Intervention of AKR1C3 on Malignant Biological Behavior of Breast Cancer Cells through PD1/PD-L1 Signaling Pathway
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摘要:
目的 探索酮还原酶家族1成员C3 （aldo-keto reductase family 1 member C3，AKR1C3）对乳腺癌恶性细胞生物学行为的干预作用及对程序性细胞死亡蛋白/程序性死亡-配体1 （ programmed cell death protein1/programmed death-ligand1，PD-1/PD-L ） 通路的影响。 方法 把MCF-7人乳腺癌细胞中NC组和AKR1C3组分别转染空质粒和AKR1C3质粒，采用MTT法检测转染后24 h、48 h、72 h细胞活力；采用流式细胞技术测定各组细胞的存活率以及早期、晚期凋亡比例；通过Transwell实验对各组细胞的迁移和侵袭能力进行检测；通过Western blot检测各组细胞PD-1、PD-L1、蛋白激酶B（protein kinase b，AKT）蛋白表达水平。使用C57BL/6小鼠构建荷瘤模型，将采用人乳腺癌MCF-7细胞转染NC质粒和AKR1C3质粒进行细胞荷瘤，每3天测量瘤体积，持续21 d，绘制两组小鼠肿瘤生长曲线及并于实验终点测量肿瘤质量。 结果 相较于NC组，AKR1C3组细胞活力降低（P < 0.05），并且具有时间依赖效应（P < 0.05），迁移和侵袭能力降低（P < 0.05），早期凋亡和晚期凋亡比例升高（P < 0.05），PD-1、PD-L1、AKT蛋白表达水平降低（P < 0.05）。小鼠实验结果表明，AKR1C3组小鼠肿瘤体积降低，肿瘤质量下降（P < 0.05）。 结论 AKR1C3可以抑制人乳腺癌细胞恶性生物学行为，抑制PD-1/PD-L1信号通路蛋白表达。 Abstract:Objective To investigate the effect of aldo-keto reductase family 1 member c3（AKR1C3） on malignant biological behavior of breast cancer cells and its influence on the programmed cell death protein 1/programmed death-ligand 1 （PD-1/PD-L1） pathway. Methods MCF-7 human breast cancer cells with NC and AKR1C3 groups transfected with NC plasmid and AKR1C3 plasmid respectively. Cell viability at 24 h/48 h/72 h post-transfection was assessed by MTT assay; flow cytometry measured cell survival rate and early/late apoptosis ratios; Transwell evaluated migration and invasion capabilities; western blot detected PD-1, PD-L1, protein kinase B （AKT） protein expression. For in vivo experiments, the C57BL/6 mice were used to establish tumor-bearing models. Human breast cancer MCF-7 cells transfected with NC plasmid and AKR1C3 plasmid were used for cell tumor-bearing. The tumor volume was measured every 3 days for 21 days, draw the tumor growth curves of the two groups of mice and measure the tumor mass at the end of the experiment. Results Compared to NC groups, the AKR1C3 group showed significantly reduced cell viability （time-dependent） （P < 0.05）, suppressed migration/invasion （P < 0.05）, increased early/late apoptosis ratios （P < 0.05）, and downregulated PD-1/PD-L1/AKT protein expression （P < 0.05）. In vivo, AKR1C3 group exhibited reduced tumor volume and weight （P < 0.05）. Conclusion AKR1C3 inhibits malignant biological behaviors in breast cancer cells and suppresses PD-1/PD-L1 signaling pathway protein expression. -
Key words:
- AKR1C3 /
- Human breast cancer /
- Apoptosis /
- Migration /
- PD-1 / PD-L1 pathway /
- Invasion /
- AKT /
- PPR5
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图 1 过表达AKR1C3转染效率验证（$\bar x \pm s $，n = 3）
与NC组相比，*P < 0.05。
Figure 1. Validation of AKR1C3 overexpression transfection efficiency in MCF-7 cells （$\bar x \pm s $，n = 3）
图 2 过表达AKR1C3对MCF-7细胞凋亡的影响（$\bar x \pm s $，n = 3）
