IL-6/STAT3炎性信号通路通过SATB1调控乳腺癌干细胞/癌非干细胞动态平衡的研究

Accumulating evidences suggest that breast cancer stem cells (CSCs)are responsible for therapy resistance and relapse of cancer. Activation of several pathways involved in inflammatory responses has recently been detected in formation of breast CSCs. And the distribution of CSCs among the different breast cancer molecular subtypes is different..The researches of ours and other's shown that activation of IL-6/STAT3 pathway induced formation of breast CSCs and their dynamic equilibrium with non-stem cancer cells. Breast CSCs drive self-renewal and tumor initiation through SATB1-mediated signaling pathways. .In this study, we intend to investigate the molecular mechanism of IL-6/STAT3 pathway drive formation of breast CSCs and their dynamic equilibrium with non-stem cancer cells through SATB1 with human breast cancer tissues, breast cancer cell lines and xenograft models. Methods in study are as follow：.Invasive breast carcinomas from distinct molecular subtypes are analysed with immunofluorescence for the expression of CD44, CD24 and ALDH1, markers of breast CSCs, with enzyme linked immunosorbent assay for the expression of IL-6, with q-RT-PCR for the expression of STAB1 mRNA, with immunoblots for the expression of STAB1, STAT3, pSTAT3 protein, with electrophoretic mobility shift assay(EMSA) for the activity of STAT3. .The distinct molecular subtype breast cancer cell lines, MCF-7, BT474 SKBR-3 and MDA-MB-231 are respectively knocked down endogenous STAT3 with RNAi or over-expressed ectopic STAT3, and then grown in medium serum-free supplemented with IL-6. Flow cytometric cell sorting was performed with FITC-conjugated CD44 antibody, PE-conjugated CD24 antibody,and FITC-conjugated ALDH1 antibody for CSCs. Mammosphere-initiating cell number and mammosphere size are evaluated for self-renewal of CSCs. Expression of STAB1 is tested with q-RT-PCR and immunoblots, expression of STAT3, pSTAT3 protein is tested with immunoblots, activity of STAT3 is tested with EMSA. Expression of miroRNAs,combination of SATA3 with SATB1 promotor are respectively tested with microRNA assay, chromatin immunoprecipitation for mechanism of SATB1 regulated by STAT3. .The xenograft models of human breast cancer stem cell from cell lines MCF-7, BT474, SKBR-3 and MDA-MB-231 are knocked down endogenous STAT3 with RNAi, and administer IL-6. Size of tumors is monitored and tumor tissues are analysed with immunofluorescence for the expression of CD44, CD24 and ALDH1, self-renewal of cancer stem cells are assessed with mammosphere-initiating cell number and mammosphere size, expression of STAB1 is tested with q-RT-PCR and immunoblots.

癌干细胞对治疗抵抗是乳腺癌复发转移的主要原因，炎性因子诱导乳癌细胞向癌干细胞转化，导致不同分子类型乳癌中癌干细胞比例不同。他人及我们前期研究发现，IL-6/STAT3炎性信号通路可诱导乳腺癌干细胞转化，乳癌干细胞与SATB1及其调控的信号通路相关。本课题拟收集不同分子亚型的人乳癌组织，检测癌干细胞比例、IL-6/STAT3信号通路激活状态、SATB1 mRNA和蛋白表达；构建STAT3表达载体和RNAi载体，转染各亚型乳癌细胞株，IL-6诱导癌干细胞转化，检测癌干细胞比例和自我更新能力、SATB1的表达；miRNA芯片、免疫共沉淀法研究STAT3调控乳癌干细胞SATB1表达的机制；建立各亚型裸鼠乳癌干细胞模型，给STAT3 RNAi载体、IL-6处理，检测肿瘤生长、SATB1表达、癌干细胞比例和自我更新能力。探讨IL-6/STAT3通路通过SATB1调控癌干细胞/癌非干细胞动态平衡的机制。

通过388例乳腺癌标本，发现在LuminalB型、Her-2+、三阴型乳腺癌中，IL-6的含量显著高于癌旁正常组织，IL-6的含量与肿瘤组织中STAT3、磷酸化的STAT3（pSTAT3）、SATB1呈正相关，也与癌干细胞的比例呈正相关。还发现新辅助化疗后IL-6高表达与三阴性乳腺癌的预后呈负相关。MB-MDA-231，SKBR-3，T474和MCF-7细胞分别转染STAT3 的siRNA慢病毒表达载体后，在添加IL-6的癌干细胞培养条件下，CD44+CD24-和ALDH1+的癌干细胞的比例显著下降，形成乳腺微球的能力显著减弱，pSTAT3，SATB1表达显著降低。在STAT3高表达癌干细胞，表达上升3倍以上的miRNA是：miR-210, miR-21, miR-181b-1，miR-23a，软件预测miR-23a的靶基因可能是STAB1，荧光素酶报告基因载体证实miR-23a可结合与SATB1基因的3′UTR区，发现了乳腺癌中调控乳腺癌干细胞/非干癌细胞平衡的一种新机制。在MCF-7和MB-MDA-231人乳腺干细胞癌的裸鼠移植模型，分别以STAT3RNAi慢病毒表达载体体内转染，并给予IL-6处理，发现STAT3的RNAi慢病毒表达载体体内转染组显著抑制肿瘤生长，pSTAT3，SATB1蛋白表达显著下降，肿瘤组织中癌干细胞比例显著下降。本研究发现了调控乳腺癌干细胞/癌非干细胞平衡的一种新的机制，可能成为乳腺癌治疗的新靶点。