The Effect Of MiR-124 On Bone Metastasis Of Breast Cancer And The Mechanism

【Backgrounds and Objectives】Breast cancer is one of the most common malignant tumors in women. The past decades have witnessed the development of the diagnosis and therapeutics techniques, so more and more patients are likely to be diagnosed as breast cancer at early stage, which results in standard treatment option and longer survival for early patients. However, there are no well-established treatment strategies for patients with advanced breast cancer who suffer from local progression and distant metastasis. So the survival for these patients has not been improved. Bone metastasis accounts for the vast majority of breast cancer metastases. It causes skeletal related events(SREs) including osteodynia, pathological fracture and spinal cord compression, which aggravate life quality and overall survival of patients.To date, in addition to systemic therapies such as chemotherapy, endocrine therapy and molecular targeted therapy as well as local therapies including operation and radiotherapy, bone modifying agents have been viewed as indispensable therapy for breast cancer patients with bone metastasis, due to its ability to prevent and treat SREs. However, bone modifying agents fail to prevent the development of bone metastasis as well as to prolong the survival of patients. Therefore, exhaustive study about the underlying molecular and cellular mechanism of bone metastasis from breast cancer will help to identify novel therapeutic target, improve life quality and prolong survival.Bone metastasis process develops in a stepwise fashion that consists a set of sequential events, in which tumor cells invade from primary site, disseminate through circulation, and reconstitute secondary tumors at bone microenvironment. In this microenvironment,tumor cells interact complicatedly with osteoblasts, osteoclasts and bone stromal cells, which leads to the imbalance of osteogenesis/osteoclastogenesis and enables breast cancer cells to form osteolytic lesions in bone, thus causing SREs. In this process, a panel of genes or molecules are deregulated, which may function as new therapy targets and diagnosis markers. micro RNA(mi RNA) is one of the research focus among them. As important posttranscriptional regulators, mi RNA could promote or suppress the occurrence of bone metastasis from breast cancer through targeting key pathways involved in breast cancer metastasis or critical chemokine or cytokine in bone microenvironment. As endogenous non-coding double-strand RNA, mi RNAs are characterized by tissue specificity and molecular targeting. Hence, externally engineering the expression of involved mi RNA may be safer than traditional chemotherapy and radiotherapy, which may turn into new therapeutics with promising application prospect for bone metastasis from breast cancer.mi R-124 was firstly cloned from the brain in mice, and then was identified to express in the human embryonic stem cells. As the most abundant mi RNA in the brain, mi R-124 was reported to be involved in the development of nervous system. And its dysregulation was related to several nervous system diseases. Recently, a growing number of researches aim to investigate the roles of mi R-124 playing in the development and progression of malignant tumors. It has been reported that mi R-124 was deregulated in breast cancer, prostate cancer, bladder cancer, gastric cancer, colon cancer, neuroblastoma, glioma, leukemia, etc. Furthermore, mi R-124 was proved to function as a tumor suppressor through negatively regulating several oncogenes. Some of these studies demonstrated that mi R-124 could inhibit the proliferation, migration and invasion of breast cancer cells, indicating its critical role in the metastasis of breast cancer. However, to date, there is no study reporting its effect on bone metastasis of breast cancer. Therefore, the present study aims to investigate the role of mi R-124 playing in bone metastasis of breast cancer and elucidate its molecular mechanism, eventually providing novel therapeutic target for bone metastasis of breast cancer.