Novel circularRNA circ-0047078 promotes pancreatic ductal adenocarcinoma progression through mircoRNA miR-11181- Chemokine (C-X-C motif) Ligand 12/Melanoma Cell Adhesion Molecule/Regulator of G-protein signaling 16 pathway.

BACKGROUND: Circular RNAs (circRNAs), new members of the noncoding RNA family, have been reported to participate in various pathological conditions, especially cancer. Pancreatic ductal adenocarcinoma (PDAC), as one of the most aggressive human solid tumors, is still with a low surgical cure rate. Exploring the role of circRNAs in PDAC is meaningful, and may offer a new therapeutic approach for PDAC. METHODS AND RESULTS: Competing endogenous RNA (ceRNA) microarray revealed that circ-0047078 was highly expressed in pancreatic ductal adenocarcinoma (PDAC) tissues compared with adjacent normal tissues, and the differential expression was further confirmed by PCR in both tissues and cell lines. Cell functional assays including cell counting kit-8 (CCK-8) assay, transwell invasion assay, flow cytometry and caspase activity assay demonstrated that circ-0047078 was positively correlated with the proliferation and invasion but negatively correlated with the apoptosis of CFPAC-1 cells. Circ-0047078 knockdown led to miR-11181, CXCL12 and MCAM downregulation and RGS16 upregulation, and the effect of circ-0047078 knockdown on CFPAC-1 cell behavior change can be reversed by miR-11181 mimic. Moreover, clinicopathological analysis indicated that circ-0047078 expression level was positively correlated with lymphatic metastasis and perineural invasion. In addition, knockdown of Chemokine (C-X-C motif) Ligand 12 (CXCL12) alone decreased proliferation, invasion, but increased apoptosis of CFPAC-1 cells, and raised the activity of caspase-3, caspase-8 and caspase-9 activity. Knockdown of Melanoma Cell Adhesion Molecule (MCAM) alone decreased invasion and increased apoptosis of CFPAC-1 cells, and both caspase-3 and caspase-9 activity increased, but no obvious change observed on caspase-8, and also no significant effect on CFPAC-1 cells proliferation. Knockdown of Regulator of G-protein signaling 16 (RGS16) alone increased invasion of CFPAC-1 cells, but had no significant effect on proliferation and apoptosis, of course, no obvious change on the activity of caspase-3, caspase-8 and caspase-9 had been observed. CONCLUSIONS: In conclusion, circ-0047078 plays a role in promoting PDAC via miR-11181 and then via CXCL12, MCAM and RGS16. Circ-0047078 may serve as a promising novel therapeutic target for PDAC patients.

SMC4 enhances the chemoresistance of hepatoma cells by promoting autophagy.

Background: Drug resistance is a major contributing factor to chemotherapy failure in hepatocellular carcinoma (HCC) patients. However, the exact mechanism underlying the chemoresistance of HCC remains unknown. Methods: HepG2 cells were incubated with different concentrations of 5-fluorouracil (5-FU), and the Cell Counting Kit-8 assay was used to test the cell survival rate. The expression level of structural maintenance of chromosome 4 (SMC4) in drug-resistant cells was analyzed by real-time quantitative polymerase chain reaction (PCR) and western blotting. To assess autophagy, immunofluorescence was applied to detect the light chain 3 beta (LC3B) level in HepG2/5-FU cells. To further study the upstream regulation of miR (microRNA)-219/SMC4, a gene chip assay was performed. A luciferase reporter assay was used to determine whether long non-coding RNA-XIST (lncRNA-XIST) functions as a competitive endogenous RNA (ceRNA) for miR-219. Cellular proliferation was evaluated using MTT [3-(4,5)-dimethylthiahiazo (-z-y1)-2,5-di-phenytetrazoliumromide] and colony formation assays, wound healing and invasion assays were performed to study the invasion and migration ability of the cells, and flow cytometry assays were carried out to evaluate cell apoptosis. Results: In the present study, we established a drug-resistant hepatoma cell line named HepG2/5-FU. We confirmed that SMC4 may play an important role in hepatoma cell autophagy and could promote autophagy to increase the drug resistance of hepatoma cells. We also demonstrated that lncRNA-XIST may competitively bind to miR-219 by acting as a miRNA sponge, thereby preventing miR-219 from effectively reducing the expression of SMC4 and further affecting the autophagy and drug resistance of hepatoma cells via the adenosine 5'-monophosphate (AMP)-activated protein kinase/mechanistic target of rapamycin (AMPK/mTOR) pathway. Conclusions: Our study suggests that SMC4 may be a potential marker of a poor HCC response to chemotherapy and a novel therapeutic target for HCC chemotherapy.

