The Study Of The Effect And Possible Mechanism For Deubiquitinase USP2 On The Proliferation,Migration And Invasion In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the most common malignant tumor,its morbidity and mortality are among the highest in the world.HCC patients with early stage could obtain better therapeutic effect though surgical resection,liver transplantation and arterial embolization.However,only a few patients could be cured(< 10%)and most patients will develop into the advanced stage.At present,there is no effective treatment for advanced HCC,which leads to the poor prognosis.Therefore,it is an urgent medical demand to develop new therapies for advanced HCC,to improve the survival time of the patients.Continuing to explore and reveal the molecular mechanism of the tumorigenesis and development in HCC is an important basis for achieving this demand.Ubiquitin-specific proteases(USPs)is the largest subgroup of the deubiquitinase(DUBs)family.USPs catalyze and remove the ubiquitin chain modification of target proteins,thereby inhibiting the degradation of target proteins through the ubiquitin-proteasome pathway,and increasing the stability of target proteins.Studies have revealed that abnormal expression of USPs will lead to abnormal expression of many key proteins,and widely involved in DNA damage repair,proliferation,apoptosis and invasion and metastasis of cancers.However,the important role and mechanism of USPs in HCC has rarely been revealed.In the present study,we hope to illuminate the important role and mechanism of USP2 in HCC through three important research parts,including:(1)Study of USP2 expression in HCC clinical tissue samples and its correlation with patients' clinicopathological features;(2)Study of the effect of USP2 on the proliferation,migration and invasion in HCC cells;(3)Study of the molecular mechanism of USP2 for affecting the proliferation,migration and invasion via Rab1 A in HCC cells.By revealing the role and molecular mechanism of USPs in HCC,it could provide a new therapeutic strategy for HCC.Part 1.Study of USP2 expression in HCC clinical tissue samples and its correlation with the patients' clinicopathological featuresObjective: To investigate the correlation between USP2 and HCC preliminarily,by detecting the expression of USP2 in clinical samples of HCC and analyzing its correlation with the patients' clinicopathological featuresMethods: 20 cases of fresh HCC tissues and the matched adjacent tissues were collected in our hospital.Western blotting assays were used to detect the expression of USP2 in HCC tissues and the matched adjacent tissues.Tissue microarray containing 100 cases of HCC tissues and the adjacent tissues was made,and immunohistochemical staining was used to detect the expression of USP2 in this tissue microarray.Chi square test and Kaplan-Meier survival analysis were used to detect the correlation between USP2 expression and the clinicopathological features and prognosis of patients.Results: In 20 fresh HCC tissues and the matched adjacent tissues,USP2 expression in the tumor tissues of 12 cases(60%)was significantly higher than that in the matched adjacent tissues.The results from tissue microarray also showed that USP2 expression in HCC tissues was significantly higher than that in the adjacent tissues.Statistical analysis revealed that USP2 expression was positively associated with the pathological grading and liver portal lymph node metastasis,but there was no significant correlation between USP2 expression and other clinicopathological features such as patient gender,age,tumor size,HBV infection.Kaplan-Meier survival analysis showed that patients with high expression of USP2 had worse prognosis.Conclusion: The expression of USP2 in HCC tissues is significantly increased,and is positive associated with the pathological grading,liver portal lymph node metastasis and poor prognosis of patients,suggesting that USP2 may be involved in the malignant progression of HCC.Part 2.Study of the effect of USP2 on the proliferation,migration and invasion in HCC cellsObjective: To investigate the effects of USP2 on the proliferation,migration and invasion in HCC cells.Methods: Western blotting assays were used to detect the expression of USP2 in HCC cell lines.Lentiviruses carrying specific interference sequence of USP2 were used to infect HCC cells,and stable cell of USP2 silencing was obtained,and CCK-8 cell proliferation assays,clone formation assays,wound healing assays,Transwell cell migration assays and matrigel invasion assays were used to detect the effects of USP2 silencing on the proliferation,migration and invasive ability of HCC cells.lentiviruses carrying USP2 overexpression sequence were used to infect HCC cells,and stable cell of USP2 overexpression was obtained.The effects of USP2 overexpression on the proliferation,migration and invasion ability of HCC cells were detected by the above-mentioned cell biofunctional experiments.The effect of USP2 silence or overexpression on the growth and metastasis of HCC cells in vivo were detected by tumor bearing nude mice.Results: Compared with human normal hepatocyte line L02,USP2 was highly expressed in HCC cell lines HepG2,SK-Hep1 and SMMC-7721,and there was no significant difference in Huh7 and MHCC97-H.After silencing USP2 expression in HepG2 and SK-Hep1 cells,the proliferation,migration and invasion of tumor cells were significantly inhibited.After overexpression of USP2 in Huh7 cells,the proliferation,migration and invasion of tumor cells were significantly promoted;The growth of subcutaneous tumors was significantly restrained after silencing USP2 expression