The Effects And Mechanism Of PIWIL1 And MiR-154-5p In Glioma Cells

Objective: PIWIL1 is an oncogene in various malignancies.The overexpression of PIWIL1 has an intimate relation to the progression of these tumors.The purpose of this study is to investigate the effects and related mechanisms of PIWIL1 and miR-154-5p on glioma cells growth,apoptosis,migration,invasion and other biological characteristics.We hope can provide new molecular markers and targets for the molecular diagnosis and treatment of glioma.Method:1.The expression of PIWIL1 in mRNA and protein levels were detected in glioma,non-tumor brain tissues and glioma cell lines(SNB19,U87,A172,LN229,U251,LN308)by RT-PCR and Western Blot.The expression of miR-154-5p were detected by RT-PCR in the samples.2.RNA interference technique was performed to study the effect of decreasing the expression of endogenous PIWIL1 in glioma cells U251 and A172.Transfection efficiency was verified by RT-PCR and Western Blot.The effect of decreasing the expression of endogenous PIWIL1 on proliferation,cell cycle,apoptosis,migration and invasion of glioma were measured by MTT,plate clone formation assay,flow cytometry,Transwell assays.3.The genes of Bcl-2,Bax,CyclinD1,p21,MMP-2 and MMP-9 were detected by RT-PCR and Western Blot in U251 and A172 with PIWIL1 decreasing cells.4.Nude mouse models of U251 glioma were established and treated with multi-point injection of PIWIL1-siRNA.The changes of PIWIL1 in tumor lump were detected by immunohistochemical technique.5.U251 and A172 cells were treated by PIWIL1-siRNA and TMZ in combination and respectively.Subsequently,the proliferation and apoptosis of cells were detected by MTT and flow cytometry.6.U251 and LN229 glioma cells were transfected with miR-154-5p mimics and inhibitor respectively.The effect on proliferation,cell cycle,apoptosis,migration and invasion of glioma were measured by MTT,plate clone formation assay,flow cytometry and Transwell assays.7.The genes of Bcl-2,Bax,CyclinD1,p21,MMP-2 and MMP-9 were measured by Western Blot in U251 and LN229 cells with the change of exogenous miR-154-5p.8.The potential target genes of miR-154-5p were predicted by bioinformatics and verified by constructing double luciferase reporter plasmids.A rescue experiment was performed to the effect of miR-154-5p on glioma cells by target PIWIL1.9.Nude mouse models of U251 glioma were established and treated with multi-point injection of miR-154-5p.The changes of miR-154-5p and PIWIL1 in tumor lump were detected by RT-PCR and immunohistochemical technique to observe the inhibitory effect in vivo.Results:1.The RT-PCR and Western Blot showed that the expression of PIWIL1 in glioma tissues and glioma cell lines were significantly higher than that in non-tumor brain tissues(P < 0.05).The expression of PIWIL1 in different grade among glioma tissues was significantly different(P < 0.05).The expression of miR-154-5p in glioma tissues and glioma cell lines were significantly lower than that in non-tumor brain tissues(P < 0.05).The expression of mi R-154-5p in different grade glioma tissues was significantly different(P < 0.05).2.The decrease of endogenous PIWIL1 in U251 and A172 cells resulted in remarkable inhibition of cell proliferation,decreasing of the rate of clone formation,arresting cell cycle in G1 phase,significant increasing of cell apoptosis and decreasing of cell invasion and migration.The related protein also had dramatically changes that the expression of Bcl-2,CyclinD1,MMP-2 and MMP-9 was decreased and the expression of Bax and p21 protein was increased.In vivo,the results showed that PIWIL1-siRNA significantly suppressed the rate of tumor growth.3.PIWIL1-siRNA combined with TMZ inhibited the proliferation of cells,increased the sensitivity of glioma cells to chemotherapeutic drugs and induced the apoptosis of glioma cells.4.The overexpression of miR-154-5p in glioma cells suppressed tumor proliferation,migration,and invasion and induces cell cycle arrest in G1 phase apoptosis.The related proteins also had dramatically changes that the expression of Bcl-2,CyclinD1,MMP-2 and MMP-9 was decreased and the expression of Bax and P21 protein was increased.Conversely,inhibition of mi R-154-5p in glioma cells led to contrary biological function change versus the overexpression of miR-154-5p.5.Bioinformatics analysis showed that PIWIL1 is a potential target gene of miR-154-5p.The dual-luciferase reporter assay was performed to verify the functional binding of miR-154-5p with PIWIL1 3 ' UTR.The rescue experiment showed that the effects of miR-154-5p on glioma was effectively reversed by pCMV-PIWIL1.Conclusions: The oncogenic role of PIWIL1 could promote the progression and metastasis of glioma,while the miR-154-5p could act as a tumor suppressor in vivo and vitro.And the tumor suppressor function of miR-154-5p may exert by inhibiting the expression of PIWIL1.Therefore,the regulation of PIWIL1 by mi R-154-5p in malignant glioma sheds great light on the diagnosis and targeted molecular therapy of malignant glioma.