miR-9 inhibits the metastatic ability of hepatocellular carcinoma via targeting beta galactoside alpha-2,6-sialyltransferase 1

Glycosylation of cell surface proteins regulates critical cellular functions, including invasion and metastasis in cancer cells. Emerging evidence has shown that microRNAs (miRNAs) are involved in regulating both the glycosylation modifications on cell surface and the progression of cancer. In this study, we investigated the role of miR-9 in α-2,6-linked sialylation and the metastasis of mouse hepatocellular carcinoma (HCC). According to array-based miRNA expression profiling data of HCC cell lines Hepa1–6, Hca-P, and Hca-F with different lymphatic metastatic capacities, reverse correlation was found between miR-9 expression levels and the metastatic potential in these HCC cells. Additionally, β-galactoside α-2,6-sialyltransferase 1 (St6gal1) expression level is associated negatively with miR-9 and positively with metastatic potential. Bioinformatics analysis indicated that miR-9 could target St6gal1, which was verified by luciferase reporter assays. miR-9 overexpression reduced expression of St6gal1, which subsequently suppressed HCC cells metastatic potential. Moreover, upregulation of miR-9 could inhibit integrin-β1/FAK-mediated cell motility and migration signaling in mouse HCC cells. Together, our results suggest that miR-9 could act as a tumor suppressor and regulate mouse HCC cells migration and invasion by inhibiting the α-2,6-linked sialylation. This finding may provide insight into the relationship between abnormal miRNA expression and aberrant cell surface glycosylation during tumor lymphatic metastasis.     

Long noncoding RNA TCL6 binds to miR-106a-5p to regulate hepatocellular carcinoma cells through PI3K/AKT signaling pathway

Long noncoding RNAs (lncRNAs) have been reported to dysregulate and involve in the pathology of hepatocellular carcinoma (HCC). Nonetheless, the functional role of lncRNA T cell leukemia/lymphoma 6 (TCL6) and its underlying mechanism in HCC remain unclear. Herein, we analyzed the expression of TCL6 and elucidated its mechanistic involvement in HCC. Bioinformatics analyses indicated TCL6 was evidently downregulated in HCC tissues compared with normal controls. TCL6 was downregulated while microRNA-106a-5p (miR-106a-5p) was upregulated in HCC cell lines. Moreover, knockdown or overexpression of TCL6 significantly raised or diminished the expression level of miR-106a-5p in HCC cells, similar to the effect of miR-106a-5p on TCL6 expression. Functionally, TCL6 inhibited the proliferative, migratory, and invasive potentials of HCC cells as analyzed by cell counting kit-8, scratch wound healing, and transwell assays, respectively. Conversely, miR-106a-5p exerted an opposite effect on the proliferative, migratory, and invasive potentials of HCC. RNA immune precipitation and luciferase reporter assays revealed TCL6 directly bound to miR-106a-5p and luciferase reporter assay verified phosphatase and tensin homolog (PTEN) was a target gene of miR-106a-5p. Mechanistically, TCL6 knockdown evidently reduced PTEN expression at both messenger RNA and protein levels, and miR-106a-5p inhibitor partially rescued this reduction effect in HCC cells. Additionally, western blot assays demonstrated miR-106a-5p downregulation or TCL6 overexpression promoted the protein level of PTEN, and suppressed the phosphorylation level of AKT, the protein level of phosphatidylinositol 3-kinase (PI3K). Collectively, these results revealed TCL6 as a tumor-suppressive lncRNA regulates PI3K/AKT signaling pathway via directly binding to miR-106a-5p in HCC. This mechanism provides a theoretical basis for HCC pathogenesis and a potential therapeutic strategy for HCC treatment.     

