The Effect Of UHRF1 Stability On The Proliferation And Migration Abilities Of Glioma Cells

Objectives 1To detect the protein levels of epigenetic regulation factor UHRF1,ubiquitin enzyme beta-TRCP1 and deubiquitin enzyme HAUSP in stage I～IV glioma tissues,and analyze the correlation between the expression of UHRF1,beta-TRCP1 and HAUSP and clinical pathological characteristics of glioma,respectively.2 To analyze the effect of beta-TRCP1 and HAUSP on the UHRF1 stability,as well as proliferation and migration abilities of glioblastoma cell.Methods 1 The protein levels of HAUSP,beta-TRCP1,UHRF1 in a total of 41 stage IIV glioma sections were dected by immunohistochemistry and the correlation between protein levels and clinicopathologic was analyzed,respectively.2 The glioblastoma cell line U251 cells were transfected with N1 plasmid containing beta-TRCP1 coding sequenc(beta-TRCP1 group)and blank N1 plasmid(N1 group),respectively.The m RNA and protein levels of UHRF1 were detected by RT-q PCR and immunocytochemistry,respectively.The proliferation and migration abilities of the two group cells were detected by cck8 test and unmatrigel-transwell assay,respectively.3 The U251 cells were transfected with si-HAUSP and negative untreated group NC,respectively.The m RNA and protein levels of UHRF1 were detected by RT-q PCR and immunocytochemistry,respectivly.The proliferation and migration abilities of the two group cells were detected by cck8 test and unmatrigel-transwell assay,respectively.Results 1 The immunohistochemistry results showed that:(1)The UHRF1 protein was localized in the nucleus.The UHRF1 protein level in high-grade gliomas(III/IV)was higher than that in low-grade gliomas(I/II).The UHRF1 protein level was significantly correlated with pathological grading(P<0.05),whereas it was not correlated with patients age,gender and tumor size(P>0.05).(2)The beta-TRCP1 protein was mostly localized in the nucleus and a few localizated in cytoplasm.The beta-TRCP1 protein level in high-grade gliomas(III/IV)was lower than that in low-grade gliomas(I/II).The beta-TRCP1 protein level was significantly correlated with pathological grading(P<0.05),whereas it was not correlated with patients age,gender and tumor size(P>0.05).(3)The HAUSP protein was localized in the nucleus.The protein level in high-grade gliomas(III/IV)was higher than that in low-grade gliomas(I/II).The HAUSP protein level was significantly correlated with pathological grading(P<0.05),whereas it was not correlateed with patients age,gender and tumor size(P>0.05).2 The results of transfecting beta-TRCP1 in U251 cells:(1)There was no siganificant difference of UHRF1 m RNA level between N1 group and beta-TRCP1 group detected by RT-q PCR(P>0.05).This showed that beta-TRCP1 overexpression has no influence on the m RNA level of UHRF1 in U251 cells.(2)The protein level of UHRF1 in beta-TRCP1 group(0.459±0.037)was significantly lower than that in N1 group detected by immunocytochemistry(P<0.05).It showed that beta-TRCP1 overexpression decreased the protein level of UHRF1 in U251 cells.(3)The cell proliferation ability in the beta-TRCP1 group was markedly lower than that in N1 group on the fifth day analyzed by cck8(P<0.05).This showed that beta-TRCP1 overexpression inhibit U251 cell proliferation.(4)Compared with in N1 group(473.33±36.11),the number of cells passing through the transwell chamber was notably reduced in beta-TRCP1 group analyzed by transwell(133.00±30.19)(P<0.05).It showed that beta-TRCP1 over-expression inhibit migration ability of U251 cell.3 The results of transfecting HAUSP in U251 cells:(1)There was no siganificant difference of UHRF1 m RNA level between NC group and HAUSP group detected by RT-q PCR(P>0.05),This showed that HAUSP knockdown has no influence on the m RNA level of UHRF1 in U251 cells.(2)The protein level of UHRF1 in HAUSP group(0.457±0.089)was significantly lower than that in NC group detected by immunocytochemistry(P<0.05).It showed that HAUSP knockdown decreased the protein level of UHRF1 in U251 cells.(3)The cell proliferation ability in the HAUSP group was markedly lower than that in NC group on 5 days,especially on the third and forth days analyzed by cck8(P<0.05).This showed that HAUSP knockdown inhibit U251 cell proliferation.(4)Compared with in NC group(340.66.00 ±102),the number of cells passing through the transwell chamber was notably reduced in HAUSP group analyzed by transwell(112.66±12.01)(P<0.05).It showed that HAUSP knockdown inhibitmigration ability of U251 cell.Conclusions 1 The protein levels of both UHRF1 and HAUSP are higher in high-grade glioma than those in low-grade gliomas,whereas the protein level of beta-TRCP1 is lower in high-grade glioma than that in low-grade gliomas.The protein levels of UHRF1,beta-TRCP1 and HAUSP are all associated with the pathological staging of glioma.2 In glioblastoma cells,the protein level of UHRF1 is negatively regulated by ubiquitin enzymes beta-TRCP1 and positively regulated by deubiquitin enzymes HAUSP.3 The beta-TRCP1 inhibit the proliferation and migration abilities of glioma cells,while HAUSP promote the proliferation and migration abilities of glioma cells.Change of UHRF1 stability is probably one of the factors involved in.Figure11;Table10;Reference 121...