Complete pathological response with diabetic ketoacidosis to the combination of sintilimab and anlotinib in an unresectable hepatocellular carcinoma patient: a case report.

Most hepatocellular carcinoma (HCC) patients have dismal prognoses because they are already in the advanced stage at the time of initial diagnosis and are unable to undergo upfront surgery. Recent studies of immune checkpoint inhibitors (ICIs) and antiangiogenic agents (AAAs) have shown encouraging results for unresectable HCC (uHCC). Here, we report a patient with uHCC who was treated with a combination of anlotinib and sintilimab (sintilimab 200 mg, intravenous glucose tolerance test, q21d and anlotinib 12 mg, orally, d1-14, q21d), an analog of the combination of lenvatinib and pembrolizumab with much lower cost. The patient with recurrent uHCC was downstaged to resectable disease by the combination therapy. After eight cycles of treatment with anlotinib and sintilimab, the patient underwent a second operation. The histology of the resected mass revealed a major and almost complete pathological response. However, this patient was diagnosed with type I diabetes mellitus with ketoacidosis after nearly 10 cycles of combination treatment with anlotinib and sintilimab. Active follow-ups revealed no signs of local recurrence or distant failure. In conclusion, this case report demonstrated that the combination of anlotinib and sintilimab, one of the strategies combining ICIs with AAAs, showed promising efficacy in the treatment of uHCC patients.

Tumor burden score dictates prognosis of patients with combined hepatocellular cholangiocarcinoma undergoing hepatectomy.

Background: The prognostic value of the tumor burden score (TBS) in patients with combined hepatocellular-cholangiocarcinoma (cHCC-CCA) remains unknown. This study aimed to investigate the impact of TBS on long-term outcomes after surgery. Methods: Patients who underwent radical-intent resection between June 2013 and December 2019 were retrospectively reviewed. Kaplan-Meier curves were used to analyze patient survival, and disease-free survival (DFS) and overall survival (OS) were examined in relation to TBS. Results: A total of 178 patients were included in this study, with 119 in the training cohort and 59 in the validation cohort. Kaplan-Meier curves showed that TBS was a strong prognostic indicator in patients with cHCC-CCA. Elevated TBS was associated with poorer DFS and OS (both P-value < 0.001) and was identified as an independent prognostic indicator. In addition, the prognostic value of TBS outperformed tumor size and number alone, microvascular invasion, and lymph node invasion. The prognostic significance of TBS was confirmed by the internal validation cohort. Conclusions: The present study suggested the significance of tumor morphology in assessing the prognosis of patients with cHCC-CCA who undergoing curative resection. The TBS is a promising prognostic index in patients with cHCC-CCA. Elevated TBS was related to a lower long-term survival rate and was identified as an independent risk factor for poor DFS and OS. Further research is needed to verify our results.

Water-cooled microwave ablation array for bloodless rapid transection of the liver.

BACKGROUND: Microwaves (MWs) deliver relatively high temperatures into biological tissue and cover a large ablation zone. This study aims to evaluate the efficacy and effectiveness of water-cooled double-needle MW ablation arrays in assisting the hepatic transection of an in vivo pig model. METHODS: Our research program comprised computer modeling, tissue-mimicking phantom experiments, and in vivo pig liver experiments. Computer modeling was based on the finite element method (FEM) to evaluate ablation temperature distributions. In tissue-mimicking phantom and in vivo pig liver ablation experiments, the performances of the water-cooled MW ablation array and conventional clamp crushing liver resection were compared. RESULTS: FEM showed that the maximum lateral ablation diameter at 100 W output and a duration of 60 s was 3 cm (assessed at 50 °C isotherm). In the phantom, the maximum transverse ablation diameter of the double-needle MW ablation increased rapidly to 3 cm in 60 s at 50 W. The blood loss and blood loss per transection area in Group A were significantly lower than those in Group B (18 (7-26) ml vs. 34 (19-57) ml, and 2.4 (2-3.1) ml/cm2 vs. 6.9 (3.2-8.3) ml/cm2, respectively) (p < 0.05). The transection speed in Group A (2.6(1.9-3.8) cm2/min) was significantly faster than that in Group B (1.7(1.1-2.2) cm2/min) (p < 0.05). CONCLUSION: In this experimental model, the new water-cooled MW array-assisted liver resection (LR) has the potential advantage of less blood loss and rapid removal than the conventional LR.

