The Mechanism Study Of Perineural Invasion In Pancreatic Cancer:Effects Of TGF-β1/ALK5Signaling On NGF Expression

Part1:The influence of TGF-β1on NGF expression in the pancreatic cancer cellsAims:The overexpression of NGF was related to the development of pancreatic cancer, especially perineural invasion. TGF-β1was reported to induce NGF expression in the nerve cells, dental pulp cells and pancreatic stellate cells. However, there was no literature about the relation between TGF-β1and NGF in pancreatic cancer cells. This study aims to reveal the effect of TGF-β1on the expression of NGF in the pancreatic cancer cells.Methods:Pancreatic cancer cell lines Panc-1, MIA PaCa-2and BxPC-3were respectively incubated with the recombined human TGF-β1protein at different concentrations (0,0.5,1.0,5.0,10.0and50.0ng/ml) and over various periods of time (24,48and72h) in serum-free Medium. Real-time RT-PCR, ELISA and Western Blot were used to evaluate the expression level of NGF in the three cell lines. The pancreatic cancer cells were then co-incubated with TGF-β1and its neutralizing antibody. The assessment of the change of the NGF level was used to prove the influence of TGF-β1on the NGF expression.Results:The NGF expression was upregulated in Panc-1and MIA PaCa-2cell lines by TGF-β1. However, it could not influence the NGF expression in BxPC-3cell line. The results showed that with the TGF-β1concentration increasing, the expression of NGF increased firstly and then decreased in these two cell lines. When the TGF-β1concentration was5.0ng/ml (Panc-1) and1.0ng/ml (MIA PaCa-2), the NGF reached the top expression level respectively after72h. Time was a positive regulator to the expression of NGF. The level of NGF in the two cell lines increased as the time passing. The neutralizing antibody decreased the expression level of NGF when co-incubating with TGF-β1.Conclusions:TGF-β1was one cause for the NGF overexpression in the pancreatic cancer cells, but a negative factor might also take a part in this mechanism at the same time. NGF might mediate the influence of TGF-β1on perineural invasion of pancreatic cancer cells. Part2:The role of ALK5in the expression of NGF induced by TGF-β1in pancreatic Panc-1cellsAims:to reveal the role of ALK5in the TGF-β1inducing expression of NGF in the Panc-1cell line.Methods:The ALK5expression level was detected by Western Blot in Panc-1cell line which was incubated with TGF-β1. The specific siRNA oligos for ALK5gene were designed and synthesized. Then we chose the most effective one on knockdown of the target gene via real-time RT-PCR and Western Blot. The recombined human TGF-β1was used to culture the siRNA transfected Panc-1cells, and then Real-time RT-PCR, ELISA and Western Blot were used to evaluate the NGF expression level. SB431542and TGF-β1were co-incubated with the Panc-1cells, and then the NGF expression level was detected by ELISA.Results:The ALK5expression level increased when the TGF-β1was used to treat the Panc-1cells (P<0.05). Two anti-ALK5siRNAs were successfully synthesized and the effects of knockdown on the ALK5gene were70.92%and42.70%respectively. The inducing expression of NGF by TGF-β1in Panc-1cells was enhanced by anti-siRNA and SB431542(P<0.05).Conlusions:ALK5negatively regulated the expression of NGF in Panc-1cell line induced by TGF-β1.Methods:A systematic literature research was performed to identify comparative studies on CP and DP. Perioperative and long-term outcomes constituted the end points. Pooled risk ratios (RR) and weighted mean differences (WMD) with95%confidence intervals (95%CI) were calculated using either fixed-effects or random-effects model.Results:Nine studies with735patients were included in this meta-analysis. Although CP cost more operative time than DP, the two groups had no significant differences in the volume of intraoperative blood loss, rate of intraoperative blood transfusion and length of postoperative hospital stay. According to the postoperative outcomes, although the CP group had higher overall complication rate (Fixed effects model; RR:1.30;95%CI:1.05-1.62; P<0.05) as well as overall pancreatic fistula rate (Fixed effects model; RR:1.58;95%CI:1.20-2.08; P<0.05), the two groups did not differ significantly in the fateful surgical complications such as clinically significant pancreatic fistula (Grade B and C), postoperative bleeding, reoperation and intra-abdominal effusion/abscess. Furthermore, the perioperative mortality rate was comparable between the two groups. During the follow-up period, the patients after DP were more likely to suffer pancreatic exocrine insufficiency (Fixed effects model; RR:0.53;95%CI:0.32-0.86; P<0.05) and endocrine impairment (Fixed effects model; RR:0.19;95%CI:0.11-0.33; P<0.05).Conclusion:CP is an acceptable and feasible procedure, and it has the advantage of preserving the pancreatic exocrine and endocrine function.