| Objective: With the acceleration of urbanization and the worsening of air pollution in China,lung cancer has become one of the highest incidence and mortality malignant tumors in the worldwide.Lung cancer includes a variety of histopathological types and Adenocarcinoma is the most common histological subtype of lung cancer,accounting for half of all lung cancer incidence.With the increasingly serious environmental pollution,the incidence of lung adenocarcinoma in women showed a significant upward trend.With the development of modern medical research on lung adenocarcinoma and the rapid development of modern surgery,radiotherapy,chemotherapy and biological targeted drug therapy,the therapeutic effect of lung adenocarcinoma has made considerable progress.However,the overall survival rate and quality of life of patients with lung adenocarcinoma are still not optimistic because of their unique molecular biology and histological specificity.The proliferation,migration and invasion of lung adenocarcinoma cells are the important reason for the rapid progression of lung adenocarcinoma.It is also one of the most difficult problems to solve in the treatment of lung adenocarcinoma.Therefore,to explore the occurrence of lung cancer and the molecular mechanism of invasion and metastasis,effectively block or inhibit metastasis of lung cancer cells and provide a new theory and targeted therapy for clinical treatment of lung cancer have become a top priority in the modern oncology research.In the previous research of our group,a number of genes related to lung cancer metastasis were screened and cloned in lung cancer and metastatic lymph node tissues.After cloning,sequencing and comparison with Genebank sequence,it was confirmed that the differentially expressed gene was ABCE1.The expression of ABCE1 protein and mRNA in lung cancer tissues and metastatic lymph node tissues was significantly higher than that in normal lung tissues by immunohistochemistry and PCR method.Further analysis of the expression of ABCE1 and the clinical characteristics of patients with the correlation of the results showed that the expression of ABCE1 protein and mRNA is closely related to the clinical pathological stage.The results of in vitro experiments showed that the expression of several genes related to lung cancer was changed and andthe biological behaviors of lung adenocarcinoma cells were affected to varying degrees through down regulation and up regulation of ABCE1 gene expression.The above results showed that the ABCE1 gene in lung adenocarcinoma,the development process plays a very important role,but the mechanism by which specific regulation remain to be further studied the occurrence and development of lung cancer.When study the 2-5A/RNase L cell antiviral pathway,foreign scholars find a RNase L inhibitory factor,known as ribonuclease L inhibitory factor.Further studies have found that it is an important member of the ATP binding cassette transporter subfamily,which is named ABCE1.ABCE1 protein is mainly synthesized in the cytoplasm,and its molecular weight is 68 KD,which can inhibit 2-5A/RNase L cells through the specific inhibition of RNase L pathway,and participate in a variety of biological processes such as cell proliferation,anti apoptosis and migration.Latest research shows that ABCE1 not only plays an important role in the occurrence,development and metastasis of some tumors,but also plays an important role in promoting protein synthesis and cell proliferation and differentiation.Acetylation modification of proteins is an important part of epigenetics,which plays a very important role in life and has many biological meanings.The main process is to combine acetyl CoA acetyl group with lysine residue under the catalysis of acetyl transferase.Acetylation of proteins involved in the regulation of a variety of biological processes,such as gene transcription,protein interaction,protein stability,cell proliferation,migration and differentiation.Protein acetylation and deacetylation modification can regulate the expression of tumor related genes,and the balance between the two is strictly controlled,once the balance mechanism is broken will become the direct cause of tumor.There is no report about the correlation between the acetylation of ABCE1 protein in lung adenocarcinoma and the correlation between the level of acetylation and clinical cases.The study of the effect of ABCE1 protein on the biological behavior of lung cancer cells is also blank.In this study,the ABCE1 protein in the lung adenocarcinoma tissue was confirmed by experiments,and the correlation between the level of acetylation of ABCE1 protein and the clinical characteristics of patients were statistically analyzed.Through the prediction of the molecular biology software and the experimental verification,we can find out the acetyl transferase Tip60 which can catalyze theacetylation of ABCE1 protein.The expression of Tip60 protein in lung adenocarcinoma were detected by immunohistochemistry and Real-PCR and the relationship between the expression of the Tip60 protein and the acetylation level of ABCE1 protein was analyzed.Based on the previous experiment,we applicate the RNA interference technology to down the Tip60 gene,then transfecte the lung adenocarcinoma cell line A549 to detecte that the ABCE1 protein whether deacetylation or not.At the same time,we observe the effects of ABCE1 protein