Computer modeling and in vitro experimental study of water-cooled microwave ablation array.

INTRODUCTION: Microwaves (MWs) quickly deliver relatively high temperatures into tumors and cover a large ablation zone. We present a research protocol for using water-cooled double-needle MW ablation arrays for tumor ablation here. MATERIAL AND METHODS: Our research program includes computer modeling, tissue-mimicking phantom experiments, and in vitro swine liver experiments. The computer modeling is based on the finite element method (FEM) to evaluate ablation temperature distributions. In tissue-mimicking phantom and in vitro swine liver ablation experiments, the performances of the new device and the single-needle MW device currently used in clinical practice are compared. RESULTS: FEM shows that the maximum transverse ablation diameter (MTAD) is 4.2 cm at 100 W output and 300 s (assessed at the 50 °C isotherm). In the tissue-mimicking phantom, the MTDA is 2.6 cm at 50 W and 300 s in single-needle MW ablation, and 4 cm in double needle MW ablation array. In in vitro swine liver experiments, the MTAD is 2.820 ± 0.127 cm at 100 W and 300 s in single-needle MW ablation, and 3.847 ± 0.103 cm in MW ablation array. CONCLUSION: A new type of water-cooled MW ablation array is designed and tested, and has potential advantages over currently used devices.

Identification of hub genes in peripheral blood mononuclear cells for the diagnosis of hepatocellular carcinoma using a weighted gene co-expression network analysis.

Human hepatocellular carcinoma (HCC) is a common malignant tumor of the digestive tract that is prevalent worldwide. Improving diagnosis methods for HCC helps to improve patient survival rate. The present study aimed to identify novel HCC biomarkers for the diagnosis of HCC through analyzing gene changes on peripheral blood mononuclear cells (PBMCs) and verifying these in additional samples. The gene expression profiles GSE49515 (including 10 specimens from normal patients and 10 specimens from patients with HCC) and GSE58208 (including 5 specimens from normal patients and 10 specimens from patients with HCC) were downloaded from the online Gene Expression Omnibus database (GEO). Differentially expressed genes (DEGs) in PBMCs between healthy controls and patients with HCC were identified using R software. A total of 935 DEGs, including 686 upregulated DEGs and 249 downregulated DEGs, were identified in the present study. In order to identify any internal associations, these DEGs were used to construct weighted gene co-expression networks (WGCNA). Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of genes in each module were conducted using the online database DAVID. Furthermore, hub genes with high module membership were identified in a co-expression network and receiver operating characteristic curves were used to verify the diagnostic values of these eight hub genes. Furthermore, the expression and diagnosis value of the eight hub genes were also verified in additional samples. The results of the present study suggested that secreted protein acidic and cysteine rich(SPARC), transmembrane protein 40 (TMEM40), solute carrier family 25 member 44, formyl peptide receptor 2 (FPR2), complement C8 ß chain, N-myristoyltransferase 1, protein kinase C δ(PRKCD) and protein phosphatase, Mg2+/Mn2+ dependent 1M(PPM1M) were hub genes. SPARC, TMEM40, FPR2, PRKCD and PPM1M had prominent diagnostic value according to the results from the GEO data and the additional samples. The present study demonstrated that these hub genes may help to improve the diagnosis of HCC.

Identification of key genes associated with the progression of intrahepatic cholangiocarcinoma using weighted gene co-expression network analysis.

