The Role Of Fibroblast Growth Factors Pathway On The Regulation Of Long Non-coding RNA Malat1 In Chondrocytes

Skeleton is the largest organ in human,play a critical role in bearing,protection,movement and endocrine.Skeletal development is a orchestrated biological processing with two major mode,i.e.intramembranous and endochondral ossification.For the developing of long bone of trunk and limbs,and a minor part of os irregulare,endochondral ossification is the major and complicated process.At first,cells condensed in zones where will developing into growth plate to forming chondral rudiment,then those rudimental cells proliferate,hypertrophy,apoptosis and mineralized in turn,finally replaced by osteoblasts,forming osseous tissue.For parietal bone,frontal bone and clavicles,intramembranous ossification is the major developing process.Endochondral ossification plays an important role in embryonic development and long bone formation,endochondral ossification defects could leads to many disorders characterized by growth retard and metaphysis structure abnormal.Through there are numerous research investigated the complicated regulate factors of endochondral ossification,the detailed mechanism is still unknown.Currently,the fibroblast growth factors(FGFs)are well-known regulators which play a critical role in endochondral ossification.Fibroblast growth factor family are consisted of 22 members,the most widely expressed factor is FGF2.FGF ligand combined with membranous fibroblast growth factor receptor(FGFR),leads to downstream biological effects.FGFR have four isomers,separately named FGFR1,FGFR2,FGFR3 and FGFR4,different FGFR have different but overlapped spatial and temporal distribution.The major FGFR in human cartilage is FGFR1 and FGFR3,which induce biological effects by activating downstream pathways including ERK/MAPK,Wnt/b-catenin,Hedgehog,and so on.In mice model and human cases,mutations in FGF gene could lead to many clinical problems characterized by dwarfism.In chondrocytes,FGF signaling pathways inhibit the cell proliferation.Many study investigated the downstream proteinic molecules influenced by FGF signaling pathway,but whether the FGF signaling pathway regulate chondrocytes by influence non-coding RNA,is still not been well-investigated.Previous conception regarding non-coding RNA as the ‘noise' produced in transcription.Recently emerging evidences suggested the important effects of non-coding RNA in physical and pathologic process.Long non-coding RNAs(Lnc RNAs)are operationally defined as transcripts which over than 200 nucleotides,poorly conserved in species,regulate genes expression by multiple ways,including transmitting signals,binding sites as decoys,guiding proteins,act as scaffolds.Lnc RNAs distinctive features conferred it's unique regulatory functions,including exquisite cell-and tissue-specific expression and the capacity to involve development and pathologic process.Metastasis-associated lung adenocarcinoma transcript 1(Malat1)is a lncRNA which was highly conserved in mammals.The MALAT1 gene of human located in chromosome 11,length 6815 nt.The Malat1 gene of mice located in chromosome 19,length 6981 nt.At first Malat1 was identified in screening investigation of non-small lung cancer.Numerous researches found that malat1 expressed abundantly in almost all cancerous cells.Multiple tumor investigations found that malat1 promoted the ability of invasion and metastasis of multiple tumorous cells.On the other hand,malat1 could promote osteoblast proliferation,and osteosarcoma invasion,those results indicate malat1 plays a role in skeletal system,but currently we still don't know whether malat1 have some effects in chondrocytes,and whether there have some relationship between FGF signaling pathway and malat1.Previously our lab performed several basical work about the FGF signaling influence on the chondrocytes,accumulated several foundation.For the downstream proteinic molecules influenced by FGF signaling we performed much work,but currently whether the FGF signaling regulate chondrocytes through influence non-coding molecules such as lncRNAs,is still unknown.So that we,at first,use the mice primary chondrocytes as a model,investigate the effects of Malat1 on chondrocytes by cellular biological measures and molecular biological techniques and methods.Second,we use the mice primary chondrocytes and mice/human chondral cell lines as model,investigate if the FGF signaling which play a role in chondrocytes could have some influences on Malat1,and if Malat1 plays a role in the regulation process of FGF signaling on chondrocytes proliferation by cellular biological measures and molecular biological techniques and methods.METHODS1.The fundamental expression level of Malat1 in chondrocytes.1.1 Isolated primary chondrocytes from C57 mice cubs(day 3-5),extract RNA after 48 h culture,and the expression