CD147通过AIM2炎症小体介导宫颈癌细胞焦亡和增殖

王玲; 秦祥川; 李金秋; 阿仙姑·哈斯木

doi:10.12259/j.issn.2095-610X.S20240103

CD147通过AIM2炎症小体介导宫颈癌细胞焦亡和增殖

doi: 10.12259/j.issn.2095-610X.S20240103

1.
新疆医科大学基础医学院/新疆地方病分子生物学重点实验室，新疆乌鲁木齐　836001

基金项目: 国家自然科学基金资助项目（82260516）

详细信息

作者简介:
王玲（1995～），女，山西运城人，在读硕士研究生，主要从事宫颈癌发病机制的研究工作

通讯作者:
阿仙姑·哈斯木，E-mail：axiangu75@126.com

中图分类号: R737.33
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出版历程
- 收稿日期: 2023-10-30
- 网络出版日期: 2024-01-09
- 刊出日期: 2024-01-25

CD147 Mediates Cervical Cancer Cell Pyroptosis and Proliferation through AIM2 Inflammasome

1.
Basic Medicine College of Xinjiang Medical University/Xinjiang Key Laboratory of Molecular Biology of Endemic Diseases，Urumqi Xinjiang 836001，China

摘要

摘要: 目的探讨跨膜蛋白CD147表达量变化对AIM2炎症小体介导的宫颈癌细胞焦亡及增殖的影响。方法采用Western blot 实验检测 CD147 在宫颈癌细胞 SiHa（HPV+）、C33a（HPV-）和正常宫颈上皮细胞 H8（HPV+）、HCer Epic（HPV-）中的表达水平；通过慢病毒转染 SiHa细胞下调CD147的表达，根据不同处理分为SiHa组、阴性对照组（shCD147-NON）、敲低组1（shCD147-1）和敲低组2（shCD147-2），通过Western blot、RT-qPCR和观察细胞绿色荧光表达验证转染效果；通过Western blot 和 RT-qPCR 检测CD147和AIM2炎症小体相关因子 AIM2、Caspase-1、IL-18、GSDMD蛋白和 mRNA 的表达；检测细胞培养上清中乳酸脱氢酶（LDH）释放度，荧光倒置显微镜下观察细胞的形态；CCK-8 实验检测细胞的增殖能力；细胞克隆实验检测细胞集落形成能力。结果 Western blot 结果显示，与 HCerEpic细胞比较，SiHa 细胞中 CD147 蛋白表达最高（P < 0.05）； CD147低表达慢病毒有效下调了SiHa细胞 CD147表达水平（P < 0.05）；Western blot 及 RT-qPCR实验结果表明，与SiHa组相比， shCD147-1 组和 shCD147-2 组AIM2、Caspase-1、IL-18、GSDMD蛋白和 mRNA 表达明显升高（P < 0.05）；乳酸脱氢酶（LDH）释放实验显示，与SiHa组相比，shCD147组LDH释放度明显升高（P < 0.05）；荧光倒置显微镜下显示，shCD147组出现肿胀和空泡化，表现出典型的细胞焦亡现象；与SiHa组相比，shCD147-1组和shCD147-2组细胞增殖能力和集落形成能力明显降低（P < 0.05）。结论 CD147低表达有效上调宫颈癌 SiHa 细胞AIM2炎症相关因子的表达，诱发细胞焦亡，抑制细胞的增殖和克隆。
- 宫颈癌 /
- CD147 /
- AIM2炎症小体 /
- 细胞焦亡 /
- 增殖
Abstract: Objective To investigate the effect of transmembrane protein CD147 expression on AIM2 inflammasome-mediated pyroptosis and proliferation of cervical cancer cells. Methods Western Blot was used to detect the expression of CD147 in cervical cancer cell lines SiHa（HPV+） and C33a（HPV-） and normal cervical epithelial cells H8（HPV+） and HCer Epic（HPV-）. SiHa cells were transfected with lentivirus to down-regulate the expression of CD147. According to the different treatments, SiHa cells were divided into SiHa group, negative control group（shCD147-NON）, knockdown group 1（shCD147-1） and knockdown group 2（shCD147-2）. The transfection effect was verified by Western Blot, RT-qPCR and green fluorescence expression. The protein and mRNA expressions of AIM2, Caspase-1, IL-18 and GSDMD were detected by Western Blot and RT-qPCR. The lactate dehydrogenase（LDH） release was measured in the cell culture supernatant, and the cell morphology was observed under the fluorescence inverted microscope; the proliferation ability of cells was measured by CCK-8 and the colony formation ability was measured by cell cloning experiments. Results Western Blot results showed that CD147 protein expression in SiHa cells was the highest compared with that in HCerEpic cells. CD147 low expression lentivirus effectively down-regulated the expression of CD147 in SiHa cells. The results of Western Blot and RT-qPCR experiments showed that the expression of AIM 2, Caspase-1, IL-18, GSDMD protein and mRNA increased in shCD147-1 and shCD147-2 group（P < 0.05）. Lactate dehydrogenase（LDH） release assay showed that compared with the SiHa group, the shCD147 group had a significant increase in LDH release（P < 0.05）. Fluorescence inverted microscope showed that the shCD147 group had swelling and vacuolization, showing typical pyroptosis. Compared with the SiHa group, the shCD147-1 and shCD147-2 groups had significantly reduced the cell proliferation and colony formation ability（P < 0.05）. Conclusion Low expression of CD147 effectively up-regulates the expression of AIM2 inflammation-related factors in cervical cancer SiHa cells, induces the pyroptosis, and inhibits the cell proliferation and cloning.
- Cervical cancer /
- CD147 /
- AIM2inflammasome /
- Pyroptosis /
- Proliferation