The present study aimed to identify the key genes that are associated with the progression of intrahepatic cholangiocarcinoma through weighted gene co-expression network analysis (WGCNA). A total of three gene datasets were downloaded from the Gene Expression Omnibus database, including GSE107943, GSE119336 and GSE26566. Differentially expressed genes (DEGs) between intrahepatic cholangiocarcinoma tissues and adjacent liver tissues were identified using GSE107943, while tissue specific genes between bile duct and liver tissues were identified using GSE26566. Following the removal of tissue-specific genes, real DEGs were used to construct the WGCNA to investigate the association between gene modules and clinical traits. Following functional analysis, pathway enrichment analysis and the construction of a protein-protein interaction (PPI) network were performed, hub genes were selected and their diagnostic value was verified in GSE119336 using a receiver operating characteristic curve. Finally, the protein levels of the hub genes were also verified in intrahepatic cholangiocarcinoma tissues. A total of 1,643 real DEGs were identified and used to construct the WGCNA. Additionally, a total of seven co-expressed gene modules were identified following WGCNA, while genes in brown and yellow modules were identified to be associated with multiple clinical traits (the number of clinical traits >3) and used as key modules. A total of 63 core key module genes were subsequently identified, and it was indicated that these genes were most enriched in the nucleus (Gene Ontology term) and the cell cycle pathway (Kyoto Encyclopedia of Genes and Genomes term). Finally, a total of eight genes, including cyclin B1, cell division cycle 20, cell division cycle associated 8, cyclin dependent kinase 1, centrosomal protein 55, kinesin family member 2C, DNA topoisomerase IIα and TPX2 microtubule nucleation factor, exhibited the highest score in PPI analysis and had a high diagnostic value for intrahepatic cholangiocarcinoma. In addition, the protein levels of these genes were also revealed to be increased in most intrahepatic cholangiocarcinoma tissues. These eight genes may be used as novel biomarkers for the diagnosis of intrahepatic cholangiocarcinoma.

Circ_0001178 regulates miR-382/VEGFA axis to facilitate hepatocellular carcinoma progression.

Circular RNAs (circRNAs) have been reported to regulate the gene expression through sponging corresponding microRNAs in multiple malignant tumors, including hepatocellular carcinoma (HCC). Up to now, the effects of circ_0001178 in HCC are barely known. In our current work, we tested circ_0001178 expression in HCC tissues and HCC cells and found it was greatly elevated. Then, we evaluated the function of circ_0001178 on HCC cell proliferation. We found HepG2 and Huh-7 cell proliferation was repressed after circ_0001178 shRNA was infected into the cells. Moreover, flow cytometry evidenced that HepG2 and Huh-7 cell apoptosis was markedly triggered and cell cycle was arrested. Meanwhile, it was shown that HCC cell migration and invasion capacity were markedly inhibited by loss of circ_0001178. Knockdown of circ_0001178 restrained HCC tumor growth in vivo. Then, miR-382 was predicted and confirmed as the target of circ_0001178. Circ_0001178 was demonstrated to modulate miR-382 expression negatively. The effect of circ_0001178 on HCC tumor was rescued by miR-382 overexpression. Furthermore, vascular epithelial growth factor A (VEGFA) is identified in various cancers. Currently, VEGFA was proved to be the downstream target of miR-382. To conclude, this research revealed that circ_0001178 induced HCC progression via modulating miR-382 and VEGFA axis.

Leucine rich repeats and calponin homology domain containing 1 inhibits NK-92 cell cytotoxicity through attenuating Src signaling.

NK-92 cell line has been used as anti-tumor cytotoxic effector cells in immunotherapy. Leucine-rich repeats and calponin homology domain containing 1 (LRCH1) is a novel gene of which the function is unclear. In the present study, we investigated the role of LRCH1 in NK-92 cell cytotoxicity. LRCH1 was ablated in NK-92 cells through CRISP-Cas9-mediated knockout. LRCH1 knockout did not influence the basal behavior of NK-92 cells such as cell survival, expression of natural cytotoxicity receptors, and proliferation. However, upon the contact with tumor cells, LRCH1 knockout promoted NK-92 cell cytotoxicity to tumor cells. Besides, LRCH1 knockout increased the production of cytotoxic mediators such as IFN-γ, TNF-α, IL-2, and granzyme B in NK-92 cells after tumor cell contact. Similarly, LRCH1 knockout increased the production of cytokines and granzyme B upon NKp30 engagement. Further experiments revealed that LRCH1 knockout enhanced the activation of Src and Lck kinase which are important for natural killer cell cytotoxicity. The in vivo assay confirmed the up-regulation of the tumoricidal activity of LRCH1-/- NK-92 cells, as demonstrated by more robust tumor cell killing. Importantly, human primary natural killer cells exhibited a similar increase in the production of IFN-γ and TNF-α when LRCH1 was knocked out. In conclusion, our study revealed the role of LRCH1 as a negative regulator of NK-92 cell cytotoxicity.

Chimeric antigen receptor modified T cell (CAR-T) co-expressed with ICOSL-41BB promote CAR-T proliferation and tumor rejection.

