Propofol-induced miR-219-5p inhibits growth and invasion of hepatocellular carcinoma through suppression of GPC3-mediated Wnt/β-catenin signalling activation

Propofol is one of the most extensively used intravenous anaesthetic agents, which has been found to improve the surgical intervention outcome of several types of cancer, including hepatocellular carcinoma (HCC). Additionally, in vitro and in vivo experiments have also indicated that propofol affects the biological behaviour of HCC. However, the underlying mechanisms of the surgical resection of HCC with propofol have not been fully understood. In the present study, we aimed to investigate the underlying mechanism of propofol inhibition of the growth and invasion of HCC cells. Our results showed that treatment with propofol suppressed the proliferation, invasion and migration of HCC in vitro. The subcutaneous xenograft tumour and orthotopic xenograft tumour experiments in nude mice showed that propofol significantly decreased tumour volumes, growth rates and the liver orthotopic xenograft tumour in vivo. Furthermore, the underlying mechanism investigations of the suppressive effects of propofol on HCC cells revealed that propofol treatment upregulated the expression levels of the candidate tumour suppressor miR-219-5p. Silencing of propofol-induced miR-219-5p using anti-miR-219-5p abrogated the inhibitory effects on the proliferation, migration and invasion of HCC cells exerted by propofol treatment. Additionally, we demonstrated that propofol reversed the epithelial-mesenchymal transition of Huh7 and SMMC7721 cells via miR-219-5p induction. The molecular mechanism behind these findings is that propofol-induced miR-219-5p inhibits HCC cell progression by targeting glypican-3 and subsequently results in the inhibition of Wnt/β-catenin signalling. Taken together, our study provides new insights into the advantages of the surgical intervention of HCC with propofol anaesthetization.     

miR-219-5p targets TBXT and inhibits breast cancer cell EMT and cell migration and invasion

miR-219-5p has been reported to act as either a tumor suppressor or a tumor promoter in different cancers by targeting different genes. In the present study, we demonstrated that miR-219-5p negatively regulated the expression of TBXT, a known epithelial–mesenchymal transition (EMT) inducer, by directly binding to TBXT 3′-untranslated region. As a result of its inhibition on TBXT expression, miR-219-5p suppressed EMT and cell migration and invasion in breast cancer cells. The re-introduction of TBXT in miR-219-5p overexpressing cells decreased the inhibitory effects of miR-219 on EMT and cell migration and invasion. Moreover, miR-219-5p decreased breast cancer stem cell (CSC) marker genes expression and reduced the mammosphere forming capability of cells. Overall, our study highlighted that TBXT is a novel target of miR-219-5p. By suppressing TBXT, miR-219-5p plays an important role in EMT and cell migration and invasion of breast cancer cells.     

miR-214-5p targets KLF5 and suppresses proliferation of human hepatocellular carcinoma cells

MicroRNAs (miRNAs) are small endogenous conserved RNAs regulating genes expression through base pairing with the 3′-untranslated region (3′-UTR) of target messenger RNAs. MiR-214-5p is a newly identified miRNA with its biological role largely unknown. In this study, we explored miR-214-5p expression status in 78 paired tumor and nontumor tissues obtained from patients with hepatocellular carcinoma (HCC) by RT-qPCR. The effects of miR-214-5p expression on HCC cell proliferation, cell cycle progression, and cell migration were measured by CCK-8 assay, flow cytometry, and wound-healing assay. A dual-luciferase activity assay was performed to identify whether KLF5 was a target of miR-214-5p. Kaplan-Meier curve and log-rank test were used to investigate the effects of miR-214-5p and KLF5 on overall survival and disease-free survival of patients with HCC. We found miR-214-5p expression was sharply reduced in HCC tissues and cell lines compared with the normal tissues and cell lines. Functional assay revealed that miR-214-5p overexpression could downregulate cell proliferation, cell migration, and arrested cell cycle at G0/G1 phase. Further, we validated Krüppel-like factor 5 (KLF5) as a direct target of miR-214-5p, and was upregulated in HCC and inversely correlated with the expression of miR-214-5p. Moreover, we found the low expression of miR-214-5p and high expression of KLF5 were correlated with tumor size, tumor stage, and poorer 5-year overall survival and disease-free survival of patients with HCC. In conclusion, our results suggested miR-214-5p functions as a tumor suppressor through targeting KLF5 in HCC. Also, miR-214-5p and KLF5 were identified as potential prognostic markers and might be therapeutic targets in 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.     

