Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model.

The cancer microenvironment plays a central role in cancer development, growth, and homeostasis. This paradigm suggests that cancer fibroblasts support cancers, probably in response to stimuli received from the cancer cells. We aimed at investigating whether extracellular vesicles (EVs) can shuttle microRNA (miR) species between cancer-associated fibroblasts (CAFs) and cancer cells. To this end, we extracted EVs according to published protocols. EVs were studied for their miR content by quantitative reverse-transcription polymerase chain reaction. EVs were transfected with select miR species and utilized in vitro as well as in vivo in a rat model of cholangiocarcinoma (CCA). We found that miR-195 is down-regulated in CCA cells, as well as in adjoining fibroblasts. Furthermore, we report that EVs shuttle miR-195 from fibroblasts to cancer cells. Last, we show that fibroblast-derived EVs, loaded with miR-195, can be administered in a rat model of CCA, concentrate within the tumor, decrease the size of cancers, and improve survival of treated rats. CONCLUSION: EVs play a salient role in trafficking miR species between cancer cells and CAFs in human CCA. Understanding of these mechanisms may allow devising of novel therapeutics. (Hepatology 2017;65:501-514).

Successful liver-directed gene delivery by ERCP-guided hydrodynamic injection (with videos).

BACKGROUND AND AIMS: A simple, safe, targeted, and efficient in vivo DNA delivery system is necessary for clinical-grade liver-targeted gene therapy in humans. Intravascular hydrodynamic gene delivery has been investigated in large animal models, but translation to humans has been hampered by its technical challenges, invasiveness, and potential for significant cardiovascular adverse events. We posited that intrabiliary delivery of DNA plasmids via ERCP-guided hydrodynamic injection could overcome these obstacles. METHODS: Twelve pigs (40-50 kg) were divided into 3 groups (4 per group) and survived 21, 30, or 60 days. ERCP was performed by inflating a balloon catheter in the common hepatic duct and creating a closed space between it and the liver parenchyma. Last, a solution composed of plasmid/sleeping beauty (SB) mix was injected under pressure through the catheter into the closed space. Swine were killed at the 3 different time points and liver tissue harvested. Plasmid DNA expression and functional translated protein expression were assessed. RESULTS: ERCP-guided hydrodynamic delivery of naked plasmid DNA facilitated by pCytomegalovirus-Sleep Beauty (pCMV-SB) transposons was technically feasible and devoid of cardiovascular and local adverse events in all 12 pigs. Furthermore, plasmid DNA (both single and combination) was successfully transferred into swine hepatocytes in all 12 pigs. Additionally, stable integration of the DNA constructs in hepatocyte genomic DNA was reliably noted at all 3 time points. In the 4 swine that were kept alive to 60 days, successful genomic integration and subsequent protein expression was observed in the targeted liver tissue. CONCLUSIONS: ERCP-guided hydrodynamic delivery of gene therapy may usher in the next chapter in gene therapy with the potential to impact a variety of single-gene, complex genetic, and epigenetic liver diseases. It also raises the possibility that other nucleic acid therapeutics (microRNA, lncRNA, siRNA, shRNA) could similarly be delivered.

Integrator complex subunit 6 (INTS6) inhibits hepatocellular carcinoma growth by Wnt pathway and serve as a prognostic marker.

