A Study Of The Implication Of Rpn10 On Prognosis Of Lung Adenocarcinoma And It’s Cell Biologic Function

Lung cancer is still one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths.In China,lung cancer is the third cause of deaths among all causes of death in 2017,only second to stroke and ischemic heart disease.The disease spectrum of lung cancer has undergone great changes,that lung adenocarcinoma has been the most common histological subtype of lung cancer,due to the tobacco control and the chest CT screening for lung cancer.Lung adenocarcinoma is highly malignant with poor prognosis.The mutation rate of EGFR in lung adenocarcinoma is high among patients in China.Targeted therapy has greatly improved the prognosis of lung adenocarcinoma,but the resistance to EGFR-TKI have been limiting further improvements in the prognosis of lung adenocarcinoma.It is urgent to explore the mechanism of lung adenocarcinoma resistance to EGFR-TKI,as well as to find new biomarkers for early diagnosis of lung adenocarcinoma and EGFRTKI resistance.Rpn10,an important subunit of the proteasome,is involved in the assembly of the proteasome and plays an important role in cell growth and development.Rpn10 is highly expressed in some malignant tumors and promotes the development and progression of tumor as well as the drug resistance.However,the role of Rpn10 in lung adenocarcinoma remains elusive.In this study,we analyzed the expression of Rpn10 in lung adenocarcinoma and its impact on prognosis.We explored the biological function of Rpn10 in the development and drug resistance of lung adenocarcinoma,and elucidated the underlying mechanisms as well.Our study seeks to find new biomarkers for the diagnosis and drug resistance monitoring of lung adenocarcinoma,as well as novel treatment for lung adenocarcinoma,especially overcoming the problem of resistance to targeted therapy.In the second chapter,by using the online bioinformatics analysis tool,we found that Rpn10 is mainly amplified mutation in lung adenocarcinoma,and Rpn10 is highly expressed in lung adenocarcinoma tissue,which is a poor prognostic factor for lung adenocarcinoma.What’s more,the pathological specimens and basic clinical information of 94 patients in our hospital were collected,and the protein expression of Rpn10 was detected by IHC And it was found that Rpn10 was highly expressed in cancer tissues,and more patients with high expression of Rpn10 in advanced lung cancer.Rpn10 is a poor prognostic factors in lung adenocarcinoma.We also detected the protein level of EGFR by IHC,and found that the proportion of patients with high expression of Rpn10 was more in patients with high EGFR expression although there is no statistical significance.In addition,we collected the serum of 26 patients with lung adenocarcinoma and 50 normal patients,and detected the protein level of serum Rpn10 using ELISA.We found that Rpn10 is highly expressed in the serum of patients with lung adenocarcinoma,and Rpn10 is expected to be a biomarker for early diagnosis of lung adenocarcinoma.In the third chapter,we generated stable cell lines with Rpn10 knockdown or overexpression by lentiviral transfection.By performing CCK-8 experiments in Rpn10 overexpressed cell line and nude mice tumor-bearing experiments,we confirmed that Rpn10 can promote the proliferation of lung adenocarcinoma cells in vitro and in vivo;by flow cytometry,we found that Rpn10 can regulate the cell cycle of lung adenocarcinoma cells resulting in regulation of the proliferation of cells;colony formation experiments showed that Rpn10 can improve the colony formation ability of lung adenocarcinoma in vitro.Flow cytometry results showed that knockdown of Rpn10 in lung adenocarcinomas resulted in increased cell apoptosis,whereas overexpression of Rpn10 could slightly reduce apoptosis in lung adenocarcinoma,demonstrating that Rpn10 enhances the ability of lung adenocarcinoma to resist apoptosis.The impaction of Rpn10 on the EMT of lung adenocarcinoma was detected by RT-qPCR,and we found that Rpn10 promoted the EMT transformation of lung adenocarcinoma;the wound-healing assay and the Transwell assay showed that the ability of lung adenocarcinoma to invade and migrate was weakened after depletion of Rpn10;Rpn10 can promote the EMT transformation of lung adenocarcinoma,which leads to the metastasis of lung cancer.In the fourth chapter,we used flow cytometry to detect the apoptosis induced by Gefitinb,and found that reduced the apoptosis of lung adenocarcinoma cells induced by Gefitinb.Rpn10 could enhance the resistance to Gefitinb induced cell apoptosis.In the fifth chapter of this study,we explored the mechanism by which Rpn10 regulates lung adenocarcinoma development and progression.EGFR was identified as the target of Rpn10 by RNA sequencing,and we confirmed that Rpn10 regulates the m RNA expression level of EGFR by RT-q PCR.Western Blot results confirmed that Rpn10 regulates the protein expression of EGFR.Using CHX to inhibit protein synthesis,we found that depletion of Rpn10 led to the down-regulation of EGFR protein,suggesting that Rpn10 may not only regulate the protein level of EGFR by regulating EGFR m RNA,but also regulate the degradation of EGFR.Co-IP experiments showed that Rpn10 does not bind to EGFR,which ruled out the possibility that Rpn10 directly binds to EGFR to regulate its degradation.Inhibition of proteasome activity by MG132 did not rescue the down-regulation of EGFR caused by Rpn10,suggesting that Rpn10 does not regulate EGFR degradation through the proteasome.After inhibiting lysosomal activity,Western Blot assay was performed to detect the changes of EGFR protein,which showed that inhibiting lysosomal activity could rescue the downregulation of EGFR caused by Rpn10.Rpn10 regulates the protein homeostasis of EGFR through the lysosomal pathway,and then positively regulates the expression of lung adenocarcinoma’s development and drug resistance.In conclusion,our study found that Rpn10 is highly expressed in lung adenocarcinoma tissue and serum of lung adenocarcinoma patients at the clinical level,and Rpn10 is expected to be a biomarker to assist in the diagnosis of lung adenocarcinoma.At the molecular cell and animal level,Rpn10 can regulate lung adenocarcinoma by regulating EMT transformation and regulate the metastasis of lung adenocarcinoma;Rpn10 can regulate the m RNA expression of EGFR,affect the synthesis of EGFR,and reduce the degradation of EGFR through the regulation of the lysosomal pathway,maintaining the high protein level of EGFR in lung adenocarcinoma,leading to enhanced proliferation and anti-apoptosis ability in adenocarcinoma;what’s mote Rpn10 promotes the targeted drug resistance of lung adenocarcinoma.Our study provides new insights into molecular mechanism of tumor development and drug resistence as well as a novel therapeutic target for the treatment of lung adenocarcinoma.