The Effects Of Differentially Expressed NOV On Proliferation, Apotosis And Migration Of Human Osteosarcoma Cell Lines And Its Mechanism Research

ObjectivesTo detect the expression level of NOV in human osteosarcoma cell lines; recombinant adenovirus mediated gene overexpression or interference was used to investigate the effects of NOV on the proliferation,apoptosis and migration of osteosarcoma 143 B and MG63 cells in vitro,and to explore its underlying mechanisms.Methods1. NOV expression level detection in human osteosarcoma cell lines: 4kinds of osteosarcoma cell lines(143B, U2 OS, MG63 and SaOS2) kept by Key Laboratory of Diagnostic Medicine Designated by the Chinese Ministry of Education, Chongqing Medical University, were selected as the research objects. RT-PCR and Western blot were used to characterize the endogenous expression of NOV in osteosarcoma cell lines.2. Infection efficiency identification of recombinant adenovirus:recombinant adenovirus expressing NOV or siNOV(Ad-NOV or Ad-siNOV) and their corresponding negative control Ad-GFP or Ad-RFPwere used to infect osteosarcoma cell lines with a relatively low or high endogenous NOV expression, infection efficiency was observed under fluorescence microscope and NOV expression in mRNA and protein level was detected by RT-PCR and Western blot.3. The role of differentially expressed NOV on proliferation, apoptosis and migration of human osteosarcoma 143 B and MG63 cell lines: after treatment with adenovirus for indicated time, cell proliferation was tested by MTT, colony forming assay and FCM; Hochest 33258 staining and FCM were used to reflect cell apoptosis level; then cell migration and invasion were detected by wound healing assay and Transwell. In addition,the expression levels of Bax and Bcl-2 related to cell apoptosis and MMP family related to migration were investigated by RT-PCR and Western blot.4. The effect of differentially expressed NOV on signal pathways of human osteosarcoma 143 B and MG63 cell lines: cells were treated with adenovirus for 48 h and integrin receptors expression levels were detected by RT-PCR and Western blot; luciferase report gene assay was used to detect the deregulated activation of signaling pathways. Then the phosphorylation levels of PI3K/Akt, MAPK and downstream AP-1 protein were measured by Western blot to determine the signal pathways which NOV acts on osteosarcoma cells.5. The role of MAPK signaling pathway on proliferation, apoptosis and migration of 143 B cell induced by NOV: 143 B cells were treated withp38 inhibitor SB203580 and JNK inhibitor SP600125, respectively.Western blot was used to test p-p38 and p-JNK expression to verify the efficiency of inhibitors; then cell viability and migration were tested by MTT assay and Transwell, respectively; Western blot was done to detect the protein expression change of Bax and Bcl-2 related to cell apoptosis.Results1. The endogenous mRNA expression of NOV in human 143 B, U2 OS,MG63 and SaOS2 osteosarcoma cell lines evaluated using RT-PCR was different. The expression level was significantly higher in MG63 cells although obviously lower in 143 B cells as compared to the remaining cells.This result was further confirmed by Western blot.2. After being infected with adenovirus for about 24-36 h, the expression of green fluorescent protein(GFP) in 143 B cell and red fluorescent protein(RFP) in MG63 cell was enhanced and the infection efficiency of adenovirus was about 60-70% under florescence microscopy.In addition, RT-PCR and Western blot results revealed an obvious increase of NOV expression in the Ad-NOV group compared with the Ad-GFP and control groups(p<0.01 and p<0.05, respectively) in 143 B cells, while a marked decrease of NOV expression in the Ad-siNOV group compared with the Ad-RFP and control groups in MG-63 cells. These data indicated that NOV was successfully overexpressed in 143 B cells and interfered in MG63 cells.3. It is indicated that the cell viability of 143 B cells treated with Ad-NOV was significantly inhibited at 48 h(p<0.05) and was evident at 72h(p<0.01), and there is a remarkable decrease in colony formation in the AdNOV group by 23% in 143 B cells compared with that of the control groups(p<0.01). FCM data showed that NOV plays a crucial role in cell cycle arrest in 143 B cells. On the contrary, the experiment results indicated an enhancement on proliferation and colony formation in MG63 cell treated with Ad-siNOV, and a promotion effect on cell cycle.It showed that the apoptotic rate of 143 B cells was significantly increased compared to that of the Ad-GFP group following treatment with Ad-NOV for 48 h. Cell staining with hoechst 33258 revealed extensive nuclear condensation in 143 B cells. Moreover, a notable increase in Bax expression and a parallel Bcl-2 decrease were observed in 143 B cells after NOV overexpression. Whereas the apoptotic rate of MG63 cells was significantly reduced as compared to that of the Ad-RFP group after Ad-siNOV treatment, and a contrary result of Bax/Bcl-2 expression.Wound healing assay and Transwell results showed that after treatment with Ad-NOV, the migration rate and number of transmembrane143 B cells increased, and MMP family expression upregulated. While the Ad-siNOV group in MG63 cell performed contrary to the aforementioned.4. According to the results of RT-PCR and Western blot, the mRNA and protein expression levels of integrin receptor αv and β3 in Ad-NOVgroup of 143 B cells increased; luciferase reporter assay detection results suggested disregulated activation of NF-κB and AP-1. In addition, the phosphorylation levels of p38 and JNK in Ad-NOV group upregulated obviously, except for p-Akt. In MG63 cells infected with Ad-siNOV, both integrin receptor and phosphorylation levels of signal molecule manifested to decline.5. After treatment with p38 inhibitor SB203580 and JNK inhibitors SP600125, respectively, Western blot results showed that the expression levels of p-p38 and p-JNK were effectively inhibited. Determination by MTT showed that both SB203580 and SP600125 significantly suppressed the NOV-induced antiproliferative effect; Transwell analysis suggests the inhibitors can partly suppress cell migration inducted by NOV. Western blot results showed NOV-induced upregulation of Bax was reversed by SB203580 and SP600125, while the expression of Bcl-2 was recovered in143 B cells, which further confirmed that apoptosis rate is inhibited.Conclusions1. The endogenous expression levels of NOV in human osteosarcoma celllines was obviously different.2. NOV overexpression could inhibit proliferation as well as promotingapoptosis and migration in osteosarcoma cell lines to some extent.3. NOV increased the expression of integrin receptors on osteosarcomacell surface, which then activated the MAPK/p38 and MAPK/JNKsignal pathways, while had no effect on PI3K/Akt pathway.4. Activation of MAPK/p38 and MAPK/JNK signal pathways inosteosarcoma cells are involved in the regulation of the cellproliferation, apoptosis and migration induced by NOV.