The Mechanism And Effect Of MALAT1/miR-124 Axis On Bone Metastasis Of Breast Cancer

Part One Analysis of prognostic factors for breast cancer spinal metastasesObjective: To investigate the differences in clinicopathological data of two palliative operations for spinal metastases in breast cancer,and to analyze the prognostic factors affecting the curative effect and the role of RANKL ex-pression.Methods:1.We collected clinical and pathological data of 56 patients with spinal metastases without spinal cord compression treated with palliative surgery in the orthopedics department of the Fourth Hospital of Hebei Medical University from January 2011 to January 2016.Inclusion criteria: 1)All the patients were female;2)The clinical,impact,and pathological data were complete;3)Multiple bone metastases and / or other visceral metastases,combined obvious bone pain,and no obvious spinal compression on imaging,and the posterior walls of the vertebra were intact,suitable for palliative vertebroplasty surgery;4)Pathological specimens were obtained during the surgery and postoperative pathology confirmed as breast cancer spine metastasis;5)The expected survival time before surgery and the actual follow-up period after surgery were more than half a year.Exclusion criteria:1)Male breast cancer;2)Oligometastasis patients which were suitable for total en-bloc surgery and existed spinal cord compression requiring spinal cord decompression surgery;3)Patients who failed to obtain tissue specimens during operation or whose pathological biopsy showed negative after surgery;4)Patients who died or lost during follow-up within six months after surgery.2.We divided all the patients into percutaneous vertebroplasty group(PVP)and percutaneous vertebroplasty combined with percutaneous pedicle screw internal fixation group(PVP+PPSF)according to the different surgical method.Clinic and pathological data such as age,Tomita classification,Tomita score,Tokuhashi score,SINS score,molecular subtypes of primary and metastatic lesions,VAS scores and Frankel grades before and 1 month after surgery were calculated respectively.The statistical differences of clinical pathological data between the two groups were compared.3.The bone metastases of the diseased vertebrae were obtained by puncture during the surgery,and conventional HE staining and ER,PR,HER2 immunohistochemistry were detected.Immunofluorescence was used to detect the expression level of RANKL in all patients after surgery.Control status,local progression-free survival time(LPFS),pain score(VAS score),Frankel’s classification,and explore the impact of different surgical methods and RANKL expression on the prognosis of patients with spinal metastases of breast cancer.4.We compared the changes of ER,PR,HER2 and other receptors in patients with primary and bone metastases,and compared the proportion of patients with molecular subtype changes in primary breast cancer and bone metastases.The influencing factors of patients’ local progression-free survival were explored through univariate analysis and multivariate analysis.Results:1.Of all the 56 patients with breast cancer underwent palliative surgery for spinal metastasis,26 cases were treated with simply percutaneous vertebroplasty,and 30 cases were treated with percutaneous vertebroplasty combined with percutaneous pedicle screw internal fixation.In different groups,Tomita score,Tokuhashi score,SINS score,preoperative VAS score,Frankel’s classification,postoperative lesion radiotherapy,HER-2,ER,PR expression and molecular subtypes of primary breast cancer lesions and bone metastases lesions,the difference was not statistically significant.The average time of surgery was 73 ± 20 min in percutaneous vertebroplasty group,and 38 ± 18 min in percutaneous vertebroplasty group,the difference was statistically significant(P<0.05).The average bleeding volume of percutaneous verteb-roplasty combined with percutaneous pedicle screw internal fixation group was 23 ± 15 ml vs 12 ± 11 ml,the difference was statistically signif-icant(P<0.05).The percutaneous pedicle screw fixation combined with percutaneous vertebroplasty group had better VAS score than the percutaneous vertebroplasty group during 1 month after surgery,the diffe-rence was statistically