BACKGROUND: Integrator complex subunit 6 (INTS6) was found to play a tumour suppressing role in certain types of solid tumours. In this study, we wanted to determine the expression level of INTS6 in hepatocellular carcinoma (HCC) and evaluate its clinical characteristics and mechanisms in HCC patients (Lui and Lu, European Journal of Cancer, 51:S94, 2015). METHODS: First, we used a microarray analysis to explore the mRNA expression levels in HCC and paired normal liver tissues; second, we used qRT-PCR to measure the INTS6 mRNA levels in a cohort of 50 HCC tissues and adjacent normal liver tissues; third, we used Western blot analyses to detect the INTS6 protein levels in 20 paired HCC and normal liver tissues; fourth, we used immunohistochemistry to determine the INTS6 expression levels in 70 archived paraffin-embedded HCC samples. Finally, we investigated the suppressive function of INTS6 in the Wnt pathway. RESULTS: Herein, according to the microarray data analysis, the expression levels of INTS6 were dramatically down-regulated in HCC tissues vs. those in normal liver tissues (p<0.05). qRT-PCR and Western blot analyses showed that the INTS6 mRNA and protein expression was significantly down-regulated in tumour tissues compared to the adjacent normal liver tissues (p<0.05). Immunohistochemical assays revealed that decreased INTS6 expression was present in 62.9% (44/70) of HCC patients. Correlation analyses showed that INTS6 expression was significantly correlated with serum alpha-fetoprotein levels (AFP, p =0.004), pathology grade (p =0.005), and tumour recurrence (p =0.04). Kaplan-Meier analysis revealed that patients with low INTS6 expression levels had shorter overall and disease-free survival rates than patients with high INTS6 expression levels (p =0.001 and p =0.001). Multivariate regression analysis indicated that INTS6 was an independent predictor of overall survival and disease-free survival rates. Mechanistically, INTS6 increased WIF-1 expression and then inhibited the Wnt/ß-catenin signalling pathway. CONCLUSION: The results of our study show that down-regulated INTS6 expression is associated with a poorer prognosis in HCC patients. This newly identified INTS6/WIF-1 axis indicates the molecular mechanism of HCC and may represent a therapeutic target in HCC patients.

Pseudogene integrator complex subunit 6 pseudogene 1 (INTS6P1) as a novel plasma-based biomarker for hepatocellular carcinoma screening.

In this study, we aimed to determine whether the pseudogene integrator complex subunit 6 pseudogene 1 (INTS6P1) in plasma could be used as a novel approach to screen for and detect hepatocellular carcinoma (HCC). We explored the clinical role of INTS6P1: First, the expression level of INTS6P1 was measured in a cohort of 33 HCC tissue samples and adjacent normal liver tissue, next, the INTS6P1 expression was detected in the culture medium and tumor cells in a cellular experiment, and last, the diagnostic performance of INTS6P1 was examined in an independent cohort of 100 people. The expression level of INTS6P1 was remarkably downregulated in the HCC tissues compared with that in the normal liver tissues (p = 0.0066). In plasma, the INTS6P1 levels were significantly decreased in HCC patients compared with non-HCC patients (p < 0.01). Additionally, we inferred that INTS6P1 might be a prospective biomarker for screening HCC patients in which the serum-AFP levels were lower than 20 ng/ml by the area under the curve-receiver operating characteristic (AUC-ROC) analysis (p < 0.05). Pseudogene INTS6P1 could be used as a novel HCC plasma-based biomarker and might improve the accuracy of HCC screening.

Retinoic acid receptor-related receptor alpha (RORalpha) is a prognostic marker for hepatocellular carcinoma.

Retinoic acid receptor-related receptor alpha (RORalpha) has been proven to play a tumor suppressive role in certain types of solid tumors. However, the clinical characteristic of RORalpha has not been reported by far. This study investigated the expression of RORalpha in hepatocellular carcinoma (HCC) and evaluated its relationship with clinical parameters and prognosis in HCC patients. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to detect RORalpha expression levels in 20 paired HCC and corresponding adjacent non-cancerous tissues. Immunohistochemistry was performed on 100 archived paraffin-embedded HCC samples. Statistical analyses evaluated the correlations between RORalpha expression and clinicopathological features. qRT-PCR showed that RORalpha mRNA expression was significantly down-regulated in tumors compared to the adjacent non-cancerous tissues, and Western blots found that RORalpha protein expression was also reduced in tumor tissues. Immunohistochemical assays revealed that decreased RORalpha expression was present in 65 % of HCC patients. Correlation analyses showed that RORalpha expression was significantly correlated with serum alpha fetoprotein (AFP, p = 0.005), pathology grade (p < 0.001), tumor recurrence (p = 0.008), and vascular invasion (p < 0.001). Kaplan-Meier analysis revealed that patients with low RORalpha expression levels had a shorter overall and disease-free survival than patients with high expression (p < 0.001 and p = 0.002, respectively). Multivariate regression analysis indicated that RORalpha was an independent predictor for overall survival and disease-free survival. In conclusion, the results of our study showed that down-regulated RORalpha expression was associated with poorer prognosis in HCC patients. RORalpha may be a new potential prognostic marker for HCC patients.