CSN1 facilitates proliferation and migration of hepatocellular carcinoma cells by upregulating cyclin A2 expression.

Constitutive photomorphogenesis 9 signalosome subunit 1 (CSN1) plays an important role in the ubiquitin-proteasome pathway and regulates various cellular processes, such as the cell cycle and DNA repair. The CSN complex consists of eight subunits (CSN1 to CSN8) and regulates the tumorigenesis of a variety of tumor types. However, the exact role of CSN1 in hepatocellular carcinoma (HCC) remains unclear. The present study evaluated the expression and biological effects of CSN1 in HCC tissue samples and cell lines. CSN1 was significantly overexpressed in HCC tissue and cell lines, compared with their normal counterparts. In patients with HCC, elevated CSN1 levels correlated with tumor size, tumor metastasis and tumor stage. Loss­of­function assays indicated that CSN1 knockdown inhibited the proliferation and migration HCC cells. In addition, CSN1 promoted the expression of cyclin A2 in a ubiquitination­independent manner. Lastly, xenograft experiments indicated that CSN1 promoted HCC tumor growth in vivo. The present study suggested that CSN1 inhibition could represent a potential approach for the prevention of HCC progression and metastasis.

Tmub1 Suppresses Hepatocellular Carcinoma by Promoting the Ubiquitination of ΔNp63 Isoforms.

Transmembrane and ubiquitin-like domain-containing 1 (Tmub1) inhibits hepatocyte proliferation during liver regeneration, but its role in hepatocellular carcinoma (HCC) has yet to be revealed. In this study, we show that the levels of Tmub1 were significantly lower in HCC tissues and cells than they were in adjacent tissues and normal hepatic cells, and the low levels of Tmub1 indicated a poor prognosis in HCC patients. Xenograft growth assay revealed that Tmub1 represses HCC growth in vivo. In addition, Tmub1 formed a protein complex with apoptosis-associated protein tumor protein 63 (p63), especially with the ΔN isoforms (ΔNp63α, ß, and γ). Further loss- and gain-of-function analyses indicated that Tmub1 promotes apoptosis of Hep3B and MHCC-LM3 cells. Tmub1 decreased the protein expression of ΔNp63, and the pro-apoptotic effect of Tmub1 can be reversed by ΔNp63 isoforms (α, ß, and γ). Additionally, we report that Tmub1 promotes the ubiquitination and degradation of ΔNp63 proteins. Finally, we confirmed in HCC tissues that Tmub1 is negatively correlated with ΔNp63 and positively correlated with the level of apoptosis. Taken together, Tmub1 suppresses HCC by enhancing the ubiquitination and degradation of ΔNp63 isoforms to induce HCC cell apoptosis. These findings provide a potential strategy for the management of HCC.

Transmembrane and Ubiquitin-Like Domain Containing 1 Protein (TMUB1) Negatively Regulates Hepatocellular Carcinoma Proliferation via Regulating Signal Transducer and Activator of Transcription 1 (STAT1).

