The Functional Analysis Of Deubiquitinase USP49 In The Development Of Non-small Cell Lung Cancer

Part 1 The study of USP49 expression in non-small cell lung cancerObjective:It is known that deubiquitinating enzymes are closely associated with the development of tumorigenesis,and it is considered that targeting deubiquitinating enzymes has become an effective strategy to develop anti-tumor drugs.In our previous studies,we found that the deubiquitinase USP49 was decreased in non-small cell lung cancer(NSCLC)by GEPIA,a public cancer database,but its function and molecular mechanism have not been reported.Therefore,based on the previous finding,this paper is aimed to analyze the expression of USP49 in NSCLC tumor tissues and cell lines,and investigate the relationship between the expression levels of USP49 and the prognosis of NSCLC patients,so as to better understand the related function and mechanism of the deubiquitinase USP49 in NSCLC,and lay a theoretical foundation for the clinical development of targeted drugs targeting USP49.Methods:(1)The expression of USP49 in lung adenocarcinoma and lung squamous cell carcinoma was analyzed by using the GEPIA tumor database.The analysis results were compared with the expression of USP49 in normal lung tissues;(2)The mRNA levels of USP49 in 20 pairs of NSCLC tumor tissues and adjacent normal tissues were detected by qRT-PCR;(3)The expression levels of USP49 in 4 NSCLC cell lines and 1 human normal bronchial epithelioid cell line were detected by immunoblotting;(4)Kaplan-Meier plotter was used to analyze the overall survival of lung patiens with different expression of USP49.Results:(1)The GEPIA tumor database showed that the expression levels of USP49 in lung adenocarcinoma and lung squamous cell carcinoma were significantly lower than in normal tissues;(2)The expression of USP49 in 20 pairs of NSCLC tumor tissues and adjacent normal tissues,and the mRNA levels of USP49 were found to be decreased in NSCLC tumor tissues;(3)Immunoblotting was used to analyze USP49 expression in NSCLC cell lines,and it was found that USP49 protein was lowly expressed in NSCLC cell lines compared with the normal bronchial epithelioid cells;(4)Kaplan-Meier plotter showed that lung cancer patients with high expression of USP49 had a longer overall survival than the patients with low expression.Conclusions:USP49 was downregulated in NSCLC.And whether through the public tumor database GEPIA,Kaplan Meier plotter,or NSCLC tumor tissues or different NSCLC cell lines,the low expression of USP49 in NSCLC could be confirmed.These data suggested that USP49 may be closely related to the development of NSCLC.In addition,USP49 could be also used as a prognostic indicator for clinical NSCLC patients.Part 2 USP49 inhibits cell growth of NSCLC by suppressing cell cycle progressionObjective:Since the expression of USP49 was decreased in lung cancer,especially in non-small cell lung cancer(NSCLC),in order to further understand the function and molecular mechanism of the deubiquitinating enzyme USP49 in NSCLC,the role of USP49 in NSCLC was analyzed by using lentivirus-delivered overexpression system,so as to elaborate the relevant function and mechanism of the deubiquitinating enzyme USP49 in NSCLC.Methods:(1)USP49 was overexpressed in NSCLC cells by using lentivirus-delivered overexpression system;(2)CCK-8 assay was used to detect the effect of overexpressed USP49 on the cell growth of NSCLC;(3)The effects of USP49 overexpression on Cyclin D1 and p53 proteins were detected by immunoblotting;(4)Flow cytometry was used to detect the effect of overexpression of USP49 on the cell cycle of NSCLC cells.Results:(1)Overexpression of USP49 significantly inhibited the cell growth of NSCLC;(2)It was found that overexpression of USP49 in NSCLC cells could significantly inhibit the expression of Cyclin D1 and induce the expression of p53 protein by