Hepatocellular carcinoma-derived exosomal miRNA-761 regulates the tumor microenvironment by targeting the SOCS2/JAK2/STAT3 pathway.

BACKGROUND: Exosomes and exosomal microRNAs have been implicated in tumor occurrence and metastasis. Our previous study showed that microRNA-761 (miR-761) is overexpressed in hepatocellular carcinoma (HCC) tissues and that its inhibition affects mitochondrial function and inhibits HCC metastasis. The mechanism by which exosomal miR-761 modulates the tumor microenvironment has not been elucidated. METHODS: Exosomal miR-761 was detected in six cell lines. Cell counting kit-8 (CCK-8) and transwell migration assays were performed to determine the function of exosomal miR-761 in HCC cells. The luciferase reporter assay was used to analyze miR-761 target genes in normal fibroblasts (NFs). The inhibitors AZD1480 and C188-9 were employed to determine the role of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway in the transformation of cancer-associated fibroblasts (CAFs). RESULTS: In this study, we characterized the mechanism by which miR-761 reprogrammed the tumor microenvironment. We found that HCC-derived exosomal miR-761 was taken up by NFs. Moreover, HCC exosomes affected the tumor microenvironment by activating NFs via suppressor of cytokine signaling 2 (SOCS2) and the JAK2/STAT3 signaling pathway. CONCLUSIONS: These results demonstrated that exosomal miR-761 modulated the tumor microenvironment via SOCS2/JAK2/STAT3 pathway-dependent activation of CAFs. Our findings may inspire new strategies for HCC prevention and therapy.

Prevalence, diagnosis, and treatment of primary hepatic gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GIST), which is the most common mesenchymal tumor of the digestive tract, account for 1%-3% of gastrointestinal tumors. Primary stromal tumors outside the gastrointestinal tract are collectively referred to as extra GISTs, and stromal tumors in different regions often have different prognoses. A primary hepatic GIST is a rare tumor with an unknown origin, which may be related to interstitial Cajal-like cells. Although primary hepatic GIST has certain characteristics on imaging, it lacks specific symptoms and signs; thus, the final diagnosis depends on pathological and genetic evidence. This review summarizes all cases of primary hepatic GIST described in the literature and comprehensively analyzes the detailed clinical data of all patients. In terms of treatment, local resection alone or with adjuvant therapy was the prioritized choice to obtain better disease-free survival and longer survival time. For advanced unresectable cases, imatinib mesylate was applied as the first-line chemotherapy agent. Moreover, transcatheter arterial chemoembolization, radiofrequency ablation, and microwave ablation were shown to improve overall survival for selected patients. Liver transplantation was a final treatment option after resistance to chemotherapy developed.

Automatic Detection and Classification of Focal Liver Lesions Based on Deep Convolutional Neural Networks: A Preliminary Study.

With the increasing daily workload of physicians, computer-aided diagnosis (CAD) systems based on deep learning play an increasingly important role in pattern recognition of diagnostic medical images. In this paper, we propose a framework based on hierarchical convolutional neural networks (CNNs) for automatic detection and classification of focal liver lesions (FLLs) in multi-phasic computed tomography (CT). A total of 616 nodules, composed of three types of malignant lesions (hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and metastasis) and benign lesions (hemangioma, focal nodular hyperplasia, and cyst), were randomly divided into training and test sets at an approximate ratio of 3:1. To evaluate the performance of our model, other commonly adopted CNN models and two physicians were included for comparison. Our model achieved the best results to detect FLLs, with an average test precision of 82.8%, recall of 93.4%, and F1-score of 87.8%. Our model initially classified FLLs into malignant and benign and then classified them into more detailed classes. For the binary and six-class classification, our model achieved average accuracy results of 82.5 and73.4%, respectively, which were better than the other three classification neural networks. Interestingly, the classification performance of the model was placed between a junior physician and a senior physician. Overall, this preliminary study demonstrates that our proposed multi-modality and multi-scale CNN structure can locate and classify FLLs accurately in a limited dataset, and would help inexperienced physicians to reach a diagnosis in clinical practice.