BACKGROUND Hepatocellular carcinoma (HCC) is a common malignancy, but the pathogenesis of HCC is unclear. TMUB1 has an inhibitory effect on normal hepatocytes, but its role in HCC has not been reported. MATERIAL AND METHODS We used immunohistochemistry to observe the expression of transmembrane and ubiquitin-like domain containing 1 protein (TMUB1) and signal transducer and activator of transcription 1 (STAT1) in 132 HCC tissue specimens. The expression of TMUB1, STAT1, and CCND1 in HCC cells were detected by quantitative polymerase chain reaction (qPCR) and western blotting. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used for detecting HCC cells proliferation, and Transwell assays were used for observing the invasion and migration of HCC cells. RESULTS TMUB1 was negatively correlated with HCC pathological malignancy; low expression of TMUB1 indicated poor prognosis. TMUB1 inhibited proliferation but not metastasis in HCC cells. TMUB1 expression was positively correlated with STAT1 in 132 HCC tissues, TMUB1 promoted the expression of STAT1, and suppressed the expression of CCND1 in HCC cells. CONCLUSIONS TMUB1 negatively regulates hepatocellular carcinoma proliferation via regulating STAT1.

Circ-IGF1R has pro-proliferative and anti-apoptotic effects in HCC by activating the PI3K/AKT pathway.

Circular RNAs (circRNAs), a novel class of widespread and diverse endogenous RNAs, have been identified as critical regulators of various cancers, including hepatocellular carcinoma (HCC). However, the specific roles of circRNAs in HCC are largely unknown. In this study, we identified a novel circRNA, circ-IGF1R, in HCC tumour tissues and cell lines. Circ-IGF1R levels were found to be significantly upregulated in HCC tissues compared with levels in paired peritumoural tissues. The high expression levels of circ-IGF1R in HCC were associated with tumour size. Moreover, knocking down circ-IGF1R with siRNA significantly attenuated cell proliferation and induced cell apoptosis and cell cycle arrest in vitro. Further investigation revealed that PI3K/AKT signalling pathway activation was involved in the oncogenic functions of circ-IGF1R in HCC. Our study suggests that circ-IGF1R may be a potential target for the prevention and treatment of HCC.

Tmub1 negatively regulates liver regeneration via inhibiting STAT3 phosphorylation.

Tmub1 (transmembrane and ubiquitin-like domain-containing 1) plays negative roles in rat hepatocyte proliferation, but its underlying molecular mechanisms in liver regeneration regulation have yet to be revealed. Here, we show that in vivo transfection of Tmub1 overexpression vectors impaired mouse liver regeneration after partial hepatectomy (PHx). Loss- and gain-of-function analyses in human hepatocyte Lo2 cells indicated that Tmub1 inhibits the phosphorylation of STAT3 and the activation of STAT3 signaling. Furthermore, the inhibitory effect of Tmub1 overexpression on hepatocyte proliferation can be reversed by the STAT3 activator OSM, while the promotive effect of Tmub1 knockdown can be abolished by the STAT3 inhibitor stattic. Coimmunoprecipitation assays revealed interaction between Tmub1 and STAT3. Finally, we present data from chromatin immunoprecipitation and luciferase reporter gene assays and report that STAT3 binds to and activates the promoter of Tmub1, suggesting a putative negative feedback loop between Tmub1 and STAT3 signaling. Taken together, the results of our study suggest that Tmub1 is an important negative regulator of hepatocyte proliferation in liver regeneration through STAT3 signaling. These findings provide a potential strategy for the management of liver regeneration.

TMUB1 Inhibits BRL-3A Hepatocyte Proliferation by Interfering with the Binding of CAML to Cyclophilin B through its TM1 Hydrophobic Domain.

Transmembrane and ubiquitin-like domain-containing 1 (Tmub1) encodes a protein (TMUB1) containing an ubiquitin-like domain and plays a negative regulatory role during hepatocyte proliferation, but its mechanism in this process is still unknown. Here, TMUB1 interfered with the binding of calcium-modulating cyclophilin ligand (CAML) to cyclophilin B, which may represent a key role in the negative regulatory process of TMUB1 in hepatocyte proliferation. Co-immunoprecipitation assays in rat BRL-3A cells confirmed the interaction between TMUB1 and CAML; significant regulation of the influx of Ca2+ ([Ca2+]i) and hepatocyte proliferation occurred following TMUB1 overexpression or knockout. Deletion of the TM1 hydrophobic domain of TMUB1 completely abolished this interaction and led to loss of TMUB1's regulatory effects on cytological behavior. Furthermore, overexpression of TMUB1 completely abolished the interaction between CAML and its downstream protein cyclophilin B, which can act upstream of calcineurin by increasing [Ca2+]i during cell proliferation. Taken together, our results indicate that TMUB1 regulates BRL-3A hepatocyte proliferation by interacting with CAML and further interferes with the binding of CAML to cyclophilin B to decrease cellular [Ca2+]i.

