Functional Role And Molecular Mechanism Of Amer1 In The Development Of Non-small Cell Lung Cancer

Objective:Lung cancer is one of the most common malignant tumors in the world,and its morbidity and mortality are among the highest in the world.In lung cancer,80%-85% are non-small cell lung cancer,mainly including lung adenocarcinoma,squamous cell carcinoma and large cell carcinoma.AMER1 is the first tumor suppressor gene discovered to be located on the X chromosome.It has been reported that AMER1 plays a role in the normal growth and development of the lung,but whether it is related to the development of lung cancer,its specific biological effects and the molecular mechanisms have been reported yet.We first used bioinformatics methods to determine the relationship between AMER1 and the prognosis of non-small cell lung cancer.We used immunohistochemistry to verify the expression of AMER1 in non-small cell lung cancer tumor tissues and its correlation with clinicopathological features.Transfection of lung adenocarcinoma A549,lung squamous cell carcinoma H226 cell line overexpressing and interfering with AMER1,to explore the molecular biological function of AMER1;further transfecting non-small cell lung cancer cell lines with AMER1 different truncations to study AMER1 protein The function of different fragments was to explore the molecular biological pathways of AMER1 regulation of invasion and metastasis of non-small cell lung cancer.This study aims to elucidate the functional role of AMER1 in non-small cell lung cancer and to reveal its molecular mechanism involved in tumor metastasis,providing a new potential molecular therapeutic target for clinical tumor therapy.Method:1.Relationship between AMER1 and clinicopathological features of non-small cell lung cancer(1)Bioinformatics analysis method:(1)Using TCGA database,analyze the expression of AMER1 in non-small cell lung cancer,and analyze the correlation between AMER1 and clinicopathological features of non-small cell lung cancer.(2)The Kaplan-Meier plot online database was used to analyze the relationship between AMER1 and patient survival prognosis.(2)Clinical pathology experimental verification:(1)Collected 121 cases of non-small cell lung cancer diagnosed in the Department of Pathology,Second Hospital of Shanxi Medical University from 2009 to 2018,including68 cases of lung adenocarcinoma and 53 cases of lung squamous cell carcinoma.The clinical data of the collected patients included gender,age,smoking history and histological type,degree of differentiation,lymph node metastasis,distant metastasis,etc;(2)121 cases of non-small cell lung cancer and adjacent tissues were made tissue chip.The expression pattern of AMER1 protein in non-small cell lung cancer was observed by immunohistochemistry.The relationship between AMER1 and clinical pathological features of NSCLC was analyzed.2.The biological function of AMER1 on non-small cell lung cancer cell lines(1)Biological function of NSCLC cell line after AMER1 overexpression(1)Biological function of NSCLC cell line after AMER1 overexpression1AMER1 overexpression plasmid was transfected into lung adenocarcinoma A549 cell line and lung squamous cell carcinoma H226 cell line respectively.The overexpression efficiency of AMER1 gene was verified by real-time PCR and western blot.(2)Using the plate colony formation and transwell migration experiments,the effects of AMER1 on the biological function of lung adenocarcinoma and lung squamous cell carcinoma,respectively,were observed.(2)Biological function of NSCLC cell line after AMER1 interference(1)The AMER1 interference fragment was transfected into lung adenocarcinoma A549 cell line and lung squamous cell carcinoma H226 cell line respectively,and the interference efficiency of AMER1 gene was identified by real-time PCR and western blot.(2)Using plate cloning and transwell migration experiments,the effects of AMER1 interference on the biological function of lung adenocarcinoma and lung squamous cell carcinoma,such as clonality and migration,were observed.3.Initial exploration of the molecular mechanism of AMER1 affecting invasion and migration of non-small cell lung cancer(1)Construction of different fragment expression plasmids of AMER1The PCR products and expression plasmids of different truncated fragments of AMER1 were digested,ligated,transformed,shaken,plasmid extracted and double digested to verify the digested products.After plasmid sequencing,each plasmid fragment was transfected into A549 cells to construct AMER1 different truncated expression vectors.(2)Effects of different truncated fragments of AMER1 on A549 and H226 cells(1)AMER1 different truncated fragment plasmids were transfected into lung adenocarcinoma A549 cell line and lung squamous cell carcinoma H226 cell line respectively,and the expression efficiency of each plasmid of AMER1 was identified by real-time PCR(2)The effects of different truncated fragments on the migration ability of lung squamous cell carcinoma and lung adenocarcinoma cells were observed by transwell method.The independent functional roles of different AMER1 fragments in NSCLC cell lines were observed.4.statistical analysisData analysis was performed using SPSS 13.0 statistical software.The chi-square test was used to analyze the relationship between AMER1 expression and clinicopathological features.Quantitative PCR results,transwell migration experiments,and plate cloning experiments were performed using two independent sample t tests(Independent-Samples T test).P < 0.05 was considered statistically significant.Result:1.Correlation between AMER1 and clinicopathological features of non-small cell lung cancer(1)Bioinformatics analysis(1)Compared with normal tissues,AMER1 expression was not differentiated in lung adenocarcinoma,but decreased in lung squamous cell carcinoma.