Molecular mechanism of Ganji Fang in the treatment of hepatocellular carcinoma based on network pharmacology, molecular docking and experimental verification technology.

Background: Hepatocellular carcinoma (HCC) is a malignant tumor harmful to human health. Ganji Fang (GJF) has good clinical efficacy in the treatment of HCC, but its mechanism is still unclear. Objective: The aim of this study was to investigate the mechanism of action of GJF in the treatment of HCC through network pharmacology, molecular docking and in vitro experiments. Methods: A series of network pharmacology methods were used to identify the potential targets and key pathways of GJF in the treatment of HCC. Then, molecular docking technology was used to explore the binding ability of key active ingredients and targets in GJF. Multiple external databases were used to validate the key targets. In in vitro experiments, we performed MTT assays, wound-healing assays, cell cycle assays, apoptosis assays and RT‒qPCR to verify the inhibitory effect of GJF on the Human hepatoma G2 (HepG2) cells. Result: A total of 162 bioactive components and 826 protein targets of GJF were screened, and 611 potential targets of HCC were identified. Finally, 63 possible targets of GJF acting on HCC were obtained. KEGG enrichment analyses showed that the top five pathways were the cell cycle, cellular senescence, p53 signaling pathway, PI3K/Akt signaling pathway, and progesterone-mediated oocyte maturation. Among them, we verified the PI3K/Akt signaling pathway. CCNE1, PKN1, CCND2, CDK4, EPHA2, FGFR3, CDK6, CDK2 and HSP90AAI were enriched in the PI3K/Akt pathway. The molecular docking results showed that the docking scores of eight active components of GJF with the two targets were all less than -5.0, indicating that they had certain binding activity. In vitro cell experiments showed that GJF could inhibit the proliferation and migration of HepG2 cells, block the cell cycle and induce apoptosis of HepG2 cells, which may be related to the PI3K/Akt signaling pathway. In summary, EPHA2 may be an important target of GJF in HCC, and pachymic acid may be an important critical active compound of GJF that exerts anticancer activity. Conclusion: In general, we demonstrated, for the first time, that the molecular mechanism of GJF in HCC may involve induction of G0/G1 phase cycle arrest through inhibition of the PI3K/Akt signaling pathway and promote apoptosis of hepatoma cell lines. This study provides a scientific basis for the subsequent clinical application of GJF and the in-depth study of its mechanism.

A prognostic model based on seven immune-related genes predicts the overall survival of patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a disease with a high incidence and a poor prognosis. Growing amounts of evidence have shown that the immune system plays a critical role in the biological processes of HCC such as progression, recurrence, and metastasis, and some have discussed using it as a weapon against a variety of cancers. However, the impact of immune-related genes (IRGs) on the prognosis of HCC remains unclear. METHODS: Based on The Cancer Gene Atlas (TCGA) and Immunology Database and Analysis Portal (ImmPort) datasets, we integrated the ribonucleic acid (RNA) sequencing profiles of 424 HCC patients with IRGs to calculate immune-related differentially expressed genes (DEGs). Survival analysis was used to establish a prognostic model of survival- and immune-related DEGs. Based on genomic and clinicopathological data, we constructed a nomogram to predict the prognosis of HCC patients. Gene set enrichment analysis further clarified the signalling pathways of the high-risk and low-risk groups constructed based on the IRGs in HCC. Next, we evaluated the correlation between the risk score and the infiltration of immune cells, and finally, we validated the prognostic performance of this model in the GSE14520 dataset. RESULTS: A total of 100 immune-related DEGs were significantly associated with the clinical outcomes of patients with HCC. We performed univariate and multivariate least absolute shrinkage and selection operator (Lasso) regression analyses on these genes to construct a prognostic model of seven IRGs (Fatty Acid Binding Protein 6 (FABP6), Microtubule-Associated Protein Tau (MAPT), Baculoviral IAP Repeat Containing 5 (BIRC5), Plexin-A1 (PLXNA1), Secreted Phosphoprotein 1 (SPP1), Stanniocalcin 2 (STC2) and Chondroitin Sulfate Proteoglycan 5 (CSPG5)), which showed better prognostic performance than the tumour/node/metastasis (TNM) staging system. Moreover, we constructed a regulatory network related to transcription factors (TFs) that further unravelled the regulatory mechanisms of these genes. According to the median value of the risk score, the entire TCGA cohort was divided into high-risk and low-risk groups, and the low-risk group had a better overall survival (OS) rate. To predict the OS rate of HCC, we established a gene- and clinical factor-related nomogram. The receiver operating characteristic (ROC) curve, concordance index (C-index) and calibration curve showed that this model had moderate accuracy. The correlation analysis between the risk score and the infiltration of six common types of immune cells showed that the model could reflect the state of the immune microenvironment in HCC tumours. CONCLUSION: Our IRG prognostic model was shown to have value in the monitoring, treatment, and prognostic assessment of HCC patients and could be used as a survival prediction tool in the near future.

