MicroRNA-199b-5p attenuates TGF-ß1-induced epithelial-mesenchymal transition in hepatocellular carcinoma.

BACKGROUND: Accumulating evidence indicates that N-cadherin is a cell adhesion molecule that has critical roles in tumour progression. However, the role of N-cadherin in hepatocellular carcinoma (HCC) remains controversial. METHODS: This study aims to investigate the expression status of N-cadherin and its molecular mechanisms in HCC. RESULTS: The expression of N-cadherin was markedly overexpressed in HCC tissues and cell lines. We identified that miR-199b-5p binds to the 3'-UTR of N-cadherin mRNA, thus decreasing N-cadherin expression in HCC cells. We also found the downregulation of miR-199b-5p in HCC specimens, which was inversely correlated with N-cadherin upregulation, predicted poor clinical outcomes in HCC patients. Next, we determined that miR-199b-5p overexpression promoted cell aggregation, suppressed cell migration and invasion in HCC cells, and inhibited xenografts tumour metastasis in nude mice. Moreover, we demonstrated that miR-199b-5p attenuated TGF-ß1 induced epithelial-mesenchymal transition (EMT) -associated traits, while its effects could be partially reversed by N-cadherin restoration. Finally, we examined that N-cadherin downregulation or miR-199b-5p overexpression suppressed TGF-ß1-induced Akt phosphorylation, and inhibition of PI3K/Akt pathway blocked TGF-ß1-induced N-cadherin overexpression in HCC cells. CONCLUSIONS: Our data demonstrate that N-Cadherin was markedly overexpressed and miR-199b-5p was significantly downregulated in HCC. MiR-199b-5p exerts inhibitory effects on EMT, and directly targets N-cadherin in HCC, supporting the potential utility of miR-199b-5p as a promising strategy to treat HCC. Also, a positive regulatory loop exists between N-cadherin and Akt signalling represents a novel mechanism of TGF-ß1-mediated EMT in HCC cells.

The research of nanoparticles as gene vector for tumor gene therapy.

With the development of molecular biology, the application of the gene therapy becomes a tendency in the development of oncotherapy. The gene therapy has been acknowledged as the major progress of modern medicine, also a focus in the oncotherapy research. Commonly vectors of the gene therapy mainly include two categories, namely, viral vectors and nonviral vectors. Nanoparticles gene vector of various different kinds of materials, which belong to non-viral carriers. It presents excellent abilities of adsorption, concentration and protection of DNA, which can be attributed as a main reason of the adsorption and operation of nano-gene vector on exogenous genes. In this article, we mainly reviewed the recent studies of the characteristics of nanoparticles, characteristics and transport mechanism of nanoparticles as gene vector, the progress on nanoparticles as gene vector in tumor gene therapy. Nano-gene vectors, as new drug and gene carriers, present characteristics such as the controlled-release, targeting, and the improvement of bioavailability. Nanoparticles for cancer imaging and therapy have evolved rapidly during the last decade and it is expected that more and more will become clinical practise. In the near future, as a new nanometer gene delivery vector will be in medical research and treatment play a bigger role.

Antiapoptotic gene BAG-1 vector structure of RNA interference and endogenous targeted screening in colon cancer cell lines.

The purposes of the present work were to construct the shRNA plasmids for BAG-1 gene of human and test the expression of mRNA and protein of BAG-1. Recombinant plasmids containing green fluorescent protein reporter genes are constructed using gene cloning methods. The shRNA plasmids for the BAG-1 gene are constructed by RNA interference technology. We applied fluorescent plasmid-transfected target cells in the cell transfection experiments and monitored the transfection rate of plasmids by observing the fluorescence amount. We transfected three synthesized shRNA in target screening cell and adopted RT-PCR and Western test to identify the difference of target gene transfection and translation level in cells. The specific shRNA plasmid for the BAG-1 gene was successfully recombined, and stably transfected colon cancer Lo Vo cell lines were obtained. The results present that the constructed shRNA plasmids significantly inhibited the expression of mRNA and protein of Lo Vo cell BAG-1, and can maintain the effect for a long term. pGPH1/GFP/Neo-BAG-1-homo-825 was screened as the optimum sequence of interference so as to lay a solid foundation to explore into the research on the BAG-1 gene and the biological behavior of colon cancer cells. It showed the remarkable advantage of RNAi in the generation of posttranscriptional gene silencing.