significant(P<0.05).The Frankel’s classification of percutaneous pedicle fixation combined with percutaneous vertebroplasty group had a higher rate of grade E than in the percutaneous vertebroplasty group during 1 month after surgery,the difference was statistically significant.Patients treated with percutaneous vertebroplasty combined with percutaneous pedicle screw fixation had a higher expression level of RANKL,the difference was statistically significant(P<0.05).2.Survival analysis of all the 56 patients showed that patients with ER and PR positive in primary breast cancer lesions and bone metastases,low expression of RANKL and radiotherapy had longer local progression free survival time,while patients with molecular subtypes diversification had shorter local progression free survival time.The difference was statistically significant.Multivariate analysis showed that ER,PR expression levl of bone metastases,RANKL expression level and whether to accept radiotherapy were independent risk factors that affected patients with local progression-free survival.Conclusions:1.In palliative surgery for spinal metastases,compared with the simple PVP group,patients in the PVP + PPSF group had better recovery of pain and neurological injury.2.The expression level of ER,RANKL in spinal metastases and radiotherapy may affect the prognosis of surgery.Part Two Relationship between expression of MALAT1 / miR-124 and clinicopathological characteristics in patients with breast cancer bone metastasesObjective: To investigate the significance of MALAT1 / miR-124 expression in bone metastasis of breast cancer.Methods: 1.We collected specimens and clinicopathological data of primary breast cancer tissues,adjacent tissues and corresponding bone metastases from 26 patients diagnosised as stage I-III breast cancer from January 2013 to January 2015 and presented bone metastases during follow-up.Real-time quantitative polymerase chain reaction(q RT-PCR)was used to detect the expression of MALAT1/miR-124 in breast cancer tissues,adjacent tissues,and bone metastasis tissue samples from patients with primary tumors.q RT-PCR results using 2-ΔΔCT method for relative quantitative analysis of gene expression.To explore expression level of MALAT1 / miR-124 in primary lesions and bone metastases,and analyzed the relationship between expression level and clinical and pathological data such as age,clinical stage,histological grade,lymph node metastasis,vascular tumor thrombus,molecular subtypes and others.And then the effect of MALAT1 / miR-124 expression on bone metastasis of breast cancer was analyzed.2.The correlation between MALAT1 and miR-124 expression levels was analyzed by comparing the expression levels of MALAT1 / miR-124 in breast cancer tissue and bone metastasis tissue.The effects of MALAT1 and miR-124 expression on prognosis were explored by survival analysis.Results: 1.26 cases of breast cancer tissues,corresponding specimens of adjacent cancer tissues and bone metastases were detected by q RT-PCR,which showed that the expression level of MALAT1 was highest in bone metastases and lowest in adjacent cancer tissues,the difference was statistically significant(P <0.05),the expression level of miR-124 was highest in the adjacent tissues and the lowest in bone metastases,and the difference was statistically significant(P <0.05).Compared in different tissues of breast cancer and bone metastases,the expressions of MALAT1 and miR-124 had a negative correlation.2.In addition,the expression level of MALAT1 in breast cancer tissues was related to the clinical stage and the number of lymph node metastases,and the expression level of miR-124 was related to the clinical stage of patients.The patients with high expression of MALAT1 and low expression of miR-124 in breast cancer tissue occurred earlier bone metastases,and patients with high expression of MALAT1 and low expression of miR-124 in breast cancer bone metastases tissue showed earlier bone metastases progression(P<0.05).Conclusions: 1.The expression level of MALAT1 and miR-124 showed negative correlation in breast cancer and bone metastasis tissues.2.High expression of MALAT1 and low expression of miR-124 may be associated with poor prognosis of breast cancer with bone metastases;Part Three