Inhibiting CBX4 efficiently protects hepatocellular carcinoma cells against sorafenib resistance.

BACKGROUND: This study aimed to investigate the possible role of inhibiting chromobox protein homologue 4 (CBX4) to deregulate of cancer stem cells (CSCs) and to evaluate the contribution of these molecules to sorafenib resistance in advanced hepatocellular carcinoma (HCC). METHODS: HCC cell lines and a xenograft mouse model with resistance to sorafenib were employed to analyse the effects of miR424 on CSC characteristics. RNA expression was analysed by RT-PCR and next-generation sequencing in a cohort of HCC cancer patients and sorafenib-resistant (SR) cell lines, respectively, to validate the key microRNAs and targets in the network. RESULTS: MicroRNA and mRNA profiles of SR cell lines identified miR424 and its direct target CBX4 as significantly associated with stem-cell-like properties, poor survival, and clinical characteristics. Functional experiments demonstrated that miR424 suppressed CBX4 and CBX4 induced nuclear translocation of YAP1 protein but was not associated with protein production. When YAP1 and CBX4 were modulated with CA3 and UNC3866, tumorigenicity and stem-like properties were extremely inhibited, thus indicating that these compounds exerted a strong anti-tumour effect in vivo against SR HCC cells. CONCLUSIONS: Our results revealed that blocking CBX4 expression is critical in response to sorafenib resistance with advanced HCC.

A network meta-analysis: The best Yiqi Fuzheng Chinese herbal injections for use based on the NP regimen to treat NSCLC.

BACKGROUND: Chinese herbal injections (CHIs) have been proven beneficial to patients with non-small cell lung cancer (NSCLC) in combination with chemotherapy. The network meta-analysis (NMA) was designed to update and expand on previous work to better evaluate the effectiveness and safety of different Yiqi Fuzheng (YQFZ) CHIs combined with the Vinorelbine plus cisplatin (NP) regimen versus NP alone for NSCLC. METHODS: We searched multiple electronic databases and identified randomized controlled trials (RCTs) concerning different YQFZ CHIs combined with the NP regimen for treating NSCLC up to March 1st, 2019. The outcomes are the objective response rate, performance status and adverse reactions (ADRs). Two individuals accomplished the quality assessment of this NMA based on the Cochrane risk of bias tool and the methodological section of the CONSORT statement. Random effects models were generated to estimate efficacy and safety outcomes. Odds ratios and corresponding 95% confidence intervals were calculated via Stata 14 software. Furthermore, the rankings for the efficacy and safety of different YQFZ CHIs for each outcome were determined by the surface under the cumulative ranking curve (SUCRA). RESULTS: Initially, a total of 4775 citations were retrieved through comprehensive searching, and 88 eligible articles involving 6695 participants and 8 CHIs were ultimately included. The cluster analysis results of the current evidence indicated that the NP regimen combined with Delisheng, Shenfu and Shenmai injections have a higher clinical effectiveness rate and better performance status compared with the NP regimen alone. Additionally, the NP regimen combined with Shenqifuzheng, Shengmai and Shenfu injections may be considered a favorable choice for reliving ADRs among patients with NSCLC. CONCLUSIONS: The current evidence demonstrated that the combination of Shenfu injection plus NP regimen could produce better outcomes than other YQFZ CHIs groups in terms of efficacy and safety. However, meticulously designed, strictly executed, high-quality trials are still required to further assess and confirm the results due to the inadequacy of the included RCTs.

