Effect of MSC-exo，a New Cell Delivery Tool，on Gene Delivery and Proliferation of Pancreatic Cancer
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摘要:
目的 观察1种新型细胞递送工具（MSC-exo）转运靶向基因调控胰腺癌增殖效应。 方法 透射电子显微镜（transmission electron microscope，TEM）和纳米颗粒跟踪分析技术（nanoparticle tracking analysis，NTA）鉴定人间充质干细胞外泌体（human mesenchymal stem cell exosomes，MSC-exo）并转运miR-450a-5p进入CFPAC-1，探讨miR-450a-5p靶向BZW2抑制胰腺癌细胞增殖效应。基因技术处理Pc-BZW2，CCK-8、EdU、细胞划痕、Transwell验证MSC-exo与MSC-exo-miR-450a-5p对细胞的抑制作用。 结果 与胰腺正常组织相比miR-450a-5p在胰腺癌组织中低表达（P<0.05），CFPAC-1细胞MSC-exo-miR-450a-5p外泌体标记蛋白CD63、TSG101表达高于MSC-exo（P<0.05）。CCK-8、EdU、细胞划痕、Transwell实验显示MSC-exo-miR-450a-5p较MSC-exo可显著抑制CFPAC-1细胞增殖、侵袭和迁移（P<0.05）。通过双荧光素酶实验证实，miR-450a-5p靶向BZW2，并且RT-qPCR和免疫印迹检测miR-450a-5p和 BZW2表达成负性相关（P<0.05）。过表达BZW2，CCK-8、EdU、细胞划痕、Transwell实验均证实，pc-BZW2逆转MSC-exo-miR-450a-5p对CFPAC-1的抑癌功能，免疫印迹检测PCNA、Ki-67、MMP2、MMP9，结果与上述实验一致（P<0.05）。 结论 hMSC-exo是1种新的递送系统，靶向BZW2转运miR-450a-5p抑制胰腺癌细胞的生物学恶性，为胰腺癌靶向治疗研究提供了重要线索。 -
关键词:
- 胰腺癌 /
- 间充质干细胞 /
- 外泌体 /
- miR-450a-5p /
- BZW2
Abstract:Objective To observe the effect of a new cell delivery tool （MSC exo） on the proliferation of pancreatic cancer by transferring targeted genes. Methods Transmission Electron Microscope （TEM） and Nanoparticle Tracking Analysis（NTA） were used to identify human mesenchymal stem cell exosomes（MSC-exo） and transport miR-450a-5p into CFPAC-1, to explore the effect of miR-450a-5p targeting BZW2 on inhibiting the proliferation of pancreatic cancer cells. Results The expression of miR-450a-5p was low in pancreatic cancer tissue （P<0.05）, and the expression of CD63 and TSG101 of MSC-exo-miR-450a-5p in CFPAC-1 cells was higher than that of MSC-exo by Western blot（P<0.05）. CCK-8 and EdU results showed that MSC-exo-miR-450a-5p significantly inhibited the proliferation of CFPAC-1 cells （P<0.05）. Cell scratch and Transwell experiments showed that MSC-exo-miR-450a-5p can inhibit the migration and invasion of CFPAC-1 cells （P<0.05）. Through dual luciferase assay, it was confirmed that miR-450a-5p targets BZW2, and RT-qPCR and Western blotting showed a negative correlation （P<0.05） between miR-450a-5p and BZW2 expression. Overexpression of BZW2, CCK-8, EdU, cell scratch, and Transwell experiments confirmed that pc-BZW2 reversed the anti-cancer function of MSC-exo-miR-450a-5p on CFPAC-1. Western blot detected PCNA,Ki-67,MMP2,MMP9, and the results were consistent with the above experiments （P<0.05）. Conclusion hMSC exo is a new delivery system, targeting BZW2 to transport miR-450a-5p to inhibit the biological malignancy of pancreatic cancer cells, which provides an important clue for the research of targeted treatment of pancreatic cancer. -
