Molecular Characterization of the Nsp2 and ORF5 (ORF5a) Genes of PRRSV Strains in Nine Provinces of China During 2016-2018.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease and brings huge economic losses to commercial pork production worldwide. PRRSV causes severe reproductive failure in sows and respiratory distress in piglets. To trace the evolution of PRRSV in pigs with respiratory diseases in some regions of China, 112 samples were collected from nine provinces in China during 2016-2018. All samples were detected by RT-PCR and analyzed by the Nsp2/ORF5 (ORF5a)-genes-phylogeny. Sequence analysis and recombination analysis were conducted on the Nsp2/ORF5 (ORF5a) genes of the identified strain in the study. The RT-PCR result shown that the positive rate of PRRSV was 50.89% (57/112). Phylogenetic analysis showed that the identified PRRSV strains were all NA genotype and belonged to lineage 1, 3, and 8. The Nsp2 gene of identified PRRSV strains exhibited nucleotide homologies of 53.0 ~ 99.8%, and amino acid homologies of 46.8 ~ 99.7%. The ORF5 gene of identified PRRSV strains exhibited nucleotide homologies of 82.4 ~ 100%, and amino acid homologies of 79.6 ~ 100%. Sequence analysis revealed that a discontinuous 30-amino-acid deletion (positions 481 and 533-561) and a 131-amino-acid discontinuity deletion (positions 323-433, 481, and 533-551) in Nsp2 of PPRSV isolates; all identified strains in this study may be wild strains, and most identified strains may be highly virulent strains. Sequence analysis of ORF5 and ORF5a revealed that the mutation sites of GP5 were mainly concentrated in the signal peptide and epitopes region, while the mutation sites of ORF5a were mainly concentrated in the transmembrane and the intramembrane region. The recombination analysis indicated that there may be multiple recombination regions in identified strains, and the recombination pattern was more complex. This study showed that the prevalent PRRSV strain in some regions of China was still HP-PRRSV, while NADC30 strain also occupied a certain proportion; different types of PRRSV strains showed different patterns and variation in China. This study suggested that the monitoring of PRRSV prevalence and genetic variation should be further strengthened.

Erratum: MicroRNA-183-5p promotes the proliferation, invasion and metastasis of human pancreatic adenocarcinoma cells.

[This corrects the article DOI: 10.3892/ol.2015.3872.].

LHPP suppresses proliferation, migration, and invasion and promotes apoptosis in pancreatic cancer.

Pancreatic cancer (PaCa) is a common malignant tumor of the digestive system with poor prognosis and no ideal treatment for inoperable patients, which is partly due to delayed diagnoses. It is recently reported that the protein histidine phosphatase LHPP is a tumor suppressor in hepatocellular carcinoma, cervical cancer, and bladder cancer. So far, there is no study on the expression level of LHPP in PaCa, and its mechanism of action on tumors is unclear. In this experiment, LHPP expression was lower in cancer tissues than that in normal pancreatic tissue, and clinicopathological results showed that LHPP expression was correlated with the degree of differentiation and lymphatic metastasis of pancreatic carcinoma. The biological characteristics of LHPP in PaCa cells were examined by the cell counting kit-8 assay, transwell assay, and monoclonal formation test. The inhibitory mechanism of LHPP in PaCa cells was determined using Western blotting and flow cytometry. The results showed that LHPP restrained PaCa cell proliferation, migration, and invasion. Increased LHPP expression promoted the apoptosis of PaCa cells through higher activation of cleaved-PARP and cleaved-Casp3 and lower activation of cIAP1. Importantly, the increase in LHPP enhanced PTEN expression and decreased the phosphorylated AKT level. Moreover, LHPP-induced apoptosis was diminished by SC79 (AKT activator) in PaCa cells. In conclusion, LHPP blocks proliferation, migration, and invasion and enhances apoptosis in PaCa cells through the PTEN/AKT signaling pathway.

Short-term postoperative complications and prognostic factors in patients with adenocarcinoma of the esophagogastric junction.

