TP53 Promotes Pyroptosis and Inhibits Cell Invasion and Migration through the MMP1 Signaling Pathway in NIH-3T3 Cells
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摘要:
目的 探究TP53通过MMP1/NLRP3信号通路调控胚胎成纤维细胞焦亡、侵袭、迁移的分子机制。 方法 对小鼠胚胎成纤维细胞（NIH-3T3）转染慢病毒，分为空白对照组（Control）、阴性对照组（Vector）、实验组（oeTP53、oeTP53+shNC、oeTP53+shMMP1）。采用CCK-8法检测细胞增殖及活力，流式细胞仪检测细胞凋亡情况，划痕及侵袭实验检测细胞迁移侵袭能力，采用免疫共沉淀（Co-IP）验证P53与MMP1蛋白相互作用，RT-qPCR检测TP53、Collagen Ⅰ、Collagen Ⅲ、α-SMA的mRNA表达水平，Westen bolt检测P53、Collagen Ⅰ、Collagen Ⅲ、α-SMA及焦亡相关蛋白的表达水平；透射电镜观察细胞焦亡小体的变化。 结果 与Control及Vector组比较，oeTP53组细胞的增殖活性降低（P < 0.01）、细胞凋亡率升高（P < 0.0001 ）、侵袭（P <0.0001 ）及迁移能力（P <0.0001 ）降低；CollagenⅠ（P < 0.001）、CollagenⅢ（P < 0.01）、α-SMA（P < 0.01）蛋白表达降低，NLRP3 （P < 0.05）及cleaved-caspase-1表达升高（P < 0.01），且焦亡小体大量出现。对oeTP53组中MMP1的蛋白水平检测发现其升高（P < 0.05），Co-IP表明p53蛋白和MMP1存在相互作用。与oeTP53组比较，oeTP53+shMMP1组中细胞活力升高（P < 0.001），细胞凋亡率降低（P < 0.01），细胞迁移侵袭能力升高（P < 0.01）；瘢痕形成相关蛋白CollagenⅠ（P < 0.01）、CollagenⅢ（P < 0.001）、α-SMA（P < 0.05）蛋白表达升高，细胞焦亡相关蛋白NLRP3（P < 0.01）及cleaved-caspase-1（P < 0.001）的表达降低，且焦亡小体数量减少。结论 过表达TP53能够抑制小鼠胚胎成纤维细胞生长增殖、迁移、侵袭能力，降低瘢痕形成相关蛋白的表达，促进细胞焦亡，其作用机制可能与MMP1/NLRP3通路有关。 Abstract:Objective To explore the molecular mechanism by which TP53 regulating pyroptosis, invasion and migration of embryonic fibroblasts through MMP1/NLRP3 signaling pathway. Methods NIH-3T3 murine embryonic fibroblasts were transfected with lentivirus and grouped as Control, Vector, oeTP53, oeTP53+shNC, and oeTP53+shMMP1. Cell proliferation and viability were assessed via CCK-8 assay, apoptosis by flow cytometry, and migration/invasion through scratch and invasion experiments. Protein interaction between P53 and MMP1 was confirmed by Co-immunoprecipitation. RT-qPCR evaluated mRNA expression of TP53, Collagen I, Collagen III, and α-SMA, while Western blot analyzed protein levels of these markers and pyroptosis-related proteins. Transmission electron microscopy was employed to examine cellular pyroptotic body modifications. Results Compared with Control and Vector groups, the oeTP53 group showed reduced cell proliferation activity （P < 0.01）, increased cell apoptosis rate （P < 0.0001 ）, decreased invasion （P <0.0001 ） and migration capabilities （P <0.0001 ）; reduced Collagen I （P < 0.001）, Collagen III （P < 0.01）, and α-SMA （P < 0.01） protein expressions; increased NLRP3 （P < 0.05） and cleaved-caspase-1 expressions （P < 0.01）; and numerous pyroptotic bodies. MMP1 protein levels were found to be elevated in the oeTP53 group （P < 0.05）, and Co-IP demonstrated an interaction between p53 and MMP1 proteins. Compared with oeTP53 group, the oeTP53+shMMP1 group showed increased cell viability （P < 0.001）, decreased cell apoptosis rate （P < 0.01）, and increased cell migration （P < 0.01） and invasion capabilities （P < 0.01）, increased scar formation-related protein expressions of Collagen I （P < 0.01）, Collagen III （P < 0.001）, and α-SMA （P < 0.05）; decreased pyroptosis-related protein expressions of NLRP3 （P < 0.01） and cleaved-caspase-1 （P < 0.001）; and reduced pyroptotic bodies.Conclusion Overexpression of TP53 can inhibit mouse embryonic fibroblast proliferation, migration, and invasion, reduce scar formation-related protein expressions, and promote cell pyroptosis, with its mechanism potentially related to the MMP1/NLRP3 pathway. -