Key words:
- Pancreatic adenocarcinoma /
- Mesenchymal stem cell /
- Exosomes /
- miR-450a-5p /
- BZW2
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图 1 胰腺癌组织和细胞中miR-450a-5p呈低表达（$\bar x \pm s$， n=6）
A：miR-126-5p在胰腺癌组织中的表达水平低于正常胰腺组织；与Normal组比较，*P<0.05；B：CFPAC-1和PANC-1细胞系的miR-450a-5p表达低于HPC-Y5细胞，与HPC-Y5组比较，***P<0.001。
Figure 1. The expression of miR-450a-5p was low in pancreatic cancer tissues and cells. （$\bar x \pm s$，n=6）
图 2 CFPAC-1细胞摄取MSC-exo-miR-450a-5p
A：使用生物透射电子显微镜（TEM）观察样品中外泌体的形态和大小；B：外泌体和MSC-exo的平均粒径和主峰；C：标记蛋白（CD63和TSG101）的表达水平在MSC-exo-miR-450a-5p组中较高；D：miR-450a-5p在MSC-exo-miR-450a-5p组中的表达高于MSC-exo组；E：CFPAC-1细胞摄取MSC-exo和MSC-exo-miR-450a-5p。与MSC-exo组比较，***P<0.001。
Figure 2. CFPAC-1 cells uptake MSC-exo-miR-450a-5p
图 3 MSC-exo-miR-450a-5p抑制CFPAC-1细胞生物学行为
A：MSC-exo-miR-450a-5p显著抑制胰腺癌细胞增殖；B：EdU测定MSC-exo-miR-450a-5P对细胞的增殖影响；C：用蛋白质印迹法检测增殖相关蛋白的水平；D～E：MSC-exo-miR-450a-5p抑制侵袭和迁移；F：MMP2和MMP9的蛋白水平被MSC-exo-miR-450a-5p抑制，与MSC-exo组比较，***P<0.001。
Figure 3. MSC-exo-miR-450a-5p inhibits the biological behavior of CFPAC-1 cells
图 4 miR-450a-5p靶向BZW2
A：PITA、miRanda和miRmap数据库中miR-450a-5p靶点的交叉点；B：miR-450a-5p和BZW2的组合；C：双荧光素酶报告基因测定，与NC mimic组比较，***P<0.001。
Figure 4. MiR-450a-5p targets BZW2
图 5 BZW2在CFPAC-1细胞中呈高表达
A：根据“StarBase”数据库，BZW2在胰腺癌组织中的表达水平高于正常组织，与Normal组比较，*P<0.05。B：BZW2在胰腺癌细胞中表达较高，与HPC-Y5组比较，***P<0.001。C：RT-qPCR检测用miR-450a-5p模拟物转染的胰腺癌细胞中BZW2的基因表达；D：免疫印迹分析检测用miR-450a-5p模拟物转染的胰腺癌细胞中BZW2的蛋白表达；E：miR-450a-5p与BZW2的相关性分析，与NC mimic组比较，***P<0.001。
Figure 5. BZW2 is highly expressed in CFPAC-1 cells
图 6 Pc-BZW2逆转MSC-exo-miR-450a-5p的增殖抑制功能
A：基于CCK-8测定，MSC-exo-miR-450a-5p抑制CFPAC-1增殖，而Pc-BZW2逆转了增殖效应；B：分组处理细胞，EdU实验检测，BZW2逆转MSC-exo-miR-450a-5p对胰腺癌的增殖效应；C：miR-450a-5p抑制PCNA和Ki-67的表达，而Pc-BZW2部分逆转了这种作用；D：分组处理，MSC-exo-miR-450a-5p抑制侵袭过程；E：分组处理， MSC-exo-miR-450a-5p抑制迁移过程；F：MMP2和MMP9的蛋白水平被MSC-exo-miR-450a-5p抑制，但Pc-BZW2可逆转，与MSC-exo组比较，***P<0.001，与MSC-exo-miR-450a-5p+pc-NC组比较，###P<0.001。
Figure 6. Pc-BZW2 reverses the proliferation inhibition function of MSC exo miR-450a-5p
表 1 引物序列
Table 1. Primer sequence
基因 上游 下游 miR-450a-5p 5'-TTTTGCGATGTGTTCC-3' 5'-GTGCAGGGTCCGAGGT-3' U6 5'-TGCTCACTGTCTAAAATTGG-3' 5'-AGAAGAAGTCTGCTGTTGAC-3' BZW2 5'-CTAACAGGCCAGCGGTTCAAA-3' 5'-GGACAAGTGTATCCCTGAAGACT-3' B-actin 5'-ACACAGTGCTGTCTGGTGGT-3' 5'-TGATCTTCATGGTGCTGGGAG-3' 
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