BACKGROUND: The study was conducted to investigate the short-term complications and prognostic factors in patients with esophagogastric junction adenocarcinoma (EGJA). METHODS: This retrospective study included 110 EGJA patients who underwent surgery from January 2010 to November 2012 at The First Affiliated Hospital of BengBu Medical College. The overall survival and short-term complications were analyzed according to the patients' clinical characteristics. RESULTS: The incidence of postoperative cardiopulmonary complications was significantly higher in patients with preoperative cardiopulmonary disease or elderly patients (P < 0.05). Four cases of upper margin cancer residue were detected using the abdominal approach and three using the thoracic approach, which indicated that the cancer residue margin was related to surgical approach. The overall five-year survival rate was 34.3% and statistically differed according to pathological stage and en block resection (Pall < 0.05). Cox regression analysis showed that lymph node metastasis (P < 0.05) and the extent of tumor invasion (P < 0.05) were independent prognostic factors. CONCLUSION: Elderly patients with preoperative cardiopulmonary disease had an increased risk of developing postoperative cardiopulmonary complications. Lymph node status and depth of tumor invasion were independent factors related to patient prognosis.

A nomogram to predict lymph node metastasis before resection in intrahepatic cholangiocarcinoma.

BACKGROUND: In this study, we developed and validated a nomogram to predict lymph node metastasis before surgery in patients with intrahepatic cholangiocarcinoma (ICC). METHODS: Using the data from January 2006 to January 2015, we enrolled a total of 218 eligible patients with clinicopathologically confirmed ICC as a primary cohort to develop the nomogram. After various variables before surgery were analyzed by multivariable logistic regression, we combined the preoperative carbohydrate antigen 19-9, primary site of tumor, lymphonodus size on computed tomography imaging, tumor growth pattern, and (if applicable) histologic grade to make two different predictive nomograms. Then, the results were validated in 62 consecutive ICC patients from February 2015 to December 2016. We also compared the performance of the different nomograms via calibration, discrimination, and clinical use. RESULTS: The nomogram displayed fine discrimination (the concordance index, 0.761) and fine calibration in the primary cohort. When applied to the validation cohort, the nomogram also showed fine discrimination (concordance index, 0.794) and fine calibration. After adding the histologic grade to the nomogram, the integrated discrimination for predictive performance improved significantly. Finally, the clinical usefulness of predictive nomogram was proven via the decision curve analysis. CONCLUSIONS: The proposed nomograms can be selectively used to achieve more accurate lymph node metastasis predictions before surgery in patients with ICC, and this information can help with clinical management.

Anti­angiogenesis gene therapy for hepatocellular carcinoma via systemic injection of mesenchymal stem cells engineered to secrete soluble Flt­1.

Anti­angiogenesis gene therapy has attracted interest as a potential treatment for hepatocellular carcinoma (HCC). Studies have indicated that soluble fms­like tyrosine kinase­1 (sFlt­1) may suppress angiogenesis by sequestering free vascular endothelial growth factor (VEGF) or by forming inactive heterodimers with VEGF receptor­2. Mesenchymal stem cells (MSCs) have been widely used as prospective delivery vehicles for therapeutic agents, owing to their ability to migrate towards tumor sites. In the present study, a subcutaneous HCC mouse model was used to assess the anti­angiogenesis effects of lentivirus­transfected MSCs engineered to secrete sFlt­1 (LV­sFlt­1­MSCs). LV­sFlt­1­MSCs effectively secreted sFlt­1, which inhibited tube formation in vitro. MSCs labeled with green fluorescence protein primarily migrated to tumor sites in vivo. An immunohistochemical assay indicated that microvessel density was reduced in mice treated with LV­sFlt­1­MSCs, compared with the control group treated with PBS. Additionally, LV­sFlt­1­MSCs inhibited tumor growth and prolonged survival in an HCC mouse model via systemic injection. Overall, the present study was designed to investigate the potential of LV­sFlt­1­MSCs for anti­angiogenesis gene therapy in HCC.

Down-regulation of ASIC1 suppressed gastric cancer via inhibiting autophagy.

