摘要
研究背景
乳腺癌是女性患病和死亡人数最高的恶性肿瘤,远处转移是乳腺癌致死的主要原因。包括乳腺癌在内的许多恶性肿瘤的转移并非随机迁徙,而是有固有路径和特定的转移器官。骨骼是乳腺癌细胞容易转移的部位,远超过肝、肺等部位转移,但是乳腺癌嗜骨转移的机理迄今为止尚待进一步阐明。
核因子κB受体活化因子(RANK)和其配体即RANKL对骨骼改建、免疫系统成熟有重要作用。骨保护素(OPG)作为游离的诱饵受体,能够和RANK竞争性结合RANKL。陆续有研究发现RANK或RANKL在原发性骨肿瘤和易发生骨转移肿瘤的细胞上表达,于是人们开始关注RANKL和RANK相互作用对肿瘤细胞生物学功能以及肿瘤嗜骨性转移的影响和作用。先前研究显示RANKL能够诱导部分RANK阳性的肿瘤细胞发生趋化迁移。我们假设骨骼中成骨细胞等细胞通过RANKL/RANK途径诱导循环系统中的肿瘤细胞迁移至骨,最终形成骨转移。目前关于RANKL对RANK阳性乳腺癌细胞诱导迁移的机制尚未完全阐明;此外,乳腺癌细胞中RANKL/RANK表达的调控因素仍鲜有报道。因此,本课题以人乳腺癌细胞为研究对象,在证实RANKL/RANK途径促进人乳腺癌MDA-MB-231细胞迁移能力的基础上,明确参与RANKL诱导乳腺癌细胞迁移的下游信号分子,并研究了缺氧微环境对乳腺癌细胞RANKL/RANK途径的调节及其可能的机制。
研究方法
第一部分RANKL/RANK途径对人乳腺癌细胞迁移能力的影响
首先通过体外细胞划痕实验和Transwell趋化小室实验,证实RANKL对MDA-MB-231细胞趋化迁移能力的影响。同时用OPG预处理RANKL后,观察对RANKL诱导的细胞迁移的影响。其次,将能够表达、分泌RANKL的人成骨细胞hFOB1.19与MDA-MB-231细胞共培养,观察前者对后者的趋化作用。最后,针对目的基因RANK设计短发卡RNA(shRNA)干扰序列,构建质粒,进行慢病毒包装后感染MDA-MB-231细胞。此外获得RANK基因(TNFRSF11A)片段并用PCR方法扩增,通过基因重组改建成穿梭质粒pLenti-TNFRSF11A-Neo,进行慢病毒包装后感染MDA-MB-231细胞。使用流式细胞仪分选或G418筛选后,RT-PCR和Western blot分别检测干扰或过表达细胞RANK mRNA以及蛋白的表达水平。通过体外Transwell小室实验观察干扰或过表达RANK对RANKL诱导细胞迁移的影响。
第二部分RANKL诱导乳腺癌细胞迁移下游信号分子的探讨
首先使用RANKL作用于MDA-MB-231细胞,在不同时间点终止培养,提取细胞总蛋白,Western blot检测细胞中磷酸化Src表达水平的变化;使用不同剂量RANKL或加入OPG分别作用于MDA-MB-231细胞,Western blot检测细胞中磷酸化Src表达水平的变化。shRNA干扰RANK表达,观察RAKL作用后磷酸化Src表达变化。使用Src抑制剂或小干扰RNA(siRNA)干扰Src表达,Transwell小室实验观察RANKL诱导的MDA-MB-231细胞迁移的变化。
Western blot检测不同作用时间和不同剂量RANKL对细胞外信号调节激酶1/2(ERK1/2)、c-Jun氨基端激酶(JNK)以及p38磷酸化表达的影响;加入OPG、Src抑制剂观察上述激酶磷酸化水平表达的改变。Western blot检测干扰Src对RANKL诱导p-ERK1/2、p-JNK以及p-p38表达的影响。使用ERK1/2, JNK, p38抑制剂预处理MDA-MB-231细胞,观察对RANKL诱导细胞迁移的影响
第三部分缺氧环境对乳腺癌细胞RANKL/RANK表达的调节及机制探讨。
分别在缺氧(0.1% O2)和常氧环境中培养人乳腺癌MDA-MB-231细胞和MCF-7细胞,RT-PCR检测缺氧不同时间RANK和RANKL mRNA表达水平的变化,同时使用Western blot检测相应时间点RANK和RANKL和缺氧诱导因子1α(HIF-1α)蛋白的表达水平。siRNA干扰MDA-MB-231细胞HIF-1α表达后,Western blot检测缺氧环境下RANK和RANKL mRNA表达水平的变化。同时,Western blot检测观察缺氧对磷酸化Akt表达的影响,抑制PI3K/Akt后对缺氧诱导RANK和RANKL和HIF-1α蛋白的表达的影响。最后使用体外细胞划痕实验观察缺氧是否增加RANKL诱导的MDA-MB-231细胞的迁移,并通过干扰HIF-1α表达或抑制PI3K/Akt,观察细胞迁移的变化。
