Expression Of Recombinant Irisin In Pichia Pastoris And Its Anticancer Activity Against Human Pancreatic Ductal Carcinoma

Modern medicine has made new progress in the control of cancer.However,due to the limitations of treatment methods and the toxic side effects and drug resistance of chemotherapy drugs,cancer is still the leading cause of death worldwide.And with the improvement of living standards of more and more Chinese people,the incidence of cancer is increasing due to the lack of exercise,the number of patients with obesity and type 2 diabetes is increasing.It has been proved that exercise and obesity can reduce the incidence of cancer and the risk of recurrence,play a positive role in tumor-assisted chemotherapy.The mechanism by which exercise benefits cancer is still being studied.Irisin is a kind of myokine that is found in recent years when skeletal muscle is activated by exercise.It can up-regulate the proliferator-activated receptor（PPAR）-activator-1 and the coupling protein 1（UCP1）,induce the brown-ing of white adipose tissue.Irisin can be used as a drug to treat obesity and some metabolic diseases,including obesity,type 2 diabetes mellitus（T2DM）,lipid metabolism and cardiovascular disease（CVD）,non-alcoholic fatty liver disease（NAFLD）,etc.As an exercise-related myokine and adipokine,irisin is widely distributed in the body.Changes in irisin levels in cancer patients and in vitro effects of irisin on various cancers and their mechanisms have been reported.As a result,irisin has the potential to become a treatment for obesity and some metabolic diseases.In this study,three parts were mainly carried out.In the first part,the recombinant irisin expression system was constructed by using the eukaryotic expression system of pichia pastoris,the glycosylated E-irisin was expressed and purified.The anti-cancer activity of recombinant irisin on several obesity-related cancers was evaluated.The second part mainly studies the inhibitory effect of recombinant irisin on the proliferation,migration and invasion of pancreatic ductal adenocarcinoma cells and its mechanism.In the fourth part,the effects of irisin and DOX on the apoptosis of pancreatic ductal adenocarcinoma and the possible mechanism were discussed.Part I:eukaryotic expression of recombinant E-irisin in P.pastoris and determination of anti-cancer activity of recombinant irisinAccording to P.pastoris codon preference,irisin sequences containing 6×His tag were synthesized.Restriction enzymes EcoR I,Not I was used to connect the target gene with the gene expression plasmid to build pPIC9K-irisin,The correct plasmid was linearized and transformed into P.pastoris strain GS115,highly expressing GS115-pPIC9K-irisin clones were were selected by G418.An approach of optimized expression of GS115-pPIC9K-irisin was established,the optimized induction conditions were as follows:pH 6.0,30°C,1%methanol,96 h.Then irisin was verified by Western blot and PAS staining.Glycosylated recombinant E-irisin was obtained after purification.Recombinant E-irisin expressed from eukaryotic and recombinant P-irisn expressed from prokaryotic were used to verify the inhibitory effect of recombinant irisin in this study on breast cancer and lung cancer,which was consistent with the report.For the first time,we found both recombinant E-irisin and recombinant P-irisn has antitumor activity against MIA PaCa-2 cells of pancreatic ductal adenocarcinoma.However,no significant effect of irisin was found on liver cancer cells BEL-7402,human acute promyelocytic leukemia NB4,and human multiple myeloma RPM18226.PartⅡ:inhibitory effect and mechanism of recombinant irisin on proliferation,migration and invasion of pancreatic ductal adenocarcinoma cell MIA PaCa-2Immunofluorescence analysis showed that irisin may have unknown receptors on the surface of MIA PaCa-2 cell membrane.MTT and colony formation assay showed that recombinant E-irisin and P-irisin inhibited the growth of MIA PaCa-2 cells and inhibited the ability of cell colony formation.Flow cytometry analysis suggested recombinant E-irisin and P-irisin can cause G0/G1 phase block of MIA PaCa-2 cell cycle and reduce the level of Cyclin D1.Wound healing assay and Tanswell experiments showed that the migration and invasion of MIA PaCa-2 cells were also inhibited by recombinant irisin,and the recombinant E-irisin and P-irisin altered the expression of marker proteins E-cadherin and vimentin in the EMT process.In addition,this study found that E-irisin and P-irisin biological activated cells energy metabolism regulating AMPK key molecules,and cause the inhibition of intracellular mTOR signaling pathways,lowered its target molecules p70S6K,4E-BP1 phosphorylation in the MIA PaCa-2 cell,effecting the metabolism of protein.The study did not found significant differences between E-irisin and P-irisin in in the process of inhibition of pancreatic ductal adenocarcinoma,which means irisin glycosylation structure does not affect its activity in MIA PaCa-2 cell.PartⅢ:effect of recombinant irisin on DOX chemotherapy sensitivity in pancreatic ductal adenocarcinoma.In this study,it was found that irisin can significantly reduce the IC₅₀ value of DOX in MIA PaCa-2 and BxPC-3 cells,enhance the anti-cancer activity of DOX at low concentration,improve the intracellular caspase-3 and PARP protein breaking of MIA PaCa-2 and BxPC-3,reduce the expression of bcl-2 and bcl-xl,and increase the apoptosis of MIA PaCa-2 and BxPC-3 cells caused by DOX.Irisin down-regulated the expression of p-AKT and p-p65,up-regulated the decrease of IkB-αinduced by DOX,and enhanced the sensitivity of MIA PaCa-2 and BxPC-3 cells to DOX by inhibiting the PI3K/AKT and NF-κB signaling pathways.