Identification of chemoresistance-related mRNAs based on gemcitabine-resistant pancreatic cancer cell lines.

Gemcitabine (GEM) alone and GEM-based chemotherapy are the preferred regimens for treating advanced unresectable and metastatic pancreatic cancer (PC). However, these treatments have limited efficacy due to acquired resistance of cancer cells to chemotherapy, the mechanisms of which are not fully understood. In this study, we established two stable multidrug-resistant cell lines, BxPC-3-GR and CFPAC-1-GR, from their corresponding parental cells through exposure to GEM following a stepwise incremental dosing strategy. The GEM IC50 values of BxPC-3-GR and CFPAC-1-GR increased 112-fold and 210-fold, respectively, compared to parental cell lines. In vitro and in vivo experiments confirmed that both GEM-resistant cell subgroups declined in proliferative capacity, but were more resistant to GEM. Unlike CFPAC-1-GR, BxPC-3-GR exhibited enhanced migratory and invasive properties compared with BxPC-3 in vitro. We also compared differentially expressed mRNA profiles between parental and GEM-resistant cells using transcriptome sequencing. RRM1, STIM1, and TRIM21 were significantly upregulated in both GEM-resistant cell lines and confirmed to be associated with the degree of GEM resistance by quantitative reverse-transcription polymerase chain reaction and western blot analysis. These three genes were more highly expressed in PC tissues and potentially regarded as prognostic biomarkers through database mining. Thus, our findings provide chemo-resistant cell models to better understand the underlying mechanisms of chemoresistance, and to explore potential biomarkers for GEM response in PC patients.

GSK343 induces autophagy and downregulates the AKT/mTOR signaling pathway in pancreatic cancer cells.

Pancreatic cancer is a common malignancy that has a poor prognosis and limited therapeutic options. Enhancer of zeste homolog 2 (EZH2) serves a key role in the progression of different types of cancers. The effect of GSK343 (a competitive inhibitor of EZH2) on pancreatic cancer cells was assessed in the present study. Cell viability was evaluated using MTT and cell counting kit-8 assays in AsPC-1 and PANC-1 cells. Flow cytometry and an EdU assay were also performed to assess the effects of GSK343 on cell proliferation, apoptosis and the cell cycle. The induction of autophagy and associated molecular mechanisms were studied using fluorescence microscopy and western blot analysis. The results demonstrated that GSK343 inhibited cell viability in a dose- and time-dependent manner. Furthermore, GSK343 suppressed cell proliferation, promoted apoptosis and blocked cell cycle progression at the G1-phase. Furthermore, GSK343 induced autophagy in pancreatic cancer via the AKT/mTOR signaling pathway. In conclusion, GSK343 exhibited an anti-cancer effect on pancreatic cancer cells, downregulating the AKT/mTOR signaling pathway.

Excipient-free nanodispersion of 7-ethyl-10-hydroxycamptothecin exerts potent therapeutic effects against pancreatic cancer cell lines and patient-derived xenografts.

Irinotecan (CPT-11) is an anti-tumor drug and formulated as nanomedicines to reduce side effects and improve efficacy. In vivo, CPT-11 must be hydrolyzed by carboxylesterase to its active form 7-ethyl-10-hydroxycamptothecin (SN-38) to exert anti-tumor activity, but the lack of this enzyme in humans causes inefficient generation of SN-38. Thus, direct delivery of SN-38, not relying on carboxylesterase, will potentially achieve higher efficacy. However, it is difficult to effectively formulate SN-38 using current excipients due to its hydrophobicity and tendency to crystallize. Herein, we report the nanodispersion of SN-38 with its amphiphilic prodrug, CPT-11, as an effective treatment for pancreatic cancer (PC). SN-38 and CPT-11 formed stable nanoparticles without any other excipients, and showed potent cytotoxicity against PC cells in vitro, slowed tumor growth in vivo, namely subcutaneously and orthotopically xenografted mice, with minimal adverse effects, and prolonged their overall survival. Even in clinically-relevant patient-derived xenograft (PDX) models, the nanodispersion showed greater anti-tumor efficacy than CPT-11. Importantly, the nanodispersion directly released SN-38, resulting in carboxylesterase-independent anti-tumor activity, in contrast to carboxylesterase-dependent CPT-11. These characteristics may enable the excipient-free nanodispersion to exert potent therapeutic effects in patients.

