Biological Function And Mechanism Of ALOX12 In The Development And Progression Of Gastric Cancer

Background and ObjectiveGastric cancer(GC) is one of the most common malignant digestive tumors,with high incidence and mortality rate.China is a country with a high incidence of GC,nearly half of the gastric cancer patients live in China.GC is the second most frequently diagnosed cancer and the leading cause of cancer in China.Although surgery combined with radiotherapy and chemotherapy has made great progress in recent years,the prognosis of patients with advanced gastric cancer is still not ideal.Metastasis is the main cause of poor prognosis in patients with advanced gastric cancer.Epithelial mesenchymal transformation(EMT)is a dynamic process in which epithelial cells acquire mesenchymal features.EMT has been associated with a variety of tumor functions,including tumor initiation,invasion,metastasis,tumor stemness and resistance to therapy.In almost all malignant tumors,malignant progression depends on the activation of EMT.Therefore,it crucial to investigate the molecular mechanism underlying the progression of GC.It is an urgent problem to find early diagnostic markers and new targets for treatment of GC.Arachidonate 12-Lipoxygenase(ALOX12)is a key member of the lipoxygenase family,which plays a vital role in many physiological and pathophysiological processes including Angiogenesis,inflammation,arteriosclerosis and cancer.It has been reported that the expression of ALOX12 is increased in a variety of tumors.Some studies revealed a close association with ALOX12 and EMT.However,the function and mechanism of ALOX12 with EMT is not clear in GC.In the first part of this study,we detected the expression of ALOX12 in clinical specimens of gastric cancer and adjacent normal tissues,further analyzed the relationship between ALOX12 expression and clinical pathologic characteristics and prognosis;Next,the RNA-sequencing method was used to detect the differentially expressed genes regulated to ALOX12 and to provide new ideas for further research.In the third part,observed the malignant biological function of ALOX12 in GC cells in vitro and in vivo;In the fourth part,we explored the molecular mechanism of ALOX12 promoting the development of GC biology.So the aim of this study was to detect the expression of ALOX12 in gastric cancer and its clinical significance,and to clarify the biological effect and molecular mechanism of ALOX12 in GC cells,and also can provide new evidence and direction for early monitoring and targeted therapy of GC.Methods(1)The expression level of ALOX12 in GEO gastric cancer database was analyzed.The expression of ALOX12 in normal gastric epithelium and gastric cancer cell lines was detected by RT-PCR and Western blot(WB).The expression of ALOX12 in gastric cancer and paracancerous tissues was detected by immunohistochemistry(IHC),and the relationship between ALOX12 expression and clinicopathological factors and prognosis was analyzed.(2)The overexpression stable GC strain of ALOX12 was constructed by lentivirus and verified by RT-PCR and WB.RNA-sequencing method was used to detect the over-expressed ALOX12 and negative control GC cells and bioinformatics analysis was performed to analyze the differential expressed genes and GO analysis,KEGG enrichment analysis and PPI network.(3)In vitro experiment,CCK8,colony formation assay and Transwell migration and invasion chamber were used to detect the effect of ALOX12 on the proliferation,migration and invasion of GC cells.The effects of ALOX12 on the proliferation,migration and invasion of GC cells in vivo study were detected by subcutaneous tumor model and tail vein metastasis in nude mice.(4)The expression of EMT markers in transfected cell was detected by RT-PCR,WB and immunofluorescence(IF),and the effect of overexpression of ALOX12 on Wnt-?-catenin pathway and EMT-related proteins were detected by WB.Results(1)GEO database analysis showed that the expression of ALOX12 in gastric cancer was significantly higher than that in paracancerous tissues,and it was related to the poor prognosis of gastric cancer.RT-PCR and WB results showed that the expression of ALOX12 in BGC823,MGC803,AGS and HGC27 cells was higher than that in GES-1.IHC detection showed that ALOX12 was highly expressed in gastric cancer tissues than paracancerous tissues.The high expression of ALOX12 was closely related to lymph node metastasis,TNM stage,vascular invasion.But there was no significant correlation with the age,sex and tumor location of the patients.The survival time of GC patients with high expression of ALOX12 was shorter than the GC patients with low level of ALOX12.ALOX12 is one of independent prognostic factor for GC patients;(2)We succeeded in constructing ALOX12 overexpression and negative control lentivirus,then BGC823 and MGC803 cell lines were transfected and selected by puromycin;Through RNA-seq of overexpressed ALOX12 and negative control MGC803 cells,258 differential expressed genes were screened,including 165 genes up-regulated DEGs and 93 down-regulated DEGs.GO analysis showed that many DEGs were involved in the positive regulation of cell proliferation and migration,cell adhesion and molecular signal transduction.KEGG analysis showed that genes were significantly enriched in adhesion junctions,extracellular matrix components and tumor-related pathways.PPI analysis showed that the TOP ten genes of key nodes were MMP9,GNG7,ITGB2,ITGAX,BDNF,NT5 E,CASP1,LOX,SAA1 and CSF1,which were mostly involved in tumor-related biological processes.(3)CCK8 experiment showed that overexpressing ALOX12 promote cell proliferation,colony assay showed that overexpression of ALOX12 promoted colone formation ability of GC cells,Transwell migration and invasion assay showed that overexpression of ALOX12 promoted migration and invasion of GC cells,and Nude mice assays revealed that faster proliferation and metastasis in BGC823-OE compared with the control group;(4)ALOX12 promoted the morphological changes of EMT in GC cells;the results of RT-PCR,WB and IF showed that overexpression of ALOX12 can significantly decrease the E-cadherin,and increase the expression of N-cadherin and Snail,which considered as EMT markers,and promote the EMT process;ALOX12 promoted EMT by activating the Wnt-?-catenin pathway and up-regulate the expression of downstream protein like TCF-1,c-MYC and Cyclin D1 in GC cellsConclusion(1)The expression level of ALOX12 in gastric cancer was higher than that in paracancerous tissues.The overall survival time of patients with high expression of ALOX12 was shorter,ALOX12 was one of independent prognostic factor for GC patients2.The expression of ALOX12 is most closely related to cell adhesion,extracellular matrix and epithelial phenotype regulation in GC cells3.Overexpression of ALOX12 in GC cells can promote the proliferation,migration and invasion in vitro and in vivo.4.ALOX12 can promotes EMT via Wnt-?-catenin pathway in GC cells.