CircRNA-Mediated Regulation of Angiogenesis: A New Chapter in Cancer Biology.

Angiogenesis is necessary for carcinoma progression and is regulated by a variety of pro- and anti-angiogenesis factors. CircRNAs are RNA molecules that do not have a 5'-cap or a 3'-polyA tail and are involved in a variety of biological functions. While circRNA-mediated regulation of tumor angiogenesis has received much attention, the detailed biological regulatory mechanism remains unclear. In this review, we investigated circRNAs in tumor angiogenesis from multiple perspectives, including its upstream and downstream factors. We believe that circRNAs have natural advantages and great potential for the diagnosis and treatment of tumors, which deserves further exploration.

Cleavage and polyadenylation-specific factor 3 induces cell cycle arrest via PI3K/Akt/GSK-3ß signaling pathways and predicts a negative prognosis in hepatocellular carcinoma.

Background: Recent studies have shown that cleavage and polyadenylation-specific factor 3 (CPSF3) is a promising antitumor therapeutic target, but its potential role in hepatocellular carcinoma (HCC) has not been reported. Materials & methods: We explored the expression pattern of CPSF3 in HCC through bioinformatics analysis, quantitative polymerase chain reaction (qPCR) and western blot. The potential role of CPSF3 as a biomarker for HCC was evaluated by Kaplan-Meier analysis. Next, changes in HCC cell lines in the CPSF3 knockdown model group and the control group were assessed by Cell Counting Kit-8, clonal formation, flow cytometry and EdU staining. Western blot detected changes in protein levels of the PI3K/Akt/GSK-3ß axis of two HCC cell lines in the knockdown group and the control group. Results: The results showed that the transcription and protein levels of CPSF3 were significantly higher in HCC tissues than in adjacent normal tissues (p < 0.05). The HCC cohort with increased expression of CPSF3 is associated with advanced stage and differentiation and predicts poorer prognosis (p < 0.05). CPSF3 knockdown significantly inhibited proliferation and clone formation of HepG2 and SMMC-7721 cell lines. Flow cytometry analysis showed G1-S cell cycle arrest in the CPSF3 knockdown group, and the results of EdU staining were consistent with this. Compared with the control group, p-Akt and cyclin D1 were decreased, and GSK-3ß was increased in the knockdown group. Conclusion: CPSF3 may be a potential diagnostic biomarker and candidate therapeutic target for HCC.

The Outcome of Primary Hepatic Carcinoid Tumor: A Retrospective Study Based on Propensity Score Matched Survival Analysis.

BACKGROUND: Primary hepatic carcinoid tumor (PHCT) is rare and has unclear clinical characteristics and prognosis. METHODS: A retrospective study using data from the SEER database for patients diagnosed with PHCT used univariate and multivariate Cox models to screen for independent prognostic factors. The outcomes of patients in the surgical and nonsurgical groups were compared, and Propensity Score Matching (PSM) analysis was used to reduce confounder bias. RESULTS: A total of 186 PHCT patients were identified and the median survival was 65 (95% CI [43.287, 86.713]) months. Tumor size(HR = 2.493, 95% CI[1.222,5.083], p = 0.012), male(HR = 1.690, 95% CI[1.144,2.497], p = 0.008), age(HR = 2.583, 95% CI[1.697,3.930], p < 0.001), SEER stage(HR = 1.555, 95% CI[1.184,2.044], p = 0.002) and surgery(HR = 0.292, 95% CI[0.135,0.634], p = 0.002) were significantly correlated with patient prognosis. In multivariate analysis, sex(HR = 3.206, 95% CI[1.311,7.834], p = 0.011) and surgery(HR = 0.204, 95% CI[0.043,0.966], p = 0.0045) were independent predictors of patient prognosis. Females are potentially susceptible to PHCT but have a better prognosis. With consistent baseline data, surgical patients have a better prognosis. CONCLUSIONS: PHCT is uncommon and survival time is longer than that of other primary liver cancers. We found that none-surgery was potentially independent risk factors for poor prognosis.

Activation of the HGF/c-MET axis promotes lenvatinib resistance in hepatocellular carcinoma cells with high c-MET expression.

