 were lowly expressed in HCC clinical specimens and cell lines using microarrays and qRT-PCR, but miR-572 inversely. Our study further verified the direct interaction between circADAMTS14 and RCAN1 with miR-572 via the dual-luciferase reporter gene assay. Overexpressed circADAMTS14 and RCAN1 induced apoptosis of HCC cells and inhibited cell proliferation and invasion. But overexpressed miR-572 could decrease apoptosis of HCC cells and promote proliferation and invasion. In vivo, circADAMTS14 inhibited the tumor growth, correlated positively with the protein expression levels of RCAN1. Our results demonstrated that circADAMTS14 might suppress HCC progression through regulating miR-572/ RCAN1 as the competing endogenous RNA.     

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.     

Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

Cancer-associated fibroblasts (CAFs) are commonly acquired activated extracellular matrix (ECM)-producing myofibroblasts, a phenotypes with multiple roles in hepatic fibrogenesis and carcinogenesis via crosstalk with cohabitating stromal/cancer cells. Here, we discovered a mechanism whereby CAF-derived cytokines enhance hepatocellular carcinoma (HCC) progression and metastasis by activating the circRNA-miRNA-mRNA axis in tumor cells. CAFs secreted significantly higher levels of CXCL11 than normal fibroblasts (NFs), and CXCL11 also had comparatively higher expressions in HCC tissues, particularly in metastatic tissues, than para-carcinoma tissues. Both CAF-derived and experimentally introduced CXCL11 promoted HCC cell migration. Likewise, CAFs promoted tumor migration in orthotopic models, as shown by an increased number of tumor nodules, whereas CXCL11 silencing triggered a decrease of it. CXCL11 stimulation upregulated circUBAP2 expression, which was significantly higher in HCC tissues than para-carcinoma tissues. Silencing circUBAP2 reversed the effects of CXCL11 on the expression of IL-1β/IL-17 and HCC cell migration. Further downstream, the IFIT1 and IFIT3 levels were significantly upregulated in HCC cells upon CXCL11 stimulation, but downregulated upon circUBAP2 silencing. IFIT1 or IFIT3 silencing reduced the expression of IL-17 and IL-1β, and attenuated the migration capability of HCC cells. Herein, circUBAP2 counteracted miR-4756-mediated inhibition on IFIT1/3 via sponging miR-4756. miR-4756 inhibition reversed the effects induced by circUBAP2 silencing on the IL-17 and IL-1β levels and HCC cell migration. In orthotopic models, miR-4756 inhibition also reversed the effects on metastatic progression induced by silencing circUBAP2.Subject terms: Tumour biomarkers, Cancer     

miR-186 modulates hepatocellular carcinoma cell proliferation and mobility via targeting MCRS1-mediated Wnt/β-catenin signaling

Previous studies have revealed that miR-186 is involved in the pathogenesis of many malignancies. However, the role of miR-186 in hepatocellular carcinoma (HCC) carcinogenesis and its detailed mechanism are poorly understood. This study was to investigate the function of miR-186 in modulating HCC cell proliferation, cell cycle, migration, and invasion. We found that miR-186 was decreased in HCC tissues and cell lines. Loss-of-function experiments showed that reduction of miR-186 dramatically enhanced tumor cell proliferation and metastasis. Besides, miR-186 also participated in the modulation of the cell cycle. In addition, luciferase reporter assays and Western blot analysis showed that MCRS1 was a novel target of miR-186 in HCC cells. Notably, upregulation of miR-186 suppressed the nuclear β-catenin accumulation and blocked the activation of Wnt/β-catenin signaling in HCC cells. Forced MCRS1 expression abrogated the inhibitory effect of miR-186 on cell growth, metastasis and Wnt/β-catenin signaling in HCC cells. Our findings may provide new insight into the pathogenesis of HCC and miR-186/ MCRS1 might function as new therapeutic targets for HCC.