HTML全文

图 1 检测 CD147在宫颈癌细胞中的表达水平及慢病毒转染效率

A：CD147蛋白在宫颈癌及正常宫颈上皮细胞中的相对表达量；B：转染慢病毒后CD147的 mRNA 的表达情况；C：转染慢病毒后CD147蛋白的相对表达量；D：慢病毒转染CD147细胞后绿色荧光表达情况 ( 标尺为100 μm)；ns：差异无统计学意义；^****P < 0.0001。

Figure 1. The expression level of CD147 in cervical cancer cells and the transfection efficiency of lentivirus were detected

下载: 全尺寸图片幻灯片

图 2 下调CD147 对 SiHa 细胞AIM2炎症小体信号通路的影响

A：下调 CD147后 AIM2、Caspase-1、IL-18、GSDMD 的 mRNA 的表达情况；B：下调 CD147后 AIM2、Caspase-1、IL-18、GSDMD 蛋白的相对表达量；ns：无统计学差异；^***P < 0.001；^****P < 0.0001。

Figure 2. Effect of down-regulating CD147 on AIM2 inflammasome signaling pathway in SiHa cells

下载: 全尺寸图片幻灯片

图 3 下调CD147对宫颈癌SiHa细胞焦亡的影响

A：荧光倒置显微镜观测下调 CD147对SiHa 细胞形态影响（标尺为100 μm）；B：下调 CD147对SiHa 细胞释放乳酸脱氢酶水平的影响；^***P < 0.001。

Figure 3. Effect of down-regulation of CD147 on pyroptosis in cervical cancer SiHa cells

下载: 全尺寸图片幻灯片

图 4 下调 CD147 后宫颈癌 SiHa 细胞增殖克隆能力情况

A：下调 CD147对SiHa 细胞增殖影响；B：下调 CD147对SiHa 细胞集落形成情况；^***P < 0.001；^****P < 0.0001。

Figure 4. The proliferation and cloning ability of cervical cancer SiHa cells after down-regulation of CD147

下载: 全尺寸图片幻灯片

表 1 RT-qPCR检测基因的引物序列

Table 1. Primer sequences of genes detected by RT-qPCR

基因	引物序列
CD147	F-TGTTCGTGCTGCTGGGATTCG R-GAGCCAAGGTCTTCTACGGTAGTG
AIM2	F-TATCGGCACAGTGGTTTCTTAGAGG R-GGGCTGAGTTTGAAGCGTGTTG
Caspase-1	F-CCCACATCCTCAGGCTCAGAAG R-TGCGGCTTGACTTGTCCATTATTG
IL-18	F-TGGCTGCTGAACCAGTAGAAGAC R-GAGGCCGATTTCCTTGGTCAATG
GSDMD	F-CCAGAAGAAGACGGTCACCATCC R-TGGAACGCTTGTGGCCTGTC
GAPDH	F-TGCACCACCAACTGCTTAGC R-GGCATGGACTGTGGTCATGAG

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