The Mechanisms Of WRAP53 And Its Interacting Proteins In The Carcinogenesis Of Lung Adenocarcinoma

SECTION 1:Mechanism of WRAP53 overexpression in the carcinogenesis of lung adenocarcinomaObjective:To investigate the role of the WD repeat containing antisense to P53(WRAP53)protein expression in the development and progression of lung adenocarcinoma via clinical,cellular and bioinformatic analyses.Methods:We observed the expression of RUVBL1 in 75 human lung adenocarcinoma specimens,individually matched with those of their adjacent non-tumor tissues from our hospital,and analyzed the clinical characteristics of patients with high expression of WRAP53.We silenced the expression of WRAP53 protein with siRNA and observed the knowdown effect on the proliferation of lung adenocarcinoma cells.We explored the reason of the lung adenocarcinoma cell-proliferation reduction by cell cycle and apoptosis arrays.Finally we obtained proteins interacted with WRAP53 during early S phase by Co-immunoprecipitation(Co-IP)and liquid chromatography/mass spectrometry(LC/MS).Results:Quantitative Real-time polymerase chain reaction(qRT-PCR)results showed a higher WRAP53 expression in tumor tissues than in adjacent tissues.The prevalence of WRAP53 overexpression was significantly higher in patients with tumors larger than 3.0 cm than in patients with tumors smaller than 3.0 cm(p<0.05).WRAP53 knockdown reduces proliferation of lung adenocarcinoma cells caused by G1/S cell cycle arrest.We obtained some promising target proteins interacted with WRAP53 during early S phase by Co-IP and LC/MS.Conclusion:These observations strongly suggested that WRAP53 and its interacter proteins should be considered a promising target in the prevention or treatment of lung adenocarcinoma.SECTION 2:Downregulation of RUVBL1 inhibits proliferation of lung adenocarcinoma cells via multiple mechanisms.Objective:We found the interaction of WRAP53 and RuvB-like AAA adenosine 5’-triphosphatase(ATPase)1(RUVBL1)protein in section 1.In this part we aimed to explore the mechanisms of RUVBL1 downregulation in inhibiting the proliferation of lung adenocarcinoma.Methods:We obtained 72 human lung adenocarcinoma specimens,individually matched with those of their adjacent non-tumor tissues from our hospital,and analyzed the expression of RUVBL1 in tissues as well as in adenocarcinoma and normal lung cell lines.We silenced the expression of RUVBL1 protein with siRNA and observed the knowdown effect on the proliferation of lung adenocarcinoma cells.We explored the reason of the lung adenocarcinoma cell-proliferation reduction by cell cycle and apoptosis arrays as well as western blot.We did the verification experiment of RUVBL1 overexpression in cells.At last,we observed the endoplasmic reticulum stress induced by RUVBL1 downregulation.Results:Our study revealed that RUVBL1 expression was higher in lung adenocarcinoma specimens than in those of adjacent non-tumor tissues as well as in lung adenocarcinoma cell lines than in normal lung cell lines.RUVBL1 knockdown via siRNA reduced proliferation and caused G1/S phase cell cycle arrest in lung adenocarcinoma cell lines.The Gl/S phase cell cycle arrest triggered by RUVBL1 down-regulation could be attributed,at least in part,to repression of the AKT/GSK-3β/cyclin D1 pathway,and probably activation of IRE1α-mediated ER stress.Conclusion:We confirmed,for the first time,that RUVBL1 played an important role in the occurrence and development of lung adenocarcinoma.It is worthy of further study as a promising clinical therapeutic target.SECTION 3:Downregulation of HSPA2 inhibits proliferation of lung adenocarcinoma cells via multiple mechanisms.Objective:We found the interaction of WRAP53 and heat shock protein A2(HSPA2)protein in section 1.In this part we aimed to explore the mechanisms of HSPA2 downregulation in inhibiting the proliferation of lung adenocarcinoma.Methods:We obtained 85 human lung adenocarcinoma specimens,individually matched with those of their adjacent non-tumor tissues from our hospital,and analyzed the expression of HSPA2 in tissues as well as in adenocarcinoma and normal lung cell lines.We silenced the expression of HSPA2 protein with siRNA and observed the knowdown effect on the proliferation of lung adenocarcinoma cells.We explored the reason of the lung adenocarcinoma cell-proliferation reduction by cell cycle and apoptosis arrays as well as western blot.At last,we observed the endoplasmic reticulum stress induced by HSPA2 downregulation.Results:Our study revealed that HSPA2 expression was higher in lung adenocarcinoma specimens than in those of adjacent non-tumor tissues as well as in lung adenocarcinoma cell lines than in normal lung cell lines.HSPA2 knockdown via siRNA reduced proliferation and caused Gl/S phase cell cycle arrest in lung adenocarcinoma cell lines.The G1/S phase cell cycle arrest triggered by HSPA2 down-regulation could be attributed,at least in part,to phosphorylation activation of the Erkl/2 pathway,and probably activation of IRE1a/PERK-mediated ER stress.Conclusion:We confirmed,for the first time,that RUVBL1 played an important role in the occurrence and development of lung adenocarcinoma.It is worthy of further study as a promising clinical therapeutic target.