MiR-214 Targets β-Catenin Pathway to Suppress Invasion, Stem-Like Traits and Recurrence of Human Hepatocellular Carcinoma

The down-regulation of miR-214 has previously been observed in human hepatocellular carcinoma (HCC). Here, we demonstrated the down-regulation of miR-214 is associated with cell invasion, stem-like traits and early recurrence of HCC. Firstly, we validated the suppression of miR-214 in human HCC by real-time quantitative RT-PCR (qRT-PCR) in 20 paired tumor and non-tumor liver tissues of HCC patients and 10 histologically normal liver tissues from colorectal cancer patients with liver metastases. Further qRT-PCR analysis of 50 HCC tissues from an independent cohort of HCC patients of whom 29 with early recurrent disease (<2 years) and 21 with late recurrent disease demonstrated that the suppression of miR-214 was significantly more suppressed in samples from HCC patients with early recurrent disease compared those from patients with no recurrence. Re-expression of miR-214 significantly suppressed the growth of HCC cells in vitro and reduced their tumorigenicity in vivo. The enhancer of zeste homologue 2 (EZH2) and β-catenin (CTNNB1) was identified as two potential direct downstream targets of miR-214 through bioinformatics analysis and experimentally validated the miRNA-target interactions with a dual-firefly luciferase reporter assay. In corroborate with this, both EZH2 and CTNNB1 are found to be significantly overexpressed in human HCC biopsies. Since EZH2 can regulate CTNNB1, CTNNB1 can also be an indirect target of miR-214 through EZH2. Silencing EZH2 or CTNNB1 expression suppressed the growth and invasion of HCC cells and induced E-cadherin (CDH1), known to inhibit cell invasion and metastasis. Furthermore, the silencing of miR-214 or overexpression of EZH2 increased EpCAM(+) stem-like cells through the activation of CTNNB1. Interestingly, the up-regulation of EZH2, CTNNB1 and the down-regulation of CDH1 in HCC patients correlated with early recurrent disease and can be an independent predictor of poor survival. Therefore, miR-214 can directly or indirectly target CTNNB1 to modulate the β-catenin signaling pathway in HCC.     

MicroRNA-144 inhibits hepatocellular carcinoma cell proliferation,invasion and migration by targeting ZFX

MicroRNA 144 (miR-144), a small non-coding RNA, is frequently dysregulated in human several tumour progression, but its role and the underlying mechanisms in hepatocellular carcinoma (HCC) is poorly investigated. In the present study, the expression of miR-144 was firstly analysed in datasets derived from GSE21362 and TCGA, and then detected in HCC tissues and cell lines by quantitative RT-PCR (qRT-PCR) analysis. MiR-144 was shown to be significantly down-regulated in HCC tissues and cell lines. Subsequently, overexpression of miR-144 was transfected into HCC cell lines so as to investigate its biological function, including MTT, colony formation, and transwell assays. Gain of function assay revealed miR-144 remarkably inhibited cell proliferation, migration and invasion. In addition, bioinformatical analysis and luciferase reporter assay identified ZFX as a novel target of miR-144 in HCC cells, as confirmed by qRT-PCR and Western blot. Furthermore, ZFX was found to be significantly up-regulated using Oncomine database analysis. Loss of function assay further indicated knockdown of ZFX had similar effects of miR-144-mediated HCC cell proliferation and invasion. Therefore, miR-144 has been demonstrated to act as a tumour suppressor in HCC cell growth and motility by directly targeting ZFX, which implicates its potential applications in the development of HCC treatment.     

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     

LncRNA FGD5-AS1 functions as an oncogene to upregulate GTPBP4 expression by sponging miR-873-5p in hepatocellular carcinoma