As autophagy has anti-apoptosis effect and accelerates cell survival, many studies start to target autophagy as a therapeutic strategy for cancer. Acid-sensing ion channels (ASICs) was reported to activate autophagy. However, whether ASICs can regulate gastric cancer through autophagy is unknown. The differentially expressed genes in normal gastric tissue and gastric cancer tissue in patients were investigated by RNA-seq. Expression of ASIC1 and autophagy related 5 (ATG5) was further confirmed by real-time PCR. Effects of knockdown expression of ASIC1 and ATG5 on the growth of gastric SGC-7901 cells were assayed by CCK-8 kit. The animal survival rate and tumor volume in murine heterotopic xenograft model was assayed. The expression of autophagy related genes was enriched in gastric cancer tissue in patients, including ASIC1 and ATG5. Knockdown expression of ASIC1 and ATG5 inhibits the growth of SGC-7901 cells, respectively. ASIC1 regulates ATG5 gene expression in SGC-7901 cells. ASIC1 knockdown extended the survival rate of animals and inhibited the tumor volume in the murine heterotopic xenograft model. This study showed that downregulation of ASIC1 inhibits gastric cancer growth via decreasing autophagy, therefore strongly suggests a therapeutic role for ASIC1 in gastric cancer.

Risk Factors for Cholangiocarcinoma After Initial Hepatectomy for Intrahepatic Stones.

BACKGROUND: Aggressive hepatectomy is effective in treating intrahepatic stones and may minimize the deleterious consequences of subsequent cholangiocarcinoma (S-CCA). The risk factors of S-CCA after different methods of hepatectomy may vary with the resection scope of stone-affected segments. METHODS: We reviewed the records of 981 patients of primary intrahepatic stones with elective hepatectomy from January 2000 to December 2010. The clinical characteristics of patients in the S-CCA group (n = 55) and the control group (n = 926) were compared. The uniformity between extent of liver resection (ELR) with stone-affected segments (SAS) was segmented into 2 varieties: ELR = SAS with ELR < SAS according to the different hepatic resection scopes. Cox regression model with forward selection was used to identify the risk factors of S-CCA. RESULTS: In the univariate analysis, significant differences were observed between the S-CCA and control groups concerning stone location (unilateral 43.6 and 65.2 %, bilateral 56.4 and 34.8 %), residual stones (32.7 and 11.6 %), hepaticojejunostomy (43.6 and 30.9 %), and uniformity between ELR with SAS (ELR = SAS 20.0 and 42.6 %, ELR < SAS 80.0 and 57.4 %). Residual stones [hazard ratio (HR) 2.101, P = 0.016], hepaticojejunostomy (HR 1.837, P = 0.026) and uniformity between ELR and SAS (HR 2.442, P = 0.013) were independent prognostic factors for S-CCA by a Cox regression analysis with forward selection. In the subsection of ELR = SAS group, the 5- and 10-year postoperative tumor occurrence rates of unilateral and bilateral stones group were 0.9 versus 1.9 % and 3.0 versus 4.1 %, respectively (P = 0.663, log-rank). In the other subsection of ELR < SAS group, the 5- and 10-year postoperative tumor occurrence rates of unilateral and bilateral stones group were 3.4 versus 3.9 % and 6.8 versus 13.2 %, respectively (P = 0.047, log-rank), and the 5- and 10-year postoperative tumor occurrence rates of residual stones and non-residual stones group were 5.8 versus 3.0 % and 16.0 versus 7.9 %, respectively (P = 0.015, log-rank). CONCLUSIONS: Patients who underwent aggressive hepatectomy and had ELR = SAS had better outcomes than those with ELR < SAS. In the patients with ELR = SAS, the S-CCA rates of unilateral and bilateral stones were low and comparable. However, patients with ELR < SAS and bilateral intrahepatic or residual stones should be monitored more carefully for high-risk factors of S-CCA.

MicroRNA-544 down-regulates both Bcl6 and Stat3 to inhibit tumor growth of human triple negative breast cancer.

