The Mechanism Of LGR6-AKT/mTOR Signaling Pathway In Gastric Cancer

ObjectiveGastric cancer(GC)is one of theimportantdiseasesthreateninghumanhealthinthe world andit is the second cause of cancer-related death in women and thethird in men in China.Although the clinical management has improved,the treatment of GC remains challenging owing to the complexity of the disease and difficulty in early diagnosis.Early diagnosisisof themost importanceto improve theprognosis of GC.Moreover,it is still needed to institute more effective therapeutic strategieswhich depends on new therapeutic targets.Investigating the molecular mechanisms of GC progression is crucial to identify new therapeutic targets.Wnt signaling pathway is an important signaling pathway whose activation is dependent on the bingding between Wnt protein and its receptors which are usually on the cell surface.As reported,the Wnt signaling is divided into two kinds according to whether it needs β-cantenin.One is canonical Wnt signaling,and the other is called non-canonical Wnt signaling.The canonical wnt signaling pathway has an important role in formation and development of embryos,and also in self-renew and differentiation of stem cells.but the recent reaearch has reported that the abnormitu of Wnt signaling is also related to some diseases,such as cancer and so on.Thus,it is of great benefit for finding new treatments against the diseases mentioned above with making an intensive study of Wnt signaling pathway.R-spondin is one important member of the agonists of Wnt signaling,it has great importang roles on gende determination,mediating limb formation,affecting embryonic bllod essel formation and nail development.So the underlying action mechanism of R-spondin and its receptors is still a hot pot of the cancer area.The focus of the research is on the occurrence and development of gastric cancer,on clarfing the transfer mechanism of gastric cancer and finding out the effective way in the prevention and treatment of gastric cancer.In this study,we have disscussed the relation between R-spondin receptor LGR6 and gastric cancer.Then we focus on the function of LGR6 in proliferation,migration and invasion.Our study maybe estabilish the basis in clinic diagose,treatment and prognosis.MethodsPart one1.The 166 gastric cancer patients were collected from the affiliated Hospital of Nantong University from 2013 to 2018.And the correlation between expression of LGR6 and pathological characteristics were analyzed using SPSS 19.0 software.2.We detect the expression level of LGR6 mRNA with Real-time PCR in 20 pairs of fresh cancer and adjacent tissues.Part two1.We construct the LGR6 over-expression plasmid and its controlled plasmid.We coated plate with the bacteric which contain the plasmid we need,then pick the monoclonal bacteria to LB solution and shake,then we use the plasmid kit to acquire the plasmid.We transfect the MKN-45 and MGC-803 cellls by LipofectamineTM 2000 regeants.2.We test the ability of proliferation in vitro used by MTT assay and the ability of apoptosis used by Annexin-V/PI Flow cytometry assays.The ability of migration in vitro was tested by wound healing and transwell assay.Finally,we test the change of apoptosis-related protein and AKT/mTOR signal pathway after overexpression and knockdown expression of LGR6.ResultsThe expression of LGR6 in gastric cancer tissue and its correlation between the clinicopathogic tissuesReal-time PCR reveal that the expression of LGR6 mRNA is higher in cancer than the cancer adjancent healthy tissues in 20 pairs of fresh gastric cancer and adjancent normal tissues.Western-blot experiment results also shows that the expression of LGR6 is higher in cancer tissues than that in adjected tissues.We analyze the correlation between LGR6 and clinicopathogic,the results show that the expression of LGR6 have no relation with Gende,Age,Tumor site and HP infection,but it has great relation with the invision,lymph node metastasis,distant metastases.The exogenous knockdown expression of LGR6We construct LGR6 siRNA to knockdown the expression of LGR6,and construct LGR6 overexpression plasmid and its controlled plasmid,then convert it into bacterias,pick monoclonal bacteria and shake it,used the qiagen kit to extracted the plasmid.We use LipofectamineTM 2000 mediated the plasmid transfer into MKN-45 and MGC-803 cells.LGR6 suppression can decrease the cell ability of proliferation,migration and invasion in vitro.We transfer LGR6 siRNA or pcDNA3.1-LGR6 plasimd into MKN-45 and MGC-803 cells used Lipofectamine2000TM regaent.The result of MTT assays showed that the ability of proliferation reduced significantly in vitro in down expression cells compared with the controlled cells.The transwell assay result revealed that the number of cells through the chamers is much less in knockdown expression cells compared with the controlled cells.Overexpression and knockdown LGR6 have an impact on AKT/mTOR signaling pathwayWe use qRT-PCR and Western-blot assay to detect the changes of apoptosis-associated protein,such as Bcl-2,Bax and Casapse-3 when knockdown or overexpression of LGR6.Meanwhile,we detect the change of AKT/mTOR signaling pathway.The results showed that Bcl-2 has a lower expression after down expression of LGR6,but Bax and Caspase-3 was increased.And the phosphorylated levels of AKT and mTOR were all reduced when down expression of LGR6,this maybe indicate that LGR6 has an impact on AKT/mTOR siganling pathway.Conclusion1.LGR6 have a high expression in gastric cancer and have a positive correlation with the invasion,lymph node metastasis,distant metastasis.2.LGR6 increased the ability of proliferation,migration and invasion in gastric cancer.3.LGR6 involved in the regulation of gastric cancer cell invasion,proliferation may by activating AKT/mTOR signaling pathways.4.LGR6 may be a valuable marker for evaluating the prognosis of gastric cancer,these data may be helpful to elucidate the molecular mechanim of LGR6 and provide new clues.