Abstract:

Objective To investigate the effect of lncRNA-MIAT on M2-type polarization of tumor-associated macrophages， and to elucidate its possible mechanism. Methods The THP-1 cells were cultured in vitro. The lentivirus particles， which over expressed （LV-MIAT） and down regulated expression of lncRNA-MIAT（LV-shMIAT），and empty vector（LV-Vector） were transfected into the THP-1 cells. The THP-1 cells were activated into the macrophages， and the activated macrophages were co-cultured with the osteosarcoma （OS） MG63 cells by using Transwell co-culture system.The above co-culture systems were divided into MG63+LV-Vector group （positive control group），MG63+LV-shMIAT group， MG63+LV-MIAT group，and IL-4+LV-Vector group. The expression levels of lncRNA-MIAT in the THP-1 cells in various groups were detected；the percentages of M2-type macrophages in various groups were detected by flow cytometry； the levels of vascular endothelial growth factor （VEGF）， interleukin-10（IL-10） ，and transforming growth factor-β1（TGF-β1） in supernatant of the THP-1 cells in various groups were detected by ELISA method；the macrophages in each culture system were co-cultured with the human umbilical vein endothelial cell （HUVEC）； EDU staining was used to detect the proliferation activities of the HUVEC in various groups；the numbers of angiogenesis in the HUVEC in various groups were detected by tube formation assay；the expression levels of Janus kinase 1 （JAK1），signal transducer and activator of transcription 6 （STAT6），and phosphorylated STAT6 proteins in the THP1 cells and the expression levels of vascular endothelial growth factor receptor 2（VEGFR2），Notch1，and delta like protein 4（DLL4）in the HUVEC in various groups were detected by Western blotting method；the p-STAT6/STAT6 ratio was calculated.The OS tumor-bearing mouse model was constructed， and 36 nude mice were divided into LV-Vector group， LV-shMIAT group，and LV-MIAT group（n=12）.The volumes and weights of the tumor and the positive expression rates of CD163 and CD31 in tumor tissue of the mice in various groups were detected after interfering lncRNA-MIAT expression. Results The THP-1 cells with stable over-expression or down-regulation of lncRNA-MIAT were successfully established by lentivirus infection. Compared with LV-Vector group， the expression level of lncRNA MIAT in the cells in LV-shMIAT group was significantly decreased （P<0.05）， and the expression level of lncRNA MIAT in the cells in LV-MIAT group was significantly increased （P<0.05）. Compared with MG63+LV-Vector group， the levels of VEGF， IL-10，and TGF- β1 in the THP-1 cells in MG63+LV-shMIAT group were significantly decreased （P<0.05），and the expression levels of VEGFR2， Notch1， and DLL4 proteins in the HUVEC were significantly decreased （P<0.05），the levels of VEGF，IL-10，and TNF-β1 in the THP-1 cells in MG63+LV-MIAT and IL-4+LV-Vector groups were increased significantly （P<0.05 or P<0.01）；the expression levels of VEGFR2， Notch1，and DLL4 proteins in HUVEC were increased（P<0.05）， the percentage of M2-type macrophages，and p-STAT6/STAT6 ratio and expression level of JAK1 protein in the THP-1 cells were increased（P<0.05）；compared with MG63+sh-MIAT group， the VEGF level in the THP-1 cells in MG63+LV MIAT group was decreased significantly （P<0.05）， the levels of IL-10 and TGF-β were decreased significantly （P<0.05），the expression levels of VEGFR2， Notch1，and DLL4 proteins in the HUVEC were increased significantly （P<0.05）；compared with MG63+LV-Vector group， the proliferation activity and number of angiogenesis in the HUVEC in MG63+LV shMIAT group were significantly decreased（P<0.05），while the proliferation activities and number of angiogenesis in the HUVEC in MG63+LV-MIAT group and IL-4+LV Vector group were significantly increased （P<0.05）.The results of in vivo tumorigenesis experiment of the nude mice showed that compared with LV-Vector group， the volume and weight of transplanted tumor of the mice in LV-MIAT group were significantly increased （P<0.05），and the positive expression rates of CD163 and CD31 in tumor tissue were significantly increased （P<0.05）； the volume and mass of transplanted tumor and the positive expression rates of CD163 and CD31 in tumor tissue of the mice in LV-shMIAT group were significantly decreased （P<0.05）. Compared with LV-shMIAT group， the volume and weight of transplanted tumor of the mice in LV-MIAT group were significantly increased （P<0.05）， and the positive expression rates of CD163 and CD31 in tumor tissue were significantly increased （P<0.05）. Conclusion LncRNA MIAT may regulate the progression of OS by promoting the M2 polarization of macrophages and up-regulating the angiogenesis in tumor tissue.

Key words: Oteosarcoma, LncRNA-MIAT, M2-type macrophage, Tumor vessel, Human umbilical vein endothelial cell