Triple negative breast cancer lacking estrogen receptor (ER), progesterone receptor and Her2 account for account for the majority of the breast cancer deaths, due to the lack of specific gene targeted therapy. Our current study aimed to investigate the role of miR-544 in triple negative breast cancer. Endogenous levels of miR-544 were significantly lower in breast cancer cell lines than in human breast non-tumorigenic and mammary epithelial cell lines. We found that miR-544 directly targeted the 3'-untranslated region (UTR) on both Bcl6 and Stat3 mRNAs, and overexpression of miR-544 in triple negative breast cancer cells significantly down-regulated expressions of Bcl6 and Stat3, which in turn severely inhibited cancer cell proliferation, migration and invasion in vitro. Employing a mouse xenograft model to examine the in vivo function of miR-544, we found that expression of miR-544 significantly repressed the growth of xenograft tumors. Our current study reported miR-544 as a tumor-suppressor microRNA particularly in triple negative breast cancer. Our data supported the role of miR-544 as a potential biomarker in developing gene targeted therapies in the clinical treatment of triple negative breast cancer.

MicroRNA-183-5p promotes the proliferation, invasion and metastasis of human pancreatic adenocarcinoma cells.

The aim of the current study was to investigate the potential role of microRNA-183-5p (miR-183-5p) in the proliferation, invasion and metastasis of pancreatic cancer, and to identify promising target genes of oncogenic miR-183-5p. Western blotting and quantitative polymerase chain reaction (qPCR) were used to investigate whether these oncogenic microRNAs may be useful as biomarkers in pancreatic carcinoma (PaCa). Potential target genes were verified using miRDB, PicTar and TargetSCAN, and qPCR was used to detect the expression of miR-183 and suppressor of cytokine signaling 6 (SOCS-6; a potential target of miR-183) in PANC-1 PaCa cells and in the HPDE6-C7 pancreatic ductal cell line for comparison. The function of miR-183 in cell proliferation, wound healing, invasion and migration was also investigated using a miR-183 inhibitor. Western blot analysis was used to confirm SOCS-6 as a tumor suppressor and qPCR was used to detect and confirm that this potential target gene is directly regulated by miR-183. The results indicated that the expression of miR-183 in PANC-1 cells was upregulated compared with that in HPDE6-C7 cells, whilst the expression of SOCS-6 was downregulated. SOCS-6 expression was also significantly lower in PaCa tissues compared with that in matched normal pancreatic tissues from PaCa patients. Furthermore, expression of miR-183 was inversely correlated with that of SOCS-6. miR-183 knockdown decreased cell growth and motility in pancreatic cancer cells and significantly increased the expression of SOCS-6. These data suggest that oncogenic miR-183 may be useful as a pancreatic cancer biomarker. In addition, inhibition of miR-183 expression may be beneficial as PaCa treatment. SOCS-6 is a potential target gene of miR-183.

Effects and mechanism of flavonoids from Astragalus complanatus on breast cancer growth.

Flavonoids from Astragalus complanatus R.Br (FAC) had anticancer effects on many tumor cells. The current study was performed to evaluate the effects of FAC on human breast cell proliferation, apoptosis, and metastasis, as well as their active mechanism. Cell viability and growth were detected using the cell counting kit (CCK)-8 assay in vitro. Assay of FAC on induced breast cancer mortality was counted as survival time of nude mice after breast cell line inoculation. The effect of FAC on cell invasion was investigated by an optimization assay that contains a 96-well Boyden chamber with wells precoated with BME at three different concentrations. The mechanism of its action on apoptosis and metastasis was determined by related gene detection. Proliferation of three breast cell lines was inhibited with FAC treatment in a dose-dependent manner. Survival time of nude mice with breast cancer cell inoculation also was prolonged with increasing FAC dose. Metastasis in FAC-treated breast cells was also significantly inhibited. Real-time polymerase chain reaction (PCR) assay demonstrated that apoptosis-related BCL-2 and caspase-9 gene expression was consistent with their phenotype change. Metastasis-related FAK and BRCA1 gene expression was inversely related to FAC treatment. Western blot analysis indicated that BCL-2 and FAK proteins were reduced, whereas caspase-9 and BRCA1 proteins were increased with a higher dose of FAC treatment. These data suggested that FAC has an important role in breast cancer growth and metastasis suppression in vitro and in vivo. Its active mechanism involves promoting programmed cancer cell death and regulates metastasis-related gene expression.

AZD-4547 exerts potent cytostatic and cytotoxic activities against fibroblast growth factor receptor (FGFR)-expressing colorectal cancer cells.