The long non-coding FGD5-AS1 (LncFGD5-AS1) has been reported to be a novel carcinogenic gene and participant in regulating tumor progression by sponging microRNAs (miRNAs). However, the pattern of expression and the biological role of FGD5-AS1 in hepatocellular carcinoma (HCC) remains largely unknown. The expression level of FGD5-AS1 in tumor tissues and cell lines was measured by RT-qPCR. CCK-8, EdU, flow cytometry, wound healing and transwell chamber assays were performed to investigate the role of FGD5-AS1 in cell proliferation, apoptosis, migration, and invasion in HCC. Dual luciferase reporter, and RNA pull-down assays were performed to identify the regulatory interactions among FGD5-AS1, miR-873-5p and GTP-binding protein 4 (GTPBP4). We found that the expression of FGD5-AS1 was upregulated in HCC tissues and cell lines. Moreover, the knockdown of FGD5-AS1 suppressed cell proliferation, migration and invasion, and induced apoptosis in HCC cells. Further studies demonstrated that FGD5-AS1 could function as a competitive RNA by sponging miR-873-5p in HCC cells. Moreover, GTPBP4 was identified as direct downstream target of miR-873-5p in HCC cells and FGD5-AS1mediated the effects of GTPBP4 by competitively binding with miR-873-5p. Taken together, this study demonstrated the regulatory role of FGD5-AS1 in the progression of HCC and identified the miR-873-5p/GTPBP4 axis as the direct downstream pathway. It represents a promising novel therapeutic strategy for HCC patients.Key words: Hepatocellular carcinoma, FGD5-AS1, miR-873-5p, GTPBP4     

Circular RNA hsa_circ_101555 promotes hepatocellular carcinoma cell proliferation and migration by sponging miR-145-5p and regulating CDCA3 expression

Circular RNAs have been reported to play significant roles in regulating pathophysiological processes while also guiding clinical diagnosis and treatment of hepatocellular carcinoma (HCC). However, only a few circRNAs have been identified thus far. Herein, we investigated the role of a specific closed-loop structure of hsa_circ_101555 that was generated by back-splicing of the host gene casein kinase 1 gamma 1 (CSNK1G1) in the development and proliferation of HCC. We investigated the expression of Hsa_circ_101555 in HCC and normal tissues using bioinformatics. The expression level of hsa_circ_101555 was further detected by fluorescence in situ hybridization and qRT-PCR in ten HCC patients. Transwell, migration, WST-1 assays, and colony formation assays were used to evaluate the role of hsa_circ_101555 in HCC development and proliferation. The regulatory mechanisms of hsa_circ_101555 in miR-145-5p and CDCA3 were determined by dual luciferase reporter assay. A mouse xenograft model was also used to determine the effect of hsa_circ_101555 on HCC growth in vivo. hsa_circ_101555 showed greater stability than the linear RNA; while in vitro and in vivo results demonstrated that hsa_circ_101555 silencing significantly suppressed cell proliferation, migration, and invasion of HCC cells. Rescue experiments further demonstrated that suppression of miR-145-5p significantly attenuated the biological effects of hsa_circ_101555 knockdown in HCC cells. We also identified a putative oncogene CDCA3 as a potential miR-145-5p target. Thus, our results demonstrated that hsa_circ_101555 might function as a competing endogenous RNA of miR-145-5p to upregulate CDCA3 expression in HCC. These findings suggest that hsa_circ_101555 may be a potential therapeutic target for patients with HCC.Subject terms: Liver cancer, Long non-coding RNAs     

Retracted: LINC00707 contributes to hepatocellular carcinoma progression via sponging miR-206 to increase CDK14

Recently, increasing numbers of long noncoding RNAs (lncRNAs) have been found to be aberrantly expressed in various cancers. However, the roles of lncRNAs in hepatocellular carcinoma (HCC) progression is largely unknown. In our current study, we identified that long intergenic nonprotein-coding RNA 707 (LINC00707) was remarkably elevated in HCC cells, indicating that LINC00707 was involved in HCC development. Subsequently, LINC00707 was significantly decreased in HepG2 and Huh7 cells. The in vitro functional assays demonstrated that knockdown of LINC00707 significantly reduced HCC cell proliferation, induced cell apoptosis, and blocked the cell cycle progression. In addition, HCC cell migration and invasion was also greatly inhibited by downregulation of LINC00707. Increasing evidence has indicated that lncRNAs can act as molecular sponges of microRNAs. Currently, we observed that microRNA-206 (miR-206) was dramatically inhibited in HCC cells and LINC00707 can modulate HCC development through sponging miR-206. The binding correlation between LINC00707 and miR-206 was confirmed by dual-luciferase reporter assay, RNA pull down and RNA immunoprecipitation assay in our study. Moreover, cyclin-dependent kinase 14 (CDK14) was predicted as a target of miR-206 and we found that miR-206 suppressed CDK14 levels in HCC cells. Finally, in vivo assays were used and it was proved that silence of LINC00707 can restrain HCC development through modulating miR-206 to upregulate CDK14. In conclusion, it was implied that LINC00707 can lead to HCC progression through sponging miR-206 and modulating CDK14.     