研究结果
(1)RANKL能够诱导乳腺癌MDA-MB-231细胞趋化迁移(P<0.05),而且这一作用随RANKL剂量的增加逐步增强,呈剂量依赖性特点。OPG能够有效抑制RANKL诱导的乳腺癌MDA-MB-231细胞的迁移,当OPG与RANKL比例达到5:1时,抑制的作用最明显。
(2)共培养人成骨细胞hFOB1.19与MDA-MB-231细胞,hFOB1.19细胞能够趋化MDA-MB-231细胞向其迁移(P<0.05),这一作用能够被OPG抑制。
(3)成功设计并筛选了针对RANK的shRNA序列,通过慢病毒载体能够有效的干扰RANK在MDA-MB-231细胞的表达。同时也构建了RANK过表达的慢病毒载体,检测显示能够明显提高RANK在MDA-MB-231细胞的表达。改变MDA-MB-231细胞RANK受体表达水平,并能够影响RANKL诱导的细胞迁移,同时慢病毒载体的构建为今后更深入的研究奠定基础。
(4)RANKL作用于MDA-MB-231细胞能够促进Src激酶的磷酸化并呈时间和剂量依赖性的特点。干扰RANK的表达可以阻断RANKL诱导的Src激酶磷酸化。使用Src抑制剂或干扰Src的表达能够抑制RANKL对MDA-MB-231细胞的迁移(P<0.05)。
(5)RANKL同样能够增加MDA-MB-231细胞中ERK1/2、p-38、JNK磷酸化的表达水平。干扰或抑制Src激酶,能够抑制ERK1/2、p-38、JNK的磷酸化水平。抑制ERK1/2、p-38、JNK同样能够抑制RANKL/RANK通路诱导的MDA-MB-231细胞迁移(P<0.05)。
(6)缺氧培养能够增加MDA-MB-231细胞、MCF-7细胞RANK和RANK的mRNA和蛋白的表达(P<0.05),同时缺氧诱导的HIF-1α蛋白的表达和RANK、RANKL表达的趋势是一致的。RNA干扰HIF-1α的表达,抑制了缺氧期间(4 h～12 h)RANK、RANKL表达的上升;缺氧能够增加Akt磷酸化的表达水平,抑制PI3K/Akt通路可以抑制缺氧诱导的HIF-1α蛋白的表达,同时也抑制了缺氧期间(4 h～12 h)RANK、RANKL蛋白表达的增加。
(7)缺氧促进了RANKL诱导的乳腺癌MDA-MB-231向划痕部位的迁移(P<0.05)。通过RNA干扰HIF-1α或抑制PI3K/Akt能够和OPG一样抑制缺氧对乳腺癌细胞迁移的影响(P<0.05)。
结论
(1)RANKL能够诱导乳腺癌MDA-MB-231细胞发生趋化迁移,这一过程参与了成骨细胞对乳腺癌细胞的趋化迁移。
(2)Src-MAPKs(ERK1/2, p38, JNK)作为下游信号分子参与了RANKL对MDA-MB-231细胞趋化迁移。
(3)缺氧可以通过PI3K/Akt-HIF-1α途径调节人乳腺癌细胞RANK、RANKL的表达水平,并能影响细胞的迁移能力。
Background
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females in worldwide, and mortality is related to metastatic spread of the primary tumor.The migration of breast cancer cells is well-orchestrated and not a random process. Bone is the most preferential metastatic target site for breast cancer metastasis, and the frequency of metastasis to bone is much higher than to the lung and liver. The exact mechanism for this preferential metastasis requires further investigation.
