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Abstract：
BACKGROUND: FLI1 is an oncogenic transcription factor that promotes diverse malignancies through mechanisms that are not fully understood. Herein, FLI1 is shown to regulate the expression of Ubiquitin Associated and SH3 Domain Containing A/B (UBASH3A/B) genes. UBASH3B and UBASH3A are found to act as an oncogene and tumor suppressor, respectively, and their combined effect determines erythroleukemia progression downstream of FLI1.
METHODS: Promoter analysis combined with luciferase assays and chromatin immunoprecipitation (ChIP) analysis were applied on the UBASH3A/B promoters. RNAseq analysis combined with bioinformatic was used to determine the effect of knocking-down UBASH3A and UBASH3B in leukemic cells. Downstream targets of UBASH3A/B were inhibited in leukemic cells either via lentivirus-shRNAs or small molecule inhibitors. Western blotting and RT-qPCR were used to determine transcription levels, MTT assays to assess proliferation rate, and flow cytometry to examine apoptotic index.
RESULTS: Knockdown of FLI1 in erythroleukemic cells identified the UBASH3A/B genes as potential downstream targets. Herein, we show that FLI1 directly binds to the UBASH3B promoter, leading to its activation and leukemic cell proliferation. In contrast, FLI1 indirectly inhibits UBASH3A transcription via GATA2, thereby antagonizing leukemic growth. These results suggest oncogenic and tumor suppressor roles for UBASH3B and UBASH3A in erythroleukemia, respectively. Mechanistically, we show that UBASH3B indirectly inhibits AP1 (FOS and JUN) expression, and that its loss leads to inhibition of apoptosis and acceleration of proliferation. UBASH3B also positively regulates the SYK gene expression and its inhibition suppresses leukemia progression. High expression of UBASH3B in diverse tumors was associated with worse prognosis. In contrast, UBASH3A knockdown in erythroleukemic cells increased proliferation; and this was associated with a dramatic induction of the HSP70 gene, HSPA1B. Accordingly, knockdown of HSPA1B in erythroleukemia cells significantly accelerated leukemic cell proliferation. Accordingly, overexpression of UBASH3A in different cancers was predominantly associated with good prognosis. These results suggest for the first time that UBASH3A plays a tumor suppressor role in part through activation of HSPA1B.
CONCLUSIONS: FLI1 promotes erythroleukemia progression in part by modulating expression of the oncogenic UBASH3B and tumor suppressor UBASH3A.
METHODS: Promoter analysis combined with luciferase assays and chromatin immunoprecipitation (ChIP) analysis were applied on the UBASH3A/B promoters. RNAseq analysis combined with bioinformatic was used to determine the effect of knocking-down UBASH3A and UBASH3B in leukemic cells. Downstream targets of UBASH3A/B were inhibited in leukemic cells either via lentivirus-shRNAs or small molecule inhibitors. Western blotting and RT-qPCR were used to determine transcription levels, MTT assays to assess proliferation rate, and flow cytometry to examine apoptotic index.
RESULTS: Knockdown of FLI1 in erythroleukemic cells identified the UBASH3A/B genes as potential downstream targets. Herein, we show that FLI1 directly binds to the UBASH3B promoter, leading to its activation and leukemic cell proliferation. In contrast, FLI1 indirectly inhibits UBASH3A transcription via GATA2, thereby antagonizing leukemic growth. These results suggest oncogenic and tumor suppressor roles for UBASH3B and UBASH3A in erythroleukemia, respectively. Mechanistically, we show that UBASH3B indirectly inhibits AP1 (FOS and JUN) expression, and that its loss leads to inhibition of apoptosis and acceleration of proliferation. UBASH3B also positively regulates the SYK gene expression and its inhibition suppresses leukemia progression. High expression of UBASH3B in diverse tumors was associated with worse prognosis. In contrast, UBASH3A knockdown in erythroleukemic cells increased proliferation; and this was associated with a dramatic induction of the HSP70 gene, HSPA1B. Accordingly, knockdown of HSPA1B in erythroleukemia cells significantly accelerated leukemic cell proliferation. Accordingly, overexpression of UBASH3A in different cancers was predominantly associated with good prognosis. These results suggest for the first time that UBASH3A plays a tumor suppressor role in part through activation of HSPA1B.
