Effects Of Hsa＿circ＿0000110 In Breast Cancer Cell On Osteoclastogenesis And Cancer Cell Invasion

BackgroundsBreast cancer is the most common type of cancer in women.It is the second leading cause of cancer death in women.Bone is the most common and earliest site of distant metastasis of breast cancer.Once bone metastasis occurs,the 5-year survival rate is only 20%.Bone metabolism is regulated by the balance between osteoblast-mediated bone formation and osteoclast mediated bone absorption.The breast cancer bone metastasis interferes the banlance and mainly causes osteolytic lesion,but mixed with osteoblastic bone lesion.Hypercalcemia,pathological fracture,osteoporosis and other complications may occur after bone metastasis,which are caused by increased osteoclast differention and osteoclastogenesis.Therefore,it is of great significance for prevention and therapy to study the osteoclastogenesis induced by breast bone metastasis.Circular RNAs(circRNAs)are a kind of circular non-coding RNAs,which can affect the cellular functions or behavior through multiple mechanisms.It has been reported that abnormal expression of specific circRNAs,e.g.,has-circ-0072309、circAGFGl and circ-ABCB10,et.al,could promote cancer proliferation and metastasis.There are research demonstrated that the expression profile of cicRNAs between bone matastasis breast cancer cell strain and its parental cell line is different.The expression of micro RNAs(miRNAs),e.g,miR-141,miR-219 and miR-34a,is related to bone metastasis of cancer.One function of circRNA is to sponge miRNA,releases the inhibition of the target mRNA by miRNA.Neverthless,it is still unexplored if the specific circRNA in breast cancer cells of bone metastasis functions to promote bone metastasis,and the corresponding mechanisms.ObjectivesTo study the circRNA expression profile of breast cancer bone metastasis cell strain,obtain the differential expression circRNAs and select one of them to study if it can promote the RANKL-induced osteoclastogenesis of the co-cultured osteoclast precursor cell.To study the possible miRNA and mRNA target of the selected circRNA through bioinformatics.Methods1.NCBI website was searched for circRNA expression profile data sets of breast cancer bone metastasis and its control.The data sets were analyzed to get the differential circular RNA expression profile of breast cancer bone metastasis cell strain and its parental cell line.The circRNA hsa-circrna-0000110 was selected as research target in this study and the differential expression level was confirmed by Q-PCR.2.The noe-contacted co-coulture:Bo-1833 strain and the osteoclast precursor cell RAW264.7 were co-cultured at non-contacted condition and induced with RANKL for 4-6 days.3.The Bo-1833 cells were infected with lentiviruses to knock down the expression of hsa-circ-0000110 and to establish the stable knockdown cell strain and the control.4.The osteoclasts were identified by tartrate-resistant acid phosphatase(TRAP)staining;The total RNA and protein of the cell were isolated to assay the molecular marker of osteoclasts by Q-PCR and Western Blotting:Q-PCR for C-fos,MMP9,Nfatcl,and DC-stamp;Western Blotting for NF-κB,MMP9 and Cathepsin K.5.The osteolysis ability of osteoclast was assayed by Osteo Assay Surface.6.Cancer cell proliferation was assayed by CCK8 experiment,clone formation ability by plate clone formation assay,and migration and invasion by Transwell Assay.Results1.The heatmap demonstrated that there were difference in the circRNA expression profile between the Bo-1833 strain and MDA-MB-231 parental cell line.The higher expression of has-circ-0000110 in Bo-1833,v.s.MDA-MB-231,was proved by Q-PCR.The circular RNA has-circ-0000110 was selected for further study in this project.2.The breast cancer bone matastasis strain Bo-1833 was a stronger promoter than its MDA-MB-231 parental cell line on RANKL-induced osteoclastogenesis from the RAW264.7co-culture.3.Knockdown of hsa-circ-0000110 in Bo-1833 cells decreased the RANKL-induced osteoclastogenesis and ostelysis by the RAW264.7 co-culture.4.Knockdown of hsa-circ-0000110 in Bo-1833 cells decreased their proliferation,clon formation,migration and invation.5.Circular RNA hsa-circ-0000110 in Bo-1833 may promote induced osteoclastogenesis through targeting eight possible miRNAs.ConclusionOur study demonstrated that the circular RNA hsa-circ-0000110 in Bo-1833 strain could promote RNAKL-induced osteoclastogenesis by osteoclast precursor RWA264.7 cell.The increased expression of specific cicular RNA in breast cancer cell can promote induced osteoclast differentiation.This study suggests that hsa-circ-0000110 may promote breast cancer metastasis,and the circRNAs in breast cancer cell and the functional pathways could be the the biological markers or threputic targets.