Incidence and survival trends for appendiceal mucinous adenocarcinoma: an analysis of 3237 patients in the Surveillance, Epidemiology, and End Results database.

Aim: To characterize the epidemiology of appendiceal mucinous adenocarcinoma. Methods: Prognostic factors were evaluated with univariate and multivariate analyses. The results were used to generate a nomogram. Results: The incidence of appendiceal mucinous adenocarcinoma showed a significant upward trend. Multivariate Cox analysis identified 11 independent prognostic factors. The nomogram was based on independent risk factors that were significant in multivariate Cox analysis, and the concordance-index for overall survival and cancer-specific survival were 0.76 (95% CI: 0.71-0.79) and 0.74 (95% CI: 0.70-0.79), respectively. Conclusion: Advanced age, single relationship status, male sex, black race, the presence of distant and regional lymph node metastases, poor differentiation or lack of differentiation, advanced SEER extent of disease, cancer-directed surgery and chemotherapy were independently associated with prognosis.

MicroRNA-200b is downregulated and suppresses metastasis by targeting LAMA4 in renal cell carcinoma.

BACKGROUND: Metastasis is the primary cause of tumor death in renal cell carcinoma (RCC). Improved diagnostic markers of metastasis are critically needed for RCC. MicoRNAs are demonstrated to be stable and significant biomarkers for several malignancies. In this study, we aimed to explore the metastasis related microRNAs and its mechanism in RCC. METHODS: The relationship between microRNAs expression and prognosis and metastasis of RCC patients were explored by data mining through expression profiles from The Cancer Genome Atlas (TCGA). A total of 80 RCC tissues and adjacent normal kidney tissues were obtained from Department of Urology, Peking University First Hospital. Expression of microRNA-200b (miR-200b) in RCC tissues and cell lines were determined by bioinformatic data mining and quantitative real-time PCR (qRT-PCR). The effects of miR-200b on cell proliferation, migration and invasion were determined by cell counting kit-8 and colony formation assay, wound healing assay and Boyden chamber assay. Mouse cell-derived xenograft and patient-derived xenograft model were also performed to evaluate the effects of miR-200b on tumor growth and metastasis in vivo. The molecular mechanism of miR-200b function was investigated using bioinformatic target predication and high-throughput cDNA sequencing (RNA-seq) and validated by luciferase reporter assay, qRT-PCR, Western blot and immunostaining in vitro and in vivo. FINDINGS: Our findings indicates that miR-200b is frequently downregulated and have potential utility as a biomarker of metastasis and prognosis in RCC. Interestingly, ectopic expression of miR-200b in the Caki-1 and OSRC-2 cell lines suppresses cell migration and invasion in vitro as well as tumor metastases in vivo. However, miR-200b has no effect on cell proliferation in vitro and tumor growth in vivo. In addition, bioinformatics target predication and RNA-seq results reveals that Laminin subunit alpha 4 (LAMA4) is one target of miR-200b and significantly inhibited by miR-200b in vitro and in vivo. INTERPRETATION: These results demonstrate a previously undescribed role of miR-200b as a suppressor of tumor metastasis in RCC by directly destabilizing LAMA4 mRNA.

Lysyl oxidase assists tumor­initiating cells to enhance angiogenesis in hepatocellular carcinoma.