MiR-27a/b Regulates Liver Regeneration by Posttranscriptional Modification of Tmub1.

BACKGROUND: Transmembrane and ubiquitin-like domain-containing 1 protein (Tmub1) negatively regulates liver regeneration. However, whether this regulation involves posttranscriptional modification of Tmub1 expression is unknown. AIM: The aim of the study was to investigate whether microRNA (miR)-27a/b regulates posttranscriptional modification of Tmub1 and cell proliferation during liver regeneration. METHODS: Tmub1 mRNA 3'-untranslated region (UTR) sequences were analyzed using online software. A luciferase assay was used to verify the relationship between miR-27a/b and the 3'-UTR of Tmub1. Rat partial hepatectomy models were used to investigate miR-27a/b and Tmub1 levels after partial hepatectomy. MiR-27a/b expression was down- and up-regulated with mimics and inhibitors, respectively, to observe the effects of miR-27a/b on Tmub1 expression. Quantitative RT-PCR and Western blot analyses were used to measure miR-27a/b and Tmub1 expression. Hepatocyte proliferation was measured using the CCK8 method for BRL-3A liver cells and proliferating cell nuclear antigen and histone H3 phosphorylation in the regenerating liver. RESULTS: A potential binding site of miR-27a/b was found in the 3'-UTR sequence of Tmub1. Our luciferase assay confirmed that the Tmub1 mRNA 3'-UTR was the target of miR-27a/b. We observed a temporal correlation between miR-27a/b and Tmub1 expression during liver regeneration. MiR-27a/b down-regulated Tmub1 expression both in vivo and in vitro. MiR-27a/b regulated hepatocyte proliferation during liver regeneration. CONCLUSION: MiR-27a/b regulates hepatocyte proliferation by controlling posttranscriptional modification of Tmub1 during liver regeneration.

The mRNA, miRNA and lncRNA networks in hepatocellular carcinoma: An integrative transcriptomic analysis from Gene Expression Omnibus.

Research advances and analysis in the non­protein coding part of the human genome have suggested that microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are associated with tumor initiation, growth and metastasis. Accumulating studies have demonstrated that a class of miRNAs and lncRNAs are dysregulated in hepatocellular carcinoma (HCC) and closely associated with tumorigenesis, diagnosis and prognosis. In the present study, integrative analysis of published data on multi­level Gene Expression Omnibus (GEO) and a bioinformatics computational approach were used to predict regulatory mechanism networks among differentially expressed mRNAs, miRNAs, and lncRNAs. Firstly, nine microarray expression data sets of mRNAs, miRNAs, and lncRNAs associated with HCC were collected from GEO datasets. Secondly, a total of 628 mRNAs, 15 miRNAs, and 49 lncRNAs were differentially expressed in this integrative analysis. Following this, mRNA, miRNA and lncRNA regulatory or co­expression networks were constructed. From the construction of the regulatory networks, five miRNAs and ten lncRNAs were identified as key differentially expressed noncoding RNAs associated with HCC progression. Finally, the regulatory effects of ten lncRNAs and miRNAs were validated. The study provides a novel insight into the understanding of the transcriptional regulation of HCC, and differentially expressed lncRNAs targeted and regulated by miRNAs were identified and validated in HCC specimens and cell lines.

Microarray analysis reveals Tmub1 as a cell cycle-associated protein in rat hepatocytes.