T cells edited by chimeric antigen receptors (CAR) have shown great potential in the treatment of tumors, especially malignant blood tumors. However, there remain many obstacles in the CAR-T therapy against solid tumors, such as the expansion of CAR-T cells ex vivo and the exhaustion of CAR-T cells in vivo. In order to solve these problems, we described a novel CAR which is targeting GPC3 by expressing CD28 co-stimulation domain and CD3z ITAM (G328z), meanwhile co-expressing ICOSL extracellular and transmembrane region fused with 41BB cytoplasmic domain (G328z-ICOSL-41BB). Compared with G328z, G328z-ICOSL-41BB fusion protein significantly reinforced the expansion ability of CAR-T cells ex vivo, and prolonged the survival time of mice with hepatocellular carcinoma. We now demonstrate that the enhancement of CAR-T cell activity is dependent on the enhanced PI3K signaling pathway and up-regulated expression of Bcl2 to inhibit apoptosis and promote proliferation of CAR-T cells. Besides, the CAR with ICOSL-41BB fusion protein have been strengthened significantly in comparison with fusing ICOSL protein only, which might be caused by the fact that ICOSL-41BB not only supplies ICOS signal for other cells, but also provides 41BB signal for itself. Consequently, CARs with ICOSL-41BB fusion protein could increase the therapeutic efficacy against solid tumors in vivo compared with the G328z CAR, which might further assist the development of potent and durable T cell therapeutics.

ODC1 promotes proliferation and mobility via the AKT/GSK3ß/ß-catenin pathway and modulation of acidotic microenvironment in human hepatocellular carcinoma.

Purpose: Ornithine decarboxylase 1 (ODC1)-an oncogene involved in the biosynthesis of polyamines-is commonly upregulated and associated with poor prognosis in numerous cancers. However, the role and mechanism of ODC1 in hepatocellular carcinoma (HCC) remains unclear. The aim of the present study was to investigate the role of ODC1 in HCC and clarify the latent molecular mechanisms. Material and methods: We used samples obtained from The Cancer Genome Atlas. The expression of ODC1 was also assessed in our additional HCC samples and HCC cell lines. The roles of ODC1 in HCC cell proliferation, migration and invasion in vitro were investigated using the cell-counting kit-8 assay, 5-ethynyl-2´-deoxyuridine assay, colony formation assay, flow cytometry, wound healing assay and transwell assay, respectively. The effect of ODC1 on HCC cell proliferation in vivo was investigated by constructing a xenotransplanted tumor model in nude mice. Quantitative real-time polymerase chain and western blotting were used to detect the expression levels of ODC1 in mimetic hypoxia, nutrient depleted, and acidotic microenvironment. The relationships between ODC1, the AKT/GSK3ß/ß-catenin pathway, and acidotic microenvironment were further investigated through western blotting, immunohistochemical staining, and immunofluorescence. Results: ODC1 was upregulated in HCC tissues and cell lines, and co-expressed with KI67 and PCNA (P<0.05). A decrease in the expression of ODC1 inhibits proliferation, migration, invasion, and induces cell cycle arrest in HCC cell lines in vitro, while suppressing HCC cell proliferation in vivo (P<0.05). Furthermore, the expression of ODC1 was increased in the mimetic acidotic microenvironment, while the interference with the expression of ODC1 reversed the effect of the acidotic microenvironment through regulation of AKT/GSK3ß/ß-catenin and related downstream proteins. Conclusion: ODC1 is an unfavorable gene in HCC patients,promoting HCC cell proliferation, migration and invasion via the AKT/GSK3ß/ß-catenin pathway and modulation of the acidotic microenvironment.

Kbtbd2 inhibits the cytotoxic activity of immortalized NK cells through down-regulating mTOR signaling in a mouse hepatocellular carcinoma model.

Natural killer cell (NK cell)-based immunotherapy is a promising therapeutic strategy for hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying the regulation of NK cell function in the tumor sites are not completely elucidated. In this study, we identified the enhanced expression of kelch repeat and BTB (POZ) domain containing 2 (Kbtbd2) in intratumoral NK cells in a mouse HCC implantation model as a negative regulator of NK cells. To investigate this interaction, we used a Tet-on inducible expression system to control Kbtbd2 expression in an immortalized mouse NK cell line KIL C.2. With this approach, we found that overexpression of Kbtbd2 reduced KIL C.2 cell proliferation, decreased expression certain of Ly49 receptor family members, and substantially impaired cytotoxic activity of KIL C.2 cells in vitro. Moreover, phosphorylation of mTOR and its target 4E-binding protein 1 was reduced in Kbtbd2-expressing KIL C.2 cells, along with down-regulated phosphorylation of Erk1/2. Adoptively transferred Kbtbd2-expressing KIL C.2 cells exhibited weaker tumoricidal effect on hepatocellular carcinoma cells in the HCC implantation model, in comparison with transferred control KIL C.2 cells. Taken together, our investigation indicates that Kbtbd2 is an inhibitory molecule for the tumoricidal activity of KIL C.2 cells and perhaps intratumoral NK cells.

