NC最近的一篇文章提供了研究腫瘤組織差異表達泛素化酶作用機制的範本，這次找到的靶蛋白是vimentin，鑑於後者在纖維化疾病中的重要作用，這樣的研究思路我們也可以借鑑。RNF208, an estrogen-inducible E3 ligase, targets soluble Vimentin to suppress metastasis in triple-negative breast cancers。https://www.nature.com/articles/s41467-019-13852-5
1、RNF208 is significantly underexpressed in aggressive TNBC，乳腺癌細胞系，RNAseq找到這個分子，是低表達的；在公共數據庫 Genomic Data Commons (GDC) datasets and public microarray datasets (GSE2034)中得到驗證。此外就是腫瘤學研究的常規思路，患者IHC、RT-PCR，預後相關性
2、ERα regulates RNF208 expression，鑑於該分子在ER陽性乳腺癌中高表達，利用公共芯片數據(GSE2034; GSE5460)分析了ER和該分子的相關性。進一步在無酚紅培養乳腺癌細胞中觀察雌激素對該分子調節，以證實該分子為雌激素靶基因，FOXOM1 and GREB1作為陽性對照。鑑於該分子promoter有ERE，所以常規實驗（熒光報告基因、CHIP）驗證。此外還做了啟動子DNA甲基化分析，這個是用了TNBC細胞 using a public genome database (GSE68379)【估計是review要求補的分析】，說明無影響。已知TNBC細胞中ESR1表達也受到表觀遺傳學機制（如甲基化）抑制，進一步實驗發現5-aza-dC能夠同時誘導ESR1和RNF208表達。ERα knockdown能夠逆轉RNF208表達，說明受體依賴調節的重要性。
3、RNF208 overexpression reduces the tumor growth and lung metastasis常規體外、在體（裸鼠皮下注射）成瘤實驗。Ki67、caspase3。常規細胞遷移和侵襲性實驗cell migration and invasiveness using Transwell migration and Matrigel invasion assays。在體觀察肺轉移結節。然而loss of RNF208 may not be sufficient to induce aggressive phenotypes and cancer progression in luminal breast cancer subtypes，僅在TNBC cells中展示出抑癌效應。
4、RNF208 overexpression decreases the stability of Vimentin protein~~採用mass spectrometry-based formaldehyde crosslinking assay觀察RNF208過表達MDA-MB-231細胞中RNF208互作蛋白，找到vimentin。RNF208過表達強烈抑制Vimentin蛋白表達，但不影響mRNA、也不影響E-cadherin表達。用cycloheximide做了蛋白穩定性實驗；用了 proteasome inhibitor MG132能夠逆轉，然而autophagy inhibitors such as bafilomycin A1 (BAF), chloroquine (CQ), and 3-methyladenine (3-MA)不能逆轉。IP驗證相互作用。RNF208過表達能夠促進vimentin泛素化。293T cells were transiently transfected with Flag-Vimentin together with HA-Ubiquitin (wild-type, K27, K29K, K48, and K63) in the presence or absence of Myc-RNF208 upon MG132 treatment. 從而驗證泛素化位點
5、RNF208 expression is inversely correlated with Vimentin expression患者we used breast cancer TMAs obtained from the Seoul National University College of Medicine in South Korea，相關性分析。另取患者和小鼠樣本驗證。
6、Activity of RNF208 is required for the degradation of Vimentin泛素化酶的RING domain是其活性核心結構域。不同片段RNF208、IP分析其與vimentin相互作用。cysteine-rich motifs是關鍵，高保守位點點突變 (C143A, C167A, and C186A三位點突變)不能結合並泛素化vimentin，也不能讓vimentin降解了。另外，vimentin突變與RNF208的IP結果顯示了vimentin的結合位點在頭部。鑑於泛素化發生在lysine殘基，將vimentin頭部保守lysine突變 (K97A) ， 也不能泛素化了，半衰期也不受RNF208影響了。整體實驗顯示，三位點突變RNF208 (3MT)過表達腫瘤細胞惡性程度增強，不再調節vimentin表達。反之，不受RNF208調節的vimentin突變（ nondegradable K97A ）則能rescue RNF208過表達的效應
7、RNF208 targets the phosphorylation of Vimentin at the Ser39 residue~~vimentin N末端頭部磷酸化調節也非常重要， phosphorylated Vimentin in turn is disassembled and present in the soluble fraction in the cytoplasm, eventually promoting cell migration and invasiveness磷酸化的vimentin才是可溶性的、與細胞侵襲相關的形式，推測RNF208可能是識別了磷酸化的vimentin來發揮後續作用。we initially confirmed the expression level of Vimentin in the soluble and insoluble fractions in RNF208-overexpressing MDA-MB-231 cells. Interestingly, RNF208 was predominantly located in the soluble fraction, and its overexpression led to a significant reduction of Vimentin in the soluble fraction compared with the insoluble fraction。製作了磷酸化位點突變的vimentin，incapable Vimentin (S39A) mutant不受RNF調節, whereas the constitutively active Vimentin (S39D) mutant仍受RNF調節（穩定性、表達、泛素化）。最後做了vimentin兩種突變+RNF過表達細胞的整體荷瘤實驗，證實RNF208 specifically targets phosphorylated Vimentin at Ser39 as a soluble form for polyubiquitination-mediated degradation, ultimately suppressing metastasis.
8、總結：RNF208 may be useful as a prognostic marker in breast cancers, and modulation of RNF208 in an ERα-dependent manner may be a therapeutic intervention against metastatic breast cancers.預後指標、治療靶點
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