Study On The Mechanism Of XIST/miR-193a-3p/RSF1 To Regulate The Occurrence And Development Of Osteosarcoma

BackgroundOsteosarcoma is one of the most common primary malignant bone tumors,mainly pathogenesis in teenagers under the age of 20,its growth is rapid,high malignant degree,strong,prone to early transfer characteristics such as oncology,high mortality prognosis.At present the treatment of osteosarcoma with neoadjuvant chemotherapy and chemotherapy during the week of extensive tumor resection method is given priority to,combined treatment of multi-mode will obviously improve the 5-year survival rate of patients,but is still not ideal.Therefore,it is urgent to find an effective diagnosis method and a new treatment or combination therapy strategy for osteosarcoma.RSF1,also known as hepatitis b x-antigen associated protein(HBXAP),is a family member of atp-dependent chromatin remodeling factor 5.RSF1 interacts with sucrose non-fermentable protein 2(hSNF2H)to form the RSF-1/hSNF2 H complex,which in turn plays a role in different biological and pathological processes by regulating nucleosome remodeling,including regulation of transcription,DNA replication and cell cycle.Recently,RSF1 has been found to have abnormal expression and dysfunction in solid tumors.Studies have shown that RSF1 is highly expressed in renal carcinoma and is significantly correlated with TNM staging of tumor and poor prognosis.RSF1 is highly expressed in lung cancer.Reducing the expression of RSF1 in lung cancer cells can lead to the stagnation of lung cancer cells at G1 stages,and at the same time increase the apoptosis of the cells and inhibit the invasion of the cells.In addition,reducing RSF1 can increase sensitivity of lung cancer cells to paclitaxel.However,the role and possible mechanism of RSF1 in osteosarcoma remain unclear.Therefore,it is of great clinical significance to explore the expression of RSF1 in osteosarcoma,its upstream genes and possible regulatory modes.With the study of the epigenetic mechanism of osteosarcoma formation,it is increasingly recognized that non-coding RNA,DNA methylation,histone modification and chromatin remodeling play an important role in the formation and progression of osteosarcoma.Non-coding RNA is a class of non-protein-coding transcripts,which can be classified according to their function or length.Short-chain non-coding RNA mainly includes microRNA,siRNA,piRNA and so on.MicroRNA plays an important role in the occurrence and development of many tumors and is an important regulator of gene expression.MicroRNAs are usually up-regulated or down-regulated in different types of human tumors,acting on different targets to regulate the proliferation and invasion of cancer cells.For example,it has been found that microRNA-154 participates in the proliferation,invasion and metabolism of many kinds of cancer cells.MicroRNA-154 inhibits bladder cancer cells by targeting RSF1.At the same time,microRNA-193a-3p is abnormally expressed in many kinds of tumors.In lung cancer,microRNA-193a-3p inhibits the metastasis of osteosarcoma cells by down-regulating ERBB4,and microRNA-193a-3p inhibits the metastasis of osteosarcoma cells by down-regulating Rab27 B.However,whether microRNA-193a-3p is involved in the regulation of RSF1 has not been reported in the literature.Long non-coding RNA(lncRNAs)constitute another major class of non-protein-coding rnas that are typically over 200 nucleotides in size,and the study identified approximately 7,000-23,000 lncRNAs.In the cytoplasm,lncRNAs regulate mRNA stability and translation,acting as precursors to tiny RNA(miRNA),or competitive endogenous RNA(ceRNA),regulating miRNA.In the nucleus,lncRNA can play a role at the epigenetic and genetic levels by interacting with the pretranscriptional initiating complex at the promoter.With the deepening of lncRNA research,researchers have gradually realized that lncRNA may have great potential in the diagnosis and treatment of tumors.The abnormal expression of lncRNA in cancer tissues is of great significance in the diagnosis of this cancer,especially in the early diagnosis of small tumors.In clinical diagnosis,the early diagnosis of the disease is made by detecting the lncRNA expression level in the blood of patients.LncRNA also plays an important role in tumor therapy.Through studies on the role of lncRNA in pathogenesis,more accurate and effective therapeutic targets and strategies are found for