Microwave-assisted liver resection vs. clamp crushing liver resection in cirrhosis patients with hepatocellular carcinoma.

PURPOSE: This study aimed to evaluate the safety and effectiveness of microwave-ablation-assisted liver resection (MW-LR) and clamp crushing liver resection (CC-LR) in cirrhotic patients with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: From July 2005 to January 2015, cirrhotic HCC patients who underwent CC-LR (n = 191) or MW-LR (n = 112) were retrospectively analysed. We compared morbidity, mortality, disease-free survival (DFS) time and overall survival time between the CC-LR and MW-LR groups. RESULTS: The blood loss volume was significantly higher in the CC-LR group (mean of 752 ml) than that in the MW-LR group (mean of 253 ml, p < 0.001). The abdominal abscess rate was higher in the MW-LR group (8.9%) than that in the CC-LR group (3.1%, p = 0.029). The 30-day mortality rate (1.5% vs. 0.8%) and postoperative complication rate (32.9% vs. 25.0%) were both similar between the CC-LR and MW-LR groups. MW-LR provided a survival benefit over CC-LR at 1, 3 and 5 years in the entire population (93.5% vs. 87.0%, 77.0% vs. 62.5% and 50.0% vs. 36.5%, respectively; p = 0.003). In a subgroup analysis, MW-LR provided a survival benefit over CC-LR for Barcelona Clinic Liver Cancer stage A (BCLC-A) HCC (p = 0.026) and stage B (BCLC-B) HCC (p = 0.035) patients and provided DFS benefits for BCLC-A HCC patients (p = 0.036). CONCLUSIONS: MW-LR is a safe and feasible procedure for HCC patients with a cirrhotic liver history.

1810011o10 Rik Inhibits the Antitumor Effect of Intratumoral CD8+ T Cells through Suppression of Notch2 Pathway in a Murine Hepatocellular Carcinoma Model.

The mechanisms by which tumor-responsive CD8+ T cells are regulated are important for understanding the tumor immunity and for developing new therapeutic strategies. In current study, we identified the expression of 1810011o10 Rik, which is the homolog of human thyroid cancer 1, in intratumoral activated CD8+ T cells in a murine hepatocellular carcinoma (HCC) implantation model. To investigate the role of 1810011o10 Rik in the regulation of antitumor activity of CD8+ T cells, normal CD8+ T cells were transduced with 1810011o10 Rik-expressing lentiviruses. Although 1810011o10 Rik overexpression did not influence agonistic antibody-induced CD8+ T cell activation in vitro, it inhibited the cytotoxic efficacy of CD8+ T cells on HCC cells in vivo. 1810011o10 Rik overexpression impeded CD8+ T cell-mediated HCC cell apoptosis and favored tumor cell growth in vivo. Further investigation revealed that 1810011o10 Rik blocked the nuclear translocation of Notch2 intracellular domain, which is crucial for CD8+ T cell activity. Furthermore, a brief in vitro experiment suggested that both antigen-presenting cells and TGF-ß might be necessary for the upregulation of Rik expression in activated CD8+ T cells. In general, our study disclosed a novel mechanism underlying the negative regulation of antitumor CD8+ T cells during HCC progression.

Simultaneous hepatectomy and splenectomy versus hepatectomy alone for hepatocellular carcinoma complicated by hypersplenism: a meta-analysis.

