Anticancer Research Of Tryptophan Side-chain Oxidase From Pseudomonas And MiR-126-5p In Regulating Tryptophan Metabolism

Hepatocellular carcinoma(HCC)is a common malignant tumor with high mortality and a serious threat to human health.Tryptophan metabolism plays a vital role in the occurrence and development of the tumors.By degrading tryptophan or regulating tryptophan metabolism,tumor growth can be limited while maintaining the body’s normal nutritional requirements.The present research is mainly about the small molecular drugs which are based on the inhibition of the tryptophan metabolism enzyme activity.It shows a certain clinical effect on cancer treatment,but its inhibition effect still has limitations.Therefore,there is need to find enzymes that directly target tryptophan degradation,or other compounds that inhibit tryptophan metabolism in vivo,in order to explore more effective treatment strategies for treating HCC.TSO(Tryptophan Side-chain oxidase)has been extracted from pseudomonas and found to be an enzyme that can degrade tryptophan in vitro,which can degrade tryptophan to β-ketol tryptophan,thus inhibiting tumor growth.In this paper,the degradation ability of TSO enzyme on tryptophan and its anticancer function were discussed.Pseudomonas was used to obtain enzyme solution containing TSO enzyme,which was concentrated by affinity chromatography and enrichment to obtain high purity TSO enzyme.The reaction products of TSO enzyme and tryptophan were identified by HPLC,HPLC-MS and enzyme activity assay.And the targeted degradation of tryptophan was verified,which revealed that the reaction products were in line with expectations.CCK-8,flow cytometry,colony formation,trans-well,q PCR and Western blot were used to verify that TSO enzyme could significantly inhibit the proliferation,invasion and migration of HCCLM3 cells and HepG2 cells in vitro.In vivo experiments,purified TSO enzyme was injected to treat the xenograft model mice by tumor injection and intraperitoneal administration.It was found that the tumor inhibition effect of the tumor injection group was better than that of the intraperitoneal injection group.Immunohistochemistry was used to detect Ki-67,BAX and VEGF,which are the biomarkers of cell invasion and proliferation in each group.And the effect of TSO enzyme on tumor formation and growth in vivo was analyzed.The results also suggested that TSO enzyme could inhibit tumor proliferation and promote tumor apoptosis.Then the TSOⅡ protein was de novo sequenced by LC-MS/MS,and the amino acid sequence of TSOⅡ protein was obtained.Then,the structure prediction and physicochemical properties of the protein were analyzed by biological software such as Swiss-model,ExPASy and TMHMM.In this paper,the anticancer effect of TSO protein was preliminarily analyzed,which provided basic conditions for the development of anticancer drugs based on TSO protein.Tryptophan-2,3-dioxygenase(TDO2)is a key enzyme in kynurenine metabolism pathway,which is involved in the occurrence and development of various tumors and has the highest expression in the liver.By regulating the expression of TDO2,it can regulate the metabolism of tryptophan in vivo to achieve the effect of inhibiting tumor growth.While miRNAs can interact with target m RNA and participate in cell metastasis.In this study,664 miRNAs that can bind to TDO2 were identified by RNA immunoprecipitation assay.miR-126-5p was identified as the target of the study by Target Scan prediction.Furthermore,qRT-PCR and Western blot analysis confirmed that overexpression of miR-126-5p promoted the expression of TDO2,PI3 K,AKT and WNT1.Meanwhile,the overexpression of miR-126-5p and TDO2 can promote the metabolism of intracellular tryptophan into kynurenine,which was verified by HPLC.In this paper,we also verified that miR-126-5p can promote the proliferation,invasion and migration of HCCLM3 cells by CCK-8,trans-well,flow cytometry and scratch tests.In vivo experiments have also verified that miR-126-5p agomir injection can significantly accelerate the formation and growth of tumor tissues.In addition,it was found by immunohistochemical detection that miR-126-5p agomir can increase the expression of Ki-67 and VEGF in tissues,and decrease the expression of BAX.Therefore,our results suggest that miR-126-5p plays a promoter role in the progression of HCC,and is a biomarker and new potential therapeutic target.Mi R-126-5p inhibitors can be used to develop anticancer drugs in the future.In summary,on the one hand,TSO enzyme can directly degrade tryptophan and block tryptophan intake from the source,and its anticancer function was verified in vivo and in vitro.On the other hand,by studying related microRNAs,miR-126-5p was found to regulate the enzymes in the tryptophan metabolic pathway,thus preventing tryptophan from being metabolized in the body according to the kynurenine pathway.So as to achieve the effect of inhibiting tumor growth.This study laid a good foundation for the exploration and development of anticancer drugs combining small molecule inhibitors and TSO enzyme in the future.