Colorectal cancer (CRC) causes significant mortalities worldwide. Fibroblast growth factor (FGF) receptor (FGFR) signaling is frequently dysregulated and/or constitutively activated in CRCs, contributing to cancer carcinogenesis and progression. Here, we studied the activity of AZD-4547, a novel and potent FGFR kinase inhibitor, on CRC cells. AZD-4547 inhibited CRC cell growth in vitro, and its activity correlated with the FGFR-1/2 expression level. AZD-4547 was cytotoxic and pro-apoptotic in FGFR-1/2-expressed CRC cell lines (NCI-H716 and HCT-116), but not in FGFR-1/2 null HT-29 cells. Further, AZD-4547 inhibited cell cycle progression and attenuated the activation of FGFR1-FGFR substrate 2 (FRS-2), ERK/mitogen-activated protein kinase (MAPK), and AKT/mammalian target of rapamycin (AKT/mTOR) signalings in NCI-H716 and HCT-116 cells. In vivo, AZD-4547 oral administration at effective doses inhibited NCI-H716 (high FGFR-1/2 expression) xenograft growth in nude mice. Phosphorylation of FGFR-1, AKT, and ERK1/2 in xenograft specimens was also inhibited by AZD-4547 administration. Thus, our preclinical studies strongly support possible clinical investigations of AZD-4547 for the treatment of CRCs harboring deregulated FGFR signalings.

Combined assays for serum carcinoembryonic antigen and microRNA-17-3p offer improved diagnostic potential for stage I/II colon cancer.

Colorectal cancer is among the leading causes of cancer-related mortality, one of the main reasons for which is the lack of an effective screening method for early-stage disease. The levels of carcinoembryonic antigen (CEA) and microRNA (miR)-17-3p in the serum of 70 patients with stage I/II colon cancer and 70 healthy volunteers were determined, and the diagnostic value of CEA plus miR-17-3p detection for colon cancer was assessed. The levels of CEA were measured by a radioimmunoassay method, and those of miR-17-3p using the reverse transcription-quantitative polymerase chain reaction method. miR-16 was used as the endogenous control, as it displayed high stability, high abundance and low variability in the analyzed serum samples. The receiver operating characteristic (ROC) curve analysis indicated the potential diagnostic value of the two markers and the area under the ROC curve (AUC) for CEA and miR-17-3p was 0.719 (95% CI: 0.658-0.843) and 0.807 (95% CI: 0.748-0.906), respectively. At a threshold of 9.6 ng/ml for CEA, the optimal sensitivity and specificity were 74.6 and 84.3%, respectively, in discriminating colon cancer patients from healthy controls. At a threshold of 2.98 for miR-17-3p, the sensitivity and the specificity were 83.6 and 72.9%, respectively. A combined ROC analysis using CEA and miR-17-3p revealed an AUC of 0.929 (95% CI: 0.834-0.978) with a sensitivity of 96.4% and a specificity of 95.7% in discriminating colon cancer patients from healthy controls. In conclusion, both CEA and miR-17-3p were highly expressed in the serum of our series of colon cancer patients. CEA plus miR-17-3p detection significantly increased the sensitivity and specificity in discriminating stage I/II colon cancer patients from healthy controls. Therefore, combined detection of serum CEA and miR-17-3p levels may have the potential to become a new laboratory method for the early clinical diagnosis of colon cancer.

