TrkB Regulates The PI3K/AKT Pathway To Promote Metastasis Of Laryngeal Carcinoma

Background:Laryngeal cancer is one of the most common malignant tumors of head and neck cancer.The disease accounts for 7.9-35%of all otorhinolaryngal malignancies and ranks third in all types of head and neck malignancies.The incidence of all types of malignancies in the body accounted for 5.7 to 7.6%.In recent years,with the aggravation of industrial pollution,the incidence of laryngeal cancer has been increasing year by year,and most of them are squamous cell carcinoma.The cause of laryngeal cancer is not yet clear,most researchers believe that smoking,drinking,contact with harmful dust and papilloma virus infection and so on.Due to laryngoscopy and imaging is more difficult to detect small and occult lesions,so the early diagnosis of laryngeal cancer is not sensitive.With the rapid development of modern medical technology,great advances have been made in the clinical diagnosis and treatment of laryngeal cancer.However,because laryngeal cancer itself has more complicated biological behaviors and has various clinical manifestations,there is still a low rate of early diagnosis.Postoperative complications,high recurrence rate,so looking for effective methods for early diagnosis of laryngeal cancer,while taking effective targeted treatment is the focus of the current medical research and problems.The occurrence and malignant progression of laryngeal carcinoma is a complex process involving multiple factors,multiple genes and multiple signaling pathways.The spread and metastasis of laryngeal carcinoma is closely related to the primary site,the degree of differentiation and tumor size.However,The specific molecular mechanism of the occurrence and development of laryngeal cancer has not been fully proved.Tyrosine kinase receptor(TrkB)is a member of tyrosine kinase family encoded by TRK.It is a specific receptor of brain-derived neurotrophic factor(BDNF)Affinity.In recent years,the study found that,BDNF-TrkB also associated with the occurrence and development of a variety of malignant tumors,including neuroblastoma,hepatocellular carcinoma,multiple myeloid and ovarian cancer,and with the above case of tumor grade,clinical Staging,distant metastasis and prognosis and other pathological conditions related.With the deepening of the research,it was found that TrkB can affect the biological behavior of tumor cells through promoting the proliferation of tumor cells,anti-apoptosis,promoting epithelial mesenchyme and tumor angiogenesis,and eventually promote the invasion of tumor And transfer.Abnormal changes in the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB)signaling pathways often occur in some human tumor tissues and are associated with the malignant progression of the tumor.Complete activation of the PI3K/AKT pathway is a multi-step,complex process in which many cytokines in the malignant tumor microenvironment are able to bind to their corresponding receptors,thereby activating PI3K.Activated PI3K/AKT can promote the proliferation,differentiation,invasion and metastasis of malignant tumor cells through the increase of various cytokines such as P-catenin,Snail,NF-?B and GLTU4.Recent studies have found that PI3K/AKT pathway abnormal activation in many human malignancies such as lung cancer,colon cancer,ovarian cancer and cervical cancer,and the expression level of PI3K and AKT in pancreatic cancer,ovarian cancer and breast cancer All abnormally increased.The PI3K/AKT signaling pathway in the occurrence and development of laryngeal cancer research is still small.Studies have found that TrkB is a key regulator of PI3K/AKT signaling pathway-mediated tumor metastasis and EMT program.TrkB can inhibit the invasion and metastasis of ovarian cancer cells by inhibiting the expression of PI3K/AKT signaling pathway related genes.Objective:This study was to detect the expression of TrkB in laryngeal squamous cell carcinoma and to analyze its clinical significance.At the same time,the effect of TrkB on the biological behavior and tumor growth of laryngeal squamous cell carcinoma was clarified through in vitro and in vivo experiments and whether it induced the above-mentioned one through PI3K/AKT signaling pathway Series of processes to explore.The aim of this study was to investigate the role of TrkB and PI3K/AKT signaling in laryngeal cancer and to provide a reliable experimental basis for the clinical diagnosis and targeted therapy of laryngeal cancer.Methods:(1)69 samples of laryngeal cancer and corresponding normal tissues were collected.The expression of TrkB mRNA was detected by fluorescence quantitative PCR.The expression of TrkB protein was detected by western blot and immunohistochemistry.The relationship between TrkB and sex,age,smoking history,clinical stage,lymph node metastasis and tumor location in patients with laryngeal cancer was analyzed.The relationship between recurrence-free survival time,total survival time and clinical parameters was also analyzed.