The interventional therapy for diabetic peripheral artery disease.

BACKGROUND: Diabetic peripheral arterial disease is the main cause of lower limb amputation in patients with diabetes. To summarize the technique and experiences and evaluate the clinical effects of blood vessel intervention operation on diabetic peripheral artery disease. METHODS: 81 patients with diabetic peripheral artery disease from October 2007 to September 2011, 81 cases of the observation group were treated by balloon PTA. By adopting the Seldinger puncture technology, intubation was placed into a cobra catheter or a pig tail artery catheter and directed to the ipsilateral lower extremity artery. A guidewire was used to reach the lesion part of patients and a long balloon with a diameter of 4-6 mm was used to expand the artery with a pressure of 6-10 atm. RESULTS: 81 patients in the observation group received the PTA surgery. The technical succesful rate was 100%, no complication happened. The skin temperature increased after treatment. The blood supply improved significantly. The pulsation of the foot dorsal artery was strengthened. The numbness and pain symptoms were moderated significantly. We observed better results in the observation group in lower limb vessel diameter and foot ulceration healing. None of the patients received amputation surgery. Its short-term effects were satisfactory. CONCLUSION: PTA is a feasible technique for diabetic peripheral artery disease. It has great clinical significance in treating diabetic peripheral arterial disease. Although its short-term effects is satisfactory, the long-term effects is necessary for follow up.

Coexpression of recombinant adenovirus carrying GDNF and EDNRB genes in neural stem cells in vitro.

Gene therapy and nerve stem cells isolated from the developing human enteric nervous system (ENS) are significant. They may open the route for the cell therapy of Hirschsprung's disease (HD). We have constructed the recombinant adenovirus-carrying glial cell line-derived neurotrophic factor (GDNF) and endothelin receptor B (EDNRB) gene, and investigated the exosomatic coexpression in neural stem cells. The recombinant adenovirus Ad-GE coexpressing GDNF and EDNRB gene was constructed by the AdEasy system and confirmed by the reverse transcription polymerase chain reaction (RT-PCR) method. Expression of exogenous genes in neural stem cells after transfection was confirmed by the flow cytometry and real-time fluorescence quantitative PCR. Fragments of pAd Track-CMV-GE were consistent with GDNF and EDNRB. The green fluorescence of the positive cells was followed by fluorescence microscopy at 24 h after NSCs had been transfected, reaching a peak at 72 h after transfection. Flow cytometry showed that the efficiency of transfection was 15.0, 23.6, and 25.4% at 24, 48 and 72 h respectively. Real-time fluorescence quantitative PCR showed the expression levels of mRNA of GDNF and EDNRB in 48 and 72 h groups were obviously higher than that in 24 and 96 h groups. Recombinant adenovirus carrying GDNF and EDNRB genes are coexpressed in neural stem cells, which may offer the possibility of a novel approach to local combination gene therapy for Hirschsprung's disease.

Preparation and characterization of nano-liposome-mediated FL gene in the Lovo cells.