A highly tumorigenic and malignant sub­population of HCC containing tumor­initiating cells (TICs) are defined by high self­renewal and sphere formation ability. Lysyl oxidase (LOX) regulates various factors involved in extracellular matrix (ECM) maintenance, migration and angiogenesis. Certain reports have demonstrated the role of LOX in ECM crosslinking, however, the cancer­promoting effects of LOX in HCC remain unclear, and whether LOX has a role in the regulation of angiogenesis in HCC TICs has not been elucidated. In the current study, RNA sequencing using next­generation sequencing technology and bioinformatics analyses revealed that LOX gene expression was significantly upregulated in cell spheres. Sphere cells may form tumors with more vascular enrichment compared with tumors produced from adherent cells, as observed in a mouse xenograft model. LOX expression is correlated with increased vascular endothelial growth factor (VEGF) and platelet­derived growth factor, as demonstrated by analyses of The Cancer Genome Atlas and Gene Expression Omnibus databases. Conditioned media obtained from LOX­overexpressing tumor cells stimulated angiogenesis via secreted VEGF and enhanced the tube formation capacity of endothelial cells. Furthermore, these functional behaviors were blocked by the LOX inhibitor ß­aminopropionitrile. These findings provide novel mechanistic insight into the pivotal role of LOX in the regulation of TICs in HCC. Combination of LOX inhibitor with sorafenib is a potentially advantageous strategy for HCC therapy.

Baduanjin Exercise for Type 2 Diabetes Mellitus: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

OBJECTIVE: To investigate the effects of Baduanjin exercise for type 2 diabetes mellitus. METHODS: Literature retrieval was performed in several databases, including PubMed, EMBASE, Cochrane Library, CNKI, Wanfang Data Information Site, CBM, and VIP from inception to April 2017. Randomized controlled trials on evaluating the effects of Baduanjin exercise were identified. The primary outcomes were glycosylated hemoglobin, fasting blood-glucose, and postprandial plasma glucose. Review Manager 5.2 (RevMan 5.2) and Stata V.13.0 software were conducted for data analysis. RESULTS: The results of the meta-analysis indicated that the effects of type 2 diabetes mellitus were favoring Baduanjin plus conventional therapy, when compared with the routine treatment. Baduanjin plus conventional therapy lowered the level of glycosylated hemoglobin, fasting blood-glucose, postprandial plasma glucose, TC, TG, and LDL-C and improved HDL-C. Adverse events were not mentioned in all included studies. No publication bias was detected by Begg's and Egger's test and no single study affected the overall result by influence analysis. CONCLUSIONS: Evidence from meta-analysis suggested that Baduanjin exercise plus conventional therapy has a positive effect on type 2 diabetes mellitus. However, more rigorously designed and large sample RCTs are required to confirm the efficacy and safety in further studies.

EVI5 is a novel independent prognostic predictor in hepatocellular carcinoma after radical hepatectomy.

The present study explored the correlation of ecotropic viral integration site 5 (EVI5) expression with clinicopathological features and prognosis in hepatocellular carcinoma (HCC). A total of 205 HCC patients were included retrospectively. Quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting were performed to detect the profile of EVI5 expression in HCC cell lines and fresh tissues. Archived paraffin-embedded specimens were investigated for EVI5 expression by immunohistochemistry (IHC). Both the mRNA and protein levels of EVI5 were obviously upregulated in HCC cell lines and tumor tissues. EVI5 protein level was closely associated with the clinicopathological characteristics, including liver function (P=0.013), venous invasion (P=0.015) and TNM stage (P=0.014). Furthermore, univariate analysis showed that the patients with high EVI5 expression indicated shorter overall survival (OS, P<0.001) and recurrence-free survival (RFS, P=0.001) than those with low EVI5 expression. Importantly, high EVI5 expression also exerts predictive power for higher postoperative recurrence rate by stratified analysis. Multivariate Cox regression analysis demonstrated that OS was correlated with both tumor number (P=0.046) and EVI5 expression (P<0.001) and that RFS was correlated with serum AFP (P=0.023), tumor number (P=0.036) and EVI5 expression (P<0.001). Taken together, EVI5 is an useful independent prognostic marker of survival and recurrence in hepatocellular carcinoma.

MicroRNA-143 inhibits tumorigenesis in hepatocellular carcinoma by downregulating GATA6.

