Experimental Studys On The Expression Of PIG11 In Human Gliomas And The Effect Of PIG11 Gene On Proliferation And Apoptosis In U251 Cells

Objective The purpose of this study was to detcet the expression level of PIG11 gene in normal brain tissue and different grade of gliomas and the effect on U251 glioma cell proliferation and apoptosis.Methods The expression level of PIG11 mRNA in 30 gliomas and 15 normal brain tissue was detected by RT-PCR and the expression difference between brain tissue and different grade of gliomas was analyzed. The cDNA of PIG11 was amplified from total RNA isolated from hman glioma by RT PCR. After enzyme cuttinged, the open reading frame (ORF) of PIG11 cDNA was cloned into eukaryotic expression vector pEGFP-N1 to form the recombinant plasmid named as pEGFP-N1-PIG11.Enzyme-cutting analysis and sequencing was used to identificated the recombinant plasmid. Then lipofectamine 2000 was used to transfected the pEGFP-N1-PIG11 into U251 cells. Subcellular localization of PIG11 gene and transfection efficacy were observed by fluorescent microscope and RT-PCR technique was applied to detected the expression of PIG11 in U251 after transfected. The effect of PIG11 on U251 cells proliferation was detected by MTT and apoptotic situation by Hoechst staining, Tunel, DNA ladder and flow cytometer (FCM) after transfection.Results PIG11 was expressed in brain tissues and all grades of gliomas. The expression intensity (OD_{PiG11/ODGapdh} of PIG11 in normal brain tissues, grade I and grade II gliomas ,grade III and grade IV gliomas were 0.95±0.20,0.71±0.15,0.39±0.16 respectively. The expression intensity decreased following the increasing of glioma grade (P<0. 05). The recombinant plasmid was correct identificated by enzyme-cutting analysis and sequencing. After transfected, green fluorescent protein (GFP) was observed by fluorescent microscope in the cytoplasm, and RT PCR showed the recombinant plasmid pEGFP-N1-PIG11 was transfected into U251 cells effectively. MTT indicated that overexpression of PIG11 gene inhibited the proliferation of U251 cells. DNA ladder showed obvious ladder-shaped electrophoresis strip in transgene group, but no ladder-shaped electrophoresis strip in blank group, vector group and liposome group. Hoechst staining Tunel showed that some cellular nucleus were stained thickly and some were splited into bits in transgene group, which was consistant with apoptotic morphological changes. It was indicated by Tunel that the apoptotic indexes were 2.1±0.1 %,2.5±0.2%,2.4±0.6% and 19.5±1.5% in blank group, vector group, liposome group and teansgene group respectively and the apoptotic index in transgene group was significantly higher than the other three groups(P< 0. 05). FCM indicated that the apoptotic ratios were 1.8±0.2%,2.2±0.2%,2.0±0.3% and 22.7±0.6%in blank group, blank-vector group, liposome group and teansgene group respectively and the apoptotic ratio in transgene group was significantly higher than the other three groups(P<0. 05 ). Conclusions PIG11mRNA was expressed in brain tissues and all grades of gliomas and the expressing intensity were negative correlated to glioma's grade. It indicated maybe PIG11 was related to glioma development. PIG11 protein was plasmosin. The overexpression of PIG11 could inhibit glioma's growth and induce apoptosis. PIG11 gene may become one of the target gene in gene therapy of gliomas.