RRAD suppresses the Warburg effect by downregulating ACTG1 in hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is a common malignancy with poor prognosis and limited therapeutic options. Ras-related associated with diabetes (RRAD) belongs to the subfamily of Ras-related GTPases and is associated with several types of cancer, including HCC, although the mechanisms involving RRAD in HCC remains unknown. PATIENTS AND METHODS: We aimed to elucidate the role of RRAD and whether it affects glucose metabolism in HCC by immunohistochemically examining tissue samples from HCC patients and assessing the effect of RRAD overexpression and knockdown on the glucose metabolism, proliferation, cell cycle, and apoptosis of HCC cell lines SK-Hep-1 and Huh7, as well as on tumor progression in vivo. RESULTS: We demonstrated that RRAD binds to actin gamma 1 (ACTG1). RRAD suppressed aerobic glycolysis in HCC by downregulating ACTG1. On the other hand, ACTG1 promoted HCC proliferation by regulating the cell cycle via downregulation of cyclins and cyclin-dependent kinases and inhibited apoptosis through the mitochondrial apoptosis pathway in vitro. In addition, RRAD retarded tumor growth by downregulating ACTG1 in vivo. ACTG1 was overexpressed in HCC tissues compared with adjacent normal tissues, whereas the expression of RRAD was low in tumor tissues. Low RRAD levels were significantly correlated with large tumor size and advanced tumor stage; high ACTG1 levels were significantly correlated with advanced tumor stage. Furthermore, Kaplan-Meier survival curves showed that HCC patients with high RRAD and low ACTG1 expression may have a better prognosis. CONCLUSION: We have shown that RRAD exhibits a tumor-suppressing role in HCC by downregulating glucose metabolism and ACTG1 expression, thus lowering cell proliferation, arresting the cell cycle, and increasing apoptosis. These findings indicate that ACTG1 may act as a downstream effector of RRAD and open a new avenue for potential HCC treatment.

High Expression of ITGA3 Promotes Proliferation and Cell Cycle Progression and Indicates Poor Prognosis in Intrahepatic Cholangiocarcinoma.

Integrin subunit alpha 3 (ITGA3) interacts with a beta 1 subunit to form a member of the integrin family. Integrins are heterodimeric integral membrane proteins that serve as cell surface adhesion proteins. In this research, we investigated the biological function of this protein in human intrahepatic cholangiocarcinoma (ICC) for the first time. Here, using Western blotting and immunohistochemistry assays, we discovered that ITGA3 was overexpressed in ICC cell lines and ICC patients. Moreover, we found ITGA3 expression correlated with several clinicopathological features, including tumor size, lymph node metastasis, and the TNM stage. Patients with high ITGA3 expression underwent a worse prognosis after complete resection compared with patients with low ITGA3 expression in terms of overall survival. Furthermore, we demonstrated that ITGA3 could significantly promote ICC cell proliferation and cell cycle progression in vitro. However, as a classical cell surface adhesion molecule, we found ITGA3 correlated negatively with the migration and invasion of ICC cell lines, which differs from other malignant tumors. Generally, these findings suggest that ITGA3 may play a role as a potential oncogene in ICC and suppression of ITGA3 expression may establish a novel target for guiding the therapy of ICC patients.

