MIR181A2HG Promote Lymph Node Metastasis Of Gastric Cancer Through Inducing Macrophage M2 Polarization

Objective Long non-coding RNAs(lncRNAs)have been shown to be involved in the development and progression of a variety of tumors,but their exact roles and molecular mechanisms in gastric cancer have not been fully elucidated.Our study aimed to investigate the role of lnc RNAs in regulating malignant progression of gastric cancer and their specific mechanisms,and to provide a new basis for targeted therapy in gastric cancer.Methods 1)Screening of significantly upregulated lnc RNAs in gastric cancer with online databases GEO(Gene Expression Omnibus)and TCGA.Real-time fluorescence quantitative PCR(q RT-PCR)was performed to detect the expression of MIR181A2HG in 108 pairs of fresh gastric and paraneoplastic tissues.The relatioships between its expression and clinicopathological characteristics or prognosis of gastric cancer were analyzed.2)The expression of MIR181A2 HG in gastric cancer cells and human gastric mucosal cells(GES-1)was detected by q RT-PCR.Two gastric cancer cell lines with the highest expression of MIR181A2 HG were selected for MIR181A2 HG knockdown.The effects of MIR181A2 HG on the proliferation and migration of gastric cancer cells were explored by CCK-8 assay,Transwell assay,colony formation assay and wound healing.3)The effect of MIR181A2 HG on lymph node metastasis of gastric cancer cells was explored by nude mice footpad-popliteal lymphatic metastasis model.4)The effects of MIR181A2 HG on macrophage aggregation and M2 polarization were evaluated by immunohistochemistry(IHC)and q RT-PCR.The effect of macrophage polarization on lymphatic tubule formation was analyed by cell co-culture and tubule formation assays.5)The molecular mechanism of the involvement of vascular endothelial growth factor-C(VEGF-C)in lymph node metastasis of gastric cancer cells induced by MIR181A2 HG was confirmed by Wastern blotting and q RT-PCR.Results 1)The expression of MIR181A2HG was significantly up-regulated in gastric cancer tissues and closely correlated with lymph node metastasis.The TCGA database and GEO microarray(GSE54129)analysis showed that MIR181A2HG was significantly up-regulated in gastric cancer tissues.Analysis of 108 gastric cancer samples revealed that the expression of MIR181A2 HG was closely correlated with TNM stage,tumor size,lymph node metastasis,and lymphatic vessel density,but not with age,gender,depth of invasion,and differentiation degree.Survival analysis showed that the expression of MIR181A2 HG was correlated with the prognosis of patients,and the overall survival of patients with high MIR181A2 HG expression was much shorter than that of patients with low MIR181A2 HG expression.2)MIR181A2HG promoted the proliferation and migration of gastric cancer cells.The results of q RT-PCR showed that the m RNA expression level of MIR181A2 HG was higher in gastric cancer cell lines.In vitro cell function experiments showed that knockdown of MIR181A2 HG with si RNA significantly inhibited the migration and proliferation of SGC-7901 and MKN-45 cells.3)MIR181A2HG promoted lymph node metastasis of gastric cancer cells.Knockdown of MIR181A2 HG significantly inhibited the lymph node metastasis of gastric cancer cells in vivo,as demonstrated in the footpad-popliteal lymphatic metastasis model of nude mice.MIR181A2 HG knockdown reduced the success rate of popliteal lymph node metastasis and prolonged the survival time of mice.4)MIR181A2HG mediates lymphangiogenesis by promoting macrophage M2 polarization.The results of IHC confirmed that the expressions of lymphatic vessel marker LYVE-1 and macrophage marker F4/80 were decreased in the tumor tissue of the footpad of MIR181A2 HG knockdown mice,suggesting that lymphangiogenesis and the number of macrophage were decreased.In vitro human lymphatic endothelial cells(HLECs)tubule formation assay confirmed that the conditioned supernatant of gastric cancer cells alone could not promote lymphatic tubule formation.THP-1 cells were induced to differentiate into macrophages by PMA.After co-culture with gastric cancer cells in vitro for 48 h,q RTPCR showed that M2-type markers of macrophages were reduced when co-cultured with MIR181A2 HG knockdown gastric cancer cell line.The conditioned supernatant after coculture was collected and treated with HLECs cells.The results of tubule formation assay showed that lymphangiogenesis was decreased after MIR181A2 HG knockdown in gastric cancer cell line.5)q RT-PCR and Western blot were used to detect the expression of VEGF-C in macrophages co-cultured with gastric cancer cells.The results showed that the m RNA and protein expression levels of VEGF-C were significantly increased after M2 polarization of macrophages.Conclusion In conclusion,MIR181A2HG upregulated VEGF-C expression by inducing M2-type polarization of macrophages,thereby promoting lymphatic vessel generation in gastric cancer and ultimately promoting lymph node metastasis of gastric cancer cells.