Fu Yun, Sun Xiaoyin, Gu Zhangyuan, Zhuang Zhigang
Department of Breast Surgery, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.
Onco Targets Ther. 2020 Jun 10;13:5323-5335. doi: 10.2147/OTT.S229076. eCollection 2020.
Triple-negative breast cancer has become an intricate part and hotspot in the clinical and experimental research. Connexins, serving as functional proteins in gap junctions, play an important role in tumorigenesis, cell proliferation and metastasis.
We constructed and employed the Connexin 43 (Cx43) overexpression lentiviral vectors and Cx43 siRNA in paclitaxel-treated MDA-MB-231 cells. We performed the experiments of clonal formation and flow cytometry to gauge the effect of paclitaxel on cellular behaviors and immunofluorescence and subsequent quantitative RT-PCR and Western blot to examine the expression of genes and corresponding proteins. Experiments of scrape loading/dye transfer were utilized to explore the gap junctions. The targets of Cx43 were identified via the experiments of co-immunoprecipitation (Co-IP), GST pull-down assays and proximal ligation assay (PLA).
The results showed that Cx43 hindered cell proliferation and promoted apoptosis in the paclitaxel-treated MDA-MB-231 cells. Overexpressed Cx43 suppressed the expression of resistance genes such as BRCP, Txr-1, α-tubulin and β-tubulin and promoted the expression of apoptosis gene as TSP-1 and Bcl-2. Cx43 was also positively related to ITGα9 and negatively related to ITGαV and ITGα11. The gap junctions altered magnificently under different expressions of Cx43, which indicated that Cx43 could promote the number of intercellular gap junctions. The immunofluorescent experiment revealed that both of Cx43 and β-tubulin were mainly localized in the cytoplasm. The assays of Co-IP and GST pull-down demonstrated that there existed a direct interaction between Cx43 and β-tubulin. Furthermore, the result of PLA also showed that Cx43 interacts with β-tubulin in MDA-MB-231 cells.
Overexpression of Cx43 could modulate the cellular resistance to paclitaxel via targeting β-tubulin in triple-negative breast cancer.
三阴性乳腺癌已成为临床和实验研究中一个复杂的部分和热点。连接蛋白作为间隙连接中的功能蛋白，在肿瘤发生、细胞增殖和转移中起重要作用。
我们构建并使用了连接蛋白43（Cx43）过表达慢病毒载体和Cx43 siRNA处理紫杉醇处理的MDA-MB-231细胞。我们进行了克隆形成和流式细胞术实验以评估紫杉醇对细胞行为的影响，并进行了免疫荧光以及随后的定量RT-PCR和蛋白质印迹实验以检测基因和相应蛋白质的表达。利用划痕加载/染料转移实验来探索间隙连接。通过免疫共沉淀（Co-IP）、GST下拉实验和邻近连接分析（PLA）实验鉴定Cx43的靶点。
结果表明，Cx43在紫杉醇处理的MDA-MB-231细胞中阻碍细胞增殖并促进细胞凋亡。过表达的Cx43抑制了如BRCP、Txr-1、α-微管蛋白和β-微管蛋白等耐药基因的表达，并促进了如TSP-1和Bcl-2等凋亡基因的表达。Cx43还与ITGα9呈正相关，与ITGαV和ITGα11呈负相关。在Cx43不同表达情况下，间隙连接发生了显著改变，这表明Cx43可以促进细胞间间隙连接的数量。免疫荧光实验显示Cx43和β-微管蛋白都主要定位于细胞质中。Co-IP和GST下拉实验表明Cx43和β-微管蛋白之间存在直接相互作用。此外，PLA结果也显示Cx43在MDA-MB-231细胞中与β-微管蛋白相互作用。
在三阴性乳腺癌中，Cx43的过表达可通过靶向β-微管蛋白来调节细胞对紫杉醇的耐药性。
