USP5 knockdown alleviates lung cancer progression via activating PARP1-mediated mTOR signaling pathway

Abstract

Background: With the rapidly increasing morbidity and mortality, lung cancer has been considered one of the serious malignant tumors, affecting millions of patients globally. Currently, the pathogenesis of lung cancer remains unclear, hindering the development of effective treatment. This study aims to investigate the mechanisms of lung cancer and develop an effective therapeutic approach for intervention in preventing lung cancer progress.

Methods: The USP5 levels are detected in lung cancerous and paracancerous tissue by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting methods to explore their roles in lung cancer progression. MTT, colony assay, and transwell chamber approaches are employed to measure cell viability, proliferation, and migration, respectively. Further, flow cytometry experiments are performed to examine the effect of USP5 on lung cancer. Finally, the investigations in vivo are executed using the mice subcutaneous tumor model to identify the effect of USP5 in promoting lung cancer development.

Results: Notably, USP5 is highly expressed in lung cancer, USP5 overexpression promoted the proliferation and migration in the lung cancer cell lines, H1299 and A549, while knockdown of USP5 inhibited these via regulating the PARP1-mediated mTOR signaling pathway. Furthermore, the subcutaneous tumors model was established in C57BL/6 mice, and the volume of subcutaneous tumors was significantly reduced after silencing USP5, while increased after USP5 overexpression and decreased significantly with shRARP1 treatment at the same time.

Conclusions: Together, USP5 could promote the progression of lung cancer cells by mTOR signaling pathway and interacting with PARP1, indicating that USP5 may become a new target for lung cancer treatment.

Keywords: Lung cancer; PARP1; USP5; mTOR.

Fig. 1

USP5 is related with the overall survival of lung cancer patients and high expressed in lung cancer tissues. (A) Expression of USP5 levels in lung cancer through GEPIA analysis based on the online TCGA database. (B) The overall survival rates of cells in correlation with the USP5 expression levels. (C) Immunohistochemical staining images showing the levels of USP5 protein in the corresponding lung cancerous and paracancerous tissues. * P < 0.05

Fig. 2

USP5 promotes H1299 and A549 cells proliferation and migration. (A) The expression levels of USP5 via RNA interference were checked by Western blotting. (B) MTT assay was performed to examine the cell viability after knocking down USP5. (C-D) Colony-forming assay represents the proliferation ability of H1299 and A549 cells after downregulation of USP5. (E) Ctrl and USP5 overexpressed H1299 and A549 cells were subjected to Western blotting analysis of USP5. (F-H) Proliferation, colony formation and migration abilities were analyzed in H1299 and A549 cells after over-expression of USP5. * P < 0.05, and ** P < 0.01

Fig. 3

USP5 knockdown suppressed the xenograft growth. Downregulation of USP5 suppressed the xenograft growth and resulted in the apparent decrease in the xenograft weights. *** P < 0.001

Fig. 4

mTOR activation of USP5 promotes the proliferation and migration of lung cancer cells. (A) Left, siCtrl, siTSC2 and siTSC2 cells treated with rapamycin were subjected to Western blotting analysis of various proteins of mTOR markers and USP5. Right, siCtrl and siRaptor cells were subjected to Western blotting analysis of various proteins of mTOR markers and USP5. (B) GSEA analysis showed that USP5 high expression was correlated with high Mtorc1 activity in lung cancer patients. (C-E) The proliferation, apoptosis, and migration efficiencies of cells were measured by colony-forming assay, flow cytometry detection, and transwell chamber, respectively, in Ctrl and USP5 overexpressed cells treated with rapamycin. * P < 0.05, ** P < 0.01

Fig. 5

USP5 promotes the progression of lung cancer by regulating PARP1. (A) USP5-Flag and PARP1-Flag were overexpressed in HEK293 cells. The cell lysates were subjected to immunoprecipitation with IgG and Flag and immunoblotting analysis of USP5 and PARP1. (B) USP5 and PARP1 protein levels were detected by immunoblotting in USP5 overexpressed and knockdown cells. (C) siCtrl and siUSP5 cells were treated with cycloheximide and subjected to immunoblotting analysis of USP5. (D) Ubiquitin was overexpressing in siCtrl and siUSP5 cells which were treated with MG132. The cells were IP with PARP1 antibody. Ubiquitionation abundance was detected by immunoblotting

Fig. 6

USP5 promotes the progression of lung cancer by targeting PARP1 in vitro and vivo. (A) Western blots showed that PARP1 was efficiently knocked down in USP5 overexpressed lung cancer cells. (B-D) Effect of PARP1 knockdown on the proliferation, colony formation, and migration abilities of USP5 overexpressing lung cancer cells. (E-F) Role of PARP1 inhibitors on colony formation, and migration abilities of USP5 overexpressing cells. (G) Effect of PARP1 knockdown on the volume of subcutaneous tumor in mice. * P < 0.05, ** P < 0.01, *** P < 0.001