MicroRNA-188-5p inhibits hepatocellular carcinoma proliferation and migration by targeting forkhead box N2.

BACKGROUND: This study aimed to identify the biological functions, expression modes, and possible mechanisms underlying the relationship between metastatic human hepatocellular carcinoma (HCC) and MicroRNA-188-5p (miR-188) dysregulation using cell lines. METHODS: A decrease in miR-188 was detected in low and high metastatic HCC cells compared to that in normal hepatic cells and non-invasive cell lines. Gain- and loss-of-function experiments were performed in vitro to investigate the role of miR-188 in cancer cell (Hep3B, HepG2, HLF, and LM3) proliferation and migration. RESULTS: miR-188 mimic transfection inhibited the proliferation of metastatic HLF and LM3 cells but not non-invasive HepG2 and Hep3B cells; nonetheless, miR-188 suppression promoted the growth of HLF and LM3 cells. miR-188 upregulation inhibited the migratory rate and invasive capacity of HLF and LM3, rather than HepG2 and Hep3B cells, whereas transfection of a miR-188 inhibitor in HLF and LM3 cells had the opposite effects. Dual-luciferase reporter assays and bioinformatics prediction confirmed that miR-188 could directly target forkhead box N2 (FOXN2) in HLF and LM3 cells. Transfection of miR-188 mimics reduced FOXN2 levels, whereas miR-188 inhibition resulted in the opposite result, in HLF and LM3 cells. Overexpression of FOXN2 in HLF and LM3 cells abrogated miR-188 mimic-induced downregulation of proliferation, migration, and invasion. In addition, we found that miR-188 upregulation impaired tumor growth in vivo. CONCLUSIONS: In summary, this study showed thatmiR-188 inhibits the proliferation and migration of metastatic HCC cells by targeting FOXN2.

Integrative analysis of DNA methylation and gene expression identify a six epigenetic driver signature for predicting prognosis in hepatocellular carcinoma.

DNA methylation is a crucial regulator of gene transcription in the etiology and pathogenesis of hepatocellular carcinoma (HCC). Thus, it is reasonable to identify DNA methylation-related prognostic markers. Currently, we aimed to make an integrative epigenetic analysis of HCC to identify the effectiveness of epigenetic drivers in predicting prognosis for HCC patients. By the software pipeline TCGA-Assembler 2, RNA-seq, and methylation data were downloaded and processed from The Cancer Genome Atlas. A bioconductor package MethylMix was utilized to incorporate gene expression and methylation data on all 363 samples and identify 589 epigenetic drivers with transcriptionally predictive. By univariate survival analysis, 72 epigenetic drivers correlated with overall survival (OS) were selected for further analysis in our training cohort. By the robust likelihood-based survival model, six epi-drivers (doublecortin domain containing 2, flavin containing monooxygenase 3, G protein-coupled receptor 171, Lck interacting transmembrane adaptor 1, S100 calcium binding protein P, small nucleolar RNA host gene 6) serving as prognostic markers was identified and then a DNA methylation signature for HCC (MSH) predicting OS was identified to stratify patients into low-risk and high-risk groups in the training cohort (p < 0.001). The capability of MSH was also assessed in the validation cohort (p = 0.002). Furthermore, a receiver operating characteristic curve confirmed MSH as an effective prognostic model for predicting OS in HCC patients in training area under curve (AUC = 0.802) and validation (AUC = 0.691) cohorts. Finally, a nomogram comprising MSH and pathologic stage was generated to predict OS in the training cohort, and it also operated effectively in the validation cohort (concordance index: 0.674). In conclusion, MSH, a six epi-drivers based signature, is a potential model to predict prognosis for HCC patients.

Activin A-Smad Signaling Mediates Connective Tissue Growth Factor Synthesis in Liver Progenitor Cells.

Liver progenitor cells (LPCs) are activated in chronic liver damage and may contribute to liver fibrosis. Our previous investigation reported that LPCs produced connective tissue growth factor (CTGF/CCN2), an inducer of liver fibrosis, yet the regulatory mechanism of the production of CTGF/CCN2 in LPCs remains elusive. In this study, we report that Activin A is an inducer of CTGF/CCN2 in LPCs. Here we show that expression of both Activin A and CTGF/CCN2 were upregulated in the cirrhotic liver, and the expression of Activin A positively correlates with that of CTGF/CCN2 in liver tissues. We go on to show that Activin A induced de novo synthesis of CTGF/CCN2 in LPC cell lines LE/6 and WB-F344. Furthermore, Activin A contributed to autonomous production of CTGF/CCN2 in liver progenitor cells (LPCs) via activation of the Smad signaling pathway. Smad2, 3 and 4 were all required for this induction. Collectively, these results provide evidence for the fibrotic role of LPCs in the liver and suggest that the Activin A-Smad-CTGF/CCN2 signaling in LPCs may be a therapeutic target of liver fibrosis.