miR-615-5p is restrictedly expressed in cirrhotic and cancerous liver tissues and its overexpression alleviates the tumorigenic effects in hepatocellular carcinoma

microRNAs aberrant behavior in heptocellular carcinoma (HCC) plays a major role in HCC pathogenesis. miR-615-5p expression has never been evaluated in HCC. We showed that miR-615-5p was preferentially expressed in HCC, cirrhotic liver tissues and HCC cell lines, but undetected in normal livers. Forced miR-615-5p expression in HCC cell lines led to significant decrease in cell growth and migration. In-silico predication revealed insulin-like growth factor-II (IGF-II) as a potential downstream target for miR-615-5p. Forcing the expression of miR-615-5p showed downregulation of IGF-II mRNA, as well as inhibition of the luciferase activity in a luciferase reporter vector harboring the IGF-II-3′UTR target sequence. miR-615-5p acts as tumor-suppressor in HCC through targeting IGF-II.     

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.

     

microRNA-485-5p inhibits the progression of hepatocellular carcinoma through blocking the WBP2/Wnt signaling pathway

microRNA-485-5p (miR-485-5p) has been shown to act as a tumor-suppressor gene in some cancers, such as ovarian epithelial tumors and oral tongue squamous cell carcinoma. However, with regard to the anti-tumor role of miR-485-5p in hepatocellular carcinoma (HCC), evidence is unexpectedly limited. In the present study, we investigated the expression and the role of miR-485-5p in the progression of HCC. Microarray analysis revealed that miR-485-5p was downregulated and WBP2 was upregulated in HCC, which was consistent with RT-qPCR and immunohistochemistry assays in the HCC tissues we collected. A negative correlation between the expression of miR-485-5p and WBP2 was also found in HCC tissues. It was predicted and confirmed that miR-485-5p could bind to WW domain binding protein 2 (WBP2) through in silico analysis of genetic sequences and an in vitro dual-luciferase reporter gene assay. Next, gain- or loss-of-function studies were applied in the HCC cell line (Huh7) to examine the effects of miR-485-5p and WBP2 on HCC cell behavior. The effects of miR-485-5p and WBP2 on the Wnt/β-catenin signaling pathway were determined by TOP/FOP flash luciferase assays. miR-485-5p was shown to downregulate WBP2 and block the Wnt/β-catenin signaling pathway. As expected, elevated miR-485-5p levels and inhibition of WBP2 protein expression exerted inhibitory effects on HCC cell proliferation, migration and invasion and, induced apoptosis. In vivo experiments were finally conducted, which confirmed that upregulation of miR-485-5p or depletion of WBP2 attenuated tumor growth. Collectively, our results suggest miR-485-5p can downregulate WBP2 to inhibit the development of HCC by the blockade of the Wnt/β-catenin signaling, providing a novel molecular target for HCC treatment.     

肝细胞癌患者血清外泌体microRNA表达鉴定

目的:探讨肝癌细胞外泌体中差异表达的microRNAs(miRNAs)在肝细胞癌(HCC)诊断中的应用价值。方法:通过高通量测序筛选肝癌细胞外泌体中差异表达的miRNAs。实时定量PCR验证差异表达分子;检测差异表达的miRNAs在健康人(Health)、慢性乙型肝炎患者(CHB)、肝硬化患者(LC)及乙型肝炎病毒阳性的肝细胞癌患者(HCC)血清外泌体中的表达。结果:高通量测序筛选到肝癌细胞外泌体中差异表达的miRNA共88种,其中58种表达上调,30种表达下调。选择其中8种差异表达的miRNAs进行q RT-PCR验证,结果显示,此8种miRNAs在细胞上清外泌体、细胞内、癌与癌旁组织中的表达趋势与测序结果一致。miR-221-3p和miR-224-5p在HCC组外泌体中的表达水平显著高于Health组、CHB组和LC组(P0.01),miR-124-3p和let-7a-5p在HCC组外泌体中的表达水平显著低于其他各组(P0.05)。四个组中,miR-21-5p、miR-191-5p、miR-34a-5p和miR-122-5p的表达水平不存在显著性差异(P0.05)。结论:血清外泌体中的miR-221-3p、miR-224-5p、miR-124-3p和let-7a-5p可能成为肝细胞癌的候选标志物。     