level of Malat1 was examined by q PCR.1.2 Mice fibroblast line NIH3T3,mice pre-chondrocyte line ATDC5,human normal chondrocyte line C28I2 and human osteosarcoma line SW1353 have been cultured for 48 h,then extracted RNA to examined the expression level of Malat1.2.The effects of Malat1 on ATDC5 proliferation2.1 Several si RNAs against mice Malat1 gene have been designed and the most effective siRNA has been screened by fluorescence indicator and q PCR.2.2 Transfect si RNA into ATDC5 cells,and examined ATDC5 proliferation by cell counting.3.The influence of FGF signaling on the expression level of Malat1 in chondrocytes.3.1 C57 mice primary chondrocytes and ATDC5 have been treated with FGF2,extracted RNA in different time point and examined Malat1 expression level by q PCR.3.2 Use the ERK inhibitor U0126 treated ATDC5 alone or treated with FGF2,extracted RNA after 24 h and examined Malat1 expression level by q PCR.3.3 Isolated primary chondrocytes from b-catenin conditional knockout mice which fed by our lab,extracted RNA after 48 h culture and examined the expression level of Malat1.3.4 Isolated primary chondrocytes from FGFR1 conditional knockout mice and FGFR3 knockout mice which fed by our lab,extracted RNA after 48 h culture and examined the expression level of Malat1.3.5 Several si RNAs against mice Fgfr1 gene have been designed and the most effective siRNA has been screened by fluorescence indicator and q PCR,pc DNA3.1-Fgfr1 plasmids have been constructed and confirmed.Then transfect siRNA-Fgfr1 and pcDNA3.1-Fgfr1 into ATDC5 separately,examined the expression level of Malat1 by q PCR.RESULTS1.The fundamental expression level of Malat1 in chondrocytes.At first we examined expression level of Malat1 in NIH3T3,regarding the level as contrast.Our results suggested that Malat1 expressed abundantly in C57 mice primary chondrocytes,mice pre-chondrocyte line ATDC5,human normal chondrocyte line C28I2 and human osteosarcoma line SW1353.In ATDC5,the fundamental expression level of Malat1 on 24 h and 48 h was no significant difference.2.The influence of Malat1 interference on the proliferation of ATDC5Co-transfected si-Malat1 with fluorescence indicator into cells,the fluorescence indicate si-Malat1 transfection was succeeded,then extracted RNA from cells and examined by q PCR,the results suggested that the expression level of Malat1 has been succeeded interfered over than 50%;In different time point after transfection,cell counting was performed and the results suggested that after Malat1 interference,the proliferation of ATDC5 significant decreased.3.The regulation of FGF signaling on Malat1We treated C57 mice primary chondrocytes,mice pre-chondrocyte line ATDC5,human normal chondrocyte line C28I2 and human osteosarcoma line SW1353 with FGF2,extracted RNA after 24 h and examined by q PCR.We found that FGF2 significantly inhibited the expression level of Malat1 in cells described above.We treated ATDC5 with FGF2 in several different concentration and time point and then we extracted RNA and examined by qPCR,we found that FGF2 inhibited Malat1 with a dose-independent manner,effect on 24 h with duration over than 24 h.Isolated primary chondrocytes from b-catenin conditional knockout mice,then examined by q PCR,results showed that the expression level of Malat1 had no significant change.Treated ATDC5 cells with ERK signaling pathway inhibitor U0126 alone or with FGF2,extracted RNA for qPCR detecting,our found that after U0126 treatment,the expression level of Malat1 significantly increased,and FGF2 couldn't inhibit the expression level of Malat1 when ATDC5 was treated by U0126 and FGF2.Our results suggested that the FGF signaling might inhibit Malat1 expression through the ERK pathway.Isolated primary chondrocytes from FGFR1 conditional knockout mice and FGFR3 knockout mice which fed by our lab,extracted RNA after 48 h culture and examined the expression level of Malat1.We found that the fundamental expression level had no significant change in chondrocytes from FGFR3 knockout mice.Moreover,FGF2 could still inhibit the expression level of Malat1.In chondrocytes from FGFR1 conditional knockout mice,the expression level of Malat1 increased significantly,and FGF2 couldn't inhibit Malat1's expression continuously.Meanwhile,we designed siRNA against Fgfr1,and constructed pcDNA3.1-Fgfr1 plasmids,then transfected siRNA and plasmids into ATDC5 separately,extracted RNA after 24 h to q PCR.We found the expression level of Malat1 increased significantly after Fgfr1 interference,and FGF2 couldn't inhibit Malat1 expression continuously.After Fgfr1 plasmids transfection,Malat1 expression level decreased significantly.CONCLUSION1.Malat1 is highly expressed in chondrocytes from both human and mice.2.Malat1 promoted proliferation of chondrocytes.3.FGF may regulated the expression of Malat1 by activating ERK through binding to FGFR1.