Objective: To investigate the relationship between the Smad4-independent pathway of TGF-?1 and drug-resistance of pancreatic cancer. Methods: The sensitivities of Smad4 homozygous-deleted pancreatic cancer cell line BxPC3 to different kinds of anti-cancer drugs (5-Fu, Gemzar, Oxaliplatin, Cisplatin, CPT-11 and Epirubicin) were observed by MTT assay before and after they were transfected with full-length cDNA of TGF-?1 or treated with TGF-?1 (5 and 10 ng/ml) solution. Western blot was used to detect p170 protein expression after stimulation with different concentrations of TGF-?1. Results: Cisplatin had the most powerful killing effect on BxPC3 cells, followed by Oxaliplatin, 5-Fu and CPT-11 with moderate effect and Gemzar and Epirubicin with the least effect. Cells transfected with full-length cDNA of TGF-?1 or treated with TGF-?1 solution became less sensitive to Cisplatin. Western blot revealed upregulation of p170 expression by TGF-?1. Conclusion: The Smad4-independent pathway of TGF-?1 can increase the drug resistance of pancreatic cancer cells through upregulating expression of p170.

Objective To study the expression profile of microRNA (miRNA) and the roles in pathogenesis of acute liver failure in mouse model. Methods Eighty-five BALB/c mice were divided into four groups: 40 in model group of acute liver failure were intraperitoneally injected with Dgalactosamine (D-GalN) and lipopolysaccharides (LPS); 20 in D-GalN group were injected with DGalN only; 20 in LPS group were injected with LPS only; 5 in control group were injected with saline.Liver histology of mouse was observed at hour 0, 5, 7 of injection, and sera and liver tissues were collected at hour 0, 1, 3, 5, 7, 9 of injection. Meanwhile, levels of inflammatory factors [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in serum and liver tissue were detected by realtime polymerase chain reaction (PCR). Lock nucleic acid (LNA)-based miRNA microarray technology was used to detect the expression profile of hepatic miRNA, and the expression of miRNA was verified by real time quantification-polymerase chain reaction (RT-PCR). Mouse macrophage Raw264.7 cells were induced by LPS in vitro and the expressions of miRNA at different time points were detected.The comparison of means among groups was analyzed using one way ANOVA and the correlation were analyzed by Pearson and Spearman correlation. Results Microarray analysis found that the expression profile of miRNA during the acute liver failure changed dramatically. There were 97 miRNA in model group changed significantly compared with control group (P＜0.01), including 21 up-regulated and 27down-regulated at hour 5 and 7 of injection. Furthermore, the expressions of miR 146a and miR-155were verified by RT-PCR and found they both increased progressively over time after injection.Correlation analysis showed that miR-155 was well correlated with both TNF-α and IL-6 expressions.It was further found that miR-146a and miR-155 were both up-regulated in activated Raw264.7 cells in vitro. Conclusions The expression profile of miRNA changes during acute liver failure in mouse model. Inflammation associated-miR-146a and miR-155 are both up-regulated significantly, which indicatcs that they may play an important regulatory role in pathogenesis of acute liver failurc.

Objective To over-express human trefoil factor 2 (hTFF2) by Escherichia coli system and an-alyze its activities in promoting migration and anchorage-independent growth in SW480 colonic cancer cells. Meth-ods hTFF2 gene encoding mature peptide was obtained by RT-PCR, and the recombinant expression vector pET32a-hTFF2 was constructed. Then pET32a-hTFF2 was transformed into E. coli BL21-32a and TrxA-hTFF2 fu-sion protein was induced to over-express. The expressed product was isolated by Ni-NTA affinity chromatography, purified by dialysis and identified by Western blotting. The activities of the recombinant hTFF2 in promoting SW480 cells migration and anchorage-independent growth were analyzed by MicroChemotaxis Chamber migration assay and Soft-agar assay,respectively. Results The TrxA-hTFF2 fusion protein was expressed to 220 mg/L at high purity. In vitro model demonstrated that recombinant hTFF2 obviously enhanced SW480 cell migration activity and anchor-age-independent growth. Conclusion The recombinant hTFF2 can be expressed in E. coli with high production, purity and biological activities. And its roles in cell migration and anchorage-independent growth suggest that up-regulation of TFF2 in colonic cancer might be involved in cancer invasion and metastases.