miR-150-5p Inhibits Hepatoma Cell Migration and Invasion by Targeting MMP14

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. Despite progress in diagnostics and treatment of HCC, its prognosis remains poor because the molecular mechanisms underlying hepatocarcinogenesis are not well understood. In the study, we focused on identifying the role of miRNAs in HCC progression. miRNA microarray was used to analyze the differentially expressed miRNAs, and the results were validated by qPCR. We found that the miR-150-5p expression is down-regulated in HCC tissues compared with pair non-tumor tissues. miR-150-5p expression is also decreased in metastatic cancer tissues compared with pair primary tissues, indicating that miR-150-5p may be involved in HCC metastasis. Functionally, miR-150-5p inhibition significantly promotes hepatoma cell migration and invasion, whereas miR-150-5p overexpression suppresses cancer cell migration and invasion in vitro. The matrix metalloproteinase 14 (MMP14) is identified as a new target gene of miR-150-5p. miR-150-5p markedly inhibits MMP14 expression in hepatoma cells, and miR-150-5p expression is negative correlation with MMP14 expression in vivo. More important, re-expression of MMP14 in hepatoma cells partially reverses the effect of miR-150-5p in inhibiting cell invasion.     

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.     

MicroRNA-219-2-3p Functions as a Tumor Suppressor in Gastric Cancer and Is Regulated by DNA Methylation

Background & Aims

Gastric cancer is the most frequent gastrointestinal tumor in adults and is the most lethal form of human cancer. Despite of the improvements in treatments, the underlying mechanism of gastric carcinogenesis is not well known. To define novel modulators that regulate susceptibility to tumorgenesis, we focused on miR-219-2-3p.

Methods

Quantitative RT-PCR was employed to investigate the level of miR-219-2-3p in gastric cancer (GC) tissues (n = 113) and their matched adjacent normal tissues (n = 113). In vitro cell proliferation, apoptosis assays, cell migration, and invasion assays were performed to elucidate biological effects of miR-219-2-3p. Since silencing of miRNA by promoter CpG island methylation may be an important mechanism in tumorgenesis, GC cells were treated with 5-aza-2′-deoxycytidine and trichostatin A, and expression changes of miR-219-2-3p were subsequently examined by quantitative RT-PCR. Finally, the methylation status of CpG island upstream of miR-219-2-3p was analyzed by methylation-specific PCR in GC tissues (n = 22).

Results

miR-219-2-3p was down-regulated in GC and cell lines. In addition, the experiments documented the lower expression of miR-219-2-3p in GC specimens with higher grade and later stage tumors. Meanwhile, miR-219-2-3p exerted antiproliferative, proapoptotic, and antimetastatic roles and reduced levels of p-ERK1/2 in GC cells. Furthermore, 5-aza-2′-deoxycytidine and trichostatin A increased the expression (∼2 fold) of miR-219-2-3p in GC cells. By methylation-specific PCR, DNA methylation in the upstream region of miR-219-2-3p was detected in both adjacent normal tissues and cancer tissues. As expected, the methylation level was considerably higher in the miR-219-2-3p down-regulated group than up-regulated group.

Conclusions

miR-219-2-3p is potentially involved in gastric cancer progression and metastasis by regulating ERK1/2-related signal pathways, which may provide a novel therapeutic strategy for treatment of gastric cancer. Methylation mechanism may be involved in modulating the expression level of miR-219-2-3p in gastric cancer.     

Knockdown of terminal differentiation induced ncRNA (TINCR) suppresses proliferation and invasion in hepatocellular carcinoma by targeting the miR-218-5p/DEAD-box helicase 5 (DDX5) axis

Terminal differentiation induced ncRNA (TINCR), a newly identified lncRNA, has been found to be associated with different human cancers including hepatocellular carcinoma (HCC). However, little is known regarding the pathological mechanisms of TINCR in HCC progression. In this study, we confirmed that TINCR expression was upregulated in HCC tumors and cell lines, and high TINCR expression was associated with larger tumor size, advanced tumor node metastasis stage, and poor prognosis. Functionally, knockdown of TINCR facilitated apoptosis and suppressed viability, colony formation and invasion in Huh7 and Hep3B cells. Mechanically, TINCR functioned as competing endogenous RNA (ceRNA) to regulate DEAD-box helicase 5 (DDX5) expression through sponging miR-218-5p. Moreover, the miR-218-5p expression was downregulated and DDX5 expression was upregulated in HCC tumors. The silencing of miR-218-5p or ectopic expression of DDX5 abated the tumor-suppressive effect of TINCR knockdown in vitro. Furthermore, si-TINCR-induced inactivation of AKT signaling was rescued by suppression of miR-218-5p or overexpression of DDX5. Also, the silencing of TINCR resulted in tumor growth inhibition in vivo. In summary, knockdown of TINCR suppressed HCC progression presumably by inactivation of AKT signaling through targeting the miR-218-5p/DDX5 axis, suggesting a novel TINCR/miR-218-5p/DDX5 pathway and therapy target for HCC.     