【Methods】(1) The expression of mi R-124 in breast cancer cells with different metastasis capacity and in bone metastasis tissues from micea. The detection of mi R-124 in normal mammary tissue from mice and in breast cancer cells with different metastasis capacity RNA was collected from normal mammary tissue of mice and from breast cancer cells with different metastasis capacity including BT549, MDA-MB-231, Hs578 t, MDA-MB-468, MDA-MB-436, MCF7, T47 D and BT474. Real time RT-PCR was used to detect the expression of mi R-124.b. The comparison of mi R-124 levels in parental breast cancer cells and bone metastasis tissues from breast cancer model in mice Model of bone metastasis from breast cancer was established by injecting luciferase-labelled MDA-MB-231 cells to the left ventricular of mice. In vivo imaging instrument was used to detect the luciferase activity of mice. Mice were sacrificed when bone metastasis was detected. RNA from metastatic bone was collected. Real time RT-PCR was used to detect the expression of mi R-124 in parental MDA-MB-231 cells and bone metastasis tissues from breast cancer model in mice.c. The detection of miR-124 level in metastatic bone tissues from patients with breast cancer and the analysis of the relationship between mi R-124 level and bone metastasis-free survival of patients Primary breast cancer tissues, adjacent noncancerous tissues and metastatic bone tissues were collected from five patients. In situ hybridization was used to test the expression of mi R-124 in these tissues and the comparison was made. Metastatic bone tissues were collected from 20 patients with breast cancer. Real time RT-PCR was used to detect the level of mi R-124 in these tissues. Patients were followed up for their bone metastasis-free survival. The correlation of mi R-124 level and the bone metastasis-free survival of patients was analyzed.(2) The effect of externally engineering the level of mi R-124 on osteoblasts and osteoclastsa. The effect of mi R-124 on the differentiation of osteoclasts from bone marrow derived macrophages(BMM) MDA-MB-231 cells were transfected with commercial synthetized mi R-124 mimic and negative control(NC) or mi R-124 inhibitor and inhibitor NC. Condition medium was collected 48 h after the transfection. Bone marrow derived macrophage(BMM) was isolated from femur of mouse and viewed as the progenitor cells of osteoclasts. The indicated condition medium was used to culture the isolated BMM. Six days later, tartrate resistant acid phosphatase(TRAP) dyeing was used to investigate the differentiation of osteoclasts.b. The effect of miR-124 on the expression of RANKL and OPG in osteoblasts MDA-MB-231 cells were transfected with mi R-124 mimic and NC or mi R-124 inhibitor and inhibitor NC. Condition medium was collected 48 h after the transfection and used to culture the progenitor cell line of osteoblasts 3T3E1. Six days later, RNA was extracted from 3T3E1. Real time RT-PCR was used to detect the expression of RANKL and OPG.c. The effect of enhanced expression of mi R-124 on the interaction between osteoclasts and osteoblasts MDA-MB-231 cells were transfected with mi R-124 mimic and NC or mi R-124 inhibitor and inhibitor NC. The indicated cells were co-cultured with 3T3E1 cells. Three days later, the indicated condition medium was collected and used to culture the progenitor cell line of osteoclasts RAW264.7. Three days later, RNA was collected from the indicated RAW264.7 cells. Real time RT-PCR was used to detect the expression of markers related to the differentiation of osteoclasts, including TRAP, c-Src, NFATc1 and c-fos.(3) Inquiring the mechanism of mi R-124 in the development of bone metastasis from breast cancera. The prediction and screening of the target of mi R-124 related to bone metastasis The software Target Scan was used to predict the potential target of mi R-124. Theresults were further screened for genes related to bone metastasis. This approach identified Interleukin 11(IL11) as a candidate.b. The effect of mi R-124 on the expression of IL11 Real time RT-PCR was used to measure the level of IL11 m RNA, and Western blot was used to detect the expression of IL11 protein in MDA-MB-231 cells transfected with mi R-124 mimic and NC or mi R-124 inhibitor and inhibitor NC.c. The binding of mi R-124 to the 3′-untranslated region(3′-UTR) of IL11 We constructed the luciferase reporter carrying the binding site of mi R-124 