MicroRNAs serve a critical role in human hepatocellular carcinoma (HCC) progression. However, the exact role of microRNA-143 (miR-143) in HCC remains unclear. The current study investigates the molecular mechanism of miR-143 in HCC. In cultured HepG2 and Bel7402 cell lines, miR-143 levels were raised by lentivirus transduction. This significantly inhibited HCC progression in terms of cell invasion and proliferation in both HepG2 and Bel7402 cell lines (P<0.05). MiR-143 also significantly decreased tumor implantation in vivo (P<0.05). Regulation of miR-143 on its direct target, GATA-binding factor 6 (GATA6), was investigated by multiple strategies, including dual-luciferase assay, quantitative polymerase chain reaction and western blot analysis. The results indicated that miR-143 was downregulated in both HCC cell lines and human tumors. GATA6 was identified as the downstream target of miR-143 in HCC, and overexpressing GATA6 was able to counter the tumor-suppressive effect of miR-143 on HCC in HepG2 and Bel7402 cells by significantly increasing proliferation and invasion rates (P<0.05). Therefore, a novel epigenetic pathway was identified in which miR-143 may suppress the malignancy of HCC by targeting GATA6.

Hoarseness due to lymph node metastasis of hepatocellular carcinoma: A case report.

Hepatocellular carcinoma (HCC) metastases in the mediastinum are rare, particularly under the arch of the aorta. The present study describes the case of a 30-year-old male patient who presented with back pain and hoarseness for 2 months due to lymph node metastasis of HCC. The patient had undergone right hepatic lobectomy for HCC 2 years prior and received transarterial chemoembolization 4 times following resection. A computed tomography scan revealed enlarged lymph nodes under the arch of the aorta that appeared to have invaded the left recurrent laryngeal nerve, causing the hoarseness. Percutaneous aspiration biopsy of the enlarged, right supraclavicular lymph node identified malignant cells consistent with HCC. Radiation administered as a therapy to treat for the metastatic lymph nodes did not diminish the tumor but relieved the symptoms.

Paradoxical role of CBX8 in proliferation and metastasis of colorectal cancer.

The effect of polycomb chromobox (Cbx) proteins in cancer is context-dependent. The Chromobox homolog 8 (CBX8) was originally characterized as a transcriptional repressor, which inhibits cell proliferation in Ink4a-Arf-dependent and -independent manner. However, the role of CBX8 in colorectal cancer remains unknown. Here, we found that high CBX8 expression was associated with a low rate of distant metastasis and good prognosis in CRC patients, even though CBX8 was up-regulated in CRC cell lines and clinical samples. Knockdown of CBX8 inhibited CRC proliferation in vitro and in vivo, mostly by increasing p53 and its downstream effectors. However, knockdown of CBX8 enhanced CRC migration, invasion and metastasis in vitro and in vivo, in part through direct up-regulation of integrin ß4 (ITGB4) that in turn decreased RhoA activity. Collectively, the knockdown of CBX8 inhibited CRC proliferation, while promoting its metastasis, thus exerting paradoxical effects in CRC progression.

Chromobox homolog 4 is correlated with prognosis and tumor cell growth in hepatocellular carcinoma.

BACKGROUND: Chromobox homolog 4 (CBX4) is a member of the chromobox family of Polycomb group proteins involved in the chromatin remodeling and transcriptional regulation. However, its clinical relevance in hepatocellular carcinoma (HCC) has not yet been explored. METHODS: Immunohistochemistry was used to analyze cytoplasmic expression of CBX4 in 246 HCC specimens. The expression of CBX4 in HCC cell lines and LO2 was detected by Western blot test. Cell cycle and MTT assays were used to determine the changes of cell growth capacity. The expression of downstream genes related to proliferation was detected by Western blot test. RESULTS: The expression of CBX4 was up-regulated in multiple HCC cell lines and clinical samples. Although the CBX4 protein was detectable in both nucleus and cytoplasm in HCC tumor tissues, the high expression of CBX4 in cytoplasm was correlated with the α-fetoprotein level in serum (P = 0.036), tumor size (P = 0.029), pathologic differentiation (P = 0.033), and tumor, node, metastasis classification system stages (P = 0.032). Moreover, HCC patients who had a high level of CBX4 in cytoplasm had a shorter overall survival (P = 0.003) and recurrence-free survival (P = 0.012). Indeed, using HCC cell line, knockdown of CBX4 led to down-regulating proliferating cell nuclear antigen and cyclin E2 as well as up-regulating p16, followed by decreased cell proliferation and impaired cell cycle progression. CONCLUSIONS: The cytoplasmic CBX4 protein may be a useful prognostic biomarker and a potential therapeutic target for HCC.