VCP/p97, down-regulated by microRNA-129-5p, could regulate the progression of hepatocellular carcinoma

Valosin containing protein (VCP)/p97 plays various important roles in cells. Moreover, elevated expression of VCP in hepatocellular carcinoma (HCC) is correlated with increased incidence of recurrence. But the role of VCP in HCC progression in vitro and in vivo is unclear. And there are few reports about the regulation mechanism on the expression of VCP in HCC. In this study, it was identified that the level of VCP was frequently increased in human HCC tissues. In addition, down-regulation of VCP with siRNAs could dramatically suppress the genesis and progression of tumor in vivo. It was found that miR-129-5p directly inhibited the expression of VCP in several HCC cell lines. Meanwhile, the level of VCP in HCC tissues was negatively associated with the level of miR-129-5p. Our further investigation showed that the enhanced expression of miR-129-5p also suppressed tumor growth in vivo. Moreover, it was revealed that miR-129-5p could inhibit the degradation of IκBα and increase the apoptosis and reduce the migration of HCC cells by suppressing the expression of VCP. Our results revealed that the expression of VCP was directly regulated by miR-129-5p and this regulation played an important role in the progression of HCC.     

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     

XIAP,commonly targeted by tumor suppressive miR-3607-5p and miR-3607-3p,promotes proliferation and inhibits apoptosis in hepatocellular carcinoma

MicroRNAs (miRNAs) are frequently aberrantly expressed in hepatocellular carcinoma (HCC) and are involved in its development. However, their role and mechanism in HCC are still not fully elucidated. Differential expression analysis and survival analysis were performed to identify potential miRNAs in HCC and miR-3607 was identified as a candidate therapeutic target and prognostic biomarker. RT-qPCR confirmed the low expression of mature miR-3607-3p and miR-3607-5p in HCC. Functional experiments suggested that both miR-3607-3p and miR-3607-5p significantly inhibited HCC proliferation and induced apoptosis. Next, the detailed mechanism of miR-3607-3p and miR-3607-5p in HCC was explored by combination of bioinformatic analysis and experimental validation, and uncovered that XIAP, a common target gene of miR-3607-3p and miR-3607-5p, was involoved in their tumor suppressive effects. Finally, a XIAP-associated protein-protein interaction network, consisting of 10 positively correlated genes, was established. Collectively, we for the first time suggest that miR-3607-3p and miR-3607-5p inhibit HCC by acting one common target XIAP.     

Retracted: Long noncoding LINC01551 promotes hepatocellular carcinoma cell proliferation,migration, and invasion by acting as a competing endogenous RNA of microRNA-122-5p to regulate ADAM10 expression

Hepatocellular carcinoma (HCC) is a severe disease with high mortality in the world. It has been shown that long noncoding RNA (lncRNA) might play a role in HCC. The aim of the present study was to identify the role of long intergenic noncoding RNA 01551 (LINC01551) in the HCC development and explore the underlying mechanism of LINC01551/miR-122-5p/ADAM10 axis. The differentially expressed lncRNAs associated with HCC were screened out by a microarray analysis. The expression of LINC01551, miR-122-5p, and ADAM10 was determined in HCC tissues and cells. The potential miRNA (miR-122-5p) regulated by LINC01551 was explored, and the target relationship between miR-122-5p and ADAM10 was confirmed. To evaluate the effect of LINC01551 and miR-122-5p on proliferation, migration, invasion, and apoptosis of HCC, different plasmids were delivered into MHCC97-H cells. High expression of LINC01551 and ADAM10 yet low-expression of miR-122-5p were revealed in HCC tissues and cells. Overexpression of miR-122-5p could downregulate ADAM10. Biological prediction websites and fluorescence in situ hybridization assay verified that LINC01551 was mainly expressed in the cytoplasm. Silencing LINC01551 reduced HCC cell viability, proliferation, migration, invasion, and cell cycle entry yet induce cell apoptosis. Upregulation of LINC01551 increased its ability of competitively binding to miR-122-5p, thus reducing miR-122-5p and upregulating ADAM10 expression, as well as promoting the proliferative, migrative, and invasive ability. Taken together the results, it is highly possible that LINC01551 functions as an competing endogenous RNA (ceRNA) to regulate the miRNA target ADAM10 by sponging miR-122-5p and therefore promotes the development of HCC, highlighting a promising competitive new target for the HCC treatment.     