BACKGROUND: This study systematically compared the efficacy and safety of simultaneous hepatectomy and splenectomy (HS) with hepatectomy (H) alone in patients with hepatocellular carcinoma (HCC) and hypersplenism. METHODS: The PubMed, Web of Science, Science Direct, EMBASE, and Cochrane Library databases were systematically searched by two independent researchers through to March 31, 2015 to identify relevant studies. All the extracted literature were managed by Bibliographic citation management software. Quality assessment of the included studies was performed using a modified Newcastle-Ottawa Scale judgment. The data were analyzed using RevMan5.2 software. RESULTS: Eight studies including a total of 761 patients with HCC and hypersplenism (360 in the HS group, 401 in the H group) were finally included in the analysis. Outcomes, including postoperative complications, perioperative mortality, operation time, 5-year survival rate, and need for blood transfusion did not differ significantly between the two groups. HS was associated with significantly more intraoperative bleeding (mean difference [MD] 57.15, 95% confidence interval [CI] 18.83-95.46, P=0.003), and CD4/CD8 ratio (MD 0.69, 95% CI 0.61-0.77, P<0.00001), CD4 subset, platelet count (MD 213.06, 95% CI 202.59-223.53, P<0.0001), white blood cell count (MD 4.85, 95% CI 4.58-5.13, P<0.0001), interferon-gamma levels (MD 18.52, 95% CI 13.93-23.11, P<0.00001), and interleukin-2 levels (MD 20.73, 95% CI 16.05-25.41, P<0.0001). In addition, lower CD8 subset (MD -7.85, 95% CI -9.07, -6.63, P<0.00001) and interleukin-10 levels (MD -18.56, 95% CI -22.61, -14.50, P<0.00001) were observed for HS. CONCLUSION: We identified that simultaneous HS do not increase postoperative complications, operation time, or perioperative mortality in patients with HCC and hypersplenism. Simultaneous splenectomy can increase postoperative white blood cell and platelet counts significantly, improve blood coagulation, reduce the incidence of postoperative bleeding, and enhance immunity. Therefore, HS is safe, effective, and feasible for patients with HCC and hypersplenism.

Regulation of tumorigenesis and metastasis of hepatocellular carcinoma tumor endothelial cells by microRNA-3178 and underlying mechanism.

This study explored the effects of microRNA-3178 (miR-3178) on hepatocellular carcinoma (HCC) tumor endothelial cells (TECs) and on the target mRNA. Real-time polymerase chain reaction (PCR) was performed to detect the differential expression of miR-3178 in hepatic sinusoidal endothelial cells (HSECs) and HCC TECs. Furthermore, HCC TECs were transfected with miR-3178 mimic/inhibitor or their respective negative controls. The expression of miR-3178 before and after transfection was confirmed through RT-PCR. The effects of miR-3178 on the proliferation, apoptosis, cell cycle, invasion, migration, and angiogenesis of HCC TECs were also investigated through methyl thiazol tetrazolium assay, flow cytometry, matrigel invasion assay, transwell migration assay, and tube formation assay. Early growth responsive gene 3 (EGR3), as the putative target of miR-3178, was detected through RT-PCR and Western blot. Compared with HSECs, HCC TECs had lower miR-3178 expression levels (P < 0.001). MiR-3178 mimic inhibited proliferation, arrested cell cycle in G1 phase, and increased apoptosis. The numbers of migrated and invaded cells and capillary-like structures were significantly less in the mimic group than in the other groups. MiR-3178 mimic significantly decreased the mRNA and protein expression levels of EGR3. By contrast, miR-3178 inhibitor induced opposite effects. We conclude that miR-3178 was lowly expressed in HCC TECs, and miR-3178 mimic specifically inhibited the proliferation, migration, invasion, and angiogenesis and promoted the apoptosis and G1 phase arrest of HCC TECs in vitro through the inhibition of EGR3 expression. Thus, miR-3178 might be a critical target in HCC therapy.

Effect of microRNA-1 on hepatocellular carcinoma tumor endothelial cells.