Reduced expression of transcriptional intermediary factor 1 gamma promotes metastasis and indicates poor prognosis of hepatocellular carcinoma.

UNLABELLED: Transcriptional intermediary factor 1 gamma (TIF1γ) may play either a potential tumor-suppressor or -promoter role in cancer. Here we report on a critical role of TIF1γ in the progression of hepatocellular carcinoma (HCC). Reduced expression of TIF1γ was detected in HCC, especially in advanced HCC tissues, compared to adjacent noncancerous tissues. HCC patients with low TIF1γ expression had shorter overall survival times and higher recurrence rates than those with high TIF1γ expression. Reduced TIF1γ expression was an independent and significant risk factor for recurrence and survival after curative resection. In HCC cells, TIF1γ played a dual role: It promoted tumor growth in early-stage HCC, but not in advanced-stage HCC, whereas it inhibited invasion and metastasis in both early- and advanced-stage HCC. Mechanistically, we confirmed that TIF1γ inhibited transforming growth factor-ß/ Drosophila mothers against decapentaplegic protein (TGF-ß/Smad) signaling through monoubiquitination of Smad4 and suppressed the formation of Smad2/3/4 complex in HCC cells. TGF-ß-inducing cytostasis and metastasis were both inhibited by TIF1γ in HCC. We further proved that TIF1γ suppressed cyotstasis-related TGF-ß/Smad downstream c-myc down-regulation, as well as p21/cip1 and p15/ink4b up-regulation in early-stage HCC. Meanwhile, TGF-ß inducible epithelial-mesenchymal transition and TGF-ß/Smad downstream metastatic cascades, including phosphatase and tensin homolog deleted on chromosome ten down-regulation, chemokine (CXC motif) receptor 4 and matrix metalloproteinase 1 induction, and epidermal growth factor receptor- and protein kinase B-signaling transactivation, were inhibited by TIF1γ. In addition, we found that the down-regulation of TIF1γ in HCC was caused by hypermethylation of CpG islands in the TIF1γ promoter, and demonstrated that the combination of TIF1γ and phosphorylated Smad2 was a more powerful predictor of poor prognosis. CONCLUSION: TIF1γ regulates tumor growth and metastasis through inhibition of TGF-ß/Smad signaling and may serve as a novel prognostic biomarker in HCC.

Smad6 suppresses the growth and self-renewal of hepatic progenitor cells.

Activation of hepatic progenitor cells (HPCs) is commonly observed in chronic liver disease and Wnt/ß-catenin signaling plays a crucial role in the expansion of HPCs. However, the molecular mechanisms that regulate the activation of Wnt/ß-catenin signaling in the liver, especially in HPCs, remain largely elusive. Here, we reported that ectopic expression of Smad6 suppressed the proliferation and self-renewal of WB-F344 cells, a HPC cell line. Mechanistically, we found that Smad6 inhibited Wnt/ß-catenin signaling through promoting the interaction of C-terminal binding protein (CtBP) with ß-catenin/T-cell factor (TCF) complex to inhibit ß-catenin mediated transcriptional activation in WB-F344 cells. We used siRNA targeting ß-catenin to demonstrate that Wnt/ß-catenin signaling was required for the proliferation and self-renewal of HPCs. Taken together, these results suggest that Smad6 is a regulatory molecule which regulates the proliferation, self-renewal and Wnt/ß-catenin signaling in HPCs.

MiR-132 prohibits proliferation, invasion, migration, and metastasis in breast cancer by targeting HN1.

Accumulating evidence indicates that miRNAs play critical roles in tumorigenesis and cancer progression. This study aims to investigate the role and the underlying mechanism of miR-132 in breast cancer. Here, we report that miR-132 is significantly down-regulated in breast cancer tissues and cancer cell lines. Additional study identifies HN1 as a novel direct target of miR-132. MiR-132 down-regulates HN1 expression by binding to the 3' UTR of HN1 transcript, thereby, suppressing multiple oncogenic traits such as cancer cell proliferation, invasion, migration and metastasis in vivo and in vitro. Overexpression of HN1 restores miR-132-suppressed malignancy. Importantly, higher HN1 expression is significantly associated with worse overall survival of breast cancer patients. Taken together, our data demonstrate a critical role of miR-132 in prohibiting cell proliferation, invasion, migration and metastasis in breast cancer through direct suppression of HN1, supporting the potential utility of miR-132 as a novel therapeutic strategy against breast cancer.