MiR-490-5p inhibits the stemness of hepatocellular carcinoma cells by targeting ECT2

To explore the targeting relationship between miR-490-5p and ECT2 in hepatocellular carcinoma (HCC) and the influences of miR-490-5p and ECT2 on the stemness of HCC cells. The expressions of miR-490-5p and ECT2 in HCC tissues and adjacent tissues were identified by quantitative real-time polymerase chain reaction (qRT-PCR). The relationships between the expression levels of miR-490-5p/ ECT2 and the overall/disease-free survival (OS/DFS) of patients with HCC were evaluated using correlative curves. In addition, the targeting relationship between miR-490-5p and ECT2 was predicted by TargetScan and verified by dual-luciferase reporter assay. Plasmid transfection was used for overexpression of ECT2 in HepG2 cells, and transfection efficiency was verified by qRT-PCR. Cell Counting Kit-8 assay and cell sphere-formation assay were conducted to detect the proliferation and sphere-formation ability of HCC cells, respectively. Cell populations with different cell transfections were sorted using flow cytometry. The expression levels of proteins in the stem cell signaling pathway were determined using Western blot analysis. MiR-490-5p was remarkably downregulated, yet ECT2 was upregulated in HCC tissues compared with adjacent tissues. MiR-490-5p expression was positively correlated with OS and DFS of patients with HCC, which were otherwise negatively correlated with ECT2 expression. ECT2 was validated to be the downstream target of miR-490-5p. Overexpression of miR-490-5p restrained the sphere formation ability, stemness, and proliferation of HCC cells. MiR-490-5p repressed the stemness of HCC cells through inhibiting the expression of ECT2. MiR-490-5p may be an underlying therapeutic target in HCC treatment.     

MicroRNA-550a Acts as a Pro-Metastatic Gene and Directly Targets Cytoplasmic Polyadenylation Element-Binding Protein 4 in Hepatocellular Carcinoma

MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that are often found at chromosomal breakpoints and play a vital role in human cancer. Our previous study found that miR-550a, a frequently amplified miRNA on 7p14.3, was upregulated in hepatocellular carcinoma (HCC). However, the possible functions and molecular mechanisms of miR-550a in HCC remain unknown. In this study, gain-of-function and loss-of-function assays revealed that miR-550a markedly promoted HCC cell migration and invasion. In addition, we discovered that cytoplasmic polyadenylation element binding protein 4 (CPEB4) was a potential target of miR-550a in HCC. Further analyses showed that knockdown of CPEB4 expression significantly facilitated HCC cell migration and invasion, which phenocopied the effects of miR-550a on HCC cells. Moreover, a decrease in CPEB4 expression mediated miR-550a-induced liver cancer cell migration and invasion. Interestingly, CPEB4 is frequently downregulated in HCC, and its expression levels correlate with the overall survival of HCC patients. Together, these results suggested that this newly identified miR-550a-CPEB4 axis may be involved in HCC cell metastasis. Moreover, the expression levels of CPEB4 could be used to predict outcomes in HCC patients. Our findings provide novel potential targets for HCC therapy and prognosis.     

