The Experimental Study Of TRIM52 Promoting Hepatocellular Carcinoma Cell Proliferation,Migration And Invasion

Part 1The effect of TRIM52 on the proliferation,migration,invasion and cell cycle of hepatocellular carcinoma cellsObjective:In order to identify the role of TRIM52 on hepatocellular carcinoma(HCC),we detected the expression of TRIM52 in HCC tissues and cell lines,investigated the effect of TRIM52 in the proliferation,migration,invasion and cell cycle of HCC cells,and studied the growth effect of TRIM52 on tumor in vivo.Methods:(1)The expression of TRIM52 in HCC tissues and adjacent non-tumor hepatic tissues was detected by immunohistochemistry(IHC)analysis.(2)The expression of TRIM52 in HCC cell lines,including MHCC-97H and MHCC-97L cells,and normal human liver cell line L02 was measured by qRT-PCR and Western blot assay.(3)shRNA targeting TRIM52 and scramble shRNA were cloned into the pLKO.1 Ientiviral vector and transfected into MHCC-97H cells,respectively,TRIM52 encoding sequence was cloned into pLVX-Puro for constructing the pLVX-Puro-TRIM52 lentiviral expressing vector and transfected into MHCC-97L cells.Blank pLVX-Puro(Vector)was used as the negative control.(4)The proliferation of HCC cells was measured by Cell Counting Kit-8(CCK-8)assay.(5)The cell cycle,migration and invasion of HCC cells were measured by flow cytometry and Transwell assay respectively.(6)A xenograft tumor-bearing model was established by inoculating pLKO.l-shRNA-TRIM52 or pLKO.l-scramble shRNA(NC)transfected MHCC-97H cells into the nude mice.The tumor sizes were measured by vernier caliper every 3 days.33 days after inoculation the mice were killed and the tumor sizes and weights were detected.Then,the tumors were examined by hematoxylin-eosin(HE)staining and IHC analysis.Results:(1)TRIM52 was significantly up-regulated in the HCC tissues compared with the adjacent non-tumor hepatic tissues.(2)TRIM52 was up-regulated in MHCC-97H and MHCC-97L cells compared with normal human liver cell line L02.(3)CCK-8 assay demonstrated that shRNA-TRIM52 significantly inhibited the proliferation of MHCC-97H cells compared with pLKO.l-scramble shRNA(NC)transfection.Whereas,TRIM52 up-regulation significantly enhanced the proliferation of MHCC-97L cells compared with pLVX-Puro(Vector)transfection.(4)shRNA-TRIM52 significantly increased the number of MHCC-97H cells in GO-G1 phase and decreased the number of MHCC-97H cells in S and G2-M phases,compared with pLKO.l-scramble shRNA(NC)transfection.Whereas,TRIM52 up-regulation significantly increased the number of MHCC-97L cells in S phase and decreased the number of MHCC-97L cells in G0-G1 and G2-M phases,compared with pLVX-Puro(Vector)transfection.(5)Transwell assay demonstrated that shRNA-TRIM52 significantly inhibited MHCC-97H cell migration and invasion compared with pLKO.l-scramble shRNA(NC)transfection.Whereas,TRIM52 up-regulation significantly enhanced MHCC-97L cell migration and invasion compared with pLVX-Puro(Vector)transfection.(6)The tumor sizes and weights of the pLKO.l-shRNA-TRIM52 transfected mice were significantly decreased compared with the pLKO.l-scramble shRNA(NC)transfected mice.Tumors in the pLKO.l-shRNA-TRIM52 transfected mice grew much slower compared with the pLKO.l-scramble shRNA(NC)transfected mice.Furthermore,shRNA-TRIM52 significantly reduced Ki67 expression compared with pLKO.l-scramble shRNA(NC)transfection measured by IHC analysis.Conclusions:TRIM52 up-regulation promotes the proliferation,migration,invasion and cell cycle progress of HCC cells.Part 2The effect of PPM1A on TRIM52-mediated enhancement of proliferation,migration and invasion in hepatocellular carcinoma cellsObjective:To elucidate the underlying mechanisms by which TRIM52 exerts its function in HCC carcinogenesis,we studied the effect of TRIM52 on p21,MMP2,PPM1A,p-Smad2/3 and Smad2/3 expression in HCC cells,investigated the interaction between TRIM52 and PPM1A in HCC cells,and examined the effect of PPM1A on TRIM52-mediated enhancement of proliferation,migration and invasion in HCC cells.Methods:(1)pLKO.1-shRNA-TRIM52 and pLKO.1-scramble shRNA(NC)were transfected into MHCC-97H cells respectively.(2)The expression of p21,MMP2,PPM1A,p-Smad2/3 and Smad2/3 was measured by Western blot assay.(3)pLVX-Puro-TRIM52 and blank pLVX-Puro(Vector)were transfected into MHCC-97L cells respectively.(4)The expression of p21,MMP2,PPM1A,p-Smad2/3 and Smad2/3 was also measured by Western blot assay.(5)The interaction between TRIM52 and PPM1A in HCC cells was identified by co-immunoprecipitation(Co-IP).(6)PPM1A encoding sequence was cloned into pLVX-Puro for constructing the pLVX-Puro-PPM1A lentiviral expressing vector and transfected into MHCC-97L cells.Blank pLVX-Puro(Vector)was used as the negative control.(7)The proliferation of MHCC-97L cells was measured by CCK-8 assay.(8)The migration and invasion of MHCC-97L cells were measured by Transwell assay.