Primary spindle cell sarcoma of gallbladder: An unusual case report and a literature review.

INTRODUCTION: Primary spindle cell sarcoma of the gallbladder is a rare condition. PATIENT CONCERNS: A 67-year-old woman was admitted to a local hospital with a chief complaint of abdominal pain in the right upper quadrant for the past 2 months. DIAGNOSIS AND INTERVENTION: Surgical resection was performed following the diagnosis of primary gallbladder sarcoma with local hepatic metastasis. Histological examination confirmed a diagnosis of primary spindle cell sarcoma and hepatic metastasis with simultaneous cholecystolithiasis. OUTCOMES: Adjuvant chemoradiation therapy was not performed because the patient refused treatment. Three months after the surgery, a relapsed lesion was diagnosed. The patient underwent transcatheter arterial chemoembolization. CONCLUSIONS: The disease should be differentially diagnosed from gallbladder carcinoma or carcinosarcoma with hepatic metastasis. An aggressive surgical approach should be based on a balance between the risk of surgery and the outcome.

An extremely atypical presentation of esophageal squamous cell carcinoma with pancreatic and hepatic metastases: A case report and overview of the literature.

RATIONALE: Esophageal carcinoma is an aggressive cancer with extremely poor therapeutic outcomes due to its high metastatic potential and a significant risk of recurrence after radical resection. Liver is the most common metastatic target organ of esophageal carcinoma, followed by the lungs, bones, and brain. Few cases of solitary pancreatic and hepatic metastases of esophageal carcinoma have been reported. PATIENT CONCERNS: We report the case of a 67-year-old male presenting with pancreatic and hepatic lesions. In addition, a friable lesion with an irregular nodular surface in the distal esophagus was detected by esophagogastroduodenoscopy. DIAGNOSIS: Pathohistological examination confirmed esophageal squamous cell carcinoma. The pancreatic lesion was also biopsied via ultrasound-guided fine needle aspiration, which also revealed squamous cell carcinoma. The hepatic lesion was also identified as metastatic carcinoma by magnetic resonance imaging, most likely of the same origin. INTERVENTIONS: Due to comorbidities that precluded surgery, the patient was administered adjuvant therapy and a multidisciplinary decision was made for palliative care. OUTCOMES: The patient died 1 month later due to multiorgan failure caused by hemorrhage from a peptic ulcer. CONCLUSION: To our knowledge, this is only the sixth case of pancreatic metastasis of esophageal squamous cell carcinoma. This case report suggests to clinicians the importance of considering potential comorbidities in every patient with advanced cancer, such as gastric ulcer and cachexia.

Downregulation of lncRNA SBF2-AS1 inhibits hepatocellular carcinoma proliferation and migration by regulating the miR-361-5p/TGF-ß1 signaling pathway.

SBF2-AS1 is an oncogenic long non-coding RNA (lncRNA). However, its role and mechanism in hepatocellular carcinoma (HCC) is still not completely clear. The HepG2, Hep3B, Bel-7402 and HL-7702 cell lines were used in our experiments. The CCK-8 kit and EdU staining were applied to detect cell viability and multiplication. The wound healing and Boyden chamber cell migration assays were employed to test the migration ability of cells. The levels of TGF-ß1 mRNA, lncRNA SBF2-AS1, and miR-361-5p were assessed by real-time PCR. TGF-ß1 protein levels were evaluated by western blotting. The direct interaction between miR-361-5p and TGF-ß1 was determined by luciferase reporter assays. A xenograft mouse model (XMM) was established to comprehensively study the effect and mechanisms of lncRNA SBF2-AS1. lncRNA SBF2-AS1 concentration in HCC cells exceeded that in a normal hepatocyte cell line. The downregulation of lncRNA SBF2-AS1 upregulated miR-361-5p levels in HCC cells. And, miR-361-5p negatively regulate TGF-ß1 expression in HCC cells. The suppression of miR-361-5p attenuated the influence of lncRNA SBF2-AS1 downregulation on the viability, proliferation, and migration capability of HCC cells. Further, the downregulation of lncRNA SBF2-AS1 inhibited neoplasm growth in an XMM of HCC. Simultaneously, miR-361-5p was upregulated and TGF-ß1 was downregulated after lncRNA SBF2-AS1 knocked down. In conclusion, downregulation of lncRNA SBF2-AS1 inhibits HCC proliferation and migration through the regulation of the miR-361-5p/TGF-ß1 signaling pathway.

