The Effect Of LOX/LOXL1 On Gastric Cancer And Its Mechanism

Objective: Gastric cancer is one of the most important cancers that threaten human health and it's the third leading cause of cancer death worldwide.Most gastric cancers have no obvious symptoms in the early stage and are easily ignored.Therefore,the early diagnosis rate of gastric cancer in China is low.Surgical resection is the only curative treatment for gastric cancer,but most patients have progressive disease at the time of diagnosis and lose their chance of surgery.Although many new treatments for gastric cancer have emerged in recent years,the prognosis of patients with advanced disease is still poor,so it is urgent to find biomarkers that have good specificity and sensitivity for the diagnosis and prognosis in gastric cancer.The lysyl oxidase(LOX)family is a group of extracellular copper-dependent amine oxidases,including five members,LOX and LOXL1-LOXL4.The primary role of the LOX family is covalently cross-linking collagen and elastin to maintain the normal structure and function of the extracellular matrix.Recent studies have found that the LOX family plays an important role in tumor advancement,invasion and metastasis.The role of LOX and LOXL2 in promoting various tumor migration and invasion has been well defined.LOXL4 has been reported to promote gastric cancer invasion and metastasis.LOXL3 has been reported to be highly expressed in highly aggressive gastric cancer cells,which is closely related to invasion and metastasis in gastric cancer.However,the role of LOXL1 in gastric cancer has not been reported,and its role remains unclear.The first part of the study used TCGA and GEO databases to analyze the relationship between the expression of LOXL1 and the prognosis,clinicopathological parameters in gastric cancer,and to determine the effect of LOXL1 on the prognosis in gastric cancer.Further cell experiments have investigated the mechanism of LOXL1 affecting the prognosis in gastric cancer.The results will provide new biomarkers for the diagnosis in gastric cancer.The second part of the study used hypoxic-resistant gastric cancer cell line to more realistically simulate the microenvironment of tumor cells in vivo,and explored the mechanism of LOX promoting gastric cancer cell migration under long-term hypoxic conditions.This result provided a new therapeutic target for gastric cancer metastasis.Methods: 1.Comparing the expression of LOXL1 in tumor tissues and normal tissues using TCGA database.2.COX univariate/multivariate regression analyses the relationship between LOXL1 expression and prognosis in gastric cancer.3.The gene expression profile gastric cancer sample data set GSE62254 was downloaded from NCBI's GEO database.The relationship between LOXL1 expression and clinicopathological features such as gender,age,TNM stage and Lauren classification was analyzed by chi-square test.4.RT-PCR detects intracellular m RNA expression levels.5.Western blot detects protein expression levels.6.Transwell detects cell transfer ability.7.MTT and colony formation assay detects cell proliferation capacity.8.Flow cytometry detects cell proliferation and apoptosis.9.Observing the morphology of parental gastric cancer cells and hypoxic resistant gastric cancer cells by microscopy.Results: 1.Compared with normal tissues,LOXL1 is highly expressed in tumor tissues,and high expression of LOXL1 is associated with poor prognosis in gastric cancer.2.LOXL1 is an independent prognostic factor in gastric cancer.3.LOXL1 tends to be highly expressed in gastric cancer patients with advanced T stage(p<0.001)and advanced N stage(p=0.046).LOXL1 tends to be high in patients with Lauren typing diffuse(p < 0.001).LOXL1 expression levels did not differ significantly between gender,age,and M stage.4.LOXL1 had no significant effect on the migration in gastric cancer.After transiently silencing/overexpressing LOXL1 in gastric cancer cells,there was no significant change in cell migration ability.5.LOXL1 promotes the proliferation in gastric cancer cells.After transiently silencing LOXL1,the proliferation capacity and colony forming ability of gastric cancer cells HGC27 were weakened.After overexpressing LOXL1,the proliferation ability and colony forming ability of gastric cancer cell MGC803 were enhanced.6.LOXL1 has no effect on apoptosis.After transiently silencing LOXL1,the apoptotic ability of HGC27 did not change significantly,and the apoptosis-related proteins caspase3 and PARP showed no significant changes.7.Silencing LOXL1 can cause G1 arrest.After transiently silencing LOXL1,HGC27 showed obvious G1 arrest,and the expression of cyclin D1 in G1 phase was significantly decreased,and the expression of cyclin B and cyclin A was not significantly changed.8.LOXL1 activates the WNT/?-catenin/cyclin D1 pathway.GSEA enrichment analysis showed that there was a correlation between LOXL1 and WNT pathway.After transiently silencing LOXL1,there was no significant change in SMAD2/3,and the phosphorylation level of ?-catenin as well as the expression of cyclin D1 were significantly decreased.After overexpressing LOXL1,?-catenin phosphorylation levels and cyclin D1 expression levels were significantly elevated.9.Compared with the parental gastric cancer cells,the metastatic ability of hypoxic-resistant gastric cancer cells were enhanced,and the expression of LOX m RNA and protein were significantly increased.10.LOX was up-regulated in hypoxic-resistant gastric cancer cells and promoted metastasis.After transiently silencing LOX m RNA and protein in hypoxic gastric cancer cells,the migration ability was reduced.11.LOX promoted hypoxic-resistant gastric cancer cell metastasis via activating the AKT pathway.Compared with the parental gastric cancer cells,the expression of P-AKT was up-regulated in hypoxic-resistant gastric cancer cells;after transiently silencing LOX,the expression of LOX in hypoxic-resistant gastric cancer cells decreased,and the phosphorylation level of AKT was also significantly decreased.And after using the AKT pathway inhibitor,the migration ability of hypoxic-resistant gastric cancer cells was significantly reduced.Conclusion: 1.The high expression of LOXL1 is associated with poor prognosis in gastric cancer,and LOXL1 is an independent prognostic factor for gastric cancer.2.The expression of LOXL1 is different between different clinicopathological features.LOXL1 tends to be highly expressed in patients with advanced T stage.3.The expression of LOXL1 does not affect the migration in gastric cancer cells;4.LOXL1 promotes proliferation in gastric cancer cells via WNT/?-catenin/cyclin D1 pathway.5.Under long-term hypoxic conditions,LOX is up-regulated and promotes hypoxic-resistant gastric cancer cell metastasis by activating AKT signaling pathway.