deacetylation to the A549 cell proliferation,apoptosis,migration and invasion.In order to look for a new target for treatment of lung adenocarcinoma and mechanisms of ABCE1 gene regulation in the development and progression of lung adenocarcinoma,the related experiment of this subject provides a new theoretical and experimental basis for the early diagnosis and treatment of lung adenocarcinoma.Methods: We select 42 cases of lung adenocarcinoma cancer tissue,paracancerous tissue and extract protein,then we detecte the ABCE1 protein acetylation level through the Co immunoprecipitation and Western blot experiment.Statistical analysis was performed to determine the relevance between the ABCE1 protein acetylation level with the patients,age,sex,smoking,tumor stage,differentiation,and lymph node metastasis.Forecast acetyltransferase which can catalyze ABCE1 protein acetylation through medical molecular biology software.(Website:Http//bioinfo.bjmu.edu.cn/huac/predict(s)).The results showed that Tip60 was the most likely acetyltransferase to catalyze the acetylation of ABCE1 protein.Lung adenocarcinoma was detected by Real Time-PCR and immunohistochemical method to detect the level of Tip60 and protein expression of mRNA,and to analyze the relevance between the expression of the Tip60 and the level of ABCE1 acetylation in the same patients.The siRNA interference sequence of Tip60 was down regulated and transfected into A549 cells.The down-regulation effect was verified by Real Time-PCR and Western Blot.Lung adenocarcinoma A549 cell line transfected with the Tip60 RNA interference sequence.The siRNA-Tip60 was used as the experimental group,si RNA-N transfected A549 cells as control group and A549 group without any treatment of N cells as blank control group.The following experiments are implemented:The effect of down regulated Tip60 on ABCE1 protein expression amount and the acetylation level of ABCE1 proteinwas detected by immunoprecipitation and Western blot method;MTT and flow cytometry were used to detect the proliferation and cell cycle of A549 cells;Cell apoptosis was detected by flow cytometry;The migration ability of A549 cells was detected by cell scratch assay.Transwell chamber were used to detect the cell invasion.Result: It was proved that ABCE1 protein was acetylated in lung adenocarcinoma tissues through immunoprecipitation and Western blot,and it is an acetylated protein.The level of ABCE1 protein acetylation and between lung adenocarcinoma tissue and paracancerous tissue have obvious difference.The level of ABCE1 protein acetylation was significantly correlated with sex,tumor stage,differentiation and lymph node metastasis in patients with lung adenocarcinoma.There was no correlation between the level of ABCE1 acetylation and the age,smoking or not.The Tip60 down si RNA interference sequence was successfully synthesized,and the results showed that Tip60 was significantly reduced through Real Time-PCR and Western blot.The A549 cell line was transfected.It was confirmed that the ABCE1 protein expression amount no difference but the level of ABCE1 protein acetylation was significantly decreased in the experimental group compared with the control group and the blank group,but there was no significant difference between the control group and the blank group.It was confirmed that cell proliferation of experimental group was significantly inhibited compared with the control group and the blank group through MTT and flow cytometry experiment.The apoptosis rate of experimental group was significantly enhanced compared with the control group and the blank group through flow cytometry experiment,but there was no significant difference between the control group and the blank group.t was confirmed that cell proliferation of experimental group was significantly inhibited compared with the control group and the blank group through MTT and flow cytometry experiment.It was confirmed that cell migration and invasion of experimental group was significantly inhibited compared with the control group and the blank group through cell scratch assay repair and Transwell chamber experiment.Conclusion: ABCE1 protein is acetylated in lung adenocarcinoma,and it is an acetylated protein.The level of ABCE1 protein acetylation was significantly correlated with sex,tumor stage,differentiation and lymph node metastasis in patients with lung adenocarcinoma.There was no correlation between the level of ABCE1 acetylation andthe age,smoking or not.These results suggest that the acetylation modification of ABCE1 may promote the occurrence and development of lung adenocarcinoma.Acetyltransferase Tip60 can catalyze the ABCE1 protein to acetylate.Acetyltransferase Tip60 plays a key role in the occurrence and development of lung adenocarcinoma.Moreover,the biological function of ABCE1 protein was enhanced after the ABCE1 protein acetylation and the occurrence and development of lung adenocarcinoma were promoted.Downregulation of Tip60 had no effect on the expression of ABCE1 protein.Downregulation of Tip60 could significantly decrease the acetylation level of ABCE1 protein.Furthermore,the deacetylation of ABCE1 protein can promote the apoptosis of lung adenocarcinoma A549 cells and inhibit cell proliferation,migration and invasion.It suggests that Tip60 may be a new target for gene targeted therapy of lung adenocarcinoma. |
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