Significant response to anti-PD-1 based immunotherapy plus lenvatinib for recurrent intrahepatic cholangiocarcinoma with bone metastasis: A case report and literature review.

INTRODUCTION: The prognosis for recurrent intrahepatic cholangiocarcinoma with bone metastasis remains dismal and its treatment poses a challenge for oncologists. To date, only 2 cases were reported in which pembrolizumab, an agent against programmed cell death protein-1 (PD-1), combined with chemotherapy led to a complete response. The safety and efficacy of nivolumab-based immunotherapy combined with lenvatinibin intrahepatic cholangiocarcinoma is unknown. PATIENT CONCERNS: A 40-year-old female was identified as having a lesion of 7.0 cm in diameter in the right lobe of the liver. In addition, calculi in the main and left hepatic bile ducts as well as the gallbladder were found. DIAGNOSIS: Based on the results of imaging studies and tumor biomarker level, the patient was initially diagnosed as having intrahepatic cholangiocellular carcinoma and cholelithiasis, after which surgery was performed. The pathological examination confirmed that the tumor was cholangiocarcinoma. Adjuvant chemotherapy was administered after surgery. However, the patient developed recurrent lesions at the 5th month after surgery, and the cholangiocarcinoma expanded to the right thoracic vertebral pedicle (T7-8) at the 6th month. INTERVENTIONS: The patient underwent percutaneous microwave ablation after recurrence in the liver was identified. After that, the patient received nivolumab plus lenvatinib. OUTCOMES: The lesions in the liver decreased in size and disappeared after treatment with nivolumab plus lenvatinib. Additionally, the metastases in the right thoracic vertebral pedicle were stable after 9 months of therapy. LESSONS: Immunotherapy has revolutionized the treatment of non-small-cell lung cancer, melanoma, and advanced renal cell carcinoma. In this case, the patient achieved an excellent radiological and symptomatic response after receiving nivolumab plus lenvatinib combination therapy. Patients suffering from cholangiocarcinoma with dMMR status and a high tumor mutation burden (TMB) may have a consistent eutherapeutic effect with anti-PD-1-directed treatment.

[Diagnostic value of DNA, RNA and proliferating cell nuclear antigen examination in malignant pleural effusions by flow cytometry].

OBJECTIVE: To study the diagnostic value of DNA, RNA and proliferating cell nuclear antigen (PCNA) examination in malignant effusion by multiparametric flow cytometry, and therefore to provide proof for clinical application. METHODS: Forty seven patients with pleural effusions in our hospital from August 2003 to February 2004 were divided into two groups: 19 suffering from benign pleural effusions and 28 from malignant effusions confirmed by pathologic examination. The cells for diagnosis were divided into four groups stained by PI (Propidium-iodide), PY (Pyronin), PCNA-FITC and PCNA-mouse-alpha-2a. The specimens were analyzed by a flow cytometer (FacS Caliber, Becton Dickinson). The sensitivity and the specificity of each examination and combined examination were calculated by statistic software SPSS 13.0. RESULTS: (1) The expression of DI, RI, and PI in benign pleural effusion was 1.03 +/- 0.06, 10.03 +/- 0.54, and (4.86 +/- 0.72)%, respectively, and those in malignant ones was 1.26 +/- 0.17, 11.65 +/- 1.45, and (11.97 +/- 1.50)%, respectively, the difference being statistically significant. The cutoff value of DI, RI and PI was 1.10%, 10.75% and 4.56%, and the sensitivity of DI examination was 89.3%, 78.6%, 75.0%, and the specificity was 89.5%, 98.5%, 84.2%, respectively. (2) In 6 cases suffering from malignant pleural effusions, RI was positive but DI was negative, indicating that DI combined with RI examination was better than DI examination alone. (3) In 5 cases suffering from malignant pleural effusions confirmed by tissue examination, the cytology was negative, but the result of DI and RI was abnormal, indicating that flow cytometry was complementary to pathologic examination. (4) The sensitivity of DI + RI, DI + PI, RI + PI and DI + RI + PI combined examination was 98.2%, 89.3%, 89.3%, 92.9%; the specificity was 84.2%, 89.5%, 84.2%, 94.2% respectively. The results demonstrated that DI + RI + PI combined examination was the best, which showed the least false negative and false positive results. The sensitivity of DI + RI combined examination was 98.2%, but the specificity was 84.2%, the false positive rate being higher than DI + RI + PI combined examination. In none of the benign pleural effusions was the DI + RI + PI higher than the cutoff value, suggesting that combined examination can exclude benign pleural effusions. CONCLUSIONS: DNA, RNA and PCNA examinations by flow cytometry are of value in the diagnosis of malignant effusion, especially for cases which can not be diagnosed by cytological examination. DI + RI + PI combined examination showed better results with the lowest false negative and false positive rates.