Risk factors of liver metastasis in patients after radical resection of pancreatic cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To analyze the risk factors of liver metastasis in patients after radical resection of pancreatic cancer.</p><p><b>METHODS</b>One hundred and twenty-four patients with non-metastatic, resectable pancreatic cancer treated in our department between 2006 and 2012 were included in this study. All of these patients underwent resection of the primary tumor combined with extensive lymph node dissection. The development of postoperative liver metastases was carefully followed up, and the clinicopathological factors and molecular characteristics were evaluated by univariate analysis and multivariate logistic regression using SPSS 16.0 software.</p><p><b>RESULTS</b>Forty-eight cases of liver metastases were found among the 124 cases of pancreatic cancer after radical surgery (38.7%). The rate of liver metastasis of pancreatic cancer after radical surgery in the age groups < 40, 40-60, and > 60 were 68.8%, 33.3% and 35.1%, respectively. The rate of liver metastasis in the body mass index (BMI) group < 20 kg/m2, 20-25 kg/m2, and > 25 kg/m2 were 21.6%, 44.1% and 52.6%, and the rate of liver metastasis in the time between the onset and diagnosis groups ≥ 3 months and < 3 months were 59.4% and 31.5%, respectively. The rate of liver metastasis in patients with preoperative fatty liver was 14.3% and it was 43.7% in patients without preoperative fatty liver. The rate of liver metastasis in patients of histological high, medium and low grade was 10.0%, 35.4% and 49.0%, respectively. The rate of liver metastasis in patients with venous tumor thrombus was 68.8% and it was 34.3% in patients without venous tumor embolus. The rate of liver metastasis in patients with postoperative chemotherapy was 31.2% and it was 51.1% in patients without postoperative chemotherapy. All those differences had statistical significance (P < 0.05). Univariate analysis revealed that age, body mass index (BMI), time between the onset and diagnosis, preoperative fatty liver, histological grading, tumor invasion depth, venous tumor embolus, and postoperative chemotherapy were significantly related to postoperative liver metastasis. Multivariate analysis revealed five statistically independent risk factors for postoperative liver metastasis: BMI, time between onset and diagnosis, preoperative fatty liver, histological grading, and venous tumor embolus.</p><p><b>CONCLUSIONS</b>Our data suggest that patient's BMI, time between onset and diagnosis, histological grade, and venous tumor embolus are significantly correlated with postoperative liver metastases in patients with pancreatic cancer. Pancreatic cancer patients with preoperative fatty liver have less postoperative liver metastasis.</p>

Analysis of HYAL3 gene mutations in Chinese lung squamous cell carcinoma patients.

PURPOSE: In a previous study, we found a hyaluronidase 3 (HYAL3) gene mutation in exon 2 at position 188 by genome sequencing in a lung squamous cell carcinoma patient. The mutation results in substitution of serine for alanine. The aim of the study was to screen the HYAL3 gene mutation in Chinese lung squamous cell carcinoma patients and explore the correlation between mutation of HYAL3 with clinical and pathological characteristics in lung squamous cell carcinoma patients in China. METHODS: We applied polymerase chain reaction to examine the HYAL3 gene mutations in cancer tissues and their adjacent normal tissues from 39 cases of lung squamous cell carcinoma patients. RESULTS: 1) The incidence rate of HYAL3 mutation in 39 cases of lung squamous cell carcinoma was 10.26% (4/39) and none in adjacent normal lung tissues (0/39). 2) The mutations of HYAL3 in the 4 cases were all heterozygous: 3 of them were located in exon 1 (G-T) and one in exon 2 (G-T). 3) Mutations of the HYAL3 gene were not correlated with the distribution of patient gender, age, tumor size, histological grade, smoking history, TNM stage or distant metastasis (P >0.05). The gene mutation was correlated with lymph node status (P = 0.044). CONCLUSION: Mutations of the HYAL3 gene are rare in Chinese lung squamous cell carcinoma patients and might contribute to lymph node metastasis.

Vascular endothelial growth factor-D promotes growth, lymphangiogenesis and lymphatic metastasis in gallbladder cancer.

Lymph node metastasis is a major prognostic factor for patients with gallbladder cancer (GBC), and greater understanding of the molecule mechanism of lymph node metastasis in GBC is needed to improve prognosis. VEGF-D has been implicated in the control of lymphangiogenesis in many carcinomas, but the biological function of VEGF-D in human GBC remains unclear. In this study, we analyzed the role of the VEGF-D in human GBC cells and addressed the functional role of VEGF-D using a xenograft mouse model. We examined the expression of VEGF-D in three human gallbladder cancer cell lines. A lentivirus-based effective VEGF-D siRNA vector was infected into GBC NOZ cells. The effect of VEGF-D siRNA on GBC NOZ cells was investigated by cell proliferation assay and invasion assay. Furthermore, we examined the role of VEGF-D-SiRNA on GBC NOZ cells in the mice of subcutaneous and orthotopic xenograft tumor. Our results are as follows: VEGF-D mRNA and protein were expressed in all three GBC cell lines (GBC-SD, NOZ, and SGC-996). We successfully selected D-3/siRNA as the most effective siRNA to silence VEGF-D expression after four VEGF-D siRNA plasmid transfection in NOZ cells. VEGF-D mRNA and protein expression were suppressed by lentivirus-mediated D-3/siRNA. D-3-RNAi-LV inhibited NOZ cells proliferation and invasion ability in vitro. D-3-RNAi-LV inhibited tumor growth and lymphangiogenesis in the NOZ cell subcutaneous xenograft model. D-3-RNAi-LV inhibited lymphangiogenesis and lymphatic metastasis in the NOZ cell orthotopic xenograft model. Furthermore, D-3-RNAi-LV inhibited tumor ascites and hepatic invasion in the NOZ cell orthotopic xenograft model. In conclusion, VEGF-D is involved and plays an important role in GBC progression, suggesting that VEGF-D may be a potential molecular target in the treatment of GBC.

