| 何佳奇,陈瑞琳,解伟,李雪健,余陈欢.异牡荆苷通过上调节律基因CRY2逆转人肺腺癌A549/GR细胞吉非替尼耐药的作用机制[J].浙江中西医结合杂志,2026,36(3): |
| 异牡荆苷通过上调节律基因CRY2逆转人肺腺癌A549/GR细胞吉非替尼耐药的作用机制 |
| Isovitexin Reversed Gefitinib Resistance of Lung Adenocarcinoma A549/GR Cells by activating CRY2 |
| 投稿时间：2024-06-05 修订日期：2024-12-23 |
| DOI： |
| 中文关键词: 非小细胞肺癌 异牡荆苷 吉非替尼 节律基因 CRY2 |
| 英文关键词:Non-small cell lung cancer Isovitexin Gefitinib Rhythm gene CRY2 |
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| 中文摘要: |
| 目的 观察异牡荆苷(IVT)对人肺腺癌吉非替尼(GEF)耐药细胞株A549/GR药物敏感性和节律基因表达的影响，揭示其逆转吉非替尼在治疗非小细胞肺癌（NSCLC）中的耐药分子机制。方法 采用CCK-8法检测异牡荆苷、吉非替尼单用及联用对A549/GR细胞增殖的影响，计算IC50和耐药指数；采用RT-PCR技术检测细胞株CLOCK、BMAL1、CRY、PER等节律基因的表达水平；采用分子对接技术预测异牡荆苷与节律基因CRY2的互作关系；采用流式细胞技术检测A549/GR的细胞凋亡率；采用Western blot检测耐药细胞中Wnt家族成员3a（Wnt3a）、β-连环蛋白(β-catenin)、骨髓细胞c-Myc基因（c-Myc）、B淋巴细胞瘤-2(Bcl-2)、B细胞淋巴瘤-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)、裂解的天冬氨酸特异性半胱氨酸蛋白酶3(clv Caspase-3)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)、磷酸化蛋白激酶B（p-AKT）的蛋白表达量。结果 CCK-8法检测结果显示，在相同浓度吉非替尼作用下，随着联合异牡荆苷给药浓度的增加，A549/GR细胞株存活率逐渐降低，IC50分别为33.98、13.00、4.34 μmol/L；吉非替尼联合不同浓度异牡荆苷作用于A549/GR细胞24h、48h及72h，随着作用时间的延长，A549/GR细胞株存活率均逐渐降低，72h后存活率分别为(30.91±2.81)%、(13.35±1.13)%、(4.92±0.48)%；提示异牡荆苷能剂量依赖性和时间依赖性逆转A549/GR细胞对吉非替尼的耐药。RT-PCR检测结果显示，异牡荆苷与吉非替尼联用可提高A549/GR细胞PER3和CRY2 mRNA表达水平，结合表达丰度及给药前后表达差异，筛选CRY2为目标节律基因。分子对接结果表明，异牡荆苷可通过π键和氢键与CRY2结合，结合能为-10.2 kcal/mol，能剂量依赖性地促进CRY2表达。流式细胞术检测结果显示，两药联合给药后A549/GR细胞的凋亡率显著增加(P<0.05)，而沉默CRY2基因后，两药联合作用显著减弱(P<0.05)。Western blot检测结果显示，两药联合给药后细胞中Wnt3a[（0.20±0.01）比(1.00±0.04）、(0.50±0.02）]、β-catenin[（0.25±0.01）比(1.00±0.04)、(0.50±0.03）]、c-Myc[（0.15±0.01）比（1.00±0.04）、（0.60±0.03）]、Bcl-2[（0.16±0.01）比（0.60±0.03）、（0.50±0.03）]、p-PI3K[（0.20±0.01）比（1.00±0.05）、（0.60±0.03）]、p-AKT[（0.10±0.01）比（1.00±0.04）、（0.72±0.04）]蛋白表达量显著降低(P<0.05)；Bax[（1.00±0.05）比（0.13±0.01）、（0.82±0.04）]、clv caspase-3[（1.00±0.05）比（0.15±0.01）、（0.39±0.02）]蛋白表达水平显著升高(P<0.05)，Bax/Bcl-2比值也明显升高；沉默CRY2基因后，两药联合作用显著减弱(P<0.05)。结论 异牡荆苷可通过上调CRY2表达，逆转A549/GR细胞对吉非替尼的耐药性。 |
| 英文摘要: |
| ABSTRACT OBJECTIVE To observe the effect of isovitexin (IVT) on drug sensitivity and rhythm gene expression of gefitinib (GEF)-resistant cell lines A549/GR, and reveal the molecular mechanism of its reversal of gefitinib resistance in human non-small cell lung cancer (NSCLC). METHODS CCK-8 method was used to detect the effects of monotherapy and combination therapy of isovitexin and gefitinib on the proliferation of A549/GR cells, IC50 and resistance index were calculated. The expression levels of rhythmic genes such as CLOCK, BMAL1, CRY, and PER were detected by RT-PCR. The interaction between isovitexin and CRY2 was predicted by molecular docking. The apoptosis rates of