the clinic.In recent years,more and more studies on lncRNA in osteosarcoma have been conducted.The study found that the expression of long non-coding RNA DNACR was significantly increased in osteosarcoma tissue specimens and osteosarcoma cell lines,and the high expression of DNACR was closely related to the poor clinical prognosis of osteosarcoma patients.Cellular functional experiments showed that low-expression DNACR could inhibit the proliferation and metastasis of rock1-mediated osteosarcoma cells.Distal location in lncRNA family is important,but its role in the development and progression of osteosarcoma is still unclear.The lncRNA counterpart(x-inactive specific transcript)is the main regulatory factor of the endowment gene.LncRNA vulgaris has been shown to play a key role in cell proliferation,differentiation and genome maintenance.In addition,it can target mir-92 b to inhibit the proliferation and metastasis of hepatoma cells,which reveals the importance of the signal axis of wings/mir-92 b /Smad7 in the occurrence and development of hepatocellular carcinoma,suggesting that senses can be used as an important biomarker for the clinical diagnosis and treatment of hepatocellular carcinoma.XIST and AR were up-regulated and positively correlated in bladder cancer.High involvement and AR expression were associated with poor TNM staging of bladder cancer.Besides,this inhibiting effect can be partially recovered through AR overexpression.The important role of the specific/mir-124 /AR regulatory axis in bladder cancer cell growth,invasion and migration was revealed.In order to clarify the role of RSF1 in the development of osteosarcoma,we conducted three parts of experiments: first,we studied the expression of RSF1 in osteosarcoma and analyzed its clinical data.Secondly,interference of RSF1 expression in osteosarcoma cells to study its biological function and possible pathways.Thirdly,the upstream regulation pathway of RSF1 is studied to determine the regulation axis.Part 1: The expression of RSF1 in osteosarcoma ObjectiveRSF1(HBXAP),is a member of ATP-dependent chromatin remodeling factor.Dysregulated RSF1 has been reported to be related to tumor progression.However,the function of RSF1 in osteosarcoma(OS)remains unclear.MethodThe clinical specimens of 41 patients with osteosarcoma were collected,and the expression of RSF1 in the osteosarcoma tissues and adjacent non-tumor tissues was detected by immunohistochemistry and quantitative real-time polymerase chain reaction(qRT-PCR).We then explored the correlation between RSF1 expression and clinicopathological characteristics and overall survival of patients by Kaplan-Meier analysis.Moreover,we used qRT-PCR and Western blot to determine the expression of RSF1 in OS cell lines.ResultsIn the present study,immunohistochemistry showed that the expression level of RSF1 protein in osteosarcoma samples was significantly increased.QRT-PCR showed that the mRNA expression level of RSF1 in osteosarcoma samples was also significantly increased than that in adjacent non-tumor tissues.Correlation analysis showed that high expression of RSF1 were associated with advanced clinical stage,and metastasis.Kaplan-Meier analysis revealed that high RSF1 expression was associated with low overall survival in osteosarcoma patients.Moreover,qRT-PCR and Western blot showed that RSF1 expression was highly expressed in osteosarcoma cell lines(U2OS?Saos2?HOS?MG63)compared to human osteoblast cell line NHOst.ConclusionThis study shows that RSF1 is highly expressed in osteosarcoma and is closely related to the advanced clinical stage,metastasis and prognosis of patients with osteosarcoma,suggesting that RSF1 plays an important role in the progression of osteosarcoma and can serve as a potential novel prognostic biomarker for osteosarcoma.Part 2:The role of RSF1 expression in the biological characteristics of osteosarcomaObjective Previous study showed that RSF1 was increased and associated with advanced clinical stage,metastasis and poor prognosis of osteosarcoma patients.However,the roles of RSF1 in osteosarcoma cells remain unclear.Method Saos2 and MG63 cells were selected and sh RNA was used to knockdown the RSF1 expression in OS cells.The effects of RSF1 on proliferation,cell cycle,apoptosis,migration and invasion of osteosarcoma cells were detected by MTT assay,cloney formation assay,flow cytometry,Wound healing assay,and Transwell invasion assay.The