on the 3′-UTR of IL11 and the mutant binding site. These reporters were co-transfected with mi R-124 mimic or NC to HEK-293 cells. Dural reporter assay was performed to detect the activity of the reporters.d. The correlation between the expression of mi R-124 and IL11 in breast cancer cells and metastatic bone tissues from breast cancer1) The correlation between the expression of mi R-124 and IL11 in normal mammary tissue from mice and breast cancer cells with different metastasis capacity Real time RT-PCR was used to detect the expression of IL11 in normal mammary tissue of mice and in breast cancer cells with different metastasis capacity including BT549, MDA-MB-231, Hs578 t, MDA-MB-468, MDA-MB-436, MCF7, T47 D and BT474. Correlation was made between its expression and the level of mi R-124.2) The correlation between the expression of miR-124 and IL11 in parental breast cancer cells and bone metastasis tissues from breast cancer model in mice Real time RT-PCR was used to detect the expression of IL11 in parental MDA-MB-231 cells and bone metastasis tissues from breast cancer model in mice. Correlation between its expression and the level of mi R-124 was made.3) The correlation between the expression of mi R-124 and IL11 in metastatic bone tissues from patients with breast cancer Primary breast cancer tissues, adjacent noncancerous tissues and metastatic bone tissues were collected from five patients. Immunohistochemistry was used to detect theexpression of IL11 in these tissues. Metastatic bone tissues were collected from 20 patients with breast cancer. Real time RT-PCR was used to detect the expression of IL11 in these tissues. Correlation between its expression and the level of mi R-124 was made.(4) Statistical analysis All experiments were biologically replicated at least three times unless otherwise stated. The data were shown as X±SD. All statistical analyses were performed using SPSS17.0 software. When the variance is homogeneous, student’s t test was used to analyze the data from two groups, otherwise a non parametric test was performed. Correlation analysis was performed using Pearson correlation test. P value was calculated. P < 0.05 was considered as significant.【Results】(1) The expression of mi R-124 was down-regulated in breast cancer cells with high metastasis capacity and in bone metastasis tissues from micea. The expression of miR-124 was down-regulated in breast cancer cells, especially those with high capacity of metastasis Real time RT-PCR shown that compared with the normal mammary tissues from mice, the expression of mi R-124 in breast cancer cells was significantly down-regulated. Furthermore, its expression was lower in the triplicately negative(estrogen receptor, progesterone receptor and HER2 were all negative) breast cancer cells with higher capacity of bone metastasis such as BT549, MDA-MB-231, Hs578 t, MDA-MB-468, MDA-MB-436 than in those with lower capacity of bone metastasis such as MCF7、T47D、BT474.b. The expression of miR-124 levels was reduced in bone metastasis tissues from breast cancer model in mice Model of bone metastasis from breast cancer was established by injecting luciferase-labelled MDA-MB-231 cells to the left ventricular of mice. Real timeRT-PCR shown that the expression of mi R-124 was lower in bone metastasis tissues from breast cancer model in mice than in parental MDA-MB-231 cells.c. The level of mi R-124 was down-regulated in metastatic bone tissues from patients with breast cancer and was significantly related to the bone metastasis-free survival of patients In situ hybridization shown that the expression of mi R-124 was down-regulated in adjacent noncancerous tissues as compared with primary breast cancer tissues, and further reduced in metastatic bone tissues collected from five patients. Real time RT-PCR shown that in 20 patients with bone metastasis from breast cancer, those with lower mi R-124 level displayed shorter bone metastasis-free survival than those with higher miR-124 level.