MicroRNA-26b-5p enhances T cell responses by targeting PIM-2 in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a common tumor malignancy threatening a significant number of people worldwide. Although microRNAs (miRNAs) have been shown to play essential role in tumorigenesis, little is known about their role in T cells functions during HCC progression.MethodsThe abundances of miR-26b-5p were detected in HCC tissues or cells, T cells and H22 cells by quantitative real-time polymerase chain reaction (qRT-PCR). Regulation effect of miR-26b-5p on proviral integrations of moloney virus 2 (PIM2) was investigated by qRT-PCR, western blot (WB) and immunohistochemical analysis. The effect of miR-26b-5p and PIM-2 on cytokines secretion in CD4+ and CD8+ cells was evaluated by commercial enzyme linked immunosorbent assays (ELISA) kit. The interaction between miR-26b-5p and PIM-2 was probed by luciferase activity and RNA immunoprecipitation (RIP). H22 subcutaneous model was established to investigate the interaction of miR-26b-5p with HCC and immune competence.ResultsThe abundance of miR-26b-5p was decreased in HCC and associated with poor survival. Addition of miR-26b-5p contributed to secretion of tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6) and IL-2 in CD4+ and CD8+ cells. Interestingly, PIM-2 was negatively regulated by miR-26b-5p and PIM-2 knockdown reversed anti-miR-26b-5p-mediated immunosuppression. Moreover, inhibitory effect of miR-26b-5p on HCC tumorigenesis was dependent on immune competence.ConclusionsmiR-26b-5p enhanced T cells responses by targeting PIM-2 in HCC, uncovering a promising therapeutic opportunity of HCC through reactivating immune system.     

Long noncoding RNA DLX6-AS1 promotes liver cancer by increasing the expression of WEE1 via targeting miR-424-5p

Long noncoding RNAs (lncRNAs) played an important role in tumorigenesis and development of hepatocellular carcinoma (HCC). In this study, we first demonstrated that lncRNA DLX6 antisense RNA 1 (DLX6-AS1) was upregulated in cancer tissues and cells lines compared with normal adjacent and cell line. Knock-down DLX6-AS1 by transfection with small interfering RNA (siRNA) suppressed cell proliferation, migration, and invasion of HCC cells. Cell cycle analysis showed that cells transfected with siRNA were arrested in G0/G1 phase. Then, we performed dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay to show that DLX6-AS1 could bind with miR-424-5p. And cotransfection inhibitor of miR-424-5p with siRNA of DLX6-AS1 could abolish the inhibitory effect of siRNA of DLX6-AS1 on cell proliferation, migration, and invasion. Moreover, we further demonstrated that the oncogene WEE1 G2 checkpoint kinase (WEE1) was the target of miR-424-5p and expression levels of WEE1 were positive correlation with that of DLX6-AS1. Taken together, these results suggested that upregulated DLX6-AS1 promoted cell proliferation, migration, and invasion of HCC through increasing expression of WEE1 via targeting miR-424-5p.     

Oncosuppressive role of MicroRNA-205–3p in gastric cancer through inhibition of proliferation and induction of senescence: Oncosuppressive role of MicroRNA-205 in gastric cancer

BackgroundOur previous study showed that CXCL11 could play an immunomodulatory role. In this study, we investigated the regulator (miR-205–3p) of CXCL11 and the mechanism of miR-205–3p as a tumor suppressor gene in gastric cancer (GC).Materials and methodsA target relationship between miR-205–3p and CXCL11 was revealed by using the bioinformatics method. This study detected the expressions of miR-205–3p and CXCL11 through qRT-PCR and Western blotting. Moreover, the expressions of Akt, PD-L1, p16, p21, and senescence-associated secretory phenotype (SASP) factor were determined. The effects of miR-205 on proliferation, invasion, and senescence of GC cells were assessed by using methods, such as transfection, Transwell assay, tablet cloning, flow cytometry, and senescence-associated beta-galactosidase (SA-β-gal) staining. Furthermore, the effects were verified using methods, like immunohistochemistry, flow cytometry and SA-β-gal in animal experiments.ResultsBased on the study, it is found that the expression of miR-205–3p is down-regulated, while that of CXCL11 is up-regulated in GC cell lines. By regulating CXCL11, miR-205–3p inhibits Akt activation, reduces the proliferation and invasion of GC cells, promotes cell apoptosis, induces senescence of GC cells, and secretes immunostimulatory SASP factor. The animal experiments confirm that miR-205–3p promotes cell senescence, down-regulates the immunosuppressive signal induced by PD-L1, and promotes secretion of immunostimulatory SASP factor, so that more T cells are recruited in blood and tumors.ConclusionsThis study revealed the molecular mechanism of miR-205–3p in inhibiting proliferation and invasion and inducing senescence of GC cells by regulating CXCL11 and Akt pathways in animal and cell experiments.Key words: microRNA-205–3p, CXCL11, Gastric cancer, Senescence, Proliferation     

