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Abstract：
The NLR family pyrin domain containing 3 (NLRP3) promotes the growth of colorectal cancer (CRC). However, the therapeutic effect of NLRP3 inhibition on CRC cell progression is controversial. This study comparatively investigated the therapeutic effect of a pharmacological NLRP3 inhibitor, glibenclamide (gli), and the post-translational suppression of NLRP3 by miR-223 on CRC cell progression in HCT-116 and HCT-15 cells. LPS and ATP were used to activate Gli-treated and LSB-hsa-miR-223-3p (WTmiR-223)-expressing HCT-116 cells. NLRP3.AB.pCCL.sin.cPPT.U6.miR-223-Decoy.hPGK.GFP.WPRE plasmid (DmiR-223) was the negative control for miR-223 expression. NLRP3, gasdermin D, and BAX expressions were analyzed using western blotting. Real-time PCR detected the RNA expression of autophagy-related genes ATG5, BECN1, and miR-223 in non-transfected cells. ELISA analyzed IL-1β and IL-18 in the medium. MTS-1, annexin V, wound-healing, and sphere-invasion assays were used to assess cell viability and progression. A multiplex cytokine assay detected proinflammatory cytokine secretion. LPS-ATP-activated NLRP3 produced gasdermin D cleavage, released IL-1b and IL-18, and activated cell migration and sphere invasion. In contrast, reduced cell growth, miR-223 expression, IFN-γ, CXCL10, and LIF secretion were found in cells after inflammasome activation. Both gli and WTmiR-223 induced autophagy genes ATG5 and BECN1 and reduced the NLRP3 activation and its downstream proteins. However, while gli had a limited effect on the production of IFN-γ, CXCL10, and LIF, WTmiR-223 increased the release of those cytokines. In addition, gli did not suppress cell growth, while WTmiR-223 promoted apoptosis. Notably, neither gli nor WTmiR-223 effectively prevented sphere invasion. These data suggest that, while WTmiR-223 could have a better anticancer effect in CRC compared to gli, the sole usage of miR-223-mediated NLRP3 suppression may not be sufficient to prevent CRC metastasis.
摘要：
NLR家族含pyrin结构域3(NLRP3)促进结直肠癌(CRC)的生长。然而,NLRP3抑制对CRC细胞进展的治疗作用存在争议.本研究比较研究了药物NLRP3抑制剂的治疗效果，格列本脲（gli），以及miR-223对HCT-116和HCT-15细胞中CRC细胞进展的NLRP3的翻译后抑制。LPS和ATP用于激活Gli处理的和表达LSB-hsa-miR-223-3p(WTmiR-223)的HCT-116细胞。NLRP3。AB.pCCL.罪。cPPT。U6.miR-223-诱饵。hPGK。GFP.WPRE质粒(DmiR-223)是miR-223表达的阴性对照。NLRP3,gasderminD,和BAX表达使用蛋白质印迹分析。实时荧光定量PCR检测自噬相关基因ATG5、BECN1和miR-223在未转染细胞中的RNA表达。ELISA分析培养基中的IL-1β和IL-18。MTS-1，附件五，伤口愈合,和球体侵袭试验用于评估细胞活力和进展。多重细胞因子测定检测促炎细胞因子分泌。LPS-ATP激活的NLRP3产生GasderminD裂解，释放IL-1b和IL-18，并激活细胞迁移和球体侵袭。相比之下,细胞生长减少,miR-223表达，IFN-γ，炎症小体激活后，细胞中发现CXCL10和LIF分泌。gli和WTmiR-223均诱导自噬基因ATG5和BECN1，并降低NLRP3激活及其下游蛋白。然而,虽然gli对IFN-γ的产生影响有限，CXCL10和LIF,WTmiR-223增加了这些细胞因子的释放。此外,gli不抑制细胞生长，WTmiR-223促进细胞凋亡。值得注意的是,gli和WTmiR-223都不能有效防止球体入侵。这些数据表明,而WTmiR-223与gli相比在CRC中具有更好的抗癌作用，仅使用miR-223介导的NLRP3抑制可能不足以预防CRC转移.
