| Gliomas, the most common tumor in the central nervous system, account for40%of all primary brain tumors, but within the malignant subset, they accountfor80%of tumors. Patients with glioma carry poor prognosis, of which only20-30%could survive5years. The average survival time of malignant gliomas isonly9-12months. In the last decades of years, although the surgical treatment,radiotherapy and chemotherapy have been greatly improved in technique,outcomes of patients with gliomas have not been significantly improved.Difficults of treating malignant glioma is mainly due to its malignant biologicalcharacteristics, such as excessive proliferation and diffuse invasion. In recentyears, with the rapid development of molecular biology, using molecularbiotechnology to research the treatment of malignant gliomas has become a focus.Therefore, it is important to find a key molecular target for curing malignantgliomas.The multifunctional growth factor hepatocyte growth factor (HGF) and itsreceptor tyrosine kinase c-Met have emerged as key determinants of brain tumorgrowth and angiogenesis. HGF and c-Met are expressed in brain tumors, theexpression levels frequently correlating with tumor grade, tumor blood vessel density, and poor prognosis. Overexpression of HGF and/or c-Met in brain tumorcells enhances their tumorigenicity, tumor growth, and tumor-associatedangiogenesis. Conversely, inhibition of HGF and c-Met in experimental tumorxenografts leads to inhibition of tumor growth and tumor angiogenesis. HGF isexpressed and secreted mainly by tumor cells and acts on c-Met receptors that areexpressed in tumor cells and vascular endothelial cells. Activation of c-Met leadsto induction of proliferation, migration, and invasion and to inhibition ofapoptosis in tumor cells as well as in tumor vascular endothelial cells. Activationof tumor endothelial c-Met also induces extracellular matrix degradation, tubuleformation, and angiogenesis in vivo. HGF induces brain tumor angiogenesisdirectly through only partly known mechanisms and indirectly by regulatingother angiogenic pathways such as VEGF. Different approaches to inhibitingHGF and c-Met have been recently developed..Ulrike Stein identified the MACC1gene by a genome-wide search fordifferentially expressed genes in human colon cancer tissues, metastases, andnormal tissues. They also evaluate the MACC1levels in the context of one of itstranscriptional targets, the receptor tyrosine kinase c-Met that activates thehepatocyte growth factor/Met signaling pathway, leading to enhanced cellmotility, invasion, and metastasis. More studies show that MACC1is a cancergene which expressed in a variety of tumors and has an important role in processof invasion and metastasis.At present, research on the relationship between MACC1and brain glioma islittle, and the role of MACC1in malignant proliferation and invasion of brainglioma is not completely clear. To observe the mechanism of MACC1in theproliferation and invasion, we performed some experiments as follows.1To confirm the expression of MACC1in human gliomas In this study, using RT-PCR and Western blot we detected expression ofMACC1in four human malignant glioma cell lines (U251, U87, SHG44andBT325) at mRNA and protein levels. The results showed that the MACC1waswidely expressed in four human glioma cell lines, and the most was the U87cells.We used immunohistochemical staining to detect the position expression ofMACC1in human brain glioma tissue of98cases with different clinical andpathological levels. We used RT-PCR to detect MACC1expression in the humanbrain glioma tissue in mRNA level. The results showed that MACC1locates innucleus and cytoplasm of glioma cells, and MACC1is expressed in gliomatissues with different pathological grades at the mRNA and protein levels, and theexpression levels of MACC1were correlated with the WHO levels.2To construct MACC1RNAi expression lentivirus to infect U87cellsIn this study, according to MACC1cDNA sequence, the specific RNAifragments targeting MACC1were dsigned and synthesized, which were clonedinto pLKO.1vector, and the eukaryotic expression vector pLKO.1-MACC1ofMACC1shRNA was constructed. After lentiviral packaging, MACC1RNAiexpression lentivirus infected U87cells. pLKO.1-MACC1shRNA-1,pLKO.1-MACC1shRNA-2and pLKO.1-NC were transfected into U87cells.After puromycin selection, stably transfected cell lines including U87-S1(MACC1shRNA group1), U87-S2(MACC1shRNIA group2) and U87-NC(negative control group) were established. Use RT-PCR and Western blot todetect expression of MACC1mRNA and protein in U87, U87-NC, U87-S1andU87-S2. The result showed that expressions of MACC1in both U87-S1andU87-S2were significantly inhibited.3To study the changes of biofeatures in U87cells after MACC1RNAi invitro and in vivo In vitro experiments, MTT assay, cell cycle assay, plate colony formation assayand soft-agar colony formation assay were used to detect the changes in U87cellsafter MACC1RNAi. The results showed that, compared with U87and U87-NCgroups, U87-S1and U87-S2cells grew significantly slow, the G1phase and G0phase cells in U87-S1and U87-S2groups significantly increased, and the colonynumber of U87-S1and U87-S2cells notablely decreased. Above studiesdemonstrated that downregulation of MACC1gene expression significantlyinhibited proliferation of glioma cells. Furthermore, cell wound healing assay andmatrigel invasion assay were used to investigate the changes in U87cells afterMACC1RNAi. The results showed that, compared with U87and U87-NCgroups, the migrations distance of cells in U87-S1and U87-S1groupssignificantly decreased, and the number of invaded cells obviously reduced.These studies demonstrated that MACC1inhibition attenuated U87cellsmigration and invasion.In vivo studies, U87, U87-NC, U87-S1and U87-S1cells were inoculatedrespectively in flank subcutaneous tissue of nude mice to establish xenograftmodels of human brain glioma. The tumor growth status was observed and tumorvolume was measured termly, and the tumor growth curve was drawn. The micewere sacrificed and tumor weight was recorded24days post inoculation. Theresults showed that the tumorigenesis time delayed, tumor grew slow, both tumorvolume and tumor weight decreased significantly in U87-S1and U87-S2groups,compared with those in U87and U87-NC. These studies demonstrated thatMACC1RNAi can significantly suppress tumorigenesis and growth of humanbrain glioma in nude mice, and also further confirmed that MACC1downregulation can inhibit proliferation and invasion in vivo.4The possible mechanisms of MACC1effects the biofeatures of U87cells We detected the expression of c-met in U87, U87-NC, U87-S1and U87-S2cell lines by Western blot. Then, we detected several genes which are involvedglioma cells’ proliferation and invasion through MAPK/ERK signal pathway. Theresults showed that downregulating of MACC1could increase p27and decreasec-met, cdk2and MMP2. It implied that MACC1may regulate HGF/c-Met viaregulating of c-Met. p27, cdk2and MMP2which are HGF/c-Met downstreammolecules were all related with proliferation and invasion of gliomas. So, MACC1could play a vital role during the growth and malignant evolution process of glioma.The results provided a theoretical basis of MACC1being a biomarkers ofmalignancy gliomas and a potential molecular target of glioma gene therapy. |
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