Long non-coding RNA CASC15 is upregulated in hepatocellular carcinoma and facilitates hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, accounting for one-sixth of all malignant tumors, and the mortality rate of HCC ranks second among all cancer-related deaths. Increasing evidence has recently shown that long non-coding RNAs (lncRNAs) play an important role in cancer occurrence and progression, including HCC. Cancer susceptibility candidate 15 (CASC15), a lncRNA, has been reported to be involved in melanoma progression and phenotype switching. However, the function of CASC15 in human HCC is still unknown. In the present study, we evaluated expression of CASC15 and its potential functions in HCC. The expression of CASC15 in HCC tissues was quantitated by the reverse-transcription quantitative polymerase chain reaction, which showed that CASC15 was overexpressed in 59% (48/82) of HCC tissues compared with corresponding adjacent normal tissues, and the CASC15 expression level was significantly correlated with metastasis (P=0.012), tumor size (P=0.037), and TNM stage (P=0.013). Kaplan-Meier survival curves showed that high CASC15 expression was associated with poor prognosis in HCC patients (P<0.05). Moreover, a knockdown model of CASC15 was established, which showed that CASC15 significantly impaired HCC cell proliferation, migration, and invasion. CASC15 knockdown also induced cell apoptosis in vitro and impaired tumor growth in vivo. In conclusion, CASC15 plays an important role in the progression of HCC, acting as an oncogene. High expression of CASC15 is correlated with a poor prognosis, suggesting that CASC15 may be a predictive biomarker of HCC.

Pseudogene PDIA3P1 promotes cell proliferation, migration and invasion, and suppresses apoptosis in hepatocellular carcinoma by regulating the p53 pathway.

Pseudogenes are a subclass of long non-coding (lnc) RNAs that arose from protein-coding genes, but have lost the ability to produce proteins. Pseudogenes play an important role in the pathogenesis of various tumors; however, the role of pseudogenes in hepatocellular carcinoma (HCC) is poorly understood. In this study, we investigated a novel pseudogene, PDIA3P1, which was upregulated in HCC tissues compared with paired normal adjacent tissues. The expression of PDIA3P1 was significantly correlated with tumor size, metastasis, TNM stage, and overall stage. Knockdown of PDIA3P1decreased proliferation, migration, and invasion of HCC cells. PDIA3P1 knockdown also promoted cell apoptosis and inhibited tumor growth in vivo. We performed a GeneChip assay to investigate the underlying mechanism of PDIA3P1 action on biological function, and our results suggested that PDIA3P1 may promote proliferation and inhibit apoptosis of liver cancer cells by inhibiting the p53 pathway. Thus, our data suggest that PDIA3P1 acts as an oncogene in HCC and could be a potential prognostic marker and therapeutic target for HCC.

Expression and Clinical Significance of the Novel Long Noncoding RNA ZNF674-AS1 in Human Hepatocellular Carcinoma.

Long noncoding RNAs (lncRNAs) play crucial roles in cancer occurrence and progression. However, the relationship between the expression levels of lncRNAs and the hepatocellular carcinoma (HCC) process is unclear. The goal of this study was to determine the expression level of ZNF674-AS1, a newly found lncRNA, in HCC and its clinical association. The expression of ZNF674-AS1 in 137 pairs of tumorous and adjacent normal tissues from patients with HCC was detected by quantitative real-time reverse transcription polymerase chain reaction. Additionally, the potential associations between its level in HCC tissue and clinicopathological features were analyzed. The expression of ZNF674-AS1 in the HCC cell lines HepG2, HCCLM3, SK-Hep1, HuH7, Hep3B, and MHCC97H was significantly downregulated compared with that in the normal liver cell line QSG-7701. The expression of ZNF674-AS1 was downregulated in 72% (99/137) of HCC tissues compared with that in paired adjacent normal tissues (p < 0.01). The results showed that the ZNF674-AS1 expression level was significantly correlated with metastasis (p = 0.041), clinical stage (p = 0.039), and histopathologic grading (p = 0.045). In addition, the Kaplan-Meier survival curves revealed that low ZNF674-AS1 expression was associated with poor prognosis in patients with HCC. Our data suggest that ZNF674-AS1 may play some role during cancer occurrence and progression and may be a new biomarker for HCC.