Circular RNA circUBE2J2 acts as the sponge of microRNA-370-5P to suppress hepatocellular carcinoma progression

Accumulating evidences indicate that circular RNAs (circRNAs), a class of non-coding RNAs, play important roles in tumorigenesis. However, the function of circRNAs in hepatocellular carcinoma is largely unknown. CircRNA microarray was performed to identify abnormally expressed circRNAs in HCC tissue samples. We conducted Kaplan–Meier survival analysis to explore the significance of circUBE2J2 in clinical prognosis. Then, we examined the functions of circUBE2J2 in HCC by cell proliferation, migration, and mouse xenograft assay. We identified miR-370-5P as a circUBE2J2-related microRNA by using biotin-labeled circUBE2J2 probe to perform RNA antisense purification (RAP) assay in HCC cells. The dual luciferase reporter assay and RNA pulldown assays were employed to verify the relationships among circUBE2J2, miRNA-370-5P, and KLF7. Microarray analysis and qRT-PCR verified a circRNA termed circUBE2J2 that was downregulated in HCC. Kaplan–Meier survival analysis showed that downregulated circUBE2J2 was correlated with poorer survival. CircUBE2J2 expression in HCC cells was selectively regulated via luciferase reporter assays; circUBE2J2 and KLF7 were observed to directly bind to miR-370-5P. Furthermore, knockdown of circUBE2J2 in HCC could downregulate KLF7, the target of miR-370-5P, thus promoting the proliferation and migration of HCC cells. Then the related experiment suggested that circUBE2J2 could regulate the expression of KLF7 by sponging miR-370-5p. In summary, we infer that circUBE2J2 may act as a competing endogenous RNA (ceRNA) to regulate KLF7 expression through sponging miR-370-5P and play a regulatory functions in HCC. CircUBE2J2 may be a diagnostic biomarker and potential target for HCC therapy.Subject terms: Tumour biomarkers, Small RNAs     

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.     

miR-155-5p modulates malignant behaviors of hepatocellular carcinoma by directly targeting CTHRC1 and indirectly regulating GSK-3β-involved Wnt/β-catenin signaling

Background

Hepatocellular carcinoma (HCC) remains one of the most lethal cancers. MicroRNA-155 (miR-155) and collagen triple helix repeat containing 1 (CTHRC1) were found to be involved in hepatocarcinogenesis, but their detailed functions in HCC are unclear. Here, we aimed to investigate the underlying role of miR-155-5p and CTHRC1 in HCC.

Methods

miR-155-5p and CTHRC1 expression levels were detected by qRT-PCR, IHC and WB in HCC patients and cell lines. Dual-luciferase assay, qRT-PCR and WB were used to validate the target interaction between miR-155-5p and CTHRC1. Biological behaviors, including apoptosis, cell cycle progression, and cell proliferation, invasion and migration, were measured by flow cytometry, CCK-8 assay and Transwell tests. A xenograft model was established to examine the effects of miR-155-5p and CTHRC1 on tumor formation. WB was finally utilized to identify the role of GSK-3β-involved Wnt/β-catenin signaling in HCC growth and metastasis.

