Mechanism Study Of LncRNA SNHG25 Promoting The Progression Of Osteosarcoma Through MiR-497-5p/SOX4 Axis

Objective To clarify the expression level of LncRNA SNHG25 in osteosarcoma tissues and cells and analyze whether the expression level of SNHG25 is correlated with the prognosis of osteosarcoma patients.To investigate the function of SNHG25 on proliferation,invasion,migration,apoptosis and epithelial-mesenchymal transition of osteosarcoma cells in vitro.To investigate whether SNHG25 promotes osteosarcoma progression through the miR-497-5p/SOX4 axis.It may provide a new way to evaluate the prognosis of osteosarcoma patients or its molecular targeted therapy in the future.Methods (1)We analyzed RNA-seq sequencing data of osteosarcoma and adjacent normal tissue in the GEO database(GSE126209)to draw a gene heat map.SNHG25 was found to be a differentially expressed noncoding RNA in osteosarcoma.Eighty-eight osteosarcoma cases were included in the high and low expression groups according to the median value of SNHG25 gene expression,respectively.The Kaplan-Meier survival curve was drawn according to patient grouping,follow-up time,and survival status.Log-rank test was used to analyze the differencein survival rate between the two groups.Ten osteosarcomas and paired para-cancer tissue were collected at the General Hospital of Ningxia Medical University.RT-q PCR detected SNHG25 expression levels in osteosarcoma tissues and adjacent normal tissue.(2)The expression level of SNHG25 in osteosarcoma cells(MG-63,U-2OS,Saos-2)was tested by RT-q PCR compared to h FOB 1.19.Lentivirus-mediated sh-SNHG25 was used to infect MG-63 and U-2OS cells.After puromycin screening,stable transfection cell lines with SNHG25 knockdown were constructed.The cell proliferation ability of the sh-SNHG25 group was detected by CCK-8and clone formation assay compared to the sh-NC group.The cell migration and invasion of the sh-SNHG25 group were analyzed by scratch assay and Transwell assay.The cell apoptosis of the sh-SNHG25 group was analyzed by flow cytometry.The expressions of E-cadherin,Vimentin and N-cadherin in the sh-SNHG25 group were detected by Western blotting compared to the sh-NC group.A subcutaneous transplantation model of nude micehas been established to investigate the effect of SNHG25 knockdown on the growth of osteosarcoma cells in vivo.The expression of SNHG25 in the tumour tissues of the sh-NC group and sh-SNHG25-2 group was detected by RT-q PCR,and the protein expression of SOX4 and PCNA in the tumour tissues of the sh-NC group and sh-SNHG25-2 group was detected by Western blotting.(3)Star Base was used to predict the miRNAs with binding sites of SNHG25,and miRNA expression was screened after SNHG25 knockdown by RT-q PCR.Combined with the results,a double luciferase assay determined and verified the miRNA that SNHG25 could bind to.The rescue experiment was performed to analyze whether transfection with a miRNA inhibitor could restore osteosarcoma cells’ proliferation,migration and invasion ability of the sh-SNHG25 group.Combined with Star Base prediction and miRNA mimics sample RNA-seq sequencing data,a Venn diagram was drawn to identify the target genes of miRNA action.The specific binding sites of miRNA and the 3 ’UTR region of the target gene were determined by the dual luciferase assay.The Pearson method analyzed the correlation between miRNA and target gene expression in osteosarcoma.Target gene m RNA and protein expression levels were detected after miRNA knockdown or overexpressed.The rescue experiment analyzed the inhibitory effect of miRNA mimics on osteosarcoma cells.RT-q PCR saw the expression of target gene m RNA,and protein expression was detected by Western blotting and immunofluorescenceafter the knockdown of SNHG25 expression.The rescue experiment was used to analyze whether the target gene overexpression plasmid could remove the inhibition effect of sh-SNHG25 on the proliferation,migration and invasion of osteosarcoma cells.Results (1)The expression of SNHG25 in osteosarcoma tissues was significantly up-regulated compared to adjacent normal tissue.The Kaplan-Meier survival curve showed the survival rate of osteosarcoma patients in the high-expression SNHG25 group(n=44)was significantly lower than that in the low-expression SNHG25 group(n=44)by analyzing the SNHG25 gene expression data and clinical information of 88 osteosarcoma patients in the TARGET database.Log-rank test analysis showed that the survival rate of osteosarcoma patients in SNHG25 high and low expression groups was statistically different,P < 0.05.(2)After SNHG25 knockdown,the proliferation of MG-63 and U-2OS cells was significantly decreased by CCK-8 and colony formation assay.The migration and invasion of osteosarcoma cells were reduced in the knockdown group,while the apoptosis rate was increased.The expression of E-cadherin protein increased,Vimentin and N-cadherin protein decreased after SNHG25 knockdown.The experimental results of nude micewith the tumour in vivo showed that the subcutaneous tumour volume of the sh-SNHG25-2 group was significantly smaller than that of the sh-NC group.The results showed that the expressions of SNHG25,SOX4 and PCAN in the sh-SNHG25-2 group were substantially lower than those in the sh-NC group by RT-q PCR and Western blotting.(3)The expression miR-497-5p was downregulated in osteosarcoma tissues compared to para cancer tissue,which inhibited proliferation,migration and invasion of osteosarcoma cells.The Pearson method negatively correlated the expression levels of SNHG25 of osteosarcoma tissue and miR-497-5p(r=-0.708 P=0.022).After SNHG25 knockdown,miR-497-p expression was increased.The rescue experiment confirmed that transfection with miR-497-4p inhibitor could partially restore osteosarcoma cells’ proliferation,migration and invasion ability in the sh-SNHG25-2group.Dual luciferase assay confirmed the presenceof binding sites between miR-497-5p and SOX4.The target gene of miR-497-5p is SOX4.The expression of miR-497-5p in osteosarcoma tissue was negatively correlated with that of SOX4(r=-0.636 P=0.048).RT-q PCR and Western blotting showed that SOX4 m RNA and protein expression decreased after transfection with miR-497-5p mimics.In contrast,SOX4 m RNA and protein expression increased after transfection with miR-497-5p inhibitor.The rescue experiment confirmed that transfection of pc DNA3.1-SOX4 could partially remove inhibition of miR-497-5p overexpression on osteosarcoma cells’ proliferation,migration and invasion.After SNHG25 knockdown,the m RNA and protein expression of SOX4 was significantly decreased.SNHG25 was positively correlated with SOX4 expression in osteosarcoma(r=-0.718P=0.019).The rescue experiment showed that transfection of pc DNA3.1-SOX4 could partially restore osteosarcoma cells’ proliferation,migration and invasion ability in the sh-SNHG25-2 group.Conclusion (1)LncRNA SNHG25 expression is up-regulated in osteosarcoma tissues and cells.Up-regulated SNHG25 expression predicted poor prognosis in osteosarcoma patients.(2)Knockdown of the expression of SNHG25 could inhibit the proliferation,migration and invasion of osteosarcoma cells and promote cell apoptosis in vitro.SNHG25 knockdown could inhibit the growth of osteosarcoma cells in vivo.(3)SNHG25 may indirectly regulate the expression of SOX4 and the EMT process of osteosarcoma cells.(4)LncRNA SNHG25 may promote proliferation,invasion and migration of osteosarcoma cells through the miR-497-5p/SOX4 axis.