(2)The expression of TrkB mRNA in Hep-2,TU686 and M4e cells was detected by real-time fluorescence quantitative PCR,and the cell line with the highest expression of TrkB mRNA was selected for further experiments.Respectively 0,100,500,600,1000 nmol/L TrkB inhibitor K252a acting laryngeal carcinoma cells,CCK-8 assay proliferation,apoptosis rate and cell cycle flow cytometry colony formation The colony forming ability of the cells was tested by the experiment,the cell migration ability was detected by the cell scratch test,and the invasion ability of the cells was detected by the Transwell chamber method.ShTrkB was used to silence the expression of TrkB in laryngeal squamous cell carcinoma to detect the cell migration ability.K252a(500 nmol/L)and SU6656(5 ?M)were respectively used to detect the expression of p-AKT and cyclin D1 in laryngeal carcinoma cells.SU6656(5?M)was applied to laryngeal carcinoma cells to detect the clonogenic capacity of the cells.The shTrkB was transfected into laryngeal carcinoma cells to detect the expression of p-c-Src and c-Src protein.The laryngeal cancer cells were randomly divided into control group,shGFP group and shTrkB group.The expression of p-AKT and cyclin D1 in each group was detected.ShTrkB was transfected into laryngeal carcinoma cells to detect the expression of RUNX3 and KEAP1 mRNA.K252a(500 nmol/L)was administered to laryngeal carcinoma cells to detect the expression of RUNX3 and KEAP1 mRNA.PI3K inhibitor LY294002(25 nM)was applied to laryngeal carcinoma cells to detect the expression of RUNX3 and KEAP1 mRNA.The shAkt was transfected into laryngeal carcinoma cells to detect the expression of RUNX3 and KEAP1 mRNA.The TrkB overexpression plasmid was transfected into laryngeal carcinoma cells to detect the expression of E-cadherin,N-cadherin,Vimentin,Twist-1 and Twist-2 in the cells.(3)The laryngeal cancer cells were transfected with control shRNA and shTrkB,respectively.Then,the transfected laryngeal carcinoma cells were inoculated subcutaneously in nude mice to establish the xenograft model of laryngeal carcinoma in nude mice.The length(L)and width(W)of the transplanted tumor in nude mice were measured.The volume of the transplanted tumor was calculated.After 6 weeks,the nude mice were sacrificed and the complete transplanted tumor tissue was collected and weighed.Fluorescent quantitative PCR was used to detect the expression of TrkB,Twist-1 and Twist-2 mRNA in each group.The nude mouse xenografts and normal laryngeal mucosa epithelial tissues were harvested and pathological sections were made.Immunohistochemical staining was used to observe the immunostaining of TrkB,cyclinD1,AKT and the expression of E-cadherin protein was detected by western blot.Results:(1)Fluorescent quantitative PCR results showed that the expression of TrkB mRNA in laryngeal carcinoma was significantly higher than that in corresponding adjacent tissues(P<0.05).The results of western blot showed that the expression of TrkB protein in laryngeal carcinoma was significantly higher than that in corresponding normal tissues(P<0.05).The results of immunohistochemistry showed that TrkB immunostaining was located in the cytoplasm with yellow or brown granules.The expression of TrkB protein in laryngeal carcinoma was significantly higher than that in corresponding adjacent tissues(P<0.05).TrkB and laryngeal cancer patients with gender,age,tumor site has nothing to do with smoking history,clinical stage and lymph node metastasis.The relapse-free survival time in patients with laryngeal cancer was not related to gender and T stage,but was related to age,N stage,M stage and TrkB expression.The overall survival time was not related with gender,age,T stage and N stage,but correlated with M stage and TrkB expression.Patients with positive TrkB expression and patients with negative TrkB expression showed significant differences in recurrence-free survival and overall survival.