CONCLUSIONS: FLI1 promotes erythroleukemia progression in part by modulating expression of the oncogenic UBASH3B and tumor suppressor UBASH3A.
摘要：
背景：FLI1是一种致癌转录因子，通过尚未完全了解的机制促进多种恶性肿瘤。在这里，FLI1显示调节泛素相关和含有SH3结构域的A/B(UBASH3A/B)基因的表达。UBASH3B和UBASH3A被发现作为癌基因和肿瘤抑制因子，分别,它们的联合作用决定了FLI1下游的红白血病进展。
方法：对UBASH3A/B启动子进行启动子分析，结合荧光素酶测定和染色质免疫沉淀（ChIP）分析。RNAseq分析结合生物信息学用于确定敲低UBASH3A和UBASH3B在白血病细胞中的作用。UBASH3A/B的下游靶标通过慢病毒-shRNA或小分子抑制剂在白血病细胞中被抑制。蛋白质印迹和RT-qPCR用于确定转录水平，MTT法评估增殖率，和流式细胞术检测细胞凋亡指数。
结果：红白血病细胞中FLI1的敲除将UBASH3A/B基因鉴定为潜在的下游靶标。在这里，我们显示FLI1直接与UBASH3B启动子结合，导致其活化和白血病细胞增殖。相比之下,FLI1通过GATA2间接抑制UBASH3A转录，从而拮抗白血病生长。这些结果表明UBASH3B和UBASH3A在红白血病中的致癌和肿瘤抑制作用。分别。机械上,我们显示UBASH3B间接抑制AP1（FOS和JUN）表达，它的丧失导致细胞凋亡的抑制和增殖的加速。UBASH3B还积极调节SYK基因表达，其抑制抑制白血病进展。UBASH3B在多种肿瘤中的高表达与预后较差相关。相比之下,红白血病细胞中UBASH3A敲低增加了增殖;这与HSP70基因的戏剧性诱导有关，HSPA1B。因此，红白血病细胞中HSPA1B的敲减显着加速了白血病细胞的增殖。因此，UBASH3A在不同癌症中的过度表达主要与良好预后相关。这些结果首次表明UBASH3A部分通过激活HSPA1B发挥肿瘤抑制作用。
结论：FLI1部分通过调节致癌基因UBASH3B和肿瘤抑制因子UBASH3A的表达促进红白血病进展。
方法：对UBASH3A/B启动子进行启动子分析，结合荧光素酶测定和染色质免疫沉淀（ChIP）分析。RNAseq分析结合生物信息学用于确定敲低UBASH3A和UBASH3B在白血病细胞中的作用。UBASH3A/B的下游靶标通过慢病毒-shRNA或小分子抑制剂在白血病细胞中被抑制。蛋白质印迹和RT-qPCR用于确定转录水平，MTT法评估增殖率，和流式细胞术检测细胞凋亡指数。
结果：红白血病细胞中FLI1的敲除将UBASH3A/B基因鉴定为潜在的下游靶标。在这里，我们显示FLI1直接与UBASH3B启动子结合，导致其活化和白血病细胞增殖。相比之下,FLI1通过GATA2间接抑制UBASH3A转录，从而拮抗白血病生长。这些结果表明UBASH3B和UBASH3A在红白血病中的致癌和肿瘤抑制作用。分别。机械上,我们显示UBASH3B间接抑制AP1（FOS和JUN）表达，它的丧失导致细胞凋亡的抑制和增殖的加速。UBASH3B还积极调节SYK基因表达，其抑制抑制白血病进展。UBASH3B在多种肿瘤中的高表达与预后较差相关。相比之下,红白血病细胞中UBASH3A敲低增加了增殖;这与HSP70基因的戏剧性诱导有关，HSPA1B。因此，红白血病细胞中HSPA1B的敲减显着加速了白血病细胞的增殖。因此，UBASH3A在不同癌症中的过度表达主要与良好预后相关。这些结果首次表明UBASH3A部分通过激活HSPA1B发挥肿瘤抑制作用。
结论：FLI1部分通过调节致癌基因UBASH3B和肿瘤抑制因子UBASH3A的表达促进红白血病进展。
