Xu Y J, Hu Y M, Qin C, Wang F, Cao W, Yu Y W, Zhao L, Li J, Chen W Q, Li N, He J
Office of Cancer Screening, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.
Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China.
Zhonghua Zhong Liu Za Zhi. 2021 Sep 23;43(9):924-931. doi: 10.3760/cma.j.cn112152-20210421-00329.
To investigate the effects and the mechanism of Calcyclin-binding protein (CacyBP) on the proliferation and invasion of non-small cell lung cancer (NSCLC) cells. Six lung cancer tissues and paired normal lung tissues were collected from NSCLC patients who underwent surgical treatment in Jinan Central Hospital during 2016. The expression of CacyBP in these tissues was examined by western blot. The protein and mRNA expression of CacyBP in human bronchial epithelial cells (16HBE), NSCLC cell lines including A549, H1299, H460 and H1975 were examined by western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. RNAi and shRNA against negative control (NC) or CacyBP were transfected into A549 cell which were denoted as siNC group, siCacyBP-1 group, sicacyBP-2 group, shNC group and shCacyBP group, respectively. Control and Flag-CacyBP plasmids were transfected into A549 cells which were denoted as NC group and Flag-CacyBP group, respectively. Cell counting kit-8 (CCK-8), plate clone formation assay and flow cytometry assay were used to assess cell proliferation ability and cycle of A549. Wound healing assay and transwell assay were used to assess abilities of A549 cells migration and invasion. The protein expressions of epithelial-mesenchymal transition (EMT) markers including E-cadherin, N-cadherin, Snail1, Vimentin, and phosphorylation of protein kinase B (p-Akt) were examined in CacyBP depleted or overexpressed A549 cells. The CacyBP protein level in NSCLC tissues was 0.41±0.23, significantly higher than 0.11±0.04 in normal lung tissues (<0.05). The CacyBP protein expression levels in different NSCLC cell lines including A549, H1299, H460 and H1975 were 0.35±0.01, 0.38±0.01, 0.32±0.01 and 0.41±0.01, respectively, which were significantly higher than 0.03±0.01 in 16HBE cells (<0.05). The result of RT-PCR was consistent with that of western blot. Compared with siNC group (absorbance was 1.54±0.03), siCacyBP-1 group and siCacyBP-2 group showed decreased cell proliferation (absorbances were 1.38±0.04 and 1.34±0.03, <0.05). The number of cell colony in shNC group was 41.33±3.21, significantly higher than 22.00±3.61 in shCacyBP group (<0.05). The proportion of G(1) phase in shCacyBP group was (61.35±5.45)%, higher than (49.61±1.54) % in shNC group (<0.05). The proportion of S phase was (25.41±3.21)%, which was lower than (38.68±0.46)% of shNC group (<0.05). The cell migration rate of shCacyBP group was (12.67±0.71)%, which was significantly lower than (35.50±2.07)% of shNC group (<0.05). The numbers of cell migration and invasion in shNC group were 406.33±7.37 and 92.33±8.50, respectively, which were significantly higher than 224.67±10.01 and 66.00±7.94 in shCacyBP group (<0.05). Compared with siNC group, the expression of epithelial marker E-cadherin was up-regulated, while the expressions of mesenchymal markers including N-cadherin, Vimentin, Snail1 and p-Akt were down-regulated in CacyBP depleted A549 cells. Compared with NC group, overexpression of CacyBP inhibited E-cadherin expression while promoted the expressions of N-cadherin, Snail1, Vimentin and p-Akt, which could be restored by LY294002. CacyBP may promote the proliferation and invasion of NSCLC cells by regulating Akt signal pathway.
探讨钙周期蛋白结合蛋白（CacyBP）对非小细胞肺癌（NSCLC）细胞增殖和侵袭的影响及其机制。收集2016年在济南市中心医院接受手术治疗的NSCLC患者的6例肺癌组织及配对的正常肺组织，采用蛋白质免疫印迹法检测这些组织中CacyBP的表达。分别采用蛋白质免疫印迹法和逆转录-聚合酶链反应（RT-PCR）检测人支气管上皮细胞（16HBE）、NSCLC细胞系A549、H1299、H460和H1975中CacyBP的蛋白和mRNA表达。将针对阴性对照（NC）或CacyBP的RNA干扰（RNAi）和短发夹RNA（shRNA）转染至A549细胞，分别记为siNC组、siCacyBP-1组、siCacyBP-2组、shNC组和shCacyBP组。将对照质粒和Flag-CacyBP质粒转染至A549细胞，分别记为NC组和Flag-CacyBP组。采用细胞计数试剂盒-8（CCK-8）、平板克隆形成实验和流式细胞术检测A549细胞的增殖能力和细胞周期。采用划痕实验和Transwell实验检测A549细胞的迁移和侵袭能力。检测CacyBP表达降低或升高的A549细胞中上皮-间质转化（EMT）标志物E-钙黏蛋白、N-钙黏蛋白、Snail1、波形蛋白的蛋白表达及蛋白激酶B（p-Akt）的磷酸化水平。NSCLC组织中CacyBP蛋白水平为0.41±0.23，显著高于正常肺组织中的0.11±0.04（<0.05）。不同NSCLC细胞系A549、H1299、H460和H1975中CacyBP蛋白表达水平分别为0.35±0.01、0.38±0.01、0.32±0.01和0.41±0.01，均显著高于16HBE细胞中的0.03±0.01（<0.05）。RT-PCR结果与蛋白质免疫印迹法结果一致。与siNC组（吸光度为1.54±0.03）相比，siCacyBP-1组和siCacyBP-2组细胞增殖能力降低（吸光度分别为1.38±0.04和1.34±0.03，<0.05）。shNC组细胞集落数为41.33±3.21，显著高于shCacyBP组的22.00±3.61（<0.05）。shCacyBP组G(1)期比例为（61.35±5.45）%，高于shNC组的（49.61±1.54）%（<0.05）。S期比例为（25.41±3.21）%，低于shNC组的（38.68±0.46）%（<0.05）。shCacyBP组细胞迁移率为（12.67±0.71）%，显著低于shNC组的（35.50±2.07）%（<0.05）。shNC组细胞迁移和侵袭数分别为406.33±7.37和92.33±8.50，均显著高于shCacyBP组的224.67±10.01和66.00±7.94（<0.05）。与siNC组相比，CacyBP表达降低的A549细胞中上皮标志物E-钙黏蛋白表达上调，而间质标志物N-钙黏蛋白、波形蛋白、Snail1和p-Akt表达下调。与NC组相比，CacyBP过表达抑制E-钙黏蛋白表达，促进N-钙黏蛋白、Snail1、波形蛋白和p-Akt表达，而LY294002可使其恢复。CacyBP可能通过调节Akt信号通路促进NSCLC细胞的增殖和侵袭。