(2)The expression of TrkB mRNA in Hep-2 cells was significantly higher than that in TU686 and M4e cells,so Hep-2 cells were used as the cells for subsequent experiments.With the increase of K252a concentration,the proliferation activity of Hep-2 cells gradually decreased at each time point.When the concentration of K252a was 1000 nmol/L,the proliferation activity of Hep-2 cells was the lowest.At the same time,Proliferative activity was no significant difference.With the increase of K252a concentration,the apoptosis rate of Hep-2 cells increased.When K252a concentration was 1000 nmol/L,the apoptosis rate of Hep-2 cells was the highest.With the increase of the concentration of K252a,the number of G2 phase cells gradually increased and the number of G1 and S phase cells gradually decreased,suggesting that TrkB inhibited the G1 phase arrest of Hep-2 cells.With the increase of K252a concentration,the colony formation rate of Hep-2 cells decreased gradually.When K252a concentration was 1000 nmol/L,the clonogenic rate of Hep-2 cells was the lowest.With the increase of K252a concentration,the migration rate of Hep-2 cells gradually decreased.When K252a concentration was 1000 nmol/L,the migration rate of Hep-2 cells was the lowest.With the increase of K252a concentration,the migration rate of Hep-2 cells gradually decreased.When K252a concentration was 1000 nmol/L,the migration rate of Hep-2 cells was the lowest.Compared with the shGFP group,the shTrkB group had a significantly lower migration ability(P<0.05).Compared with the control group,the expression of p-AKT protein in K252a and SU6656 cells was significantly decreased(P<0.01);the expression of cyclin D1 protein in K252a cells was significantly decreased(P<0.01)The expression of cyclin D1 protein had significant change(P<0.05).Compared with SU6656(-)group,the number of colonies in SU6656(+)group was significantly decreased(P<0.01).Compared with control group,the expression of p-c-Src protein in shTrkB group was significantly decreased(P<0.05),while the expression of c-Src protein had no significant change(P>0.05).The expression of p-AKT and cyclin D1 in shTrkB group was significantly decreased(P<0.01).Compared with the control group,the expression levels of RUNX3 and KEAP1 mRNA in shTrkB group were significantly increased(P<0.01).Compared with K252(-)group,the expression of RUNX3 and KEAP1 mRNA in K252(+)group were significantly increased(P<0.01).Compared with LY294002(-)group,the expression of RUNX3 and KEAP1 mRNA in LY294002(+)group were significantly decreased(P<0.05).Compared with the control group,the expressions of RUNX3 and KEAP1 mRNA in shAkt group were significantly increased(P<0.01).Compared with the control group,the expression of E-cadherin protein in Overexpressed group was significantly decreased(P<0.05),the expression of N-cadherin ad and Vimentin protein was significantly increased(P<0.05),and the expression of Twist-1 And Twist-2 protein expression were significantly increased(P<0.05).(3)Fadu cells were inoculated into the right shoulder of nude mice.On the 8th day,the nude mice could see the size of the xenotransplanted tumor in this part.All nude mice in this experiment all formed tumors.Nude mice in the tumor,the nude mice in each group were normal in eating and drinking,urine and urine were not abnormal.Nude mice in each group in good condition,normal activity,rapid response.In addition,nude mice in each group had normal skin color and no ulceration occurred in the xenografted tumor tissue.Compared with the control group,the tumor volume and weight of shTrkB group were significantly increased(P<0.05).Compared with control group,the expression levels of TrkB,Twist-1 and Twist-2 mRNA in shTrkB group were significantly decreased(P<0.05).The positive staining of TrkB,cyclinD1 and AKT in the xenografts of the control group was also significantly increased compared with shTrkB group,and the expression of E-cadherin protein was significantly decreased(P<0.05).Conclusion:(1)The expression of TrkB in cancer tissues of laryngeal cancer is abnormally increased,which is related to smoking history,clinical stage and lymph node metastasis of laryngeal cancer patients.(2)TrkB can promote the growth,invasion and migration of laryngeal carcinoma cells and inhibit its apoptosis;TrkB activates AKT via c-Src to promote the proliferation of laryngeal carcinoma cells and induce the development and progression of EMT by up-regulating EMT-associated transcription factor expression.(3)TrkB can promote the occurrence and development of laryngeal carcinoma by regulating the PI3K/AKT pathway to promote the expression of EMT,invasion and migration related genes.