Hamza Shaimaa, Garanina Ekaterina E, Shkair Layaly, Alsaadi Mohammad, Khaiboullina Svetlana F, Tezcan Gulcin
Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia.
Department of Fundamental Sciences, Faculty of Dentistry, Bursa Uludag University, Bursa 16059, Turkey.
Pharmaceuticals (Basel). 2024 Feb 26;17(3):299. doi: 10.3390/ph17030299.
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 (WT)-expressing HCT-116 cells. NLRP3.AB.pCCL.sin.cPPT.U6.miR-223-Decoy.hPGK.GFP.WPRE plasmid (D) 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 WT 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 WT 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.
含NLR家族pyrin结构域蛋白3（NLRP3）促进结直肠癌（CRC）的生长。然而，抑制NLRP3对CRC细胞进展的治疗效果存在争议。本研究比较了药理学NLRP3抑制剂格列本脲（gli）和miR - 223对NLRP3的翻译后抑制作用对HCT - 116和HCT - 15细胞中CRC细胞进展的治疗效果。使用脂多糖（LPS）和三磷酸腺苷（ATP）激活经Gli处理和表达LSB - hsa - miR - 223 - 3p（野生型，WT）的HCT - 116细胞。NLRP3.AB.pCCL.sin.cPPT.U6.miR - 223 - Decoy.hPGK.GFP.WPRE质粒（D）是miR - 223表达的阴性对照。使用蛋白质免疫印迹法分析NLRP3、gasdermin D和BAX的表达。实时定量聚合酶链反应（Real - time PCR）检测未转染细胞中自噬相关基因ATG5、BECN1和miR - 223的RNA表达。酶联免疫吸附测定（ELISA）分析培养基中的白细胞介素 - 1β（IL - 1β）和白细胞介素 - 18（IL - 18）。使用MTS - 1、膜联蛋白V、伤口愈合和球体侵袭试验评估细胞活力和进展。多重细胞因子检测法检测促炎细胞因子的分泌。LPS - ATP激活的NLRP3产生gasdermin D裂解，释放IL - 1β和IL - 18，并激活细胞迁移和球体侵袭。相反，在炎性小体激活后的细胞中发现细胞生长、miR - 223表达、干扰素 - γ（IFN - γ）、CXC趋化因子配体10（CXCL10）和白血病抑制因子（LIF）分泌减少。gli和WT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、CXCL10和LIF的产生影响有限，但WT miR - 223增加了这些细胞因子的释放。此外，gli不抑制细胞生长，而WT miR - 223促进细胞凋亡。值得注意的是，gli和WT miR - 223均不能有效阻止球体侵袭。这些数据表明，虽然与gli相比，WT在CRC中可能具有更好的抗癌效果，但单独使用miR - 223介导的NLRP3抑制可能不足以预防CRC转移。