Key words:
- TP53 /
- MMP1 /
- Post-burn scar formation /
- Scar formation /
- Pyroptosis
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图 1 TP53过表达对NIH-3T3细胞增殖的影响
A: Western bolt检测结果；B: CCK-8法检测结果；*P < 0.05；**P < 0.01；***P < 0.001；ns P > 0.05。
Figure 1. Effect of TP53 overexpression on proliferation of NIH-3T3 cells
图 2 TP53过表达对NIH-3T3细胞凋亡的影响
A: Control组；B: Vector组；C: oeTP53组；D: 各组细胞凋亡率统计图；****P < 0.0001; ns P > 0.05。
Figure 2. The effect of TP53 overexpression on apoptosis in NIH-3T3 cells
图 3 TP53过表达对NIH-3T3细胞侵袭、迁移能力的影响
A: 细胞侵袭结果（×40）；B: 各组细胞数统计图；C: 细胞划痕实验结果（×40）；D: 各组细胞伤口愈合率；****P < 0.0001。
Figure 3. Effect of TP53 overexpression on invasion and migration ability of NIH-3T3 cells
图 4 TP53过表达对瘢痕形成相关蛋白的影响
A: Western blot检测条带图；B: 灰度分析柱状图；**P < 0.01；***P < 0.001；ns P > 0.05。
Figure 4. Effect of TP53 overexpression on scarring related proteins
图 5 TP53过表达对NIH-3T3细胞焦亡的影响
A: Western blot 检测条带图；B: 透射电镜观察焦亡小体；*P < 0.05；**P < 0.01。
Figure 5. Effect of TP53 overexpression on pyroptosis of NIH-3T3 cells
图 6 TP53过表达调控MMP1的表达
A: PPI数据库蛋白互作分析；B: RT-qPCR检测mRNA表达；C：Western blot检测蛋白水平；D: p53蛋白和MMPA蛋白的Co-IP实验结果。*P < 0.05；**P < 0.01；ns P > 0.05。
Figure 6. TP53 overexpression regulates the expression of MMP1
图 7 MMP1干扰质粒载体的构建
A: RT-qPCR检测mRNA表达；B: Western blot 检测蛋白表达；***P < 0.001，****P < 0.0001；ns P > 0.05。
Figure 7. MMP1 interferes with the construction of lentiviral vectors
图 8 MMP1对NIH-3T3细胞活力的影响
A: Western blot 检测蛋白表达；B: CCK-8检测细胞活力；***P < 0.001；****P < 0.0001；ns P > 0.05。
Figure 8. Effect of MMP1 on the viability of NIH-3T3 cells
图 9 流式细胞仪检测细胞凋亡情况
A: 流式检测细胞凋亡荧光强度散点图；B: 流式检测细胞凋亡柱形图；**P < 0.01；ns P > 0.05。
Figure 9. The apoptosis detected by flow cytometry
图 10 Western blot检测蛋白表达
A: Western blot 检测条带图；B: 灰度分析柱状图；*P < 0.05；**P < 0.01；***P < 0.001；ns P > 0.05。
Figure 10. The protein expression detected by Western blot
图 11 干扰MMP1对NIH-3T3细胞侵袭、迁移能力的影响
A: 细胞侵袭结果图（×40）；B: 侵袭的细胞率；C: 细胞迁移结果图（×40）；D: 伤口愈合率；***P < 0.001；****P < 0.0001。
Figure 11. Effect of interference with MMP1 on invasion and migration ability of NIH-3T3 cells
图 12 干扰MMP1对NIH-3T3细胞焦亡的影响
A: Western blot 检测焦亡相关蛋白表达；B: 透射电镜观察焦亡小体；**P < 0.01；***P < 0.001。
Figure 12. Effect of interference with MMP1 on pyroptosis of NIH-3T3 cells
表 1 sh RNA序列表
Table 1. sh RNA sequences
序列编号 引物序列（5'-3'） shMMP1#1 CACATGACTTTCCTGGAAT shMMP1#2 TTGTGGCTTATGGATTCAT shMMP1#3 AAGATGAAAGGTGGACCAA shNC TTCTCCGAACGTGTCACGT 
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表 2 荧光定量PCR扩增引物列表
Table 2. Nucleotide sequences of the primers used for real-time quantitative PCR
引物名称 引物序列（5'-3'） MMP1 forward GGACACCAACTATTGCTTCAG MMP1 reverse ATGTCCTTGGGGTATCCGTGTAG β-actin forward CCTAAGGCCAACCGTGAAAAG β-actin reverse AGGCATACAGGGACAGCACAG
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表 3 Western blot中所用一抗
Table 3. Primary antibodies used in Western blot
一抗 稀释比例 种属 MMP1 1∶ 1000 Rabbit Collagen Ⅰ 1∶ 1000 Rabbit Collagen Ⅲ 1∶ 1000 Rabbit α-SMA 1∶ 1000 Rabbit β-actin 1∶ 1000 Mouse NLRP3 1∶ 1000 Rabbit pro-caspase-1 1∶ 5000 Rabbit cleaved-caspase-1 1∶ 1000 Rabbit
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