AIM: To investigate the effect of microRNA-1 (miR-1) on tumor endothelial cells (TECs) of human hepatocellular carcinoma (HCC). METHODS: MiR-1 specific short hairpin RNA (shRNA) was synthesized and cloned into a recombinant lentiviral vector. TECs were then infected by the miRNA-1-shRNA recombinant lentivirus. TECs were divided into three groups: a control (CON) group consisting of normal TECs without lentiviral infection, a negative control (NC) group consisting of normal TECs infected with a negative control virus, and a micro-down (MD) group consisting of normal TECs infected with the miR-1-inhibition virus containing the target gene. Silencing of miR-1 expression was quantified via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The proliferation of TECs was detected using MTT (Thiazolyl Blue Tetrazolium Bromide) assay; the observations were continued for 5 d, and the optical density value at 490 nm was detected every day. Apoptosis was detected via flow cytometry using Annexin V-APC single staining. The migration and invasion of TECs were detected using transwell assays. RESULTS: Lentiviral miR-1 shRNA was successfully transduced into TECs, and specifically silenced the expression of miR-1. The results of qRT-PCR showed that the expression of miR-1 was significantly decreased in the MD group (2(-ΔΔCt) = 0.57 ± 0.14) compared with the CON group (2(-ΔΔCt) = 1) and the NC group (2(-ΔΔCt) = 1.05 ± 0.13) (P < 0.01). The results of MTT assay showed that the cell proliferation was all significantly inhibited in the MD group in the 5 days compared with the CON and NC groups (P < 0.01). The results of flow cytometry showed that the apoptosis was significantly increased in the MD group (6.32% ± 0.33%) compared with the CON group (2.03% ± 0.30%) and the NC group (2.18% ± 0.15%) (P < 0.01). The ability of cell migration was significantly inhibited in the MD group (62.0 ± 5.48) compared with the CON group (99.8 ± 3.11) and the NC group (97.2 ± 3.70) (P < 0.01). The ability of invasion of TECs was also significantly inhibited in the MD group (29.8 ± 2.39) compared with the CON group (44.6 ± 3.36) and the NC group (44.4 ± 5.17) (P < 0.01). CONCLUSION: MiR-1 might be a potential tumor activator. Inhibiting its expression could decrease proliferation, induce apoptosis, and inhibit the migration and invasion of TECs of human HCC.

[Protective effect of magnesium isoglycyrrhizinate combinated with verapamil on liver ischemia-reperfusion injury after semi-hepatectomy in rats].

OBJECTIVE: To investigate protective effect of isoglycyrrhizinate combinated with verapamil on liver ischemia-reperfusion injury after semi-hepatectomy in rats. METHODS: Male SD rats were randomly divided into five groups: false operation (group A); rats subjected to 50％hepatectomy and 45 min of hepatic ischemia (group B); rats subjected to 50% hepatectomy and 45 min of hepatic ischemia, and treated with isoglycyrrhizinate (group C); rats subjected to 50% hepatectomy and 45 min of hepatic ischemia, and treated with verapamil (group D); rats subjected to 50% hepatectomy and 45 min of hepatic ischemia, and treated with isoglycyrrhizinate and verapamil (group E). Liver function, as well as morphology, SOD activity, FasL, caspase 3, and ß-catenin expression of rats were analyzed. RESULTS: Compared to group B, the levels of serum ALT of group E were significantly lower (P<0.05), SOD activity of liver tissue (98.8±7.1) were significantly higher (P<0.01), pathological changes were milder, FasL gene (1.327±0.193) and protein (0.010 9±0.001 4) expression were significantly lower (P<0.01). Caspase 3 protein expression levels (0.141 0±0.005 3) were significantly lower (P<0.001), ß-catenin protein expression levels (0.079 1±0.008 2) were higher significantly (P<0.01). CONCLUSIONS: Isoglycyrrhizinate combinated with verapamil can effectively reduce rats liver ischemia-reperfusion injury. The protective effect on liver cells may be mediated by inhibiting apoptosis caused by Fas/FasL system, increasing ß-catenin protein expression, promoting liver regeneration and relieving liver peroxidation damage.

Growth inhibition of hepatocellular carcinoma tumor endothelial cells by miR-204-3p and underlying mechanism.