The Receptor Activator of NF-κB (RANK) and its ligand, known as RANKL, was originally shown to be crucial for osteoclastogenesis and lymph node development. Osteoprotegerin (OPG) acts as a soluble decoy receptor and competes with RANK for binding to RANKL. Accumulating studies have shown that the expression of both RANKL and RANK have been observed in primary and secondary bone tumors, it has been proposed that the RANKL/RANK system can regulate bone metastases of epithelial tumours. So we speculated that RANKL might act as a‘soil’factor facilitating cancer cell metastasis to bone. Although the mechanism of RANKL/RANK pathway in regulation of bone was well established, the role of RANKL/RANK signal transduction pathways in inducing the migration of human breast cancer cells is still not well understood. Moreover, less is known about the mechanisms that what stimulies controls the expression of RANKL and RANK in breast cancer cells. In this study, we confirmed that RANKL could trigger the migration of human breast cancer cells in vitro, and explored the downstream signaling pathways of RANKL/RANK in MDA-MB-231 cells to induce cell migration. We also determined for the first time that the regulation of RANKL/RANK by hypoxia led to increased migration of breast cancer cells in vitro.
Methods
PartⅠ. Effects of RANKL/RANK pathway on the migration of MDA-MB-231 cells
The migration of MDA-MB-231 human breast cancer cells induced by RANKL was confirmed using wound-healing and transwell chemotaxis assays. In order to inhibit RANKL-induced migration, RANKL was incubated with OPG before adding into the plate. MDA-MB-231 cells were also co-cultured with osteoblastic cells (hFOB 1.19) in the transwell assay, and then the migration of MDA-MB-231 from the upper insert towards the osteoblastic cells in lower insert were examined. To silence RANK gene expression, short hairpin RNAs (shRNAs) targeting mRNA regions of RANK were designed. The sequences were inserted into the plasmid, and lentiviral vectors expressing shRNA were constructed. Another HIV-derived lentiviral vector was packaged using the pLenti-TNFRSF11A-Neo which contained the full length cDNA sequence of the human RANK gene. The RT-PCR and Western blot assays were performed to test the expression of RANK in the transfected cells. The transwell assays to test the migration of the transfected cells.
PartⅡ. The related signal transduction pathways in RANKL-induced cell migration
After the MDA-MB-231cells were treated with RANKL at different times, the levels of phosphorylated Src were tested by Western blot, and after the cells were treated with different doses of RANKL or co-incubated with OPG/RANKL the levels of phosphorylated Src by Western blot were tested. The change of phosphorylated Src in cells transduced with RANK-targeted shRNA was also tested. To confirm the involvement of Src kinases in RANKL-induced MDA-MB-231 cell migration, MDA-MB-231 cells were pretreated with the specific Src inhibitor or transfected with Src small interfering RNA (siRNA). After the MDA-MB-231cells were treated with RANKL at different times or in different doses, the levels of phosphorylated MAPKs (p-ERK1/2, p-JNK, and p-p38) were tested by Western blot. The phosphorylation levels of ERK1/2, JNK, and p38 were tested after the cells were pretreated with specific Src inhibitor or transfected with Src siRNA. The cell migration was examined via treatment with PD98059 (30μM), SB203580 (20μM), SP600125 (20μM)
PartⅢ. RANK and RANKL expressions regulated by hypoxia and its mechanism.