in SK-Hep1 cells;The growth ability of subcutaneous tumors was significantly enhanced after overexpression of USP2 in Huh7 cells;In addition,the metastasis of subcutaneous tumors was significantly inhibited after SK-Hep1 cells silencing USP2 expression.Conclusion: USP2 can significantly promote the proliferation,migration and invasion of HCC cells.Part 3.Study of the molecular mechanism of USP2 for affecting the proliferation,migration and invasion via Rab1 A in HCC cellsObjective: To reveal the molecular mechanism of USP2 promoting the proliferation,migration and invasion in HCC cells.Methods: HCC cells HepG2 and SK-Hep1 were infected with flag-labeled overexpression lentiviruses of USP2,respectively.Immunoprecipitation(IP)assays with antibody against Flag-USP2 were performed to precipitate the proteins that might directly bind with USP2.LC-MS/MS(liquid chromatography-mass spectrometry)was performed to identify the precipitated proteins,and screening the target proteins for subsequent study.HepG2 and SK-Hep1 cells were co-infected with the overexpression lentiviruses of USP2 and target protein,respectively.Co-immunoprecipitation(CO-IP)and cell immunofluorescence(ICC)assays were performed to verify the direct binding between USP2 and target protein.qRT-PCR and western blotting assays were performed to detect the effects of USP2 silencing or overexpression on the mRNA and protein level of target protein.CHX protein stability tests were performed to detect the effects of USP2 silencing or overexpression on the stability of target protein.SK-Hep1 cells were co-infected with the overexpression lentiviruses of target protein and the interfere lentiviruses of USP2,or Huh7 cells co-infected with the overexpression lentiviruses of target protein and the overexpression lentiviruses of USP2,after MG132 treatment,the effect of USP2 silencing or overexpression on the ubiquitin modification of target protein was detected by IP assays.SK-Hep1 cells were co-infected with the interfere lentivirus of USP2 and the overexpression lentivirus of target protein.CCK-8 assays,Transwell migration assays and matrigel invasion assays were performed to verify that USP2 promoted the malignant progression of HCC via the target protein.Western blotting assays were used to detect the expression of target protein in HCC cell lines and clinical samples,and to analyze the correlation between USP2 and target protein expression.Results: In total,210 proteins from HepG2 cells and 279 proteins from SK-Hep1 cells were identified as the potential targets of USP2,and 121 proteins in the two cell lines were found to overlap.In this study,we choose the top ten overlapping proteins to further analysis,basing on the background analysis and preliminary verification from these proteins,it was determined that Rab1 A protein might be a downstream target of USP2.Rab1 A is a member of the Rab small GTPase family,it is known to mediate dynamic membrane trafficking between ER and Golgi,and this transport system involves various important signaling transduction such as cell growth,lipid metabolism and autophagy.Studies have confirmed that increased Rab1 A expression promotes tumor proliferation,migration and invasion.CO-IP assays have confirmed that USP2 and Rab1 A bound directly in HCC cells.ICC assays also showed that USP2 and Rab1 A were colocalization in HCC cells.qRT-PCR results show that there was no significant difference of Rab1 A mRNA expression after USP2 silencing in HepG2 and SK-Hep1 cells,and no significant difference in Rab1 A mRNA expression after USP2 overexpression in Huh7 cells.Western blotting results showed that Rab1 A protein expression decreased significantly after USP2 silencing in HepG2 and SK-Hep1 cells,and increased significantly after USP2 overexpression in Huh7 cells.CHX protein stability tests showed that the stability of Rab1 A protein decreased significantly after silencing USP2 expression in SK-Hep1 cells,and increased significantly after overexpressing USP2 in Huh7 cells.When detecting the effect of USP2 on the ubiquitination modification of Rab1 A,it was found that the K48-linkage specific polyubiquitin modification of Rab1 A was increased significantly after silencing USP2 expression in SK-Hep1 cells,while that modification was decreased after overexpressing USP2 in Huh7 cells.Functional recovery experiments showed that the inhibitory effect of USP2 silencing on the proliferation,migration and invasion of SK-Hep1 cells was significantly restored after overexpression of Rab1 A.Western blotting results showed that there was a significant positive correlation between the expression of USP2 and Rab1 A in HCC cell lines and tissue samples.Conclusion:(1)There is a direct binding between USP2 and Rab1 A protein in HCC cells;(2)USP2 affects Rab1 A expression through protein level rather than transcriptional level;(3)USP2 increases the stability of Rab1 A protein;(4)USP2 deubiquitinates Rab1 A protein and removes the K48-linkage specific polyubiquitin modification of Rab1 A protein;(5)USP2 promotes the malignant progression of HCC via Rab1A;(6)USP2 and Rab1 A expression in HCC cell lines and tissue samples are significantly positively correlated,which could further confirmed the above conclusions.In brief,USP2 can deubiquitinate and stabilize Rab1 A protein to promote the proliferation,migration and invasion of HCC.Through the above three parts of study,we make a conclusion that:USP2 is a clear oncogene in HCC,the highly expressed USP2 promotes the proliferation,migration and invasion of tumor cells by deubiquitinating and stabilizing Rab1 A protein,which leads to the decrease of survival time of patients.