AIM: To investigate the mechanism by which miR-204-3p inhibits the growth of hepatocellular carcinoma (HCC) tumor endothelial cells (TECs). METHODS: Flow cytometry was used to identify HCCTECs and analyze their purity. Differentially expressed miRNAs in HCC TECs as compared to normal hepatic sinusoidal endothelial cells (HSECs) were examined using the HmiOA v4 Human miRNA OneArray microarray. miR-204-3p showed the most significant decrease in expression and was further studied. Over-expression of miR-204-3p was achieved using lentiviral transduction into TECs of HCC. The biological changes in HCC TECs before and after transduction were detected using MTT and apoptosis assays. The association between miR-204-3p and fibronectin 1 (FN1) was determined using the dual luciferase activity assay. Changes in FN1 protein expression before and after transduction were detected using Western blot analysis. RESULTS: Microarray results showed that compared to normal HSECs, 15 miRNAs were differentially expressed in HCC TECs, including 6 miRNAs with increased expression and 9 miRNAs with decreased expression. Among them, miR-204-3p showed the most significant decrease in expression (log2 = -1.233477, P = 0.000307). Over-expression of miR-204-3p in HCC TECs via lentiviral transduction significantly inhibited the proliferation of HCC TECs and promoted apoptosis. Results from the dual luciferase activity experiment showed that the luciferase intensity in the wild type FN1 group was significantly inhibited (P < 0.05), while that in the mutant FN1 group was not obviously affected. This observation indicated that FN1 was one of the potential targets of miR-204-3p. After over-expression of miR-204-3p in HCC TECs, Western blot analysis showed that the expression of FN1 protein was significantly inhibited. CONCLUSION: MiR-204-3p acts on its potential target gene, FN1, and inhibits its expression, thus blocking the adhesion function of FN1 in promoting the growth of TECs.

The role of prophylactic antibiotics in laparoscopic cholecystectomy in preventing postoperative infection: a meta-analysis.

BACKGROUND: Although laparoscopic cholecystectomy (LC) is a common and widely applied technique, the use of antibiotics during the perioperative period in infection prevention remains controversial. In our study, a meta-analysis was performed to assess the impact of antibiotic prophylaxis on the postoperative infection rate in LC. METHODS: A literature search was conducted on studies published between January 1966 and March 2010 that involved LC and prophylactic administration of antibiotics. Only randomized trials that compared perioperative antibiotic prophylaxis with placebo or no treatment in low-risk patients undergoing LC were selected. Eighteen studies qualified according to the inclusion criteria, but only 12 were of adequate quality according to the Jadad scale to be included for the meta-analysis. Data were analyzed via the Peto odds ratio (OR) method and run using RevMan 4.2 software. The precision of the estimation of OR by individual studies was used to calculate their contribution (or weighting) to the pooled OR. RESULTS: The results of the 12 studies did not have significant heterogeneity, and thus, the fixed effect model was used for data analysis. Compared with placebo or no treatment, there was no significant risk reduction in the antibiotic prophylaxis group with regard to overall infections (OR=1.11; 95% confidence interval [CI], 0.68-1.82; P=.67), wound infections (OR=1.07; 95% CI, 0.59-1.94; P=.99), major infections (OR=2.88; 95% CI, 0.3-28.09; P=.36), distant infections (OR=1.01; 95% CI, 0.43-2.36; P=.99), or positive bile cultures (OR=0.76; 95% CI, 0.54-1.08; P=.12). However, prophylactic antibiotics did shorten length of hospital stay (weighted mean difference=-0.16; 95% CI, -0.22 to -0.09; P<.01). CONCLUSION: Prophylactic antibiotics are not necessary for elective LC in low-risk patients.

Intratumoural GM-CSF microspheres and CTLA-4 blockade enhance the antitumour immunity induced by thermal ablation in a subcutaneous murine hepatoma model.

PURPOSE: We evaluated the effect of a new antitumour immunity regimen that included microwave ablation, intratumoural microspheres encapsulating granulocyte-macrophage colony stimulating factor (GM-CSF), and blockade of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). MATERIALS AND METHODS: C57BL6 mice with an established subcutaneous Hepa 1-6 hepatoma underwent microwave ablation, followed by intratumoural injection of GM-CSF microspheres, and intraperitoneal injection of anti-CTLA-4 antibodies. The therapeutic effects were evaluated by tumour growth, survival analysis, and cytotoxicity of T lymphocytes against Hepa 1-6. RESULTS: The co-administration of microwave thermal ablation, GM-CSF microspheres, and anti-CTLA-4 rejected tumour rechallenge in 90% of treated mice in a subcutaneous murine Hepa 1-6 model, and cured established distant tumour in 50% of the treated mice. This antitumour immune response was tumour-specific and mediated by natural killer (NK), CD4+, and CD8+ T cells. CONCLUSIONS: Microwave ablation, followed by intratumoural GM-CSF microspheres, and anti-CTLA-4 antibodies results in the local eradication of tumours, rejection of tumours following rechallenge, and cures established distant tumours, suggesting that this is a promising regimen and one that is readily applicable in the clinic.