Basic Pathology Department, Pathology College, Qiqihar Medical University, Qiqihar City, Heilongjiang Province, China.
Bull Exp Biol Med. 2023 Jul;175(3):376-381. doi: 10.1007/s10517-023-05871-6. Epub 2023 Aug 11.
The study examined the mechanisms of action of signal protein claudin 6 (CLDN6) on migration and invasion of breast cancer cell lines MCF-7 and SKBR-3. To this end, the signal proteins SMAD were blocked with their inhibitor SB431542, the genes CLDN6 and SNAIL were knocked down with short hairpin RNAs, and MMP2 and MMP9 were inhibited with TIMP-1. Expressions of MMP2 and MMP9 mRNAs were evaluated by reverse transcription PCR, Expressions of MMP-2, MMP-9, E-cadherin, N-cadherin, and vimentin were examined by Western blotting. Migration and invasion were analyzed by scratch test and Matrigel invasion assay. SB431542 inhibited expression of MMP2 and MMP9 in both cell lines. Single use of SB431542 inhibited expression of MMP-2/MMP-9 and corresponding mRNAs, but subsequent silencing of CLDN6 gene reversed this effect. TIMP-1 reversed down-regulation of E-cadherin, upregulation of N-cadherin and vimentin, facilitation of migration and invasion evoked by CLDN6 knocking down. Silencing of SNAIL gene inhibited migration and invasion, upregulated the expression of E-cadherin, and down-regulated expression of MMP2, MMP 9, N-cadherin, and vimentin. Thus, CLDN6 suppresses the epithelial-mesenchymal transition, migration, and invasion via blocking SMAD/Snail/MMP-2/9 signaling pathway in MCF-7 and SKBR-3 cancer cell lines.
该研究探讨了信号蛋白紧密连接蛋白 6（CLDN6）对乳腺癌细胞系 MCF-7 和 SKBR-3 迁移和侵袭的作用机制。为此，用其抑制剂 SB431542 阻断信号蛋白 SMAD，用短发夹 RNA 敲低 CLDN6 和 SNAIL 基因，并使用 TIMP-1 抑制 MMP2 和 MMP9。通过逆转录 PCR 评估 MMP2 和 MMP9 mRNA 的表达，通过 Western blot 检测 MMP-2、MMP-9、E-钙黏蛋白、N-钙黏蛋白和波形蛋白的表达。通过划痕试验和 Matrigel 侵袭试验分析迁移和侵袭。SB431542 抑制了两种细胞系中 MMP2 和 MMP9 的表达。单独使用 SB431542 抑制 MMP-2/MMP-9 及其相应 mRNA 的表达，但随后沉默 CLDN6 基因逆转了这种作用。TIMP-1 逆转了 CLDN6 敲低引起的 E-钙黏蛋白下调、N-钙黏蛋白和波形蛋白上调、迁移和侵袭的促进作用。沉默 SNAIL 基因抑制了迁移和侵袭，上调了 E-钙黏蛋白的表达，并下调了 MMP2、MMP9、N-钙黏蛋白和波形蛋白的表达。因此，CLDN6 通过阻断 MCF-7 和 SKBR-3 癌细胞系中的 SMAD/Snail/MMP-2/9 信号通路，抑制上皮-间充质转化、迁移和侵袭。