Objective To observe the regulatory role of microRNA-1187(miR-1187)in hepatocyte apoptosis through miR-1187 targeting regulation of caspase-8 mRNA expression.Methods The acute liver failure model was established by injection of D-galactosamine plus lipopolysaccharides(LPS)in BALB/c mice.The liver tissues were collected for LNA-miRNA array analysis and functional analysis of genes targeted by miRNA.Embryonic murine hepatocyte cell line 2(BNL-CL2)was cultivated in vitro and treated with tumor necrosis factor(TNF)-α and D-galactosamine to induce the transfection of miR-1187 in transfected group or untransfected group.The expressions of miR-1187 and caspase-8 mRNA were detected by real-time polymeramse chain reaction(PCR)and caspase-8 protein was determined by Western blot.The apoptosis rate was detected by flow cytometry.The comparison of means between groups was done by t test.Results The miR-1187 signal was deceased with the development of acute liver failure.The 3'UTR of caspase-8 mRNA had direct binding sites with miR1187.In BNL-CL2 cell experiments,miR-1187 was down-regulated in untransfected group and decreased more slowly in transfected group(t=6.371,P<0.01).The expression of caspase-8 mRNA was up-regulated in untransfected group and increased less in transfected group(t=4.539,P<0.01).The apoptosis rate in transfected group was significantly lower than untransfected group(t=3.365,P<0.05).Conclusios miR-1187 is one of inhibitors of hepatocyte apoptosis.High expression of miR-1187 could regulate the expression of caspase-8 mRNA,thus inhibit the apoptosis of hepatocytes.

Objective To explore the expressions of circulating microRNAs (miRNAs) in acute liver failure mice induced by D-galactosamine (GalN)/lipopolysaccharides (LPS) and the correlation with miRNAs in the liver.Methods Forty clean grade Balb/C mice,with 32 in the model group and 8 in the control group were enrolled in the study.Liver failure was induced by intraperitoneally injection of D-GalN and LPS in mice of the model group,while mice of the control group were intraperitoneally injected with 1 mL 0.9 ％ sodium chloride solution.Serum and liver samples were collected at 0,3,5,7 hours following administration,and eight mice should be supplied to each sample,and changes of alanine aminotransferase (ALT),aspartate aminotransferase (AST) and histopathology of the liver were observed.miRNA from both the serum and the liver was extracted,miRNA expression profile in the liver at 0,5,7 hours by locked nucleic acid (LNA)-miRNA microarray was analyzed and miRNA by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was detected.Means of the two groups were compared using one-way ANOVA and correlation analyses were performed using Pearson and Spearman correlation.Results Expression of miRNAs in the liver tissue changed significantly over time with the occurrence of acute liver failure in the mice.Twenty-one miRNAs were up-regulated and 27 were down-regulated,among which miRNA-122 and miRNA-1187 were down-regulated while miRNA-146a and miRNA-155 were up-regulated.It was confirmed by the PCR assay that the expression of miRNA-122 and miRNA-1187 in the liver gradually decreased,while those in the serum were up-regulated over time.However,the expressions of inflammation associated miRNA-155 and miRNA-146a were up-regulated both in the serum and the liver after administration.The expressions of miRNA-122 and miRNA-1187 were negatively correlated between serum and liver (r=-0.477,P=0.0089,r=-0.420,P=0.231),while the expressions of miRNA-155 in serum and liver were positively correlated (r=0.678,P=0.0001).Moreover,the expressions of miRNA-122 (r=0.571,0.554) and miRNA-1187 (r=0.471,0.542) were also positively correlated with serum levels of ALT and AST (all P＜0.05).Liver and serum levels of miRNA-122 and miRNA-1187 changed significantly at 5 hours after administration,which preceded the changes of ALT/AST.Conclusions The expressions of miRNA-122 and miRNA-1187 in serum are well inversely correlated with the corresponding expressions in liver tissues during acute liver failure in mice.The changes of miRNA-122 and miRNA-1187 in the serum precede those of ALT/AST.These data suggest that serum miRNA-122 and miRNA-1187 might be the candidate serum biomarkers for early prediction of liver injury.