(2) Overexpression of mi R-124 could promote the differentiation of osteoclasts directly or through regulating osteoblastsa. Overexpression of miR-124 suppressed while inhibition of mi R-124 enhanced the differentiation of osteoclasts from BMM MDA-MB-231 cells were transfected with mi R-124 mimic and NC or mi R-124 inhibitor and inhibitor NC. Condition medium was collected and used to culture BMM. TRAP dyeing shown that overexpression of mi R-124 suppressed while inhibition of mi R-124 enhanced the differentiation of osteoclasts from BMM.b. Overexpression of mi R-124 increased while inhibition of mi R-124 decreased the ratio of OPG to RANKL in 3T3E1 cells MDA-MB-231 cells were transfected with mi R-124 mimic and NC or mi R-124 inhibitor and inhibitor NC. Condition medium was collected and used to culture the 3T3E1 cells. Real time RT-PCR shown that overexpression of mi R-124 increased while inhibition of mi R-124 decreased the ratio of OPG to RANKL in 3T3E1 cells.c. Enhanced expression of mi R-124 suppressed the ability of osteoblasts to promote the differentiation of osteoclasts MDA-MB-231 cells were transfected with mi R-124 mimic and NC or mi R-124inhibitor and inhibitor NC and then co-cultured with 3T3E1 cells. Condition medium was collected and used to culture RAW264.7 cells. Real time RT-PCR shown that TRAP, c-Src, NFATc1 and c-fos were significantly down-regulated in the RAW264.7 cells co-cultured with MDA-MB-231 cells transfected with mi R-124 mimic.(3) mi R-124 suppressed bone metastasis from breast cancer partially through regulating IL11a. mi R-124 inhibited the expression of IL11 Real time RT-PCR shown that mi R-124 mimic inhibited while mi R-124 inhibitor enhanced the level of IL11 m RNA. Western blot shown that mi R-124 mimic inhibited while mi R-124 inhibitor enhanced the expression of IL11 protein.b. miR-124 bound to the 3′-UTR of IL11 The luciferase reporter carrying the binding site of mi R-124 on the 3′-UTR of IL11(wild-type) and the mutant binding site(mutant) were constructed and co-transfected with mi R-124 mimic or NC to HEK-293 cells. Dural reporter assay shown that mi R-124 mimic decreased the luciferase activity of the wild-type IL11 3′-UTR. Mutation of the target sequence on the IL11 3′-UTR diminished the effect of mi R-124 on IL11, indicating that IL11 is a direct downstream target of mi R-124.c. The expression of mi R-124 was negatively related to that of IL11 in breast cancer cells and metastatic bone tissues from breast cancer1) The expression of mi R-124 was negatively related to that of IL11 in normal mammary tissue from mice and breast cancer cells with different metastasis capacity Real time RT-PCR was used to detect the expression of IL11 in normal mammary tissue of mice and in breast cancer cells with different metastasis capacity including BT549, MDA-MB-231, Hs578 t, MDA-MB-468, MDA-MB-436, MCF7, T47 D and BT474. Correlation analysis shown that the level of mi R-124 was negatively related to that of IL11.2) The expression of mi R-124 was negatively related to that of IL11 in parental breast cancer cells and bone metastasis tissues from breast cancer model in miceReal time RT-PCR was used to detect the expression of IL11 in parental MDA-MB-231 cells and bone metastasis tissues from breast cancer model in mice. Person analysis shown that the expression of miR-124 was negatively related to that of IL11.3) The expression of mi R-124 was negatively related to that of IL11 in metastatic bone tissues from patients with breast cancer Primary breast cancer tissues, adjacent noncancerous tissues and metastatic bone tissues were collected from five patients. Immunohistochemistry showed that the expression of IL11 was upregulated in primary breast cancer tissues as compared with that in adjacent noncancerous tissues, and was futher enhanced in metastatic bone tissues. Metastatic bone tissues were collected from 20 patients with breast cancer. Real time RT-PCR and the subsequent correlation analysis shown that the expression of mi R-124 was negatively related to that of IL11.【Conclusions】1. The expression of mi R-124 was down-regulated in breast cancer cells and metastatic bone tissues from breast cancer and related to the bone metastasis-free survival of patients with breast cancer, indicating that mi R-124 plays important roles in bone metastasis of breast cancer.2. mi R-124 from breast cancer cells inhibited the differentiation and activation of osteoclasts.3. mi R-124 may function in bone metastasis of breast cancer by directly regulating IL11 in breast cancer cells.