effects of RSF1 on MAPK/ERK signaling pathway in osteosarcoma cells was detected by Western blot.Furthermore,we explored the roles of RSF1 in nude mouse model.Results In this study,we successfully constructed interfering RNA and effectively inhibited the expression of RSF1 in Saos2 and MG63 cells.MTT assay results showed that reducing RSF1 expression in osteosarcoma cells significantly inhibited cell proliferation.Clonal formation experiments also showed similar results.Further flow cytometry techniques have shown that reducing RSF1 expression in osteosarcoma cells can arrest osteosarcoma cells in the G0/G1 phase and promote apoptosis in osteosarcoma cells.Further wound experiments and Transwell invasion experiments showed that reducing RSF1 expression in osteosarcoma cells significantly inhibited the migration and invasion of osteosarcoma cells.Mechanism studies suggest that reducing RSF1 expression in osteosarcoma cells can significantly inhibit phosphorylated ERK1/2,p38 and MEK in osteosarcoma cells,suggesting that low expression of RSF1 can inhibit MAPK/ERK pathway and thereby regulate osteosarcoma cell growth and Transfer.In vivo experiments in nude mice showed that reducing the expression of RSF1 significantly inhibited the growth of osteosarcoma cells in vivo.Conclusion RSF1 promotes proliferation,invasion and metastasis of osteosarcoma cells by inducing and activating MAPK/ERK signaling pathways.It is suggested that RSF1 is an important molecule in the development of osteosarcoma and proposes new treatment ideas for future research.Part3:XIST/mi R-193a-3p targeting regulates RSF1 expression in osteosarcoma cellsObjective Non-coding RNA plays an important role in tumorigenesis and metastasis.It is necessary to search for mi RNAs that may regulate RSF1 and upstream lnc RNA genes that may regulate mi RNAs,so that the entire regulatory mechanism is more clear,looking for a regulation axis from lnc RNA to mi RNA to target genes.The axis provides a new theoretical and experimental basis for the future targeted therapy of osteosarcoma.Methods The upstream mi RNA of RSF1 was explored by database search and luciferase reporter assay,and the effect of mi RNA on the expression of RSF1 in osteosarcoma cells was detected by q RT-PCR and Western blot.The expression of mi RNA in osteosarcoma was detected by q RT-PCR.We then explored the correlation between mi RNA expression and clinicopathological characteristics and overall survival of patients by Kaplan-Meier analysis,and correlated the expression of mi RNA and RSF1 to determine the targeting relationship between them.Further through bioinformatics analysis,pull down experiments and luciferase reporter experiments to find lnc RNA that can bind to mi RNA,and determine the targeting relationship between them,by blocking the lnc RNA in osteosarcoma cells by rescue experiments,to understand its dependence on mi RNA to RSF1 Regulatory role.Results The public database(Target Scan,micro RNA,mi RDB)showed that mi R-193a-3p has a binding site in the 3'-UTR region of RSF1 m RNA.Luciferase assay experiments showed that mi R-193a-3p binds to RSF1 and inhibits its expression.q RT-PCR and Western blot experiments further confirmed that mi R-193a-3p can specifically inhibit the expression of RSF1,Further studies showed that mi R-193a-3p was down-regulated in osteosarcoma and was closely associated with advanced clinical stage and metastasis in patients with osteosarcoma,and survival was significantly reduced.Correlation analysis also showed a negative correlation between mi R-193a-3p and RSF1 expression in osteosarcoma.Further,the lnc RNA XIST capable of binding to mi R-193a-3p was found by searching the database,and the targeting relationship between XIST and mi R-193a-3p was confirmed by pull-down experiments and luciferase reporter experiments.Rescue experiments showed that XIST can achieve positive regulation of RSF1 by inhibiting mi R-193a-3p,thus confirming that XIST plays a ce RNA in regulating osteosarcoma progression.Conclusion Our study showed that XIST may act as an oncogene in osteosarcoma,negatively regulating the expression level of mi R-193a-3p,thereby attenuating its negative regulation of RSF1 expression,resulting in increased expression of RSF1.The XIST/mi R-193a-3p/RSF1 axis provides novel insight into the molecular mechanisms of osteosarcoma carcinogenesis,which provides a new direction for the future treatment of osteosarcoma.