Effects Of Atp1a1 Gene Silencing On Invasion Of Human U251 Glioma Cells In Vitro And Its

ObjectiveATP1A1 belongs to the family of Na+/K+ATPase enzymes.ATP1A1 overexpression has been reported in a variety of cancers,including esophageal cancer,melanoma,liver cancer and so on.It has been reported that ATP1A1 plays a key role in the biological processes associated with cancer.The inhibition of tumor growth and metastasis after silencing ATP1A1 was also reported in a variety of human cancers but not in glioma.In order to confirm this assumption,we used RNA interference technology to silence the ATP1A1 gene in U251 glioma cells,to observe the changes of cell proliferation,migration,invasion and tumorigenicity,and finding out whether the regulation of the expression of matrix metalloproteinase 2/9(MMP-2/9)is the key target.MethodWe build ATP1A1/sh RNA plasmids,then package lentivirus by three plasmid system and transfect U251 cell lines to knock-out ATP1A1 gene expression.The positive cloneswere obtained by the screen of Puromycin.The expression of ATP1A1 m RNA and protein in stable transfectants U251 cell lines was detected by im-Munofluorescence,RT-q PCR and Western blot respectively.The U251 cells were divided into three groups: blank group without any treatment;control group were transfected by control/sh RNA;gene-silencing group were transfected by ATP1A1/sh RNA.The proliferation of U251 glioma cells was determined by MTT assay.The migration and invasion ability were detected by cell scratch test and Transwell chamber.The tumorigenicity of U251 glioma cells was determined by Subcutaneous tumor formation in nude mice.The protein expression of MMP-2 and MMP-9 in U251 glioma cells were detected by Western blot.ResultsAfter transfected and screened by Puromycin,the glioma cells with Silencing ATP1A1 gene were accounted for 90%-95% of all the cells under fluorescence microscope.The m RNA and protein expression of ATP1A1 in the silence group were significantly inhibited(P<0.05).Compared to the control groups,the ability of proliferation,migration,invasion and tumorigenicity were also significantly inhibited(P<0.05),The expression of MMP-2 and MMP-9 were also significantly reduced,there was a significant difference(P<0.05).ConclusionRNA interference targeting ATP1A1 gene can significantly inhibit the proliferation,migration,invasion and tumorigenicity of glioma U251 cells.The mechanism might be related to the down-regulation of MMP-9 and MMP-2.ATP1A1 can be used as a potential target for the treatment of glioma.