AIMS: The purpose of the present work was to formulate and evaluate cationic nano-liposomes as novel nonviral gene delivery for colon cancer treatment. METHODS: Recombinant pEGFP-c1-Fms-like tyrosine kinase receptor 3 ligand (FL) plasmids containing human FL gene and green fluorescent protein (GFP) reporter genes were constructed. FL and GFP Gene-carrying cationic nano-liposomes were prepared based on the electrostatic adherence principle and then transfected into Lovo cells. The morphology, particle size, and zeta potential of gene-carrying cationic nano-liposomes were observed using an electron microscope. GFP expression was observed by fluorescence microscopy to assay the transfection efficiency. The cytotoxicity of FL/nano-liposomes was evaluated by the MTT method. RESULTS: Recombinant plasmids pEGFP-c1-FL are successfully constructed using gene cloning methods and confirmed by restriction enzyme digestion and sequencing. The cationic nano-liposomes carrying pEGFP-cl-FL were observed by an electron micrograph and showed uniform spherical or elliptical shapes and many pores. The fluorescence microscopy images of gene-carrying cationic nano-liposomes showed good expression of GFP in pEGFP and pEGFP-cl-FL groups. The MTT assay of cell death indicated a significantly higher level of cell death between the FL group and the control group at 24, 48, and 96 hours after transplantation. CONCLUSION: Cationic nano-liposomes show safe and high-performance transfection as gene carriers. Gene therapy has significant implications for colon cancer treatment in future.

Nanoliposome-mediated FL/TRAIL double-gene therapy for colon cancer: in vitro and in vivo evaluation.

OBJECTIVE: To investigate the therapeutic effects of cationic nanoliposome-mediated gene therapy combined with immunotherapy for colon cancer treatment. METHODS: Recombinant plasmids containing green and red fluorescent protein reporter genes were constructed using gene cloning methods. Gene-carrying cationic nanoliposomes were prepared based on the electrostatic adherence principle and then transfected into dendritic cells (DC), which were transplanted into colon cancer cells. RESULTS: Recombinant plasmids containing green or red fluorescent protein reporter genes were successfully constructed by gene cloning and confirmed by restriction enzyme digestion and sequencing. Gene-carrying cationic nanoliposomes were transfected into colon cancer cells, and good gene expression was detected. A better level of apoptosis was observed in the combined group of tyrosine kinase receptor 3 ligand (FL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), while the lowest level was detected in the control group. The parameters in the FL and TRAIL groups were between the above-mentioned combined group. CONCLUSION: Cationic nanoliposomes have the advantage of being gene carriers. The joint therapeutic effects of the two genes are superior to those of a single gene. Gene therapy combined with immunotherapy has significant implications for cancer treatment.

Endovascular treatment of Budd-Chiari syndrome.

BACKGROUND: Budd-Chiari syndrome (BCS) is a posthepatic portal hypertension caused by the obstruction of the lumen of the hepatic veins or the proximal inferior vena cava (IVC). This study aimed to evaluate the clinical experience of interventional therapy for Budd-Chiari syndrome. METHODS: IVC venography was carried out first, the obliteration or stenosis in the IVC was opened or dilated with the hard guided wire or Rups100 puncture needle and balloon, then a stent was routinely implanted for the type of obliteration or stenosis. RESULTS: The procedure was successful in 821 out of 903 cases including IVC intervention in 760 cases, and hepatic vein intervention in 61 cases. An IVC stent was used in 517 cases and hepatic vein stent in 19 cases. There were no pulmonary embolisms, but acute renal failure occurred in eight cases, hepatic coma in two cases and acute heart failure in 43 cases. Two patients died in this group and five cases were complicated with acute IVC thrombosis. Follow up of 7 to 124 months was made in 679 cases with recurrence found in 59 cases. CONCLUSIONS: Interventional therapy is safe and effective with a fast recovery for most types of BCS. It is gradually becoming the first therapeutic choice.

Correlation between the expression of the BAG-1 gene and clinicopathologic factors in colorectal cancer.