A549/GR were detected by flow cytometry. The expressions of proteins (Wnt3a, β-catenin, c-Myc, Bax, Bcl-2, caspase-3, clv caspase-3, p-PI3K, p-AKT) were detected by Western blot analysis. RESULTS The CCK-8 method detection results showed that under the same concentration of gefitinib, the survival rate of A549/GR cell lines gradually decreased with the increase of the combined isovitexin administration concentration, with IC50 values of 33.98, 13.00, and 4.34 μmol/L, respectively. The combination of gefitinib and different concentrations of isovitexin was applied to A549/GR cells for 24 h, 48 h, and 72 h. As the treatment time increased, the survival rate of A549/GR cell lines gradually decreased. After 72 h, the survival rates were (30.91±2.81)%, (13.35±1.13)%, and (4.92±0.48)%, respectively. Suggesting that isovitexin can dose - and time-dependent reverse the resistance of A549/GR cells to gefitinib. The RT-PCR technology detection results showed that the combination of isovitexin and gefitinib can increase the mRNA expression levels of PER3 and CRY2 in A549/GR cells. Based on the expression abundance and the difference in expression before and after administration, CRY2 was selected as the target rhythm gene. The molecular docking results showed that isovitexin can bind to CRY2 through π - and hydrogen bonds, with a binding energy of -10.2 kcal/mol, and can dose dependently promote CRY2 expression. The results of flow cytometry analysis showed that the apoptosis rate of A549/GR cells significantly increased after combined administration of the two drugs (P<0.05), while the combined effect of the two drugs was significantly weakened after silencing the CRY2 gene (P<0.05). Western blot analysis showed that the expression levels of Wnt3a[0.20±0.01） vs. （1.00±0.04）,（0.50±0.02）], β-catenin[（0.25±0.01） vs. (1.00±0.04) ,（0.50±0.03）], c-Myc[（0.15±0.01） vs. （1.00±0.04）,（0.60±0.03）], Bcl-2[（0.16±0.01） vs. （0.60±0.03）,（0.50±0.03）], p-PI3K[（0.20±0.01） vs. （1.00±0.05）,（0.60±0.03）], and p-AKT[（0.10±0.01） vs. （1.00±0.04）,（0.72±0.04）] proteins in cells were significantly reduced after combined administration of the two drugs; The expression levels of Bax[（1.00±0.05） vs. （0.13±0.01）,（0.82±0.04）] and clv caspase-3[（1.00±0.05） vs. （0.15±0.01）,（0.39±0.02）] proteins were significantly increased, and the Bax/Bcl-2 ratio was also significantly increased; After silencing the CRY2 gene, the combined effect of the two drugs was significantly reduced (P<0.05). CONCLUSION Isovitexin reversed the resistance of A549/GR cells to gefitinib via activating CRY2 expression. |
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