Hepatitis B core protein promotes liver cancer metastasis through miR-382-5p/DLC-1 axis

The hepatitis B virus core protein (HBc), also named core antigen, is well-known for its key role in viral capsid formation and virus replication. Recently, studies showed that HBc has the potential to control cell biology activity by regulating host gene expression. Here, we utilized miRNA microarray to identify 24 upregulated miRNAs and 21 downregulated miRNAs in HBc-expressed HCC cells, which were involved in multiple biological processes, including cell motility. Consistently, the in vitro transwell assay and the in vivo tail-vein injection model showed HBc promotion on HCC metastasis. Further, the miRNA-target gene network analysis displayed that the deleted in liver cancer (DLC-1) gene, an important negative regulator for cell motility, was potentially targeted by several differentially expressed miRNAs in HBc-introduced cells. Introduction of miRNAs mimics or inhibitors and 3′UTR luciferase activity assay proved that miR-382-5p efficiently suppressed DLC-1 expression and its 3′-UTR luciferase reporter activity. Importantly, cotransfection of miR-382-5p mimics/inhibitors and the DLC-1 expression vector almost abrogated HBc promotion on cell motility, indicating that the miR-382-5p/DLC-1 axis is important for mediating HBc-enhanced HCC motility. Clinical HCC samples also showed a negative correlation between miR-382-5p and DLC-1 expression level. Furthermore, HBc-positive HCC tissues showed high miR-382-5p level and reduced DLC-1 expression. In conclusion, our findings revealed that HBc promoted HCC motility by regulating the miR-382-5p/DLC-1 axis, which might provide a novel target for clinical diagnosis and treatment.     

miR-219-5p inhibits tau phosphorylation by targeting TTBK1 and GSK-3β in Alzheimer's disease

As the most common neurodegenerative disease, Alzheimer's disease (AD) is characterized by memory, perception, and behavioral damage, which may ultimately lead to emotional fluctuation and even lethal delirium. Increasing studies indicate that microRNAs (miRNAs) are associated with pathological features of AD. However, the role of miR-219-5p in AD progression is still unclear. In this study, the functions of miR-219-5p were analyzed in vitro and in vivo. miR-219-5p was notably overexpressed in brain tissues of patients with AD. The overexpression of miR-219-5p activated the phosphorylation of Tau-Ser198, Tau-Ser199, Tau-Ser201, and Tau-Ser422. We further showed that miR-219-5p could mediate a decrease in the protein levels of tau-tubulin kinase 1 (TTBK1) and glycogen synthase kinase 3β (GSK-3β) by directly binding to their 3′-untranslated region, thereby promoting the phosphorylation of tau in SH-SY5Y Cells. Rescue experiments further revealed that the phosphorylation of tau-mediated by miR-219-5p was dependent on the inhibition of TTBK1 and GSK-3β. Moreover, suppressing the expression of both TTBK1 and GSK-3β using miR-219-5p remarkably rescued AD-like symptoms in amyloid precursor protein/presenilin 1 mice. Our findings indicate that the upregulation of TTBK1 and GSK-3β mediated by the loss of miR-219-5p is a possible mechanism that contributes to tau phosphorylation and AD progression.     

miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines

Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines. To determine if miR-199a-3p has a tumor suppressive role, pre-miR-199a-3p oligonucleotides were transfected into the HCC cell lines. Pre-miR-199a-3p oligonucleotide reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two cell lines (SNU449 and SNU423); the proliferation of the other 5 treated cell lines was similar to control oligonucleotide. A pre-miR-199a-3p oligonucleotide formulated with chemical modifications to enhance stability while preserving processing, reduced cell proliferation in SNU449 and SNU423 to the same extent as the commercially available pre-miR-199a-3p oligonucleotide. Furthermore, only the duplex miR-199a-3p oligonucleotide, and not the guide strand alone, was effective at reducing cell viability. Since a CD44 variant was essential for c-Met signaling [V. Orian-Rousseau, L. Chen, J.P. Sleeman, P. Herrlich, H. Ponta, CD44 is required for two consecutive steps in HGF/c-Met signaling, Genes Dev. 16 (2002) 3074-3086] and c-Met is a known miR-199a-3p target, we hypothesized that miR-199a-3p may also target CD44. Immunoblotting confirmed that only the two HCC lines that were sensitive to the effects of pre-miR-199a-3p were CD44+. Direct targeting of CD44 by miR-199a-3p was confirmed using luciferase reporter assays and immunoblotting. Transfection of miR-199a-3p into SNU449 cells reduced in vitro invasion and sensitized the cells to doxorubicin; both effects were enhanced when hyaluronic acid (HA) was added to the cell cultures. An inverse correlation between the expression of miR-199a-3p and CD44 protein was noted in primary HCC specimens. The ability of miR-199a-3p to selectively kill CD44+ HCC may be a useful targeted therapy for CD44+ HCC.