| Background&Aims: Osteosarcoma (osteosarcoma, OS) is the most common skeletal systemmalignant tumors, frequently occurring in children and adolescents. Metastasis-associated incolon cancer-1(MACC1) was originally identified in primary and metastatic coloncarcinoma, which was confirmed to be an independent prognostic factor of metastasisformation and non-metastatic survival rate. Many studies have demonstrated thatcombinations of MACC1with many tumor-related signaling pathways controlproliferation, differentiation, apoptosis and metastasis. In this study, we sought to studythe expression, regulation, molecular mechanism and clinical implication of MACC1inOS.Methods:(1) MACC1protein expression was detected by immunohistochemistry (IHC) in OS and paired normal bone tissues; Semi-quantitative RT-PCR and real-timequantitative PCR were utilized to detect the expression of relative MACC1mRNA levelin different clinical stage, metastatic status and fresh tumor tissue; Kaplan-Meiersurvival curve was used to investigate the correlation between MACC1level andprognosis of the patients with OS.(2) MACC1mRNA and protein levels were detectedin4of human OS cell lines (MG-63, HOS, SaOS-2and U2OS) and human osteoblasthFOB1.19by real-time quantitative PCR and Western blotting methods; Eukaryoticexpression vector pcDNA-MACC1was constructed by molecular cloning technology,and pcDNA-MACC1was transfected to MG-63cells, meanwhile, MACC1siRNA andcontrol siRNA were also used to transfect to U2OS cells, MACC1mRNA and proteinlevels were identified by real-time quantitative PCR and Western blotting methods invarious treatment cells; cell proliferation was investigated by CCK-8; colony formationability was analyzed by soft agar colony formation experiment; cell invasion ability wasdetected by Boyden chamber; cell cycle and apoptosis were investigated by Flowcytometry; Caspase-3activity was detected by fluorogenic substrate (Ac-DEVDAMC);expressions of key components of Akt signaling pathway p-Akt and total Akt as well asits downstream target genes E-cadherin, MMP-2and MMP-9were detected by Westernblotting.(3) MG-63and U2OS cells with various treatments were injectedsubcutaneously into nude mice, which will establish subcutaneous implanted human OSmodel in nude mice; Tumor size was monitored, tumor growth curve was made andtumor weight was measured; MACC1protein expression was investigated by Westernblotting in various OS xenografted tumors; Ki-67proliferation index was evaluated byIHC in various OS xenografted tumors.Results:(1) The result of semi-quantitative RT-PCR demonstrated that the relative levelof MACC1mRNA in OS tissues was significantly higher than that in normal bonetissues (P<0.01), which was further confirmed by real-time quantitative PCR; therelative MACC1level in clinical stage III was obviously higher than that in I and IIstages; the relative MACC1level in metastatic OS tissues were significantly higher thanthat in non-metastatic OS tissues (P<0.01); the differences positive MACC1protein expression in OS tissues and normal bone tissues were statistical significance (P=0.000),and MACC1protein expression was not related to OS patients’ sex, age, tumor size andanatomical location (P>0.05), but closely correlated with clinical stage and metastasis(P<0.05); as determined by the log-rank test (Mantel–Cox), the survival rates of thepatients with high MACC1protein level were significantly lower than those with lowMACC1protein level (P<0.01).(2) MACC1mRNA and protein levels in four OS celllines (MG-63, HOS, SaOS-2, and U2OS) were markedly higher than those in humanosteoblast cell hFOB1.19(P<0.05); expressions of MACC1mRNA and protein inpcDNA-MACC1group were significantly higher than those in untreated group andempty vector group (P<0.05), whereas the result in MACC1siRNA group was opposite(P<0.05); MACC1overexpression significantly improved the proliferation, colonyformation ability and invasion ability of MG-63cells, suppressed cell apoptosis andreduced the activity of caspase-3, activated Akt signaling pathway, downregulated thelevel of E-cadherin and increased the levels of MMP-2and MMP-9proteins (P<0.05),whereas MACC1downregulation had the opposite results (P<0.05); cell numberpercentage at G0/G1phase in pcDNA-MACC1groupwas significantly lower than thosein untreated group and empty vector group (P=0.000), whereas cell number percentageat S phase in pcDNA-MACC1group was significantly higher than those in untreatedgroup and empty vector group (P=0.000), whereas the results in MACC1siRNA groupwere opposite (P=0.000).(3) Compared with untreated group and empty vector group,tumor weight and size, MACC1expression, the number of Ki-67positive cells and theproliferation index was significantly increased in nude mice tumor tissues inpcDNA-MACC1group (P<0.05), whereas the results in MACC1siRNA group wereopposite(P<0.05).Conclusions: MACC1at high level play a pivotal role in the occurrence, developmentand growth of OS, and its high expression may be an essential molecular marker ofmalignancy, metastasis phenotype and prognosis of OS patients, alteration of MACC1level mediated the changes of biological behavior of OS may be closely associated withactivity status of Akt signaling pathway, and thus may be considered MACC1as a novel molecular target for therapy of the patients with OS。... |