An integrin beta4-EGFR unit promotes hepatocellular carcinoma lung metastases by enhancing anchorage independence through activation of FAK-AKT pathway.

Anoikis, a form of programmed cell death, occurs when the cells are detached from the appropriate extracellular matrix. Anoikis resistance or anchorage independence is necessary for distant metastases of cancer. The mechanisms by which hepatocellular carcinoma (HCC) cells become resistant to anoikis are not fully understood. Integrin beta4 (ITGB4, also known as CD104) is associated with progression of many human cancers. In this study, we demonstrate that ITGB4 is over-expressed in HCC tissues and aggressive HCC cell lines. To explore the role of ITGB4 in HCC, we inhibited its expression using small interfering RNA in two HCC cell lines: HCCLM3 and HLF. We show that knockdown of ITGB4 significantly enhanced susceptibility to anoikis through inhibition of AKT/PKB signaling. Moreover, ITGB4 interacts with epidermal growth factor receptor (EGFR) in a ligand independent manner. Inactivation of EGFR inhibits the anchorage independence and AKT pathway promoted by ITGB4. Further investigation proved that the ITGB4-EGFR unit triggers the focal adhesion kinase (FAK) to activate the AKT signaling pathway. Finally, we demonstrate that over-expression of ITGB4 is positively associated with tumor growth and lung metastases of HCC in vivo. Collectively, we demonstrate for the first time that ITGB4 is overexpressed in HCC tissues and promotes metastases of HCC by conferring anchorage independence through EGFR-dependent FAK-AKT activation.

MicroRNA-630 suppresses tumor metastasis through the TGF-ß- miR-630-Slug signaling pathway and correlates inversely with poor prognosis in hepatocellular carcinoma.

The epithelial-mesenchymal transition (EMT) is the key process that drives tumor metastasis. Accumulating evidence suggests that the deregulation of some microRNAs (miRNAs), is implicated in this process. Here, we highlight the function and molecular mechanism of miR-630 and its potential clinical application in hepatocellular carcinoma (HCC). First, we identified the clinical relevance of miR-630 expression in a screen of 97 HCC patient tissues. Patients with low miR-630 expression had higher recurrence rates and shorter overall survival than those with high miR-630 expression. Functional studies demonstrated the overexpression of miR-630 in HCC cells attenuated the EMT phenotype in vitro. Conversely, inhibition of miR-630 promoted EMT in HCC cells. Mechanistically, our data revealed that miR-630 suppressed EMT by targeting Slug. Knockdown of Slug expression reversed miR-630 inhibitor-mediated EMT progression. Furthermore, we found that the TGF-ß-Erk/SP1 and JNK/c-Jun signaling pathways repressed miR-630 transcription through occupying transcription factor binding sites. Ectopic expression of miR-630 restored the TGF-ß-activated EMT process. Taken together, these findings demonstrate, in HCC cells, miR-630 exerts its tumor-suppressor functions through the TGF-ß-miR-630-Slug axis and provides a potential prognostic predictor for HCC patients.

Transforming growth factor ß induces expression of connective tissue growth factor in hepatic progenitor cells through Smad independent signaling.

Hepatic progenitor cells (HPCs) are activated in the chronic liver injury and are found to participate in the progression of liver fibrosis, while the precise role of HPCs in liver fibrosis remains largely elusive. In this study, by immunostaining of human liver sections, we confirmed that HPCs were activated in the cirrhotic liver and secreted transforming growth factor ß (TGF-ß) and connective tissue growth factor (CTGF), both of which were important inducers of liver fibrosis. Besides, we used HPC cell lines LE/6 and WB-F344 as in vitro models and found that TGF-ß induced secretion of CTGF in HPCs. Moreover, TGF-ß signaling was intracrine activated and contributed to autonomous secretion of CTGF in HPCs. Furthermore, we found that TGF-ß induced expression of CTGF was not mediated by TGF-ß activated Smad signaling but mediated by TGF-ß activated Erk, JNK and p38 MAPK signaling. Taken together, our results provide evidence for the role of HPCs in liver fibrosis and suggest that the production of CTGF by TGF-ß activated MAPK signaling in HPCs may be a therapeutic target of liver fibrosis.