The Mechanism And Regulation Of CAMK? On Promoting The Invasion And Matastasis Of Lung Adenocarcinoma

Objective: To study the role of CAMK? in the invasion and metastasis of lung adenocarcinoma,to explore its relationship with the activation of EGFR pathway,to preliminarily define the mechanism of its transcriptional regulation,and to provide a new diagnosis for patients with EGFR-resistant lung adenocarcinoma.Prognosis and therapeutic targets provide a further theoretical basis for drug resistance progression in patients with EGFR mutant lung adenocarcinoma.Research content:(1)To explore the relationship between CAMK? expression and invasion and metastasis of lung adenocarcinoma,and to analyze the clinicopathological significance and prognostic value of CAMK?.(2)Explore the relationship between CAMK? and the EGFR pathway.(3)Analysis of transcriptional regulation of transcription factor CREB on CAMK?.Research method:(1)A total of 160 paraffin-embedded specimens from patients with lung adenocarcinoma with complete clinical prognosis data from June to December 2011 in Tianjin Medical University Cancer Hospital were collected.Immunohistochemical staining was used to analyze the expression of CAMK? in lung adenocarcinoma tissues with different histological subtypes.The difference between the levels,analysis of the relationship between CAMK? and clinical information such as age,gender,smoking history,analysis of the relationship between CAMK? expression and gene mutation,analysis of the interaction between CAMK? and lymph node metastasis and distant metastasis The relationship between CAMK? expression and patient survival was analyzed.Paraffin-embedded specimens of 21 patients with lymph node metastasis from lung adenocarcinoma were collected.The expression of CAMK? in primary and metastatic lesions was compared by immunohistochemical staining.Transwell and wound healing experiments were used to analyze the effects of lung adenocarcinoma cell lines H1299 and Calu3 on the invasion of lung adenocarcinoma before and after high expression of CAMK?.(2)Eight paraffin-embedded specimens of EGFR-resistant patients were collected and analyzed by immunohistochemical staining for CAMK? expression before and after drug resistance;lung adenocarcinoma cell lines H1299 and Calu3 induced epidermal growth factor(EGF)-induced EGFR pathway The expression of p-CAMK?,CAMK?,p-EGFR and EGFR protein at 15',30',45',60' were detected by Western Blot assay,and the activation and regulation of EGFR-CAMK? pathway were explored.The lung adenocarcinoma cell lines H1299 and Calu3 were pre-cultured with different concentration gradients and different time gradients of EGFR small molecule inhibitor AG1478,and then stimulated with EGF.The expression of p-CAMK?,CAMK?,p-EGFR and EGFR protein was detected by Western Blot assay.To explore the regulation mechanism of activation and inhibition of EGFR-CAMK? pathway;(1)Bioinformatics predicts the transcription factor of CAMK? promoter region;Pearson method is used to analyze the correlation between CREB and CAMK? expression in lung adenocarcinoma patients.The lung adenocarcinoma cell lines H1299 and Calu3 were stimulated with epidermal growth factor(EGF),and the expression of CREB and CAMK? at different time points was detected by Western Blot test.The lung adenocarcinoma cell lines H1299 and Calu3 cultured according to the conventional method were separately transferred.The method of staining CREB high expression plasmid and blank plasmid was divided into experimental group(CREB high expression group)and control group.Western Blot method was used to analyze the change of CAMK? expression after high expression of CREB;lung adenocarcinoma transfected with CREB overexpression plasmid Cell lines H1299 and Calu3 were inhibited with different concentration gradients and different time gradients of CREB inhibitor SGC.Western blot was used to analyze the expression of CAMK? after CREB expression.Research result:(1)The high expression of CAMK? was correlated with the pathological type of histological subtype of lung adenocarcinoma(p<0.05),and the expression of CAMK? was significantly higher in lung adenocarcinoma with poor differentiation(micropapilla-based,solid-type).Highly differentiated(Fubo-based,acinar-based)lung adenocarcinoma.CAMK? expression was associated with lymph node metastasis,distant metastasis,and TNM staging(p<0.05).High expression of CAMK? is prone to metastasis.CAMK? was associated with survival(p<0.05),and patients with high expression of CAMK? had significantly lower survival than patients with low expression of CAMK?.CAMK? was associated with EGFR mutations(p < 0.05).The expression of CAMK? in lymph node metastasis of lung adenocarcinoma was significantly higher than that of primary tumor(p<0.05).The scratch test showed that the wound healing increased significantly after the high expression of CAMK?,and the number of transmembrane cells in the transwell invasion experiment increased significantly(p<0.05).(2)The expression of CAMK? in EGFR-resistant patients was significantly higher than before the use of targeted drugs.After stimulation with EGF,the lung adenocarcinoma cell line H1299 and Calu3 EGFR-CAMK? pathway were activated within 1 hour,p-EGFR and p-CAMK? increased first and then decreased,p-EGFR and p-CAMK? protein expression was the highest at 30 minutes,EGFR And CAMK? expression is unchanged.After pretreatment with AG1478,EGF was stimulated.The expression of p-EGFR and p-CAMK? decreased first and then remained unchanged.0.75?? AG1478 was the optimal concentration,and 3 hours was the optimal inhibition time of AG1478.(3)CREB expression was positively correlated with CAMK? expression in patients with lung adenocarcinoma.LGF was used to stimulate lung adenocarcinoma cells H1299 and Calu3,with a rise in CREB for 48 hours,and an overall increase in p-CAMK? and CAMK? expression.The CREB inhibitor SGC was used to act on the lung adenocarcinoma cell lines H1299 and Calu3,and the 24-hour CAMK? expression was decreased.Lung adenocarcinoma cells H1299 and Calu3 were transfected with CREB plasmid and CAMK? expression was increased.Analysis conclusion:(1)The high expression of CAMK? is closely related to the invasion and metastasis process of lung adenocarcinoma;(2)High expression of CAMK? is associated with activation of the EGFR pathway;(3)CREB is involved in the regulation of transcriptional regulation of CAMK?.