| Objective:Osteosarcoma(OS)originates from mesenchymal tissue and is the most common malignant bone tumor in adolescents.It has the characteristics of high recurrence rate,early strong aggressivity,easy metastasis,especially pulmonary metastasis.The early diagnosis of osteosarcoma is relatively difficult and its cure rate is low.At present,the main treatment of osteosarcoma is a combination of neoadjuvant chemotherapy and surgical resection,but the effect is still not satisfactory.With the development of the molecular biology and the research on molecular genetics of tumors,non-surgical treatments such as gene-targeted therapy provide a new way for the treatment of osteosarcoma.URG4 is a gene that up-regulates cell proliferation and has recently been found to play an important role in tumorigenesis,development,invasion and metastasis.The purpose of this study was to investigate the expression and clinical significance of URG4 in osteosarcoma,as well as its effects on osteosarcoma proliferation,migration,invasion and tumorigenicity and related mechanisms.Method:1.After collecting osteosarcoma specimens and clinical data of related patients,Immunohistochemical staining was performed to detect the protein expression of URG4 in 40 cases of osteosarcoma and adjacent tissue to the corresponding tumor,and then analyzed the relationship of URG4 expression and its clinical pathology data.2.QPCR and Western-blot was applied to detect the m RNA level and the protein expression of URG4 gene in human osteosarcoma cell lines MG63,HOS,143 B,U2OS,Saos-2 and human osteoblast h FOB1.19.After being established stable osteosarcoma cell models of being knockdown of URG4(si URG4-1,si URG4-2)and empty carrier(NC),CCK8,plate cloning,flow cytometry,scratch,Transwell and nude mice subcutaneous tumorigenesis experiments was used to detect cell proliferation,apoptosis,cycle,migration,invasion and tumorigenic ability.These are to clarify the effect of URG4 gene on the biological function of osteosarcoma cells.3.Firstly,the co-expressed genes with URG4 were screened through the MEM database and the TARGET database;GO analysis on the URG4 coexpressed genes was performed to explore the possible effects of co-expressed genes by using the DAVID6.8 online database;The different signal pathways Enrichment of URG4 co-expressed genes were analyzed to explore the signal pathways that it may participate in by KEGG database;URG4 interacting proteins were analyzed by STRING database.Secondly,Nuclear staining was used to observe the key protein ?-catenin expression of the classic Wnt pathway in the osteosarcoma cell nucleus after URG4 knockdown.Finally,Western-blot was used to detect the total protein expressions of GSK3?,P-GSK3? and the nuclear protein expression of ?-catenin in Wnt passway and the protein expressions of its downstream target genes(c-Myc,Cyclin D1,MMP-2,MMP-9)after URG4 being knocked down.Result:1.It was showed in the clinical relevance:(1)URG4 expression in 40 osteosarcoma tissues(0 negative,1 weak positive,17 positive,22 strong positive)positive and strong positive rate is 97.50%,and its expression in the adjacent tissue to the tumor(2 negative cases,37 weak cases,1 positive case,0strong case)positive and strong positive rate is 2.50%,they have statistically significant(P<0.001);(2)The expression of URG4 is closely related to the size,metastasis and Enneking stage of osteosarcoma: URG4 high expression rate of less than 5 cm tumor is 53.85%,its high expression rate of the tumor with ?5cm is 85.71%,their comparisons have statistical significance(P<0.05).The high expression rate of URG4 in metastatic tumors was 92.31%,and the high expression rate of non-metastatic tumors was 74.07%.The comparison between the two was P=0.012,which was statistically significant(P<0.05).The high expression rates of URG4 in patients with Enneking stage I,II,and III were22.22%,76.00%,and 83.33%,respectively.The difference between stage I and II and III was significant and statistically significant(P<0.05).There was no statistically significant between phase II and phase III(P>0.05).With the development of the tumor,URG4 high expression rate also increases;(3)The relationship between URG4 expression and gender,age,location,tumor necrosis rate,and tissue type is not different,there was not statistically significant(P>0.05).2.The results of QPCR and Western Blot confirmed that the expression