Macrophages have been shown to demonstrate a high level of plasticity, with the ability to undergo dynamic transition between M1 and M2 polarized phenotypes. We investigate long non-coding RNA (lncRNA) cox-2 in macrophage polarization and the regulatory mechanism functions in hepatocellular carcinoma (HCC). Lipopolysaccharide (LPS) was used to induce RAW264.7 macrophages into M1 type, and IL-4 was to induce RAW264.7 macrophages into M2 type. We selected mouse hepatic cell line Hepal-6 and hepatoma cell line HepG2 for co-incubation with M1 or M2 macrophages. Quantitative real-time PCR was used to detect the expressions of lncRNA cox-2 and mRNAs. ELISA was conducted for testing IL-12 and IL-10 expressions; Western blotting for epithelial mesenchymal transition related factors (E-cadherin and Vimentin). An MTT, colony formation assay, flow cytometry, transwell assay, and stretch test were conducted to test cell abilities. The M1 macrophages had higher lncRNA cox-2 expression than that in the non-polarized macrophages and M2 macrophages. The lncRNA cox-2 siRNA decreased the expression levels of IL-12, iNOS, and TNF-α in M1 macrophages, increased the expression levels of IL-10, Arg-1, and Fizz-1 in M2 macrophages (all P < 0.05). The lncRNA cox-2 siRNA reduces the ability of M1 macrophages to inhibit HCC cell proliferation, invasion, migration, EMT, angiogenesis and facilitate apoptosis while strengthening the ability of M2 macrophages to promote proliferation HCC cell growth and inhibit apoptosis. These findings indicate that lncRNA cox-2 inhibits HCC immune evasion and tumor growth by inhibiting the polarization of M2 macrophages.

Background/Aims: To investigate the biological roles and underlying molecular mechanisms of long non-coding RNA (lncRNA) PVT1 in Hepatocellular carcinoma (HCC). Methods: qRT-PCR was performed to measure the expression of miRNA and mRNA. Western blot was performed to measure the protein expression. CCK-8 assay was performed to determine cell proliferation. Flow cytometry was performed to detect cell apoptosis. Wounding-healing assay and Transwell assay was performed to detect cell migration and invasion. Dual luciferase reporter assay was performed to verify the target relationship. Quantichrom iron assay was performed to check uptake level of cellular iron. Results: PVT1 expression was up-regulated in HCC tissues and cell lines. Function studies revealed that PVT1 knockdown significantly suppressed cell proliferation, migration and invasion, and induced cell apoptosis in vitro. Furthermore, PVT1 could directly bind to microRNA (miR)-150 and down-regulate miR-150 expression. Hypoxia-inducible protein 2 (HIG2) was found to be one target gene of miR-150, and PVT1 knockdown could inhibit the expression of HIG2 through up-regulating miR-150 expression. In addition, the expression of miR-150 was down-regulated, while the expression of HIG2 was up-regulated in HCC tissues and cell lines. Moreover, inhibition of miR-150 could partly reverse the biological effects of PVT1 knockdown on proliferation, motility, apoptosis and iron metabolism in vitro, which might be associated with dysregulation of HIG2. In vivo results showed that PVT1 knockdown suppressed tumorigenesis and iron metabolism disorder by regulating the expression of miR-150 and HIG2. Conclusion: Taken together, the present study demonstrates that PVT1/miR-150/HIG2 axis may lead to a better understanding of HCC pathogenesis and provide potential therapeutic targets for HCC.