| Osteosarcoma is the most common primary malignant bone tumor in children and adolescents.It is highly aggressive and easy to metastasize early,so that the patient has a fast course of disease and a poor prognosis,which often poses a fatal threat.Although the treatment of tumors has been greatly improved in recent years,it is difficult to break through the complex pathogenesis of osteosarcoma and to achieve breakthrough progress in the treatment effect.Therefore,in-depth exploration of the molecular mechanism of osteosarcoma and finding the key functional molecules(therapeutic targets)in osteosarcoma are extremely important for the early diagnosis and effective treatment of osteosarcoma.Long non-coding RNAs(lnc RNAs)are a class of large molecules that constitute non-protein coding RNAs.Lnc RNAs can be involved in a variety of cellular biological processes(signaling,decoys,guidance,scaffolds)through interactions with different molecules.In addition,lnc RNAs not only play a role in the regulation of some normal physiological processes,but also participate in the regulation of tumor occurrence,development,distant metastasis,etc.Some lnc RNAs have been proved to be biomarkers(prognostic indicators,therapeutic targets),and even can regulate the sensitivity of patients to chemotherapy drugs.Currently,it has been proved that lnc RNAs play an important regulatory role in the occurrence and development of osteosarcoma.Therefore,continue to explore the lnc RNAs,has not yet been "unlocked" system of multidimensional exploring lnc RNAs in the role of osteosarcoma,screening of lnc RNAs is critical,to dig deeper into its specific regulatory role in osteosarcoma,not only helps to find related pathogenic mechanism of osteosarcoma,can also provide theoretical basis for the accurate treatment of osteosarcoma,contribute to the development of health.In the study of osteosarcoma tissue adjacent to carcinoma and whole transcriptome sequencing analysis of samples,to detect differentially expressed lnc RNAs,miRNAs and m RNAs expression spectrum data,using the biological information statistics research methods,get three differences between gene regulation network data,through the GO functional annotations and KEGG metabolic pathway analysis,to the differences in gene function analysis and data extraction module,filter core lnc RNAs,Lnc RNAs /miRNAs/m RNAs co-regulation networks were constructed and functional analysis of core lnc RNAs was performed,and LINC01614(core lnc RNAs)was verified by cytology and histology.Validation,after having confirmed the regulation of the biological function of osteosarcoma(proliferation,migration,invasion,and apoptosis,etc.)experiment,combining LINC01614/miR-520a-3p/SNX3 relationship between regulation and verify further to the control shaft and determine its regulation function in osteosarcoma,thus clear LINC01614 by miR-520a-3p/SNX3 control shaft promote the progress of osteosarcoma.Part 1 Construction of lnc RNAs as the core regulatory network and screening of key genes in osteosarcomaWe use three sets of osteosarcoma tissue adjacent to carcinoma and samples whole transcriptome sequencing for the molecular expression spectrum data,through the comparative analysis of screen 163 differentially expressed lnc RNAs cut express(raised express,67,96),207 differentially expressed miRNAs cut express(raised express,92,115)and 4247 different differences m RNAs(cut express 2019 raised expression,2228).Integrated application of LNCipedia,DAVID,a variety of database such as STRING,using the method of statistical analysis of biological information,obtain lnc RNA/miRNA/m RNA lnc RANs control network and the difference expression of target genes for raw letter function analysis,found that differences in osteosarcoma expression lnc RNAs may have a regulation of bone cell differentiation,protein,biological macromolecular synthetic function,and may participate in the PI3K-Akt,AMPK is closely related to the development of tumor metabolic pathways.Additionally we expressed significant difference through selecting the LINC01614,MALAT1 two lnc RNAs,respectively built with both as the core of lnc RNA/miRNA/m RNA control network,and is applied to the target gene regulation network to the raw letter function analysis and analysis of protein interaction network,find LINC01614 target genes may be involved in regulating the Wnt signaling pathways,cell migration,cancer biology process;The target gene of MALAT1 may be involved in the regulation of osteogenic differentiation,protein transport and other biological processes.Further we use osteosarcoma cells and normal osteoblasts and specimens of osteosarcoma tissue adjacent to carcinoma and to select the expression profile of these two kinds of lnc RNAs q RT-PCR detection,found in a variety of osteosarcoma cells and osteosarcoma were showing high expression in cancerous tissue,which are consistent with the results of the letter before we born,but also for our further digging lnc RNAs in osteosarcoma control function and the related molecular mechanism provides a direction.Part2 Study on the regulatory function of LINC01614 in osteosarcomaThrough the analysis and verification in the first part,LINC01614 was selected as the breakthrough point of this study,aiming to explore its related regulatory functions in osteosarcoma.Firstly,we used the clinical data of patients with osteosarcoma obtained from the "Target" database for survival analysis.It was found that LINC01614 was negatively correlated with patient survival and positively correlated with the case analysis of osteosarcoma,suggesting that LINC01614 may play a negative and adverse role in the occurrence and development of osteosarcoma.Next,in vitro experiments were conducted to detect cell proliferation by CCK-8 assay.The results suggested that silencing LINC01614 could significantly inhibit the proliferation of osteosarcoma cells.Cell migration and invasion ability were detected by Transwell cell invasion assay and cell scratch assay.Silencing LINC01614 was found to inhibit the invasion and migration ability of osteosarcoma cells.Western Blot was used to detect the protein expressions of PCNA,caspase-3 and E-cadherin in the silenced LINC01614 osteosarcoma cells,which confirmed that silencing LINC01614 can inhibit the proliferation,invasion and migration of osteosarcoma cells,and suggested that LINC01614 may be related to the apoptosis of osteosarcoma cells.Then,cell apoptosis was detected by flow cytometry,and silencing LINC01614 was found to promote the apoptosis of osteosarcoma cells.In vivo experiments,it was found that silencing LINC01614 could effectively reduce the tumor formation rate and volume of osteosarcoma in vivo through tumor transplantation experiments in mice.In general,LINC01614 has been shown to promote the progression of osteosarcoma in vitro and in vivo.Part3 Study on the Molecular Mechanism of LINC01614 Regulating the Occurrence and Development of OsteosarcomaBased on the analysis of the regulatory network of LINC01614 in the first part and the regulatory role of LINC01614 in osteosarcoma in the second part,we proposed the hypothesis that LINC01614 may promote the progression of osteosarcoma through the miR-520a-3p/SNX3 regulatory axis.Next,q RT-PCR and Western Blot experiments were used to verify the binding relationship between the three.The binding sites of miR-520a-3p and LINC01614 as well as miR-520a-3p and SNX3 were predicted by using the bioinformatics database,and the binding sites were verified by luciferase reporter gene assay.The results suggested that LINC01614 could regulate the expression of SNX3 by competitively binding with miR-520a-3p.Then through the expression of silence and the miR-520-a-3 p,silent SNX3 and ck of osteosarcoma cells CCK8 cell proliferation experiment,transwell cell invasion and scratched cell experiment and flow cell apoptosis,etc.,the experimental results suggest LINC01614 can indeed by miR-520-a-3 p/SNX3 control shaft effect of osteosarcoma cells proliferation,invasion,migration and apoptosis process of the development of osteosarcoma has certain regulation effect.Finally,we detected the expressions of ?-catenin and Wnt1 proteins in the silenced LINC01614,silenced SNX3,silenced and overexpressed miR-520a-3p osteosarcoma cells by Western Blot.The results suggested that the biological function of LINC01614/miR-520a-3p/SNX3 regulating osteosarcoma may be related to its involvement in the regulation of Wnt signaling pathway,which also provided some ideas for the research group in the future. |
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