Transmembrane and ubiquitin-like domain containing protein 1 (Tmub1), formerly known as hepatocyte odd protein shuttling (HOPS) has been recognized as a ubiquitously expressed shuttling protein that moves between the nucleus and cytoplasm in hepatocytes. Tmub1 is involved in liver regeneration and functions as a bridging protein in tumor cell proliferation. To investigate the transcriptional profile and potential biological processes affected by Tmub1 expression in normal rat hepatocytes, microarray and bioinformatics experiments were used to identify 127 mRNAs differentially expressed between Tmub1­overexpression, Tmub1­knockdown and normal BRL­3A cells (fold­change ≥2.5). The expression levels of 17 key node genes associated with the cell cycle were confirmed by reverse transcription-quantitative polymerase chain reaction analysis. Flow cytometry, 5­Ethynyl­20­deoxyuridine, Cell Counting Kit­8 and western blotting experiments revealed the effects on the cell cycle and the inhibition of proliferation in BRL­3A cells overexpressing Tmub1. Further co­immunoprecipitation assays demonstrated that Tmub1 interacts with cyclin A2 during the cell cycle and that the overexpression of Tmub1 may postpone cyclin A2 and cyclin B1 degradation in the M phase. The results of the present study indicated that Tmub1 functions as a cell proliferation inhibitor and cell cycle­associated protein.

Upregulated long non-coding RNA LOC90784 promotes cell proliferation and invasion and is associated with poor clinical features in HCC.

A growing amount of literature has indicated that long non-coding RNAs (lncRNAs) are important factors in hepatocellular carcinoma (HCC) progression. However, the significance of lncRNAs in the progression and prognosis of liver cancer is largely unknown. In the present study, upregulated lncRNA LOC90784 was identified through integrative analysis of GSE58043 and GSE55191. Furthermore, associations between LOC90784 expression and the clinicopathological characteristics of patients were analyzed with a validated cohort 1 and the Cancer Genome Atlas (TCGA) cohort 2. We investigated the mechanisms by which this highly expressed lncRNA promotes HCC proliferation, invasion and migration via qRT-PCR, fluorescence in situ hybridization (FISH) staining, siRNA transfection, cell proliferation assays, Transwell and colony formation assays, flow cytometry analysis and Western blot. The results showed that LOC90784 expression levels were significantly higher in HCC cell lines and tissues and mainly localized in the cytoplasm. Knockdown of lncRNA LOC90784 expression inhibited proliferation and induced apoptosis and cell cycle arrest by promoting Bax and repressing CDK4 and Cyclin D1 protein expression; it also inhibited invasion and migration by repressing MMP2 and MMP9 expression in HCC cells. LOC90784 overexpression was associated with poor clinical features in the two cohorts and poor overall survival rates in HCC patients with clear resection margins (R0) in cohort 2. These results indicated that LOC90784 upregulation may be a critical oncogene and potential new biomarker in HCC.

MiR-377 inhibits the proliferation of pancreatic cancer by targeting Pim-3.

MicroRNAs (miRNAs) play important roles in the regulation of various tumor biological processes including proliferation and apoptosis. MiR-377 has been implicated in many types of cancer, whereas its expressional feature and potential biological function in pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, we scanned the global miRNA expression profiles in PDAC from The Cancer Genome Atlas (TCGA) and found miR-377 was down-regulated significantly in PDAC. Then, its expression was measured in both pancreatic cancer tissues and cells; the data showed that miR-377 was de-regulated and inversely correlated with pathologic parameters of tumor growth or metastasis. We generated PDAC cell lines with stable overexpression or inhibition of miR-377, and our results indicated that miR-377 up-regulation significantly promoted cell viability, proliferation, and migration in PDAC cells, and also induced cell apoptosis and cell cycle arrest simultaneously. Binding-site predictions by bioinformatics showed that Pim-3 might be a potential target of miR-377. Luciferase reporter assay ulteriorly identified that miR-377 suppressed Pim-3 expression by binding the 3'-UTR. In tumor tissues, we also showed that the Pim-3 expression was inversely correlated with that of miR-377. Furthermore, stable ectopic miR-377 expression in pancreatic cancer cell lines suppressed Pim-3 expression, leading to the attenuation of Bad phosphorylation level at its Ser112 and promoting cell apoptosis. Overall, these results reveal that miR-377 may have tumor growth suppression function by down-regulating Pim-3 kinase expression to inhibit both pancreatic tumor growth and migration, and induce cell apoptosis. Hence, miR-377 may be a potential diagnostic marker and therapeutic target.