[Effect of human hepatocyte growth factor gene-modified bone marrow mesenchymal stem cells on immunological rejection after allograft liver transplantation in rats].

OBJECTIVE: To study the effect of recombinant lentiviral vector mediated human hepatocyte growth factor (hHGF) gene-modified bone marrow mesenchymal stem cells (BMSCs) on the immunological rejection after allograft liver transplantation in rats, and to reveal the mechanism of immune tolerance. METHODS: Eight male Sprague Dawley (SD) rats of clean grade (aged 3 to 4 weeks, weighing 75-85 g) were selected for the isolation and culture of BMSCs; 64 adult male SD rats of clean grade (weighing 200-250 g) were used as donors; and 64 adult male Wistar rats of clean grade (weighing 230-280 g) were used as receptors. After establishing a stable model of rat allogeneic liver transplantation, 1 mL saline, 2 x10(6)/mL of BMSCs 1 mL, 2 x 106/mL of BMSCs/green fluorescent protein 1 mL, and 2 x 10(6)/mL of BMSCs/hHGF 1 mL were injected via the portal vein in groups A, B, C, and D respectively. Then the survival time of the rats was observed. The hepatic function was determined and the histological observation of the liver was performed. The hHGF mRNA expression was detected by RT-PCR, the level of cytokine including hHGF, interleukin 2 (IL-2), IL-4, IL-10, and interferon gamma (IFN-gamma) by ELISA assay, the level of apoptosis by TUNEL method, and the expression level of proliferating cell nuclear antigen (PCNA) by immunohistochemical method. RESULTS: The survival time of group D was significantly higher than that of groups A, B, and C (P < 0.01); the survival time of groups B and C was significantly higher than that of group A (P < 0.01), but there was no significant difference between group B and group C (P > 0.05). RT-PCR demonstrated the transcription of hHGF mRNA in the grafts of group D; the serum cytokine hHGF reached to (6.2 +/- 1.0) ng/mL. Compared with groups B and C, group D exhibited significant inhibitory effect, significantly improved liver function, and showed mild acute rejection. In addition, the levels of cytokine IL-2 and IFN-gamma decreased; the levels of cytokine IL-4 and IL-10 increased; the level of apoptosis reduced; and the expression level of PCNA increased. Except for the expression of IL-4 (P > 0.05), there were significant differences in the other indexes between group D and groups B, C (P < 0.05). CONCLUSION: BMSCs/hHGF implanting to rat liver allograft via portal vein can induce immune tolerance. Compared with injection of BMSCs alone, BMSCs/hHGF treatment can alleviate acute rejection and prolong the survival time significantly. The immunosuppressive effect of BMSCs/hHGF is correlated with Th2 shifts up of Th1/Th2 shift, reduced apoptosis, promoted liver regeneration.

[Research of lentiviral vector mediated growth factor gene-modified bone marrow mesenchymal stem cells].

