The Regulation Role And Mechanism Study Of MFAP5 In Breast Cancer Bone Metastasis

Background and objective Breast cancer is the most commonly diagnosed malignant tumor in female,which accounts about 30% of all cancers among women.Despite the developments of the treatment of cancer,including surgery,radiotherapy,chemotherapy,and endocrine therapy,have evidently reduced the rates of premature mortality,breast cancer still ranks second among the most common causes of cancer death in women.Tumorigenesis is caused by various carcinogenic factors and is characterized by uncontrolled cell proliferation and high metastasis rates.Bone is the most common site of advanced breast cancer and the reported incident rate is 60-80%,that increased such complications as pathologic fracture and spinal cord compression which decrease the quality of life.But the strategies targeting bone metastasis of breast cancer are still limited.Therefore,it is urgent to explore the molecular mechanisms underlying breast cancer bone metastasis for developing new effective therapeutic targets.Microfibrillar-associated protein 5(MFAP5),also known as microfibril-associated glycoprotein 2(MAGP2),is a component of extracellular elastic microfibril which functioned in bone growth,cardiovascular development,alveolar elastogenesis and Marfan syndrome.Study showed that MFAP5 was secreted by bone marrow mesenchymal stromal cell(BMSC),and functioned in hematopoiesis and immune systems,while loss of MFAP5 is protective against bone loss in mice.These results indicated that MFAP5 worked as a crucial factor in bone microenvironment.Recent studies further showed that MFAP5 was overexpressed in head and neck,pancreatic,lung,and tongue cancers.But the role of MFAP5 in these cancers remains to be elucidated.It has been shown that serum MFAP5 level negatively correlated with prognosis in patients with ovarian cancer,while cancer cell secreted MFAP5 promoted tumor proliferation,endothelial cell motility,chemoresistance and angiogenesis.However,the role of MFAP5 in breast cancer or bone metastasis has still not been reported.Due to the important roles of MFAP5 in both bone and tumor microenvironment,we wondered the function of MFAP5 in breast cancer bone metastasis.In the present study,we firstly demonstrated that MFAP5 was highly expressed in breast cancer bone metastasis and studied the effect of MFAP5 on cell proliferation and migration of breast cancer cells,as well as explored the possible underlying molecular mechanisms,especially about the pathway of ERK/MMP.Research contents and methods Primary breast cancer specimens and bone metastasis tissues were collected from patients with breast cancer who received surgical resection in Changzheng Hospital of the Second Military Medical University(Shanghai,China).Immunohistochemical(IHC)staining for MFAP5 was carried out using standard histological procedures described.Total RNA was isolated and reverse transcribed into c DNA.Gene transcripts were quantified on 7900 HT Fast Real-Time PCR System.For MFAP5 overexpression plasmid,vector pcDNA3.1+ plasmid was enzyme digested and the code sequence of MFAP5 amplified by PCR was inserted into pcDNA3.1+.Two breast cancer cell lines MCF7 and MDA-MB-231 were routinely maintained in DMEM medium supplemented with 10% fetal bovine serum(FBS).Cells of logarithmic growth were transfected with plasmids using DNA Transfection Reagent or with si RNA.MCF7 and MDA-MB-231 cells transfected with overexpression plasmid or si RNA were seeded in 96-well plates at an initial density of 5 × 103 per well,cultured for 48 hours,and assessed using the Cell Counting Kit 8.The results were measured by absorbance at 450 nm using an ELx800 microplate reader.An 8-?m pore size transwell chamber was used for transwell migration assay.MCF7 and MDA-MB-231 cells transfected with overexpression(OE)plasmid or si RNA were digested and counted.The blotted membranes were incubated with antibodies for MFAP5,MMP2,MMP9,p-FAK,FAK,p-Erk1/2,Erk1/2,p-c Jun(Ser63),p-c Jun(Ser73),c Jun,and actin diluted at 1:1000.The blots were developed using Easy See Western Blot Kit.Beta-actin protein was also determined by using the specific antibody as a loading control.All experiments were carried out in triplicate.SPSS 19.0 statistical software was used for statistical analysis.Data are presented as mean ± standard error of the mean(SEM).Statistics of the mean value between groups were assessed using independent Student t test,assuming double-sided variance.All experiments were repeated at least three times,and representative experiments are shown.P values of < 0.05 were considered statistically significant.Results IHC staining showed that MFAP5 was mainly expressed in intercellular space,and significantly up-regulated in bone metastasis compared with that in primary breast cancers.Western blot assay further confirmed the overexpression of MFAP5 in bone metastasis than primary cancers.In addition,the results of q RT-PCR assay showed that the m RNA level of MFAP5 was significantly evaluated in breast cancer bone metastasis than the primary ones(p < 0.05).The expression level of MFAP5 was highest in MDA-MB-231 cell,and lowest in MCF7 cell.The efficiencies of the overexpression plasmid and si RNAs were confirmed by western blot assay.CCK8 assay showed that cell viability was significantly evaluated after the transfection of MFAP5 overexpression plasmid,while decreased when transfected with either si RNA of MFAP5 in both MCF7 and MDA-MB-231 cells,indicated that MFAP5 mediated cell proliferation in breast cancer cells.Transwell assay showed that the number of MCF7 and MDA-MB-231 cells migrating through the chamber membrane in OE-MFAP5 plasmid transfection group was decreased significantly as compared with that in the control group,while inhibition of MFAP5 by si RNAs transfection obviously decreased the ability of migration.These results suggested that MFAP5 might promote cancer metastasis through the accelerating of cell migration.The m RNA levels of MMP2 and MMP9 were significantly increased,while the m RNA levels of MMP7 and MMP14 hardly changed when MFAP5 overexpressed in both MCF7 and MDA-MB-231 cells.Western blot assay further showed that the expression of MMP2 and MMP9 obviously up-regulated MCF7 and MDA-MB-231 cells transfected with OE-MFAP5 plasmid compared to OE-Ctrl plasmid.On the contrary,inhibition of MFAP5 by si RNAs clearly suppressed the expression of MMP2 and MMP9.These results indicated that MFAP5 might functioned in breast cancer bone metastasis through the regulation of MMP2 and MMP9.No significant fluctuation was observed in the expression of FAK,Erk1/2 and c Jun after MFAP5 overexpression,but MFAP5 obviously activated the phosphorylation of FAK,Erk1/2 and c Jun.Conversely,inhibition of MFAP5 by si RNAs clearly suppressed the expression of p-FAK,p-Erk1/2 and p-c Jun,but not FAK,Erk1/2 or c Jun.These results suggested that ERK signaling pathway possibly mediated the regulation of MMP2 and MMP9 by MFAP5.Conclusion In this article,we firstly reported the up-regulation of MFAP5 in breast cancer bone metastatic tissues versus primary breast cancer tissues,and further found that MFAP5 overexpression accelerated breast cancer cell proliferation and migration,while an opposite effect was observed when MFAP5 was knocked down.In addition,up-regulation of MFAP5 increased the expression of MMP2 and MMP9,as well as activated ERK signaling pathway.Conversely,inhibition of MFAP5 suppressed the expression of MMP2,MMP9,p-FAK,p-Erk1/2 and p-c Jun.These results suggest that MFAP5 acts as an oncogene in breast bone metastasis.The above findings of our study may contribute to the development of novel diagnostic and treatment strategies for breast cancer bone metastasis.