1Department of Respiratory and Critical Care Medicine, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi, China.
Department of Respiratory and Critical Care Medicine, Nanxishan Hospital of Guangxi Zhuang Autonomous Region, Guilin, Guangxi, China.
Acta Biochim Pol. 2021 Dec 21;69(1):31-36. doi: 10.18388/abp.2020_5543.
The paper aimed to explore the mechanism of cellular retinoic acid binding protein 2 (CRABP2) involvement in Golgi stress and tumor dryness in non-small cell lung cancer (NSCLC) cells through the estrogen receptor (ER) dependent Hippo pathway.
Human NSCLC cell line A549 was purchased from ATCC andcultured in RPMI-1640 with 10% FBS. Attractene reagent was used for plasmid transfection. ER (sh) RNA was designed using RNAi Designer. Seventy-six hours after infection, stable cells were obtained after treated with puromycin for 3 weeks. ER silencing cells (with inhibited ER expression) were compared to the control cells (normal cultured NSCLC cell line A549, CRABP2 normal expression). CRABP2 and ER expression levels were detected by RT-PCR. MTT assay was used to detect cell proliferation, and the cell localization of ER and Golgi was observed by confocal microscopy. The invasion and metastasis of cells were analyzed by Boden chamber invasion and migration assays. Western blotting assays was used for detecting the protein expression of E-cadherin, vimentin, ZO-1 protein and epithelial-mesenchymal transition (EMT) related factors.
The lower expression level of mRNA was detected in the ER-silencing group compared to the control group (P<0.05). We also found a higher proliferation level of cells, the number of invading and metastatic cells, the expression of vimentin, p-Lats1T1079, Lats1 and p-YAPS127 mRNA in the control group compared to the ER silencing group (P<0.05). And the expression level of protein kinase RNA-like endoplasmic reticulum kinase (PERK), phosphorylate eukaryotic initiation factor 2 (p-eIF2 alpha), activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP) in the control group was higher than that in the ER silencing group (P<0.05). Adversely, a lower expression level of E-cadherin and ZO-1 protein was found in the control group compared to the ER silencing group (P<0.05).
The expression of CRABP2 in NSCLC cells was regulated by ER, and cell proliferation and invasion were regulated by the Hippo pathway. At the same time, it was found that decreased expression of CRABP2 enhanced endoplasmic reticulum/Golgi stress response.
本研究旨在通过雌激素受体（ER）依赖性 Hippo 通路探索细胞视黄酸结合蛋白 2（CRABP2）参与非小细胞肺癌（NSCLC）细胞高尔基体应激和肿瘤干燥的机制。
人非小细胞肺癌细胞系 A549 购自 ATCC，在含 10%胎牛血清的 RPMI-1640 中培养。使用 Attractene 试剂进行质粒转染。使用 RNAi Designer 设计 ER（sh）RNA。感染 76 小时后，用嘌呤霉素处理 3 周以获得稳定细胞。将 ER 沉默细胞（抑制 ER 表达）与对照细胞（正常培养的 NSCLC 细胞系 A549，CRABP2 正常表达）进行比较。通过 RT-PCR 检测 CRABP2 和 ER 的表达水平。通过 MTT 测定法检测细胞增殖，通过共聚焦显微镜观察 ER 和高尔基体的细胞定位。通过 Boden 室侵袭和迁移测定分析细胞的侵袭和转移。通过 Western blot 测定法检测 E-钙粘蛋白、波形蛋白、ZO-1 蛋白和上皮-间充质转化（EMT）相关因子的蛋白表达。
与对照组相比，ER 沉默组的 mRNA 表达水平较低（P<0.05）。我们还发现对照组细胞的增殖水平较高，侵袭和转移细胞的数量较多，波形蛋白、p-Lats1T1079、Lats1 和 p-YAPS127mRNA 的表达水平也较高（P<0.05）。与 ER 沉默组相比，对照组蛋白激酶 RNA 样内质网激酶（PERK）、磷酸化真核起始因子 2（p-eIF2alpha）、激活转录因子 4（ATF4）和 C/EBP 同源蛋白（CHOP）的表达水平较高（P<0.05）。相反，对照组 E-钙粘蛋白和 ZO-1 蛋白的表达水平低于 ER 沉默组（P<0.05）。
CRABP2 在 NSCLC 细胞中的表达受 ER 调节，Hippo 通路调节细胞增殖和侵袭。同时，发现 CRABP2 表达降低增强了内质网/高尔基体应激反应。