immunoblotting;(3)Flow cytometry was used to further analyze the effect of overexpression of USP49 on the cell cycle of NSCLC cells,and it was found that overexpression of USP49 prevented the transformation from G0 phase to G1 phase in NSCLC cells.Conclusions:Overexpression of USP49 in NSCLC cells can significantly inhibit cell growth,and we further evaluated the relative protein levels of cell survival signaling,and it was found that USP49 markedly inhibited the expression of cell-cycle associated proteins,including downregulating Cyclin D1 expression,and increasing the expression of tumor suppressor protein p53.And the flow cytometry showed that USP49 overexpression prevented the transformation of cells from G0 phase to G1 phase,thereby inhibiting the growth of NSCLC cells.Part 3 USP49 regulates PI3K/AKT signaling in NSCLC cellsObjective:It is known that PI3K is involved in mediating cell growth,proliferation and migration in tumors.And our previous study found that USP49 could regulate PI3K signal in NSCLC cells.Therefore,in this part,we would confirm this phenomenon,and further clarify the mechanism of USP49 in NSCLC cells.In specific,we would evaluate the expression of some proteins involved in PI3K signaling by lentivirus-delivered overexpression and knockdown systems..Methods:(1)Immunoblotting was used to detect the phosphorylation of PI3K p85 when cells were overexpressed with USP49 in NSCLC cells;(2)The phosphorylation levels of AKT and mTOR proteins were detected by immunoblotting when cells were overexpressed with USP49 in NSCLC cells;(3)The phosphorylation of PI3K p85 was measured by Immunoblotting when cells USP49 was silenced by shRNAs in NSCLC cells;(4)The phosphorylation levels of AKT and mTOR were measured by Immunoblotting when cells USP49 was silenced by shRNAs in NSCLC cells.Results:(1)Overexpression of USP49 can significantly inhibit the phosphorylation level of PI3K p85 in NSCLC cells.(2)Overexpression of USP49 in NSCLC significantly inhibited the phosphorylation levels of AKT and mTOR proteins;(3)Silence of USP49 by shRNA upregulated the phosphorylation of PI3K p85 in NSCLC cells;(4)Silence of USP49 by shRNA upregulated the phosphorylation of AKT and mTOR in NSCLC cells.Conclusions:Deubiquitinating enzyme USP49 can affect the cell growth and function of NSCLC by regulating the PI3K/AKT signaling pathway,which has been well verified by overexpressing or silencing USP49 in NSCLC cells.This result further explained the mechanism and the signaling pathway involved in USP49 in NSCLC.Part 4 USP49 stabilizes PTEN by inhibiting its polyubiquitination in NSCLCObjective:In NSCLC,the mechanism of ubiquitinating enzyme USP49 has not been reported.Since overexpression of USP49 can upregulate the expression of PTEN protein,this part will explore whether USP49 can deubiquitinate PTEN,so as to further elaborate the regulation of USP49 in the ubiquitination process of PTEN in NSCLC.Methods:(1)The expression correlation between USP49 and PTEN was analyzed by GEPIA database;(2)Co-IP was used to analyze whether USP49 could deubiquitinate PTEN;(3)Transfection was carried out to detect whether USP49 upregulated PTEN in NSCLC cells;(4)CHX chase assay was carried out to investigate the effect of USP49 on the half-life of PTEN protein.Results:(1)GEPIA database analysis found that the expression of USP49 and PTEN have a great correlation;(2)Co-IP experiment showed that USP49 could deubiquitinate PTEN protein;(3)Co-transfection of USP49,PTEN and Ub in vitro showed that USP49 could stabilize PTEN protein;(4)CHX chase assay showed that USP49 could prolong the half-life of PTEN protein.Conclusions:In NSCLC,USP49 can decrease the polyubiquitination of PTEN protein,and stabilize PTEN protein.Additionally,the CHX chase assay showed that USP49 prolonged the half-life of PTEN protein.This mechanism can enrich the ubiquitination mechanism of USP49 in NSCLC cells.