LSD1调控hiPSCs高效定向分化为IPCs的作用及机制研究

周淑艳，孙　倓，桑金凤，张根葆(皖南医学院病理生理学教研室，口腔正畸教研室，安徽芜湖400)

LSD1调控hiPSCs高效定向分化为IPCs的作用及机制研究

周淑艳1△，孙倓2，桑金凤1，张根葆1
(皖南医学院1病理生理学教研室，2口腔正畸教研室，安徽芜湖241002)

目的:通过抑制或沉默人皮肤成纤维细胞来源的诱导性多能干细胞(hiPSCs)内赖氨酸特异性去甲基化酶1(LSD1)基因的表达，建立一种hiPSCs高效、定向分化为胰岛素分泌细胞(IPCs)的方案，并初步探讨其调控分化机制。方法:采用shRNA 或LSD1抑制剂，沉默或抑制hiPSCs内LSD1基因表达，筛选出利于hiPSCs向定型内胚层分化的LSD1活性;利用四步法将hiPSCs诱导分化为IPCs，并对IPCs分化效率及成熟度进行检测;将IPCs移植到糖尿病模型免疫缺陷小鼠肾包膜下，评估其体内降糖疗效; ChIP-qPCR检测抑制LSD1前后hiPSCs细胞核内相应组蛋白水平的变化情况。结果:抑制LSD1可促使hiPSCs提前进入定型内胚层分化，最终IPCs的效率由21. 52%提高至39. 32%;体外胰岛素分泌量由1/8提高到1/6;移植的IPCs可在1周内使糖尿病小鼠血糖恢复至正常水平; ChIP-PCR结果显示hiPSCs的分化基因启动子区域H3K4me2/me3和H3K9act水平提高，H3K9/H3K27me3和HDAC1水平下降，而多潜能基因表达情况相反。结论:适当抑制LSD1活性可显著提高hiPSCs 向IPCs的分化效率和成熟度，并在体内发挥有效降糖作用; LSD1通过调控hiPSCs多能性基因和分化基因启动子区域组蛋甲基化、乙酰化修饰水平来影响IPCs分化效率。

AIM: To systematically discover and compare the misregulated long noncoding RNAs (lncRNAs) in tamoxifen-and trastuzumabresistant breast cancer cells in order to shed light on the molecular basis of lncRNA involvement in anti-cancer drug resistance.METHODS: The breast cancer cells were divided into tamoxifen-sensitive MCF-7/WT，tamoxifen-treated MCF-7/TamT and tamoxifen-resistant MCF-7/TamR cells; trastuzumab-sensitive SK-BR-3/WT，trastuzumab-treated SK-BR-3/TraT and trastuzumab-resistant SK-BR-3/TraR cells.RNA isolated from the corresponding cell samples was amplified and hybridized to 40 485 human lncRNAs microarrays followed by qRT-PCR validation.Comprehensive bioinformatic analysis was performed to identify the misregulated lncRNAs，lncRNA categories as well as lncRNA-miRNA interaction network in the 2 different types of resistant lines.RESULTS: 2 107 and 611 lncRNAs were either up-or down-regulated respectively in the tamoxifen-resistant cells as compared with the sensitive cells (≥2-fold change).1 154 and 2 141 lncRNAs were either up-or down-regulated respectively in the trastuzumab-resistant cells as compared with the sensitive cells (≥2-fold change).Besides the previously reported resistance-related lncRNAs such as MEG3，H19 and PVT1 in other cancer types，we also uncovered novel lncRNA regulators in tamoxifen-or trastuzumab-resistant breast cancer cells.Based on each 2 up/down-regulated sets of data，we further found 93 common lncRNAs (69 up-regulated and 24 down-regulated) between the 2 tested lines.Among these，some were epigenetic regulators in cancer methylome，and others were involved in malignancy development，mitotic checkpoint and genotoxic stress-induced cell death.CONCLUSION: A large number of lncRNAs are dysregulated in either tamoxifen-or trastuzumab-resistant cells，but with a small number of lncRNAs overlapped，suggesting that the lncRNAs to initiate resistance may possess strong specificity in different types of anti-cancer drug resistance.The dysregulated lncRNAs in resistant cancer cells are tend to be previously identified cell cycle regulators and DNA damage-related genes.

*［Foundation item］Supported by National Natural Science Foundation of China (No.31471226; No.91440108 )，the funds from Chinese Academy of Sciences (No.KJ2070000031) and the funds from University of Science and Technology of China (No.KY2070000024)

△Corresponding author Tel: 0551-63600080; E-mail: wangxt11@ustc.edu.cn

EMT phenotypes，Rho activation，cellular senescence and antioxidant response after hormone ablation in breast and prostate cancers

Akira TANAKA
(Department of Pathology，Jichi Medical University，Shimotuke，Tochigi 329-0498，Japan)

Prostate and breast cancers are the second and the most common cancers in men and women，respectively.The progression to hormone-unresponsive tumors is the most important issue to prevent cancer death of these cancers.Our group mainly investigates cellular changes under hormone-unresponsive cancer progression using a model system of Shionogi carcinoma cells.Androgens markedly stimulated cell growth of the cloned cell line of SC-3 cells，whereas scarcely promoted that of SC-4 cells，both of which were established from the Shionogi carcinoma tumor.We first compared gene expression profiles between SC-3 and SC-4 cells，and found that epithelial-mesenchymal transition (EMT) along with Rho activation took place in hormone-unresponsive SC-4 cells.EMT and RhoC upregulation were also found in human prostate cancer specimens resected after endocrine therapy，and in human breast cancer specimens resected after chemotherapy.We also found that cellular senescence was triggered in response to hormone ablation in androgen-sensitive cultured cells and in murine castration models.Importantly，senescence-associated secretory phenotypes and antioxidant response were prominently found in human prostate cancer specimens after endocrine therapy.In conclusion，these cellular changes might facilitate therapyresistant tumor progression in prostate and breast cancers.

Hypoxia-regulated apoptosis-inducing factor protects mitochondrial PTEN from oxidation to modulate epithelial-mesenchymal transition and metastasis of cancers

SHEN Shao-ming，GUO Meng，YU Yun，XIONG Zhong，CHEN Guo-qiang
(Department of Pathophysiology，Key Laboratory of Cell Differentiation and Apoptosis，Chinese Ministry of Education，Shanghai Jiaotong University School of Medicine，Shanghai 200025，China)

Apoptosis-inducing factor (AIF)，a FAD-containing，NADH-dependent oxidoreductase residing in the mitochondria，has dualroles in cell death and survival，but its specific enzymatic activity and potential roles in the pathogenesis of cancers remain unknown.Here we report that the tumor suppressor PTEN is localized into mitochondria，and AIF physically interacts with and protects PTEN from oxidation through its oxidoreductase activity.Hypoxia represses AIF expression via a HIF-1-dependent mechanism，and the reduced AIF triggers cancer cells to undergo epithelial-mesenchymal transition and metastasis of in vitro and xenografts through inactivating PTEN，thus activating Akt/GSK-3 signaling.Moreover，re-expression of AIF partially blocks hypoxia-induced EMT.In conclusion，these findings suggest that AIF functions as an oxidoreductase of PTEN and its decrease favors cancer metastasis.

Long noncoding RNA expression profiling of tamoxifen-and trastuzumab-resistant breast cancer cells*

ZHANG Xiu-lei，ZHU Tao，WANG Xiang-ting△
(School of Life Sciences，University of Science and Technology of China，Hefei 230027，China)

△E-mail: zhoushuyan_xju2005@126.com