A Preliminary Study On The Inhibition Of Tumor Cell Invasion And Metastatic Colonization By Intracellular IL-37b

Objective: To investigate the expression of intracellular IL-37 b in tumor cells and its influences on the invasion and metastatic colonization capacity of tumor cell.Methods:(1) Immunohistochemistry was used to detect the expression of IL-37 in tumor tissue from surgery patients who had been diagnosed with liver cancer, lung cancer and prostate cancer.(2) Cultured and collected human SK-Hep-1 cells, Hep G2 cells, PC3 cells, LNCap cells, 95 D cells and 95 C cells, then total RNA, total protein, cytoplasmic protein and nucleoprotein were extracted. Used RT-PCR to detect the expression of IL-37a-e m RNA and used Western Blot to detect the expression of IL-37 protein in total protein, cytoplasmic protein and nuclear protein.(3) Real-time PCR was applied to inspect the expression of IL- 37 b in mankind SK-Hep-1 cells, PC3 cells and 95 D cells which were cultured in 6-well plates and had been stimulated with TGF-?1, LPS and LPS/TGF-?1. Eukaryotic expression plasmid with p IL-37 b were recombined and then transformed into PC3 cells, SK-Hep-1 cells and 95 D cells, stable transfection cells were selected. Transfecting Hep G2 cells, LNCap cells and 95 C cells with IL-37 b sh RNA lentvirus and stable transfected cells were selected.(4) Invasion of human SK-Hep-1 cells, Hep G2 cells, PC3 cells, LNCap cells, 95 D cells and 95 C cells were detected with Transwell experiment. The anti-anoikis ability of these tumor cells were detected with Annexin V-FITC/Propidium Iodide(PI) Apoptosis assay. Soft Agar assay was used to detect the metastatic colonization capacity of these tumor cells.(5) Cultured normal and transferred SK-Hep-1 cells and Hep G2 cells in 6-well plates. Used Real-time PCR to detect the expression of tumor cell invasion related genes MMP9, TPM1, SERPINB5, MIR21, and anoikis-related genes BAX, BIM, BCL2,MCL1, BID, CFLAR, and Clone colony forming related genes ITGB and EDG-2. Western blot was used to detect protein expression of these genes.(6) Cultured normal and transferred SK-Hep-1 cells, PC3 cells, 95 D cells, Hep G2 cells,LNCap cell and 95 C cells. Calculated the number of cells after collected, and inoculated nude mice with tumor cells though the tail vein and then killed them after 35 days. The lung tissue of those nude mice was dissected and watched under dissecting microscope and then calculated the number of metastatic lesions of the lung surface.Results:(1) IL-37 expressed in human liver cancer, lung cancer and prostate cancer tissues.(2) High invasion and metastasis SK-Hep-1 cells,PC3 cells,95 D cells expressing IL-37 b lower than low invasion and metastasis Hep G2, LNCap, 95 C cells, immature IL-37 b expression in the cytoplasm, and mature IL-37 b enters the nucleus, IL-37 was secreted in the culture supernatant.(3) IL-37 b expressed in SK-Hep-1 cells, Hep G2 cells, PC3 cells, LNCap cells, 95 D cells and 95 C cells couldn't be regulated by TGF-?1and LPS..(4) Human SK-Hep-1 cells, PC3 cells and 95 D cells with overexpression of IL-37 b, its capability for invasion, anoikis, and clone colony formation in vitro were significantly decreased. Hep G2 cells, LNCap cells, 95 C cell silencing IL-37 b, its capability for invasion, anoikis, and clone colony formation in vitro were significantly enhanced.(5) After SK-Hep-1 cells overexpressed IL-37 b, the expression of tumor cell invasion related genes MMP9, MIR21 were significantly down-regulated while TPM1, SERPINB5 had no significant change; the expression of anti-anoikis related genes BCL2, MCL1, CFLAR, were significantly reduced, while the expression of promoting anoikis related genes BAX, BIM, BID were significantly increased; the expression of colony cloning formation related genes ITGB1 and EDG-2 were also significantly reduced. Protein levels further shown that the expression ofTPM1, maspin promoting migration and invasion were unregulated; the expression of anti-anoikis BCL2, MCL1, c-FLIP were significantly reduced, while the expression of promoting anoikis BAX, BIM, BID were significantly increased; ?1 and EDG-2 is also significantly reduced. Compared with SK-Hep-1 cells overexpressed IL-37 b, the m RNA and protein levels of the Hep G2 cells silenced IL-37 b, appeared completely opposite results.(6) Lung metastasis model in nude mice tests showed that, the ability of forming metastatic lesions on the lung surface of SK-Hep-1 cells, PC3 cells and 95 D cells overexpressed IL-37 b was significantly inhibited. And Hep G2 cells, LNCap cells, 95 C cells silenced IL-37 b could form metastatic lesions on the lung surface.Conclusions: IL-37 b was differentially expressed in tumor cells, and played a role in inhibiting tumor cell invasion and metastatic colony forming ability through inhibiting the expression of tumor invasion, anoikis and colony formation related gene.