Denosumab inhibits spontaneous osteoclast differentiation induced by the MCF-7 cell line via the RANKL / MALAT1 / miR-124 axisObjective: To investigate the significance and mechanism of RANKL / MALAT1 / miR-124 axis in MCF-7 and RAW264.7 cell co-culture system,and the effect of Denosumab on MCF-7 cell line-induced spontaneous osteoclast differentiation In order to explore the potential drug resistance mechanism and prognostic factors of Denosumab in bone metastasis of breast cancer.Methods: 1.We established a MCF-7-induced co-cultured cell line system of RAW264.7 spontaneous osteoclast differentiation as an in vitro breast cancer bone metastasis model.The MCF-7 cell line was cultured in the upper layer and the RAW264.7 cell line was placed in the lower layer.2.The co-culture system of MCF-7 cell line and RAW264.7 cell line was divided into an experimental group and a blank control group.The experimental group treated the lower chamber Raw264.7 cell line with Denosumab 0.1 mg / ml.TRAP staining was performed on the two groups of immunohistochemical methods.In addition,we tested the expression levels of bone resorption genes and osteoclast phenotype genes in the experimental and control groups,including cathepsin K,CAII,integrin av and interinβ3,and RANK,TRAP,TRAF6,and MMP-9.3.To explore the effect of MALAT1 knockdown on the effect of Denosumab: Silent MALAT1(si-MALAT1)was transfected into RAW 264.7 cells with a plasmid to knock down the expression level of Lnc RNA MALAT1.The lower chamber of the co-culture system is used for cultivation.The 4 groups were divided into negative control group(NC),Denosumab group,si-NC group and si-MALAT1 group,and TARP + labeled osteoclasts were used to evaluate osteoclast differentiation.The expression level of MALAT1 gene was detected by q RT-PCR,and the expression level of RANKL was detected by immunofluorescence.The effect of MALAT1 knockdown on the effect of Denosumab was further evaluated.4.To explore the effect of miR-124 overexpression on the effect of Denosumab: we transfected miR-124 mimic into RAW264.7 cells by plasmid transfection to above-regulated miR-124 expression level,then the miR-124 overexpressing cells were placed in the lower chamber of the co-culture system for culture.We divided into four groups for comparison,involved NC group,denosumab group,miR-NC group and miR-124-mimic group.TARP + osteoclasts were detected by TARP staining to evaluate the osteoclast differentiation.The expression level of miR-124 gene was detected by Western Blot and q RT-PCR,while the expression of RANKL was detected by immunofluorescence.The effect of miR-124 overexpression on the effect of Denosumab was further evaluated.5.To analyze the regulation of osteoclast differentiation by MALAT1 and miR-124: analyze the complementary pairing sequences of the base sequences of MALAT1 and miR-124,and verify the miR-124-mimic transfection by verifying the luciferase gene reporting system Effect on WT 3’-UTR luciferase activity in RAW264.7 cells.q RT-PCR was used to analyze the effect of MALAT1 expression down-regulation on miR-124 expression induced by si-MALAT1 transfection.The effect of miR-124 overexpression on the expression levels of Rab27 a,IL-11,NFATc1 and TARP protein was detected by Western Blot.Results: 1.Denosumab could inhibit the spontaneous differentiation of RAW264.7 cell line induced by MCF-7 cell line to osteoclasts,and the inhibitory effect of RAW264.7 cell line spontaneous differentiation to osteoclasts was more obvious when Lnc RNA MALAT1 low expressed and / or miR-124 overexpressed,and the difference was statistically significant(P<0.01).2.However,RANKL protein expression was not altered when MALAT1 expression was knocked down or miR-124 was overexpressed.In addition,the dual luciferase gene reporting system showed that miR-124 could inhibit the expression level of MALAT1 by targeting,and si-MALAT1 could up-regulate the expression of miR-124.At the same time,miR-124-mimic could also reduce the expression levels of Rab27 a,IL-11,NFATc1 and TARP proteins.Conclusion: Denosumab could inhibit MALAT1 expression through targeted binding to RANKL,thereby up-regulating the expression level of miR-124,and finally inhibiting MCF-7 cell line to induce spontaneous osteoclast differentiation.