关键词: R-脊椎蛋白3, 自然杀伤细胞, 结直肠癌, 肿瘤生长

Abstract:

Background and purpose: The occurrence and development of colorectal cancer involve the activation of multiple oncogenes and the inactivation of tumor suppressor genes. At present, the role of wild-type R-spondin 3 (RSPO3) in the growth of colorectal cancer is still unclear. This study aimed to investigate the effect of RSPO3 on the growth of colorectal cancer and explore the underlying mechanisms. Methods: RSPO3 expression in various tumor tissues and adjacent tissues and the relationships between RSPO3 expression and natural killer (NK) cell infiltration and activating molecules in colorectal cancer tissues were analyzed by bioinformatics. RSPO3-knockdown SW480 (SW480-RSPO3-KD) and RSPO3-overexpressed HCT116 (HCT116-RSPO3-OE) gene modification cell line as well as their control cell lines were established by short hairpin RNA (shRNA) and lentiviral infection. Cell proliferation in vitro were detected using cell counting kit-8 (CCK-8). Cell cycle of colon cancer stable cells and the proportion of NK cells in spleen and transplanted tumor tissues of nude mice were determined by flow cytometry. The growth of SW480-RSPO3-KD and HCT116-RSPO3-OE stable cell subcutaneous xenografts in vivo was observed in BALB/c nude mice. The Wnt gene activity was detected by dual-luciferase reporter system. Results: Expression level of RSPO3 was low in a variety of solid tumor tissues including colorectal cancer tissues compared to their adjacent normal tissues. RSPO3 knockdown or overexpression did not affect cell proliferation (P>0.05) and cell cycle in colon cancer cells in vitro (P>0.05). Surprisingly, RSPO3 knockdown significantly promoted the growth of SW480 subcutaneous xenograft tumor (260.2±162.4 vs 1 311.7±570.1, P<0.05). RSPO3 overexpression significantly inhibited the growth of HCT116 subcutaneous xenograft tumor (1 549.0±241.2 vs 512.1±250.0, P<0.05). Flow cytometry analysis showed that the percentages of NK cells in spleen and xenograft tissues were significantly decreased in mice transplanted with RSPO3-KD cells compared with control nude mice transplanted with control cells (spleen:6.42±0.94 vs 5.25±0.59, P=0.04; transplanted tumor: 8.27±0.29 vs 6.48±1.48, P=0.04). The percentage of NK cells was significantly increased in mice transplanted with RSPO3-OE cells compared with control mice transplanted with control cells (spleen: 5.29±0.16 vs 7.02±0.49, P=0.01; transplanted tumor: 6.39±0.39 vs 8.14 ±0.34, P<0.05). The Cancer Genome Atlas (TCGA) data analysis showed that expression of RSPO3 was positively correlated with the expressions of NK cell markers CD56 (R=0.58, P<0.05) and CD16 (R=0.64, P<0.05), and with the expressions of NK cell activation markers CD69 (R=0.51, P<0.05) and KLRB1 (R=0.37, P<0.05). RSPO3 knockdown inhibited the activity of Wnt luciferase (1.00±0.00 vs 0.45±0.09, P<0.05), and RSPO3 overexpression increased the Wnt luciferase activity in colon cancer cells (1.00±0.00 vs 1.75±0.14, P<0.05). Conclusion: RSPO3 can significantly inhibit the growth of colorectal cancer xenograft in vivo and increase NK cell frequency and Wnt activity in colon cancer cells. RSPO3 may be a potential colorectal cancer suppressor.

Key words: R-spondin 3, Natural killer cell, Colorectal cancer, Tumor growth