The Mechanism Of Hsa＿circ＿0008928 In Osteosarcoma Migration Regulation

Osteosarcoma（osteosarcoma,OS）is a type of malignant tumor of mesenchymal tissue originating from mesenchymal cells.It is the most common type of malignant bone tumor,accounting for 20%of primary bone tumors.The annual incidence is about 3%.It occurs in children and adolescents and is the main cause of death in children and adolescents.Osteosarcoma itself has high malignant degree,strong invasiveness,easy metastasis and other biological characteristics.It often shows a fast growth rate in human body,and it is extremely easy to metastasize beyond bone tissues.Up to 20%of patients with osteosarcoma have distant metastases at the time of diagnosis,and lung metastases are more common,accounting for about 90%.Existing treatments include amputation surgery and chemotherapy,which have very limited effects on patients with lung metastasis,and the 5-year survival rate is only 20%to 30%.Although it seriously threatens the health of adolescents and imposes a fatal burden on countless families,what is worse is that since the 1970s and the present,the research and clinical treatment of osteosarcoma has gone through a deserted period of 50 years,and it still cannot explain The biological behavior of the tumor,and the current treatment is still not satisfactory for improving the prognosis of osteosarcoma patients.With the popularity of second-generation sequencing technology and the exponential growth of global biomedical big data,non-coding RNA provides new ideas for malignant tumor research.circular RNA（circRNA）is a large class of non-coding RNA.It is more common in the cytoplasm or exosomes.Due to the lack of a 5’ cap and 3’ tail structure,it is mistaken for an abnormal by-product and has a small functional potential.However,with the advancement of detection methods and quantitative algorithms,the potential biological value of a large number of circRNAs in various tumors has gradually received attention.This provides new possibilities for fully exploring the molecular events of osteosarcoma under the slow progress of the existing exploration mode.Based on the above considerations,this paper is the first to construct the expression profile of circRNA in osteosarcoma in order to fully understand the situation of this new type of small molecule in osteosarcoma.Not only that,we have passed multiple verifications through strict screening,cell,tissue and big data,confirming the robustness of our candidate circSATB2 in the importance of osteosarcoma.In the work of this thesis,we have for the first time fully demonstrated the possibility of high expression of circSATB2 from many aspects such as ceRNA hypothesis mechanism,changes in transcriptome level,and epigenetic regulation of histone could promote the metastasis of osteosarcoma.Not only that,based on the new use of old medicines and the promotion of traditional Chinese medicine,we first discovered at the therapeutic level that nitidine chloride can inhibit the expression of circSATB2 and restrain the migration of osteosarcoma.Specifically,we have completed the following parts:In the first part,we constructed the circRNA expression profile of osteosarcoma tissue for the first time.In the in house circRNA chip we found a total of 500 significantly differentially expressed circRNA in osteosarcoma tissue,which fully shows that circRNA is very likely to participate in the development of osteosarcoma.Therefore,we conducted an in-depth analysis of the expression profile.Through strict quality control,the significantly high expression of the three circRNAs derived from the host gene SATB2（hsa_circ₀003915,hsa_circ₀007422 and hsa_circ₀008928）aroused our interest.By screening suitable osteosarcoma cell lines,we further confirmed the important value of hsa_circ₀008928 in further cell overexpression and knock down experiments.Through a large number of repeatable experiments,we have identified that hsa_circ₀008928 is significantly overexpressed in osteosarcoma tissues and promotes osteosarcoma cell migration.In the second part,we further explored the molecular mechanism of hsa_circ₀008928 to promote osteosarcoma cell migration.In this part of the work,we fully explored the possibility of hsa_circ₀008928 functioning based on the classic ceRNA hypothesis.The FISH test confirmed that circSATB2 is expressed in the nucleus and cytoplasm,which fully shows that circSATB2 has the prerequisites for ceRNA regulation.Through effective integration of database resources,we preliminarily presume the hsa_circ₀008928/miR-330-5p/SRF regulatory axis,and further verified the targeted binding relationship between the three genes by dual luciferase.Not only that,after overexpressing hsa_circ₀008928,the target gene SRF protein was significantly increased,which provided further evidence support for the regulatory relationship between the two.We finally confirmed the joint effect of hsa_circ₀008928 and miR-330-5p on migration behavior through cell migration function recovery experiments.In summary,this part systematically verifies that in osteosarcoma hsa_circ₀008928 may play one of the important biological function mechanisms--ceRNA regulation mode.In the third part,we comprehensively observed the possible impact of hsa_circ₀008928 on the overall level of transcriptome and genome in osteosarcoma,in order to understand the main ways in which it plays a role.We first obtained the differentially expressed genes in the transcriptome after overexpression of hsa_circ₀008928 by RNA-seq,and also found significant enrichment of pathways related to migration regulation.And GESA analysis suggests that hsa_circ₀008928 is closely related to histone modification H3K27ac.We speculate that hsa_circ₀008928 may participate in the regulation of osteosarcoma migration through more complex epigenetic regulation.Based on the above considerations,we tested H3K27ac modification changes in the hsa_circ₀008928 overexpression cell line.ChIP-seq differently peak related genes are also significantly enriched in the cell migration pathway.This further confirms that the high expression of hsa_circ₀008928 promotes the migration of osteosarcoma,which may specifically affect the migration of downstream target genes by affecting the modification of H3K27ac.In the fourth part,we first explored other potential molecular mechanisms that hsa_circ₀008928 affects the migration of osteosarcoma,in order to obtain more accurate information on the role of hsa circ 0008928 in osteosarcoma.We integrated the translation element ORF and IRES for the first time to discover the polypeptide translation potential of hsa_circ₀008928,and through the construction of hsa_circ₀008928 translation potential tag-peptide fusion protein recombinant vector construction,protein level detection of peptide products,to explore hsa_circ₀008928 coding ability.The experimental results did not detect the translation product of hsa_circ₀008928,which fully indicates that hsa_circ₀008928 may not have a regulatory mechanism about polypeptide encoding,and further narrows the exploration of the hsa_circ₀008928 mechanism.In the fifth part,we fully combined with the clinical difficulties of osteosarcoma metastasis,and discussed the impact of intervention on osteosarcoma.This part of the work is based on the new use of old medicines and the promotion of traditional Chinese medicine.In other tumor treatment studies our team found that the small molecule nitidine chloride derived from traditional Chinese medicine has inhibitory effects on tumors.It was found that nitidine chloride could down-regulate the expression of hsa_circ₀008928 and it also has the function of inhibiting the migration of osteosarcoma cells.The transcriptome level sequencing data after the treatment of nitidine chloride suggests that nitidine chloride is very likely to act on DRD1,CCL24,SEMA3C,BMP4 and SYNE2 by hsa_circ₀008928 to affect the migration of osteosarcoma.Through the work in this part,we first explored the possible mechanism of nitidine chloride inhibiting osteosarcoma migration through hsa_circ₀008928,providing new ideas for clinical research on osteosarcoma treatment,and providing a richer molecular theoretical basis for traditional Chinese medicine anti-tumor.In summary,this project selected the key small molecule hsa_circ₀008928 by constructing the osteosarcoma circRNA expression profile,and discussed the main ways that hsa_circ₀008928 may play a role from the transcriptome level,genomic level,and protein level.Through a large number of molecular experiments,we identified the possibility of ceRNA as a key regulatory mechanism.Not only that,based on clinical difficulties,we first explored the molecular mechanism by which nitidine chloride may inhibit osteosarcoma migration,providing a rich theoretical basis for the promotion of traditional Chinese medicine.