Figure 2. Effect of AKR1C3 overexpression on apoptosis in MCF-7 cells（$\bar x \pm s $，n = 3）
图 3 过表达AKR1C3对MCF-7细胞迁移和侵袭的影响（$\bar x \pm s $，n = 3）
A：细胞迁移侵袭图；B：细胞迁移统计图；C：细胞侵袭统计图；与NC组比较，*P < 0.05。
Figure 3. Effect of AKR1C3 overexpression on migration and invasion in MCF-7 cells （$\bar x \pm s $，n = 3）
图 4 过表达AKR1C3对MCF-7细胞PD-1/PD-L1信号通路的影响（$\bar x \pm s $，n = 3）
A：AKT、PD-L1、PD-1蛋白条带图；B：PD-1蛋白统计图；C：PD-L1蛋白统计图；D：AKT蛋白统计图；与NC组相比，*P < 0.05。
Figure 4. Effect of AKR1C3 overexpression on the PD-1/PD-L1 signaling pathway in MCF-7 cells （$\bar x \pm s $，n = 3）
图 5 过表达AKR1C3对肿瘤生长的影响（$\bar x \pm s $，n = 3）
A：肿瘤代表性图片；B：肿瘤体积统计图；C：肿瘤质量统计图；D：对PD-1/PD-L1蛋白的影响；与NC组相比，*P < 0.05
Figure 5. Effect of AKR1C3 overexpression on tumor growth （$\bar x \pm s $，n = 3）
表 1 AKR1C3引物序列信息
Table 1. AKR1C3 primer sequence information
基因 引物序列 引物长度（bp） AKR1C3 F 5′-CAA GCG TCC TAA TTG TGG TCA-3′
R 5′-CCC TGC TAG ATG TCC AAC TGA TT-3′22 GAPDH F 5′-AGA AGG CTG GGG CTC ATT TG-3′
R 5′ AGG GGC CAT CCA CAG TCT TC-3′20 
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表 2 过表达AKR1C3对人乳腺癌MCF-7细胞活力的影响（$\bar x \pm s $，n = 3）
Table 2. Effect of AKR1C3 overexpression on cell viability in MCF-7 cells （$\bar x \pm s $，n = 3）
组别 24 h 48 h 72 h 组内时间比较 对照组 98.65 ± 0.71 97.81 ± 0.88 96.46 ± 0.61 F=1.32，P=0.312 NC组 97.92 ± 0.91 95.61 ± 1.71 95.98 ± 1.02 F=2.15，P=0.152 AKR1C3组 77.12 ± 1.41* 70.61 ± 1.73*# 61.21 ± 1.52*# F=86.34，P < 0.001 P <0.001* <0.001* <0.001* 与NC组比较，*P < 0.05；与24 h相比，#P < 0.05。
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表 3 过表达AKR1C3对人乳腺癌MCF-7细胞凋亡的影响（$\bar x \pm s $，n = 3）
Table 3. Effect of AKR1C3 overexpression on apoptosis in human breast cancer MCF-7 cells （$\bar x \pm s $，n = 3）
组别 细胞存活（%） 早期凋亡（%） 晚期凋亡（%） 对照组 94.51 ± 0.91 3.21 ± 0.18 1.06 ± 0.05 NC组NC 93.21 ± 0.98 3.16 ± 0.47 1.28 ± 0.52 AKR1C3组 61.71 ± 5.73* 13.21 ± 0.41* 25.21 ± 1.02* F 89.675 715.490 1320.242 P <0.001* <0.001* <0.001* 与NC组比较，*P < 0.05。
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表 4 过表达AKR1C3对人乳腺癌MCF-7细胞PD-1/PD-L1信号通路的影响（$\bar x \pm s $，n = 3）
Table 4. Effect of AKR1C3 overexpression on the PD-1/PD-L1 signaling pathway in human breast cancer MCF-7 cells （$\bar x \pm s $，n = 3）
组别 PD-1 PD-L1 AKT 对照组 1.21 ± 0.15 1.50 ± 0.43 1.50 ± 0.11 NC组 1.22 ± 0.34 1.56 ± 0.32 1.59 ± 0.30 AKR1C3组 0.81 ± 0.12* 0.65 ± 0.09* 0.79 ± 0.21* F 8.828 7.892 11.821 P 0.015* 0.021* 0.008* 与NC组比较，*P < 0.05。
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表 5 过表达AKR1C3对肿瘤生长的影响（$\bar x \pm s $，n = 3）
Table 5. Effect of AKR1C3 overexpression on tumor growth （$\bar x \pm s $，n = 3）
组别 肿瘤体积（cm3） 肿瘤质量（g） PD-1 PD-L1 AKT NC组 1.22 ± 0.34 2.36 ± 0.32 2.21 ± 0.22 1.56 ± 0.33 2.51 ± 0.34 AKR1C3组 1.01 ± 0.12* 1.15 ± 0.09* 1.21 ± 0.18* 0.78 ± 0.25* 1.11 ± 0.34* t 6.781 7.892 15.88 16.78 19.21 P 0.0078 *0.0088 *<0.001* <0.001* <0.001* 与NC组比较，*P < 0.05。
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