Results

Our results showed that miR-155-5p and CTHRC1 were down-regulated and up-regulated, respectively, in HCC patients and cell lines. Dual-luciferase assay verified that CTHRC1 was the direct target of miR-155-5p. Moreover, elevated miR-155-5p expression promoted apoptosis but suppressed cell cycle progression and cell proliferation, invasion and migration in vitro and facilitated tumor formation in vivo; elevated CTHRC1 expression abolished these biological effects. Additionally, miR-155-5p overexpression increased metastasis- and anti-apoptosis-related protein expression and decreased pro-apoptosis-related protein expression, while forced CTHRC1 expression conserved the expression of these proteins.

Conclusion

Altogether, our data suggested that miR-155-5p modulated the malignant behaviors of HCC by targeting CTHRC1 and regulating GSK-3β-involved Wnt/β-catenin signaling; thereby, miR-155-5p and CTHRC1 might be promising therapeutic targets for HCC patients.

     

Circular RNA circMTO1 suppressed the progression of renal cell carcinoma progression by sponging miR-211 and miR-204

Despite considerable improvements in renal cell carcinoma (RCC) diagnostic and therapeutic strategy, the clinical prognosis of patients is far from satisfactory due to its recurrence and metastasis. Here, we attempted to explore the role of circMTO1 in RCC progression, and the underlying mechanism was further elucidated. We first detected the expression of circMTO1 in 90 pairs of RCC tissues and adjacent normal tissues using qRT-PCR. Besides, circMTO1, miR-211, miR-204 and KLF6 expression levels in RCC cells were also measured using qRT-PCR. MTT assay, cell migration, flow cytometry analysis, qRT-PCR and western blotting analysis were applied to evaluating the effect of circMTO1 in RCC cells. The bioinformatics analysis and the rescue experiment were devoted to the underlying mechanism. The results demonstrated CircMTO1 expression was significantly down-regulated in RCC tissues and cell lines. Besides, CircMTO1 inhibited cell proliferation, migration and invasion, induced apoptosis in RCC cells. In addition, CircMTO1 serves as a sponge for miR-211 and miR-204, KLF6 is a direct target of miR-211 and miR-204. Furthermore, circMTO1 and KLF6 overexpression rescued the suppression of miR-211/204 in RCC cell proliferation. In short, circMTO1 repressed RCC progression by regulating KLF6 via sponging miR-211 and miR-204, which may provide new idea of diagnosis and treatment in renal cell carcinoma.

     

MicroRNA-520b inhibits growth of hepatoma cells by targeting MEKK2 and cyclin D1

Growing evidence indicates that the deregulation of microRNAs (miRNAs) contributes to the tumorigenesis. We previously revealed that microRNA-520b (miR-520b) was involved in the complement attack and migration of breast cancer cells. In this report, we show that miR-520b is an important miRNA in the development of hepatocellular carcinoma (HCC). Our data showed that the expression levels of miR-520b were significantly reduced in clinical HCC tissues and hepatoma cell lines. We observed that the introduction of miR-520b dramatically suppressed the growth of hepatoma cells by colony formation assays, 5-ethynyl-2-deoxyuridine (EdU) incorporation assays and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Moreover, ectopic expression of miR-520b was able to inhibit the growth of hepatoma cells in nude mice. Further studies revealed that the mitogen-activated protein kinase kinase kinase 2 (MEKK2) and cyclin D1 were two of direct target genes of miR-520b. Silencing of MEKK2 or cyclin D1 was able to inhibit the growth of hepatoma cells in vitro and in vivo, which is consistent with the effect of miR-520b overexpression on the growth of hepatoma cells. In addition, miR-520b significantly decreased the phosphorylation levels of c-Jun N-terminal kinase (p-JNK, a downstream effector of MEKK2) or retinoblastoma (p-Rb, a downstream effector of cyclin D1). In conclusion, miR-520b is able to inhibit the growth of hepatoma cells by targeting MEKK2 or cyclin D1 in vitro and in vivo. Our findings provide new insights into the role of miR-520b in the development of HCC, and implicate the potential application of miR-520b in cancer therapy.