(2)Bioinformatics student analysis found that the expression of AMER1 is opposite to that of lung squamous cell carcinoma and lung adenocarcinoma.In lung squamous cell carcinoma,the prognosis of high expression group is better than that of low expression,while in lung adenocarcinoma,AMER1 is high.The prognosis of the expression group is worse than the prognosis of low expression.(3)Bioinformatics analysis found that AMER1 expression in female patients,AMER1 expression rate was more than male patients,the difference was statistically significant(P=0.000),between the smoking,AMER1 expression difference was statistically significant(P= 0.028),and there was no significant difference between the type,race and stage of lung cancer(P>0.05).(2)Clinical pathology experimental verification(1)121 cases of non-small cell lung cancer were collected.The majority of lung cancer occurred in the elderly aged over 60 years(69,57.02%),including 53 cases of lung squamous cell carcinoma(43.80%)and 68 cases of lung adenocarcinoma(56.20%).The incidence of smoking in lung squamous cell carcinoma(47,88.68%)was higher than that in non-smoking patients(6,11.32%),while the incidence of female lung adenocarcinoma(36,52.94%)was higher than that of males(32,47.06%).(2)The positive rate of AMER1 protein expression in lung adenocarcinoma(52,76.5%)and lung squamous cell carcinoma(37,69.8%),the difference was not statistically significant(P>0.05);AMER1 expression in lung squamous cell carcinoma and lung adenocarcinoma Differently located,in all positive patients,the expression of AMER1 in lung adenocarcinoma was mainly in the nucleus(43,63.24%),and the expression of AMER1 in lung squamous cell carcinoma was mainly in cell membrane and cytoplasm(24,45.28%);the clinical analysis of AMER1 Pathological features showed that in patients with lung squamous cell carcinoma and lung adenocarcinoma,the expression of AMER1 was statistically different from gender and differentiation(P<0.05).The higher the degree of tumor differentiation,the higher the positive rate of AMER1 expression.2.Effect of AMER1 on the biological function of lung cancer A549 and H226 cells(1)The biological function of overexpression AMER1 on NSCLC(1)The biological function of NSCLC after overexpression of AMER1,PCR and western blot results showed that the expression of AMER1 in the experimental group was higher than that in the control group,and the difference was statistically significant,suggesting that the AMER1 gene was successfully overexpressed.(2)After AMER1 overexpression,the clone formation ability of A549 cells was lower than that of the control group,the difference is statistically significant,suggesting that AMER1 can inhibit the clonality of A549 cells.(3)After AMER1 overexpression,the number of cells transfected into the transwell membrane of A549 cells was significantly reduced,the difference is statistically significant,suggesting that AMER1 can inhibit the migration ability of A549 cells.(4)After AMER1 overexpression treatment,the number of cells passing through the transwell membrane of H226 cells was significantly reduced,the difference is statistically significant,suggesting that AMER1 can inhibit the migration ability of H226 cells.(2)The biological function of AMER1 interference on NSCLC(1)The results of PCR and Western blot showed that the expression of AMER1 in the experimental group was lower than that in the control group(P < 0.05),suggesting that AMER1 gene successfully interfered with the expression.(2)After the interference of AMER1 expression,the cloning ability of A549 cells was improved,the difference was statistically significant(P < 0.05),suggesting that AMER1 interference could promote the cloning ability of A549 cells.(3)After AMER1 interference,the number of A549 cells passing through Transwell membrane increased significantly(P < 0.05),suggesting that AMER1 interference can promote the migration of A549 cells.(4)After the interference of AMER1 expression,the number of H226 cells passing through the Transwell membrane of the ventricle increased significantly(P < 0.05),suggesting that the interference of AMER1 expression can promote the migration of H226 cells.3.Effects of different truncated fragments of AMER1 on A549 and H226 cells(1)The sequencing results of each fragment were aligned by BLAST,and the sequencing product was identical to the sequence published by NCBI,suggesting that the plasmids of different functional fragments of AMER1 were successfully constructed.(2)Each plasmid was transfected into A549 and H226 cells respectively.It was verified by PCR that each plasmid could be overexpressed in cells.Experiments on the effects of different functional fragments of AMER1 on lung cancer cell function are currently underway.In conclusion:(1)Bioinformatics analysis found that AMER1 has different expression levels and different prognosis in non-small cell lung cancer.Immunohistochemistry results confirmed that AMER1 has different expression localization in different histological types of non-small cell lung cancer,suggesting that AMER1 is involved in squamous cell carcinoma and adenocarcinoma.The mechanism of action is different.(2)The biological function of AMER1 in lung adenocarcinoma A549 and squamous cell carcinoma cell line H226 suggests that AMER1 plays a potential role in tumorigenesis in the development of different histological types of non-small cell lung cancer,in order to further study AMER1 in non-small cells.The role of lung cancer provides a theoretical basis.(3)The different functional fragment plasmids of AMER1 were successfully constructed,which provided a basic tool for further study of the function of full-length and truncated fragments of AMER1 and analysis of the molecular mechanism of AMER1 affecting the progression of lung cancer.