Lenvatinib is a long-awaited alternative to sorafenib for the first-line targeted therapy of patients with advanced hepatocellular carcinoma (HCC). However, resistance to lenvatinib has also become a major obstacle to improving the prognosis of HCC patients. The underlying molecular mechanisms contributing to lenvatinib resistance in HCC are largely unknown. HGF/c-MET axis activation is related to tumor progression and several hallmarks of cancer and is considered as the key contributor to drug resistance. In the present study, we focused on the role of the HGF/c-MET axis in mediating lenvatinib resistance in HCC cells. We showed that HGF reduced the antiproliferative, proapoptotic, and anti-invasive effects of lenvatinib on HCC cells with high c-MET expression but did not significantly affect HCC cells with low c-MET expression. The c-MET inhibitor PHA-665752 rescued HCC cells from HGF-induced lenvatinib resistance. Furthermore, HGF/c-MET activated the downstream PI3K/AKT and MAPK/ERK pathways and promoted epithelial-mesenchymal transition (EMT) in HCC cells. Collectively, our results suggested that combining lenvatinib treatment with a c-MET inhibitor may improve its systemic therapeutic efficacy in HCC patients with high c-MET expression.

Comprehensive analysis of the expression of chaperonin containing TCP1 subunits (CCTs) and their influence on prognosis in hepatocellular carcinoma.

BACKGROUND: Chaperonin containing TCP1 subunits (CCTs) are important components in the folding of newly synthesized proteins and are involved in cell growth, proliferation, and apoptosis in eukaryotes. Accumulating evidence indicates that dysregulation of CCTs is involved in tumorigenesis. However, the roles of distinct CCTs in the occurrence and development of hepatocellular carcinoma (HCC) are largely unknown. To address this issue, the mRNA expression and the prognostic value of different CCTs in HCC patients were analyzed. METHODS: The mRNA expression levels of CCTs in tumors and the relationship between clinical parameters and CCTs in patients with HCC were analyzed by using ONCOMINE and Gene Expression Profiling Interactive Analysis (GEPIA) databases. The prognostic values of CCTs in HCC patients were determined by using the Kaplan-Meier plotter. The genetic alteration, coexpression, and interaction of CCTs and their frequently altered neighboring genes in HCC patients were analyzed by c-BioPortal. Gene functional enrichment and signaling pathways affected by CCTs in patients with HCC were investigated by using R software. RESULTS: The mRNA expression levels of CCTs were significantly upregulated in HCC tissues. Upregulated expression of CCTs was found to be significantly associated with alpha-fetoprotein (AFP), pathological grade, and macro- and microvascular invasion, but there was no correlation with the Child-Pugh classification. Moreover, survival analysis showed that the upregulated expression of CCTs correlated with shorter overall survival (OS) and disease-free survival (DFS) in patients with HCC. The observed genetic alteration rate of CCTs was as high as 51.39% in HCC and was associated with a poorer prognosis in HCC patients. Pathway analysis confirmed that the expression levels of PI3K/AKT pathway genes were affected by CCT genetic alterations. CONCLUSIONS: Our results suggest that CCTs could be promising prognostic biomarkers and potential therapeutic targets for HCC patients. However, further studies are required to validate our findings and promote the clinical utility of CCTs in HCC patients.

Construction of a potential microRNA, transcription factor and mRNA regulatory network in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the third leading cause of cancer-related death. MicroRNAs and transcription factors (TFs) cooperate to regulate the same target gene, thus affecting the progression of HCC. METHODS: Differentially expressed miRNAs and mRNAs were screened. Functional enrichment analysis of these HCC-related mRNAs was performed, and a protein-protein interaction network was constructed. TFs that regulate these miRNAs and hub genes were also screened. RESULTS: Ten differentially upregulated miRNAs and 5 differentially downregulated miRNAs were screened. Additionally, 183 downregulated mRNAs and 303 upregulated mRNAs that are potentially bound to these differentially expressed miRNAs were identified. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that the differentially expressed mRNAs were significantly enriched in pathways in cancer, the Wnt signaling pathway, and the Rap1 signaling pathway. Then, 220 TFs were identified for 5 candidate genes of the downregulated mRNAs, and 258 TFs were identified for 9 candidate genes of the upregulated mRNAs. Finally, the 9 upregulated hub genes were related to higher overall survival (OS) in the low-expression group, and 4/5 downregulated hub genes were related to higher OS in the high-expression group. CONCLUSIONS: This study constructed a potential regulatory network between candidate molecules and that need to be further verified. These regulatory relationships are expected to clarify the new molecular mechanisms of the occurrence and development of HCC.