Ras-related associated with diabetes gene acts as a suppressor and inhibits Warburg effect in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is rapidly becoming one of the most prevalent cancers worldwide and is a prominent source of mortality. Ras-related associated with diabetes (RRAD), one of the first members of the 35-39 kDa class of novel Ras-related GTPases, is linked to several types of cancer, although its function in HCC remains unclear. In this study, we observed that RRAD was downregulated in HCC compared with adjacent normal tissues. This change was associated with a poor prognosis. Furthermore, knockdown of RRAD in SK-Hep-1 cells facilitated cell proliferation, accelerated the G1/S transition during the cell cycle, induced cell migration, and reduced apoptosis. In contrast, overexpression of RRAD in Huh7 cells had the opposite effects. Moreover, we demonstrated that RRAD induced cell proliferation through regulation of the cell cycle by downregulating cyclins and cyclin-dependent kinases. RRAD induced tumor cell apoptosis through the mitochondrial apoptosis pathway. In addition, we confirmed that knockdown of RRAD promoted aerobic glycolysis by upregulating glucose transporter 1, whereas overexpression of RRAD inhibited aerobic glycolysis. In conclusion, RRAD plays a pivotal role as a potential tumor suppressor in HCC. An improved understanding of the roles of RRAD in tumor metabolism may provide insights into its potential as a novel molecular target in HCC therapy.

MicroRNA-761 is upregulated in hepatocellular carcinoma and regulates tumorigenesis by targeting Mitofusin-2.

Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the third leading cause of cancer-related deaths worldwide. The fate of a cell is determined by the balance between the processes of fission and fusion that constantly occur in the mitochondria of cells. We previously showed that overexpression of Mitofusin-2 can induce apoptosis in HCC cells by triggering an influx of Ca(2+) into the mitochondria from the ER. The function of Mitofusin-2 has been studied extensively, but the mechanism underlying the post-transcriptional regulation of Mitofusin-2 has not been elucidated. In the present study, we aimed to identify the mechanism of Mitofusin-2 regulation in HCC. We demonstrated that Mitofusin-2 is a direct target of miR-761, which was found to be upregulated in HCC tissues. Furthermore, a miR-761 inhibitor impaired mitochondrial function by upregulating Mitofusin-2 and effectively repressed tumor growth and metastasis both in vivo and in vitro. Our findings provide new insight into the mechanism underlying Mitofusin-2 regulation and the potential role of miR-761 in HCC, making it a potential candidate for use in HCC therapy in the future.

Effects of salicylic acid on alternative pathway respiration and alternative oxidase expression in tobacco calli.

The alternative pathway (AP) respiration of plants is a cyanide-resistant and non-phosphorylating electron transport pathway in mitochondria. Alternative oxidase (AOX) is the terminal oxidase of the AP and exists in plant mitochondria as two states: the reduced, noncovalently linked state or the oxidized, covalently cross-linked state. In the present study, the effects of 20 microM exogenous salicylic acid (SA) on both AP activity and AOX expression in mitochondria of tobacco (Nicotiana rustica L. cv. yellow flower) calli were investigated. The results showed that SA treatment enhanced the AP activity. During the process of SA treatment, the AP activity increased dramatically and achieved the peak value after 8 h of treatment. Then it declined until 16 h, and maintained a steady level between 16 and 24 h. Changes in both the total AOX protein level and the reduced state were in accordance with the AP activity, but the oxidized state changed differently. The aox1 gene transcript level also showed a similar change as the AP activity and AOX protein level. The induction of AOX expression by low concentrations of SA was inferred through a reactive oxygen species (ROS)-independent pathway. These results indicate that the enhancement of AP activity in response to SA is correlated to the expression of AOX, and the reduced, non-covalently linked state of AOX plays an important role during this process.