RSPO3抑制体内结直肠癌移植瘤生长和增加NK细胞浸润的作用研究

doi:10.19401/j.cnki.1007-3639.2023.07.004

摘要/Abstract

摘要：

背景与目的：结直肠癌的发生、发展涉及多个癌基因的激活和抑癌基因的失活，野生型R-脊椎蛋白3（R-spondin 3，RSPO3）在结直肠癌生长中的作用目前尚不清楚，本研究旨在探讨RSPO3对结直肠癌生长的影响并探索其潜在机制。方法：采用生物信息学分析RSPO3在结直肠癌及泛癌组织中的表达，分析结直肠癌中RSPO3表达与自然杀伤（natural killer，NK）细胞浸润、NK细胞激活分子表达的相关性。利用短发夹RNA（short hairpin RNA，shRNA）和慢病毒感染建立RSPO3敲减的SW480-RSPO3-KD细胞株、RSPO3过表达的HCT116-RSPO3-OE细胞株及相应的对照细胞株。采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）检测体外各稳定转染细胞株的细胞增殖。采用流式细胞术分析各稳定转染细胞株的细胞周期、裸小鼠脾脏和移植瘤组织中NK细胞的比例。通过裸小鼠皮下移植瘤模型观察RSPO3敲减或过表达的结肠癌细胞在裸小鼠体内的生长。利用双荧光素酶报告基因系统检测RSPO3敲减或过表达对结肠癌Wnt基因转录活性的影响。结果：生物信息学分析显示，RSPO3在多种实体瘤肿瘤组织包括结直肠癌组织中的表达显著低于相应的癌旁组织。RSPO3敲减或过表达不影响体外SW480和HCT116结肠癌细胞的增殖（P>0.05）和细胞周期（P>0.05）。但在裸小鼠体内，与对照细胞相比，RSPO3敲减显著促进SW480细胞移植瘤的生长（260.2±162.4 vs 1 311.7±570.1，P<0.05），而RSPO3过表达则显著抑制HCT116细胞移植瘤的生长（1 549.0±241.2 vs 512.1±250.0，P<0.05）。流式细胞术分析发现，在荷移植瘤裸小鼠体内，RSPO3敲减显著减少了脾脏和移植瘤组织中NK细胞的比例（脾脏：6.42±0.94 vs 5.25±0.59，P=0.04；移植瘤：8.27±0.29 vs 6.48±1.48，P=0.04）；而RSPO3过表达显著增加了脾脏和移植瘤组织中NK细胞的比例（脾脏：5.29±0.16 vs 7.02±0.49，P=0.01；移植瘤：6.39±0.39 vs 8.14±0.34，P<0.05）。癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据相关性分析显示，RSPO3表达与NK细胞表面标志物CD56（r=0.58，P<0.05）和CD16（r=0.64，P<0.05）的表达显著正相关，并与NK细胞激活标志物CD69（r=0.51，P<0.05）和KLRB1（r=0.37，P<0.05）的表达显著正相关。双荧光素酶报告基因实验结果显示，RSPO3敲减后Wnt荧光素酶活性下调（1.0±0.0 vs 0.45±0.09，P<0.05），而RSPO3过表达后Wnt荧光素酶活性上调（1.0±0.0 vs 1.75±0.14，P<0.05）。结论：RSPO3能在体内显著抑制结直肠癌移植瘤的生长，并能增加移植瘤组织中NK细胞浸润，RSPO3是一个潜在的结直肠癌的抑制基因。

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

中图分类号:

周聪, 何丽娜, 成小姣, 黄廷磊, 涂水平. RSPO3抑制体内结直肠癌移植瘤生长和增加NK细胞浸润的作用研究[J]. 中国癌症杂志, 2023, 33(7): 664-672.

ZHOU Cong, HE Lina, CHENG Xiaojiao, HUANG Tinglei, TU Shuiping. Effect of RSPO3 on inhibiting the growth of colorectal cancer transplanted tumors and increasing NK cell infiltration in vivo[J]. China Oncology, 2023, 33(7): 664-672.

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