OBJECTIVE: To construct lentiviral vector carrying the human hepatocyte growth factor (hHGF) gene, and then to get hHGF gene/modified bone marrow mesenchymal stem cells (BMSCs) by infecting the BMSCs. METHODS: The hHGF gene was obtained with PCR from pcDNA-hHGF plasmid. The recombination lentiviral vector plasmid hHGF was constructed with Age I digestion and gene recombinant, then was identified with PCR and sequencing. Mediated by Lipofectamine 2000, the three plasmids system of lentiviral vector including pGC-E1-hHGF, pHelper 1.0, and pHelper 2.0 was co-transfected to 293T cells to produce hHGF gene. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus was measured by real-time quantitative PCR. The BMSCs were infected by the constructed lentivirus and the multiplicities of infection (MOI) was identified with fluorescent microscope, the efficiency of infection with flow cytometry (FCM) analysis, the hHGF level with ELISA analysis, and the expression of hHGF gene with RT-PCR. RESULTS: Lentiviral vector carrying hHGF gene was constructed successfully. The titer of lentivirus was 1 x 10(8) TU/mL. The infection efficiency of BMSCs by hHGF lentiviral was high and reached 98% by FCM, and the best MOI was 10. A great mount of green fluorescence was observed with the fluorescent microscope at 28 days after infection. Peak concentration of hHGF secreted by BMSCs/hHGF reached 40.5 ng/mL at 5 days. The concentration could maintain a high level until 28 days after infection. RT-PCR showed that BMSCs/hHGF could express hHGF gene. CONCLUSION: By lentiviral vector, hHGF gene was integrated into BMSCs genome, and it can express stably.

Vascular endothelial growth factor-C promotes the growth and invasion of gallbladder cancer via an autocrine mechanism.

Vascular endothelial growth factor-C (VEGF-C) has a well-defined action on neoplastic lymphangiogenesis and angiogenesis through VEGF receptor-3 (VEGFR-3) and VEGFR-2, respectively, which are generally expressed in endothelial cells. The function of the VEGF-C/receptors pathway in tumor cell types is largely unknown. In this study, we examined the expression and role of VEGF-C/receptors in gallbladder cancer (GBC) cells. We examined the expression of VEGF-C in 50 surgical specimens from gallbladder cancer and three human gallbladder cancer cell lines. Both siRNA and neutralizing antibody to deplete the expression of VEGF-C were used to characterize the biological effect of VEGF-C in GBC NOZ cells. Furthermore, we examined the expression of its receptors, VEGFR-3 and VEGFR-2, in three human GBC cell lines. Our results are as follows: The expression of VEGF-C in the invasive marginal portion was significantly higher than the expression in the central portions. All the three GBC cell lines expressed VEGF-C. Treatment of NOZ cells with VEGF-C siRNA or a neutralizing antibody suppressed cell proliferation and invasion. Moreover, all the three GBC cell lines expressed VEGFR3, but only the NOZ cells expressed VEGFR-2 mRNA. Treatment of NOZ cells with a VEGFR-3 neutralizing antibody suppressed cell invasion, but treatment of NOZ cells with a VEGFR-2 neutralizing antibody suppressed cell proliferation and invasion. In conclusion, GBC cells express both VEGF-C and its receptors. VEGF-C may have a role in the progressive growth and invasion of human GBC through an autocrine mechanism.

Reduced expression of SK3 and IK1 channel proteins in the cavernous tissue of diabetic rats.

The small (SK3) and intermediate (IK1) conductance calcium-activated potassium channels could have key roles in the endothelium-dependent hyperpolarization factor pathway, which is believed to contribute to normal penile erection function. We aimed to investigate the expression of SK3 and IK1 in diabetic rodents. The experimental diabetes model was induced in 8-week-old male Sprague-Dawley rats (250-300 g) by a single administration of streptozotocin. Both the diabetes mellitus group (DM group, n = 20) and the control group (NDM group, n = 10) were injected with a low dose of apomorphine to allow for the measurement and comparison of the corresponding penile erections. The mRNA and protein expression levels of SK3 and IK1 were measured by reverse transcription polymerase chain reaction and western blot, respectively. Erectile function was significantly decreased in the DM group compared with control group (P < 0.05). The mRNA and protein expression levels of SK3 and IK1 were reduced in the cavernous tissue of diabetic rats compared with the control group (P < 0.05). Diabetes inhibits mRNA and protein expression of both SK3 and IK1 in the cavernous tissue of diabetic rats. This could play a key role in the development of erectile dysfunction in diabetic rats.