Study On The ARHI Expression And Clone In Human Gliomas And The Inhibitory Effect Of ARHI Transfected On Proliferation And Invasion Of Gliomas

ARHI(aplysia ras homolog I)/NOEY2 is a new tumor suppressor gene.In a number of recent researches,it has been demonstrated that ARHI gene charged with the regulation and control of the growth and apoptosis of the cell and has been implicated in signaling pathway control.Moreover,ARHI gene had been verified to play an important role in infiltration and metastasis of tumor cell.To our knowledge,however,the expression and ARHI and its role in glioma progression has not been investigated.For further studying the aberration and the role of ARHI in gliomas,we began with examining the expression of ARHI in a large number of glioma specimens in the present study.A recombinant vector carrying ARHI cDNA was then constructed and transfected into glioma cells with lower expression of ARHI for observing its biological effect on gliomas in vitro.In the first part of this study,ARHI expression was examined in 59 freshly resected glioma samples and 10 normal brain tissues at mRNA level by RT PCR.Meanwhile,ARHI expression was also detected by Western blot in the same 59 resected glioma samples,10 normal brain tissues and 4 malignant glioma cell lines.It was found that the normal brain tissues had constitutional expression of ARHI while the expression of ARHI mRNA and protein was reduced in most of the gliomas and its expression was positively correlated with the tumor grade.The expression level of ARHI in high grade gliomas was significantly decreased than that of low grade ones.Moreover,there was also a lower expression of ARHI in human glioblastoma cell lines(U87MG,U251, SHG44 and BT325).This result further confirmed that ARHI expression was significantly reduced in majority of gliomas,especially in high grade ones.Based on this finding,an eukaryotic expression vector of ARHI was constructed for studying its biological effect on glioma cells. Recombinant vector was identified by DNA sequencing.In the second part of this study,ARHI constructs was transfected to human glioblastoma U251 cell line with reduced expression of ARHI.The positive clones were selected by G418 and identified by Real time Quantitative PCR and Western blot analysis.For observing the phenotypic changes of the cells transfected with ARHI,in the third part of this study,the cell proliferation was determined by MTT assay and flowcytometry.Cell apoptosis was detected by TUNEL procedure.Moreover,cell invasion was evaluated by Transwell migration assay and Wound-healing method test.It was found that the cell proliferation activity was inhibited after transfection with ARHI,the G0/G1 phase faction was lowered and cell cycle was arrested in G2/M phase.The other results revealed that ARHI exerted the suppressive effect on the glioma cell migration and invasion,and induced apoptosis.In conclusion,the results of the current study demonstrate that normal brain tissues have constitutive expression of ARHI.However, ARHI expression is significantly reduced in the majority of the gliomas and its expression is positively correlated with the tumor grade. Transfection of ARHI constructs into the glioma cells with lower expression of ARHI could inhibit the glioma cell proliferation activity and invasive ability and induce cell apoptosis.Consequently,ARHI may be considered as a tumor suppressor gene and a promising candidate for gene therapy of glioma.To our knowledge,the present research is the only comprehensive study on the biological effect of human ARHI on gliomas,and provides the first molecular evidence that ARHI is a tumor suppressor gene.