Objective To investigate the establishment of focal cerebral ischemia model in rabbits with the improved suture method.Methods A total of 45 healthy and clean adult New Zealand rabbits were divided into either a sham operation group (n =5) or a model group (n =40) using random number table method before modeling,and the sex was not limited.The self-made head ends of 2-0 fishing lines dipped in paraffin were used as the sutures.The external carotid artery was cut and inserted into a intracranial artery through the internal carotid artery and blocked the origin of middle cerebral artery.The neurological function score was performed after 6 h.The neurological deficit scores ≥2 was successful modeling.The rabbits were killed by anesthesia.The brain slices were stained with 2％ 2,3,5-triphenyl tetrazolium chloride solution.The infarct foci were observed.The diameters of suture head and the depth of suture insertion were compared in the model rabbits with successful modeling,failure,and death in the model group.Results There were 40 rabbits in the model group,six of them died,including 4 died of subarachnoid hemorrhage within 4 h after operation,and 2 died from anesthetic accident.The mortality rate was 15.0％.Seven rabbits failed,mainly because of cerebral vasospasm and the insertion depth of suture was insufficient.Twenty-seven had successful modeling,and the success rate was 67.5％.All the rabbits in the sham operation group survived.The diameter of the suture head and insertion depth in the successful modeling rabbits were compared with the death and failure outcome in rabbits.The difference was statistically significant (diameter:0.52 ± 0.14 mm vs.0.45 ±0.40 mm and 0.58 ±0.17 mm;depth:5.49 ±0.17 cm vs.6.04 ± 0.11 cm and 4.26 ±0.30 cm;all P ＜ 0.05).Conclusions The improved suture method can successfully prepare the focal cerebral ischemia model of middle cerebral artery in rabbits.The method is simple.Its repeatability and practicability are better.

Objective To screen the secretory factor-related, mechanoresponsive microRNAs (miRNA) of osteocytes. Methods Cyclic mechanical tensile strain (ε=2.5,f=0.5 Hz) was applied to osteocytes and osteoblasts cultured in vitro respectively, and the differentially expressed miRNAs only in the osteocytes were screened out by using miRNA chip. Through bioinformatics technology, in these differentially expressed miRNAs, the target genes of secretory factors including insulin-like growth factor-1(IGF-1), nitric oxide synthesase (NOS), fibroblast growth factor 23 (FGF23) and sclerostin (SOST) were further screened out. Then the selected miRNAs were compared with the biochip detected, differentially expressed miRNAs in femur bone of the mice which were trained on treadmill, and four of these miRNAs were randomly selected for quantitative PCR verification. Results For the 77 differentially expressed miRNAs only in the mechanically strained osteocytes in vitro, 22 miRNAs whose target genes were the 4 secreted factors (IGF-1, NOS, FGF23 and SOST), were screened out. Moreover, a total of 11 miRNAs in the 22 miRNAs were differentially expressed in femur bone of the treadmill trained mice with the same trend as those in osteocytes in vitro, and the randomly selected miR-361-3p, miR-3082-5p, miR-6348 and miR-706 were confirmed to be differentially expressed with the same trend in femur bone and osteocytes. Conclusions These mechanoresponsive miRNAs differentially expressed only in osteocytes, such as miR-361-3p, miR-3082-5p, miR-6348 and miR-706, probably influence osteoblastic differentiation or bone metabolism through regulating the secretory factors.

Objective: To investigate the mechanism of resveratrol promoting fibronectin type Ⅲ domain-containing 5 (FNDC5) degradation in skeletal muscle of male obese mice． Methods: Six-week-old male C57BL /6 mice were randomly divided into three groups : standard control diet ( SCD) ，high-fat diet ( HFD) and high-fat diet treated with resveratrol (HFD + RES) ．HFD + RES group was intervened with resveratrol via gavage ［400 mg / kg · d) ］ while fed HFD for 20 weeks．The body mass，serum TG，TC，LDL-C and HDL-C levels were detected．The pathological changes in skeletal muscle were detected by HE staining．The expression of FNDC5，SIRT1，SIRT2，LC3， p62，Beclin-1，ATG5，ATG7 was assessed by immunohistochemistry，RT-PCR and Western blot respectively. Results: The body mass ，serum TG ，TC and LDL-C levels increased significantly ，meanwhile HDL-C levels decreased in HFD group．Lipid deposition between skeletal muscle fibers were obvious in HFD group．The immuno- histochemistry results showed that protein expression levels of SIRT1，SIRT2 and LC3 obviously decreased，while the protein levels of FNDC5 and p62 obviously increased．The expression levels of FNDC5 significantly increased， while the gene expression levels of SIRT1，SIRT2，LC3，Atg7 and Beclin-1 obviously decreased．All these responses were attenuated by treatment with RES． Conclusion RES has obvious effects of lipid-lowering and promoting FNDC5 degradation in skeletal muscle tissues，which may be related with SIRT1 and SIRT2-induced autophagy， thus resulting in degradation of FNDC5 .