Study On The Matrix Metalloproteinase-2 Expression In U2OS Cells Transfected With HTERT

Telomere is an especial DNA construction residing in terminal end of a chromosome of eucaryotic cells, follow the cell division of normal human body cells every time, terminal end of chromosomes shorten gradually,until the cells died at the last. Telomerase is a kind of nuclear ribonuclear protein that can synthesis the reverse transcription tandem repetitive sequence of the terminal end of telomere .It's most important function is synthesis telomere DNA fragments to extend the abbreviated telomeres of terminal end of a chromosomes. Due to telomerase activation is seen in majority malignant tumors, its considered to a high specificity malignant tumor marker. hTERT is human telomerase reversetranscriptase,its expression is high positive correlation to telomerase activation. Therefore hTERT and telomerase activation are considered to be the foundation of the infinite reproductive activity of malignant tumor cells.The infiltration and extension processes of tumor are very complicated . Although there are many reports about telomerase and malignant tumor's genesis,but there are very few reports about the influence of telomerase activation to extracellular matrix.To make further approach of telomerase activation's effect in tumor infiltration and extension,and the possible mechanism of action,our study chose non-telomerase activation, hTERT negative express-human osteosarcoma line U2OS as study target ,observe the influence of hTERT transfection to MMP-2's secretion.We hope this study could provide reference for telomerase activation in mechanism of action of the infiltration and extension processes of tumor.We utilized hTERT gene eukaryotic expression vector PCI-Neo-hTERT, and extracted plasmid DNA after enzyme-cutting identify; liposome infection protocol to transfect U2OS cells,and made G418 bolting .At last we chose two well growth positive clonings(positive cloning 1 and positive cloning 2) for study. We used hTERT RT-PCR to detect the transfection effect of positive cloning 1 and positive cloning 2,the result show that there were a GAPDH amplification linear vector strip which was about 108bp through electrophoresis analysis,while there were a hTERT amplification linear vector strip which was about 111bp ,but there was not hTERT amplification linear vector strip in the control group. Relative expression levels of hTRER mRNA in the two positive cloning groups were higher than the control guoup(P<0.05),this indicate that we succeed to obtain hTERT expression cell clonings.Base this foundation,we used TRAP-ELISA to detect telomerase activation of the two positive clonings,the result show that compared to control group,the two positive clonings show obvious telomerase activation. TRAP-ELISA detection show that the OD450-690 were:control group 0.069±0.012;positive cloning1 0.560±0.024;positive cloning2 0.589±0.016.This data illustrate that U2OS cells stable transfected with hTERT could generate telomerase activation.We use RT-PCR and zymography to observe two positive MMP clonings. In the RT-PCR of the MMP-2 RNA, we discovered that there were GAPDH amplification strap in the control group and the two positive clonings, and the brightness of these straps is similar. There was no difference between the MMP-2 mRNA of two positive clonings and that of the control group. At the same time, we discovered that there were proMMP-2 and proMMP-9 in the supernatant of the control U2OS cells in the result of zymography.And there was only proMMP-2 in the hTERT transfected U2OS positive cloning cells. In contrast of the control group, the proMMP-2 in the hTERT transfected U2OS positive cloning cells was very low.In a word,by utilizing the pCI-Neo-hTERT plasmid as vector,we transfeced the hTERT into U2OS cells,and successfully bolting the positive cloning,which established the foundation for the further research of telomere and telomerase.The stable transfection of hTERT to the U2OS cell line ectopic expressed hTERT could generate telomerase activity.The stable transfection of hTERT into U2OS cells made the cells'proMMP-9 completely disappeared,and made the proMMP-2 obviously decreased.While the mRNA of MMP-2 did not change obviously . hTERT Ectopic expression generated telomerase activation caused variation of MMP-2 and MMP-9, whether or not unity of MMPs between telomerase positive immortalized cells and telomerase activation tumour cells ,and the mechanism of action of telomerase to MMP-2's secretion has to make further comparison and research.