MiR-16 inhibits glioma cell invasion through regulating NF-κB1/MMP-9 signaling pathway

Abstract

Objective To explore the microRNA-16 (miR-16) and nuclear-transcription factor-κB1 (NF-κB1) expressions in human brain gliomas and their correlations with cell invasion and growth of malignant gliomas SHG44,U87 and U373.Methods (1) Twenty-nine cases of human glioma tissue samples and 6 normal brain tissues,collected in our hospital from January 2000 to January 2011,were chosen in our study; quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expressions ofmiR-16 and NF-κB1 in these tissues.(2) In vitro cultured U87,U373 and SHG44 cells were divided into blank-control group,nonsense sequence transfected group and miR-16 mimics transfected group; 48 h after the transfection,qRT-PCR was used to detect the expressions of miR-16 and NF-κB1; tmnswell assay was used to observe the cell invasion capability; 72 h after the transfection,Western blotting was employed to detect the protein expressions of NF-κB1,matrix metallopeptidase 9 (MMP-9) and MMP-2.(3) Luciferase reporter assay was used to detect the target regulating role of miR-16 in NF-κB1 gene.(4) U87 cells were used as negative control group,and U87 cells carried stably expressed miR-16 gene were implanted into intracranial and subcutaneous nude mice (U87-miR-16 group); immunofluorescence was used to detect the MMP-9 expression,and immunohistochemical staining was used to detect the protein expressions of Ki-67,NF-κ B1 and MMP-9; subcutaneous tumor volume was measured and the growth curve was drawn.Results (1) The qPCR results showed that the expression of miR-16 in human brain glioma tissues was significantly lower than that in normal brain tissues; and gradually decreased miR-16 expressions were noted in gliomas of graded Ⅰ,Ⅱ,Ⅲ and Ⅳ (P＜0.05); NF-κB1 expression in human brain glioma tissues was significantly higher than that in normal brain tissues; and gradually increased NF-κB1 expressions were noted in gliomas of graded Ⅰ,Ⅱ,Ⅲ and Ⅳ (P＜0.05).(2) As compared with those in the blank-control group and nonsense sequence transfected group,miR-16 mimics transfected group had significantly increased miR-16 expression,decreased NF-κB1 mRNA expression,decreased invasiveness,and decreased protein expressions of NF-κB1 and MMP-9 (P＜0.05).(3) Luciferase reporter assay showed that the fluorescence normalized ratio in the pMIR-NF-κB1 group was signfcaintly higher than that in the pMIR-NF-κB1+pre-miR-16 group (P＜0.05).(4) As compared with the negative control group,the U87-miR-16 group on the 24-42 d of implantation had significantly smaller volume of tumors (P＜0.05),and lower MMP9 expression,and NF-κB1,MMP-9 and Ki-67 expressions (the proliferation index of Ki-67:13.91％ and 32.98％).Conclusion MiR-16 inhibits glioma cell invasion and growth through down-mgulating NF-κB1 and MMP-9 expressions. Key words: MicroRNA-16; Nuclear-transcription factor-κB1; Matrix metallopeptidase 9; Glioma; Invasion