Study On The Mechanism Of Lncrna FOXD2-AS1 In Regulating The Biological Behavior Of Osteosarcoma

Osteosarcoma is a high malignant primary bone neoplasm in adolescents,which with 90% pulmonary metastasis.The emergence of neoadjuvant chemotherapy combined with limb salvage surgery significantly prolongs the survival period of patients with osteosarcoma.However,the therapeutic effects and prognosis of patients with osteosarcoma with pulmonary metastasis are still counterproductive.To elucidate transfer related molecular mechanism becomes a breakthrough in improving osteosarcoma survival rate,and also becomes the key for the treatment of plateau bottleneck.LncRNAs are a class of RNA molecules that are longer than 200 nt and lack of open reading frame,which is involved in many diseases development.A novel oncogenic FOXD2-AS1 has been found to play an important role in a variety of tumors including liver,bladder and non-small cell lung cancer.However,the biological function and potential molecular mechanisms of FOXD2-AS1 in human OS are unclear.In the early stage of this study,tissues and para-tumor tissues from 5 pairs of osteosarcoma patients were sequenced by using high-throughput sequencing technology,LncRNAs in terms of research valued were screened out.Through literature retrieval,combined with further research needs differential multiples,FOXD2-AS1 was selected as the research object.In the present study,FOXD2-AS1 expression was significantly increased in OS tissues and cell lines.In addition,the Kaplan-Meier analysis showed that FOXD2-AS1 expression was closely related to the survival of patients with OS,which indicated that FOXD2-AS1 may become a new molecular marker affecting the biological behavior of osteosarcoma cells.If potential mechanism can be revealed,it may become a new potential therapeutic target as well as an effective approach on the treatment of osteosarcoma patients.Objective1.The expression level of FOXD2-AS1 in osteosarcoma tissues,para-tumor tissues,osteosarcoma cells and Human SV40 transfected osteoblasts(hFOB1.19)wasdetected by real-time polymerase chain reaction(RT-PCR),and the correlationbetween the expression level of FOXD2-AS1 and the survival of patients wasanalyzed by kaplan-meier analysis.2.The effects of FOXD2-AS1 on proliferation,cloning,migration,invasion andcycle of osteosarcoma cells in vitro and in vivo were studied by lentivirusinterference.3.The distribution of FOXD2-AS1 in cells was determined by fluorescence in situhybridization,and the expression of downstream proteins that might be affectedwas detected by RT-PCR and western blot.Method1.During July 2012-August 2014,a total of 40 pairs of osteosarcoma tissues andpara-tumor tissues resected by surgery were selected.The expression ofFOXD2-AS1 in osteosarcoma tissues,para-tumor tissues,osteosarcoma cells andhuman sv40-transfected osteoblasts were detected by RT-PCR,and therelationship between FOXD2-AS1 and the survival period of osteosarcomapatients was analyzed by kaplan-meier analysis.2.The expression of FOXD2-AS1(shRNA FOXD2-AS1)in SOSP-9607 and U20Swas stably interfered by lentivirus interference method,and the effects ofsilencing FOXD2-AS1 was detected by RT-PCR.3.The effects of shRNA FOXD2-AS1 silencing on proliferation,migration,invasion and cloning of osteosarcoma cells were verified by CCK8,cell cycle,clone formation assay,cell migration,invasion,and tumor formation in nudemice.4.Fluorescence in situ hybridization was used to verify the sublocalization ofFOXD2-AS1 in cells,and the intracellular distribution of FOXD2-AS1 wasdetected by RNA nucleoplasmic separation.5.After silencing FOXD2-AS1,the expression of downstream proteins weredetected by RT-PCR and Western blot.Results1.RT-PCR results showed that FOXD2-AS1 was significantly higher inosteosarcoma tissues than in para-tumor tissues(32/40),which with a positiveexpression rate of 80%.FOXD2-AS1 was significantly higher in osteosarcomatissues and cells than in para-tumor tissues and Human SV40 osteoblasts.Kaplan-meier analysis showed that the survival period of patients with highFOXD2-AS1 expression was significantly lower than that of patients with lowFOXD2-AS1 expression.2.FOXD2-AS1(shRNA)lentivirus was successfully constructed to stably interferewith FOXD2-AS1 and to stably transfected osteosarcoma cells SOSP-9607 andU20S.The expression of FOXD2-AS1 was significantly down-regulated afterstably transfected SOSP-9607 and U20 S,which was suggested by RT-PCRresults.3.CCK8,cell cycle,clone formation assay,cell migration,invasion into experimentand tumor formation in nude mice found after silence FOXD2-AS1 gene,osteosarcoma cell proliferation,migration,invasion,and cloning abilitysignificantly suppressed compared with the control group,nude mice after silenceFOXD2-AS1 genes into tumor experiment found that,in situ tumor significantlysmaller than the control group,the pulmonary metastasis nodule is also reduced.4.Fluorescence in situ hybridization and RNA nucleoplasmic separationexperiments showed that FOXD2-AS1 was mainly expressed in the cytoplasmcompared with the nucleus.RT-PCR and Western blot experiments showed that,after down-regulating FOXD2-AS1 expression in cells,the expression levels ofdownstream proteins RRM2 and PHGDH decreased significantly.Conclusions1.Compared with normal tissues,FOXD2-AS1 is highly expressed in osteosarcomatissues,and the expression level of FOXD2-AS1 is significantly correlated withthe survival of patients with osteosarcoma patients.The expression ofFOXD2-AS1 was significantly higher in osteosarcoma cells than hFOB1.19.2.After the stable and low expression of FOXD2-AS1,the proliferation,cloneformation assay,migration and invasion abilities of osteosarcoma cells weresignificantly reduced.Tumorigenesis experiments in nude mice showed that thegrowth of tumor in situ slowed down and the metastatic nodules of lung werealso reduced.3.The expression of FOXD2-AS1 was mainly located in the cytoplasm,and theexpression of downstream proteins RRM2 and PHGDH was significantlyreduced after down-regulation of FOXD2-AS1.4.FOXD2-AS1 may be a potential target for the diagnosis and treatment ofosteosarcoma,and may provide a research basis for further exploring themolecular mechanism of FOXD2-AS1 involved in pulmonary metastasis ofosteosarcoma.