(9)The expression of p21,MMP2,PPM1A,p-Smad2/3 and Smad2/3 was measured by Western blot assay.Results:(1)shRNA-TRIM52 significantly increased p21 and PPM1A expression,decreased MMP2 expression and induced Smad2/3 dephosphorylation in MHCC-97H cells compared with pLKO.1-scramble shRNA(NC)transfection.(2)TRIM52 up-regulation significantly decreased p21 and PPM1A expression,increased MMP2 expression and induced Smad2/3 phosphorylation in MHCC-97L cells compared with pLVX-Puro(Vector)transfection.(3)TRIM52 interacted with PPM1A in MHCC-97H and MHCC-97L cells.shRNA-TRIM52 significantly inhibited the ubiquitination of PPM1A in MHCC-97H cells.(4)PPM1A up-regulation significantly inhibited the proliferation,migration and invasion of MHCC-97L cells compared with pLVX-Puro(Vector)or pLVX-Puro-TRIM52 transfection.Both TRIM52 and PPM1A up-regulation significantly inhibited the proliferation,migration and invasion of MHCC-97L cells compared with only pLVX-Puro-TRIM52 transfection.(5)PPM1A up-regulation significantly increased p21 and PPM 1A expression,decreased MMP2 expression and induced Smad2/3 dephosphorylation in MHCC-97L cells compared with pLVX-Puro(Vector)or pLVX-Puro-TR1M52 transfection.Both TRIM52 and PPM1A up-regulation significantly increased p21 and PPM1A expression,decreased MMP2 expression and induced Smad2/3 dephosphorylation in MHCC-97L cells compared with only pLVX-Puro-TRIM52 transfection.Conclusions:PPM1A up-regulation inhibits TRIM52-mediated enhancement of proliferation,migration and invasion in HCC cells.TRIM52 up-regulation promotes HCC cell proliferation,migration and invasion through the ubiquitination of PPM1A.Part 3 The experimental study of TRIM52 promoting cell proliferation in hepatitis B virus-associated hepatocellular carcinomaObjective:In order to identify the role of TRIM52 on HBV-associated HCC,we detected the expression of TRIM52 and HBx in HBV-associated HCC tissues,investigated the effect of HBx on TRIM52 and NF-?B p65 expression,studied the effect of TRIM52 on HepG2.2.15 cell proliferation,and measured the effect of NF-?B inhibitor on TRIM52 expression and HepG2.2.15 cell proliferation.Methods:(1)HBV DNA levels in the peripheral blood samples of the HBV-associated HCC patients were detected by FQ-PCR.(2)The expression of TRIM52 in HBV-associated HCC tissues,HBV-associated cirrhotic liver tissues and adjacent normal liver tissues was measured by qRT-PCR and Western blot assay.(3)The expression of HBx in HBV-associated HCC tissues,HBV-associated cirrhotic liver tissues and adjacent normal liver tissues was measured by Western blot assay.(4)The ectopic HBx-expressing cellular model was established by transfecting HBx-expressing vector(HBx-pcDNA3.1)into HepG2 cells.(5)The expression of TRIM52 in HepG2 cells transfected with HBx-pcDNA3.1 was measured by qRT-PCR and Western blot assay.(6)The expression of TRIM52 and NF-?B p65 in LO2,HepG2 and HepG2.2.15 cells was measured by qRT-PCR and Western blot assay repectively.(7)pLK0.1-shRNA-TRIM52 was transfected into HepG2.2.15 cells.Then,the expression of TRIM52 in HepG2.2.15 cells was measured by qRT-PCR and Western blot assay.HepG2.2.15 cell proliferation was measured by CCK-8 assay.(8)In the presence of PDTC(NF-?B inhibitor),the expression of TRIM52 in HepG2.2.15 cells was measured by Western blot assay.HepG2.2.15 cell proliferation was measured by CCK-8 assay.Results:(1)The serum HBV DNA levels of all the HBV-associated HCC patients were above 103IU/mL.(2)HBV-associated HCC tissues had the highest level of TRIM52 and adjacent normal liver tissues had the lowest level.(3)HBV-associated HCC tissues had the highest level of HBx and adjacent normal liver tissues had the lowest level.(4)The expression of TRIM52 was significantly increased in HBx-pcDNA3.1 transfected HepG2 cells compared with untransfected HepG2 cells or negative control plasmid(NC)transfected HepG2 cells.(5)The expression of TRIM52 and NF-?B p65 was up-regulated in HepG2 and HepG2.2.15 cells.Moreover,the elevation was more obvious in HepG2.2.15 cells.(6)The expression of TRIM52 was specifically inhibited by TRIM52-shRNA compared with the untransfected group or the negative control plasmid(NC)transfected group.(7)CCK-8 assay demonstrated that after transfection with TRIM52-shRNA for 24,48 and 72 h,HepG2.2.15 cell proliferation was significantly attenuated.Furthermore,the anti-proliferation effect was time dependent.(8)In the presence of PDTC,TRIM52 expression was significantly reduced in HepG2.2.15 cells.(9)CCK-8 assay demonstrated that HepG2.2.15 cell proliferation was significantly inhibited after pretreatment with varying concentrations of PDTC for 24,48 and 72 h.Furthermore,the effect of PDTC was dose dependent.Conclusions:TRIM52 can promote cell proliferation and HBx may regulate TRIM52 expression via the NF-?B signaling pathway in HBV-associated HCC.