The human breast cancer MDA-MB-231 cells and MCF-7 cells were cultured in normoxia or hypoxic conditions. Hypoxia-dependent expressions of RANK and RANKL in different time were investigated by RT-PCR. Western blot assay tests was used to test the protein level of RANK, RANKL and hypoxia inducing factor 1α. (HIF-1α). To determine the effect of HIF-1αon hypoxia-induced expression of RANK and RANKL of MDA-MB-231 cells, siRNA against HIF-1αwas used to downregulate the expression of HIF-1αin MDA-MB-231 cells. We further investigated the role of PI3K/Akt signaling on HIF-1α-mediated expressions of RANK and RANKL in MDA-MB-231 cells by Western blot. Finally, the wound-healing assay was used to test the effect of hypoxia on RANKL-induced cell migration.
Results
(1)RANKL could induce MDA-MB-231 cell migration in a dose-dependent manner(P<0.05). OPG could inhibit RANKL-induced cell migration, especially when the ratio between OPG and RANKL was 5: 1.
(2)After co-culturing MDA-MB-231 cells with hFOB 1.19 cells, the migreation of MDA-MB-231 cells was increased(P<0.05). OPG could suppress the migration of MDA-MB-231cells in this assay.
(3)The lentiviral vectors containing RANK-targeted shRNA were constructed and packaged successfully and the silencing effect of lentivirus-mediated RANK-targeted shRNA was confirmed. Another lentiviral vector was packaged using the pLenti- TNFRSF11A-Neo which containing the full length cDNA sequence of the human RANK gene. With to the changes of the expression of RANK, the ability of cell migration toward RANKL was changed too.
(4)The levels of phosphorylated Src was increased after treated with RANKL in a concentration-dependent and time-dependent manner. The RANKL-induced expression of phosphorylated Src was significantly lower in the MDA-MB-231-I002 cells transduced with lentivirus-mediated RANK-targeted shRNA than in the MDA-MB-231 cells. The cells transfected with Src siRNA and treated with Src inhibitor antagonized RANKL-induced migration(P<0.05)
(5)The levels of phosphorylated ERK1/2, p-38 and JNK were increased after treatment with RANKL in a concentration-dependent and time-dependent manner. The cells transfected with Src siRNA and treated with Src inhibitor suppressed the expression of phosphorylated ERK1/2, p-38 and JNK induced by RANKL. Cell migration induced by RANKL was also effectively decreased by treatment with inhibitors of ERK1/2, p-38 and JNK.
(6)Hypoxia upregulated the expressions of RANK and RANKL in human breast cancer cells(P<0.05),which was similar to the findings of the upregulation of HIF-1αprotein induced by hypoxia. The treatment with HIF-1αsiRNA not only blocked the upregulation of HIF-1α, but also suppressed the upregulation of RANK and RANKL protein levels after the cells were exposed to hypoxic conditions at 4 h, 8 h, and 12 h. Exposure of MDA-MB-231 cells to hypoxia led to increased phosphorylation of Akt. The PI3K/Akt inhibitor abolished hypoxia- mediated upregulation of RANK and RANKL protein expressions.
(7)In wound healing assay, exposure of MDA-MB-231 cells to hypoxic conditions resulted in significantly increased RANKL-induced migration compared with normoxia(P<0.05). These effects were blocked by additional treatment of OPG. Moreover, the rate of wound recovery was decreased following downregulation of HIF-1αby siRNA against HIF-1α. Pretreatment with PI3K/Akt inhibitor also blocked the RANKL-induced migration of MDA-MB-231cells under hypoxia(P<0.05).
Conclusion
(1) RANKL can induce MDA-MB-231 cell migration and the RANKL/RANK pathway mediates the process of MDA-MB-231 cells migrated towards osteoblasts.
(2) Src-MAPKs (ERK1/2, p38, and JNK) signaling pathway is involved in RANKL-induced cell migration of MDA-MB-231 cells.
(3) The expression of RANK and RANKL in breast cancer cells can be upregulated by hypoxia. The hypoxia-PI3K/Akt -HIF-1α-RANKL-RANK pathway is likely to be involved in the migration of breast cancer cells in hypoxic environments.
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