Prognostic significance of BRCA1-associated protein 1 in colorectal cancer.

Mutations of BRCA1-associated protein 1 (BAP1), a nuclear-localized deubiquitinating enzyme, had been documented in multiple human cancers. However, its role and clinical relevance in colorectal cancer is unknown. The purpose of this study was to reveal the prognostic significance of BAP1 in colorectal cancer. We performed quantitative PCR and Western blotting analyses to examine BAP1 expression in 8 cases of CRC tissues and matched adjacent non-cancerous tissues. And immunohistochemistry was used to evaluate BAP1 expression in archived 252 paraffin-embedded CRC specimens. We found that the mRNA and protein levels of BAP1 were down-regulated in 6 out of 8 cases of CRC tissues compared with their adjacent non-cancerous tissues. The BAP1 expression was closely correlated with age (p = 0.037), clinical stage (p = 0.001), T classification (p < 0.001), N classification (p < 0.001), and pathologic differentiation (p = 0.008) and histological type (p = 0.047) in CRC. The CRC patients with lower BAP1 expression survived shorter than those with higher BAP1 expression. Importantly, multivariate analysis demonstrated that BAP1 expression was an independent prognostic factor for CRC (p = 0.037). Collectively, we provide the first evidence that reduced BAP1 expression is associated with poor prognosis of CRC and BAP1 may serve as a novel prognostic biomarker for CRC.

Evaluating the effectiveness of Washington state repeated job search services on the employment rate of prime-age female welfare recipients.

This paper uses an unbalanced panel dataset to evaluate how repeated job search services (JSS) and personal characteristics affect the employment rate of the prime-age female welfare recipients in the State of Washington. We propose a transition probability model to take into account issues of sample attrition, sample refreshment and duration dependence. We also generalize Honoré and Kyriazidou's [Honoré, B.E., Kyriazidou, E., 2000. Panel data discrete choice models with lagged dependent variables. Econometrica 68 (4), 839-874] conditional maximum likelihood estimator to allow for the presence of individual-specific effects. A limited information test is suggested to test for selection issues in non-experimental data. The specification tests indicate that the (conditional on the set of the confounding variables considered) assumptions of no selection due to unobservables and/or no unobserved individual-specific effects are not violated. Our findings indicate that the first job search service does have positive and significant impacts on the employment rate. However, providing repeated JSS to the same client has no significant impact. Further, we find that there are significant experience-enhancing effects. These findings suggest that providing one job search services training to individuals may have a lasting impact on raising their employment rates.

[Clinicopathological and immunohistochemical features of pulmonary blastoma: analysis of 4 cases and review of the literature].

OBJECTIVE: To analyze the clinicopathological and immunohistochemical features of pulmonary blastoma (PB). METHODS: Four patients with PB, 1 male and 3 females, with the onset age of 2 months approximately 80 years, underwent resection of the tumor and were followed up for 32 months at most. The clinical data were analyzed retrospectively. RESULTS: Pathology showed that the 4 cases all suffered from epithelial type PB. Under microscope, epithelial cells were atypical, mostly in mitogenic phase, and were lined up as many dense tubes. All of the patients underwent surgical resection and 3 - 5 cycles of chemotherapy and one had radiotherapy following surgery. The survival time was 5 - 32 months. One of the four patients died during follow-up due to metastasis 5 months after operation. The other patients still survived. Immunohistochemistry showed that cytokeratin and thyroid transcription factor-1 were positive, and vimentin, epithelial membrane antigen, and S-100 protein were negative in the tumor tissues; and a part of tumor cell presented positive Cg-A. CONCLUSION: PB is rare and presents different clinical features. It is difficult to determine the diagnosis before operation. The modular structures and expression of neuroendocrine markers are helpful in differentiating epithelial type PB from usual adenocarcinoma in immunohistochemical staining.