OBJECTIVE: The present study aims to investigate the expression and significance of the anti-apoptotic gene Bag-1 in colorectal cancer and to evaluate the relationship between the gene and the disease. METHODS: Bag-1 expression was examined in 320 colorectal cancer and 30 normal colorectal tissue samples using reverse transcriptase polymerase chain reaction (RT-PCR) and the immunohistochemical staining (streptavidin-biotin-peroxidase complex method. RESULTS: Using RT-PCR, Bag-1 was observed to be expressed in colorectal cancer tissues, but not in normal colorectal tissues. The expression of Bag-1 in colorectal cancer was closely correlated with pathologic grade, distant metastasis, Dukes stage, and prognosis, but it was not correlated with the pathologic type, tumor diameter, depth of invasion, and lymph node metastasis. CONCLUSION: Bag-1 protein was found to be overexpressed in colorectal cancer. They might be regarded as a biomarker for the diagnosis of the early stages of colorectal cancer. In addition, they have particular significance for the prognosis of colorectal cancer.

[Treatment of severe acute biliary pancreatitis in the elderly by integrated traditional Chinese and Western medicine].

OBJECTIVE: To summarize the therapeutic effect of integrated traditional Chinese and Western medicine (ICWM) on severe acute biliary pancreatitis (SABP), and to discuss the opportunity of operation. METHODS: The hospitalization duration, incidence of complications, operation transmitting rate and mortality were analyzed in 96 senile SABP patients (Group A) treated by ICWM, and 32 senile SABP patients treated by conventional Western medicine, they were hospitalized from January 2000 to December 2007. RESULTS: (1) The average hospitalization duration in Group A and B was 28.2 +/- 11.3 days and 32.7 +/- 14.3 days respectively, showing insignificant difference between them (P>0.05); (2) The early stage incidence of complications being 29.2% (28/96) in Group A and 34.4% (11/32) in Group B, no significant difference between groups was shown, but a significant difference did show at the late stage, 36.5% (35/96) vs 53.1% (17/32), the incidence in Group A was lower significantly (P<0.05). (3) The two groups were not different in operation transmitting rate 36.4% (35/96) vs 43.8% (14/32), P>0.05. (4) The mortality in Group A, 21.9% (21/96) was lower than that in Group B, 37.5% (12/32), P <0.05. CONCLUSION: ICWM has good effect in treating SABP, and the opportunity of operation transmitting should be decided according to whether there obstruction of biliary tract exists or not.

[Inhibitory effects of high mobility group box 1 antisense nucleotide on invasion of human pancreatic cancer cell line PCNA-1].

BACKGROUND & OBJECTIVE: High mobility group box 1 (HMGB1) is abundantly expressed in most of immature, and malignant cells, and is closely related to cell proliferation, invasion, and migration. We have previously proved HMGB1 over-expressed in pancreatic cancer tissues. This study was to evaluate the in vitro effects of HMGB1 antisense nucleotide on human pancreatic cancer cells invasion, and explore the underlying mechanism. METHODS: An eukaryotic expression vector containing antisense-HMGB1 was transfected into human pancreatic cancer cell line PCNA-1. The expression of HMGB1, matrix metalloproteinase-2 (MMP-2),and MMP-9 mRNA before and after transfection was detected by reverse transcriptase-polymerase chain reaction(RT-PCR); HMGB1 protein expression was examined by Western blot. The activities of MMP-2, and MMP-9 were analyzed by gelatin zymography. The in vitro invasive ability of PCNA-1 cells was determined by Boyden chambers method. RESULTS: Antisense-HMGB1 eukaryotic expression vector was successfully constructed. HMGB1 mRNA expression in PCNA-1 cells was effectively inhibited by antisense-HMGB1. HMGB1 protein expression was also suppressed upon transfecting (P< 0.05). Antisense-HMGB1 transfected cells showed lower MMP-2 and MMP-9 mRNA expression (P< 0.01). MMP-2 and MMP-9 activities were also inhibited by antisens-HMGB1 (P< 0.01). Cells migrating through the Boyden chamber membrane was significantly reduced as compared with the control (P< 0.01). CONCLUSIONS: HMGB1 plays a crucial role in the invasion of pancreatic cancer, and blocking HMGB1 may be a potential strategy in preventing the migration of pancreatic cancer.