Right Hemihepatectomy via an Anterior Approach for Hepatocellular Carcinoma in a Situs Inversus Totalis Patient.

Situs inversus totalis (SIT) is a rare congenital condition in which the usual position of the organs is reversed from left to right as a mirror image of the normal situation. Due to the abnormal transposition, this represents a technical challenge for the surgeon. In the present study, right hemihepatectomy via the anterior approach was performed for a 68-year-old hepatocellular carcinoma (HCC) patient with SIT. SIT was diagnosed by chest X-ray and computed tomography. The tumors were located in segments VIII and VI of the liver, and there was no metastasis to the lymph nodes and distant organs. Hemihepatic vascular inflow occlusion was performed using the selective intra-Glissonian approach. The middle hepatic vein was preserved under the guidance with intraoperative ultrasonography. The present case suggests that right hemihepatectomy via the anterior approach may be a safe, feasible, and effective procedure for HCC patients with SIT.

Zygomatic bone metastasis from hepatocellular carcinoma and the therapeutic efficacy of apatinib: A case report and literature review.

RATIONALE: Hepatocellular carcinoma (HCC) metastases to the zygomatic bone are extremely uncommon, and the treatment of target drugs against such case is unknown. PATIENT CONCERNS: A 48-year-old male patient was admitted to our hospital under suspicion of an advanced liver tumor due to an increase in levels of alpha-fetoprotein (AFP) after radiofrequency ablation for independent nodule in his liver 1 month before. He had a hepatitis B virus (HBV) history for 20 years without treatment. DIAGNOSIS AND INTERVENTIONS: A diagnosis of primary HCC was made based on pathological examination following right hepatectomy. Seven months after the surgery, a mass in S8 was identified and treated by ARF. Twenty days later, a right zygomatic mass was observed and the incisional biopsy revealed metastasis from HCC. Due to side effects of chemotherapy, the metastatic zygomatic mass was treated with radioactive seed implantation. Despite these interventions, there was steady increase in AFP values as well as increase in size of the zygomatic mass. Hence, the patient was started on apatinib with a dose of 500 mg/day from 1 to 28 days per cycle for a duration of 10 months. OUTCOMES: The AFP values were significantly decreased but the size of the zygomatic mass continued to increase indicating progression of disease. But the progression-free survival was more than 10 months. The patient exhibited adverse reactions which were controllable by symptomatic treatments. As of last follow-up, the patient is unwell with pain in the face, blurred vision in the right eye, dyscrasia, and exhibited difficulty in opening his mouth. LESSONS: HCC metastases to the zygomatic bone are very aggressive with a very low incidence and immunohistochemistry is useful diagnostic indicators. Still now, there is no optimal treatment strategy for these patients. Apatinib may be a promising drug in the treatment of HCC metastases to the zygomatic bone.

Pseudogene INTS6P1 regulates its cognate gene INTS6 through competitive binding of miR-17-5p in hepatocellular carcinoma.

The complex regulation of tumor suppressive gene and its pseudogenes play key roles in the pathogenesis of hepatocellular cancer (HCC). However, the roles played by pseudogenes in the pathogenesis of HCC are still incompletely elucidated. This study identifies the putative tumor suppressor INTS6 and its pseudogene INTS6P1 in HCC through the whole genome microarray expression. Furthermore, the functional studies - include growth curves, cell death, migration assays and in vivo studies - verify the tumor suppressive roles of INTS6 and INTS6P1 in HCC. Finally, the mechanistic experiments indicate that INTS6 and INTS6P1 are reciprocally regulated through competition for oncomiR-17-5p. Taken together, these findings demonstrate INTS6P1 and INTS6 exert the tumor suppressive roles through competing for oncomiR-17-5p. Our investigation of this regulatory circuit reveals novel insights into the underlying mechanisms of hepatocarcinogenesis.