of URG4 in five human osteosarcoma cell lines was higher than that of normal osteoblasts.3.Knockdown of URG4 can inhibit the proliferation of osteosarcoma cells:CCK8 showed that the proliferation of two types of osteosarcoma cells is significantly inhibited after 3 and 4 days after knockdown of URG4,which is statistically significant compared with the negative control group(NC group)(P<0.001).Plate cloning experiments showed that the cloning ability of the two URG4 knockdown osteosarcoma cells was significantly lower than that of the NC group,and the difference was statistically significant(P<0.01).4.The flow cytometry test showed that the apoptosis rate of osteosarcoma cells of the two URG4 knockdowns was significantly higher than that of the control group,and the difference was statistically significant(P<0.01).Knocking down URG4 can make osteosarcoma cells stay in the G0/G1 phase of the cell cycle,so that the number of cells entering the S phase is less,thereby inhibiting the growth cycle of osteosarcoma cells,and thus inhibiting cell growth(P<0.05).5.The wound healing experiment proved that the cell migration repair area of the cell scratches in the URG4 knockdown group was significantly smaller than that in the NC group,and the difference was statistically significant(P<0.01).6.The Transwell migration experiment showed that knocking down URG4 can reduce the migration ability of the two osteosarcoma cells,and the difference is statistically significant compared with the control group(P<0.01).7.The tumor formation experiment in nude mice proved that the tumor growth of URG4 knockdown group was slower,the tumor volume was significantly reduced,and the tumor weight was significantly reduced.The difference was statistically significant compared with the control group(P<0.01).8.8319 and 784 genes related to URG4 expression were screened by the MEM database and TARGET database,and 396 genes co-expressed with URG4 were obtained at the intersection of two parts.9.GO analysis showed that URG4 co-expressed genes were mainly involved in biological processes such as protein transport,intracellular protein transport,and cage clathrin-mediated endocytosis,and cellular components such as nucleocytoplasm,cytosol,and cytoplasm.protein binding,poly(A)RNA binding and ATP binding and other molecular functions were Involved.10.The results of KEGG suggest that URG4 co-expressed genes are mainly involved in the regulation of Wnt signaling pathway,cell cycle,Hippo signaling pathway and other signaling pathways,and Wnt signaling pathway is the most meaningful(P=0.012891).11.STRING database shows that there are 10 kinds of proteins interacting with URG4(including Cyclin D1,MYC,MMP9).12.The results of nuclear staining showed that ?-catenin expression in the Osteosarcoma cell nucleus nucleus decreased significantly after URG4 being knocked down;Western blot results showed that the total GSK3? protein expression was not Significant difference(P>0.05)and the expression of GSK3?ser9 phosphorylated protein and ?-catenin protein in the nucleus was significantly reduced(P<0.001)in the osteosarcoma cells after URG4 being knocked down.The expression levels of Cyclin D1,c-Myc,MMP-2,and MMP-9 in osteosarcoma cells with URG4 knockdown were significantly lower than those in the control group.Conclusion:1.URG4 is highly expressed in osteosarcoma tissue,but it is not expressed or lowly expressed in adjacent tissue to the tumor.The expression of URG4 in osteosarcoma is positively correlated with tumor size,tumor metastasis,and degree of Enneking stage,but has no significant correlation with gender,age,location,tumor necrosis rate,and tissue type.2.URG4 is highly expressed in human osteosarcoma cell lines.Knockdown of osteosarcoma cells URG4 expression can promote apoptosis,block the cell cycle,inhibit the proliferation of osteosarcoma cells,reduce cell migration and invasion,and reduce the tumorigenic ability of nude mice.These prove that URG4 can promote the development,invasion and metastasis of osteosarcoma in turn.3.The nucleus protein expression of ?-catenin in the osteosarcoma cells was reduced after knockdown of its URG4 expression,that inhibits the activity of the classical Wnt pathway,and thus the expression of its downstream target genes Cyclin D1,c-Myc,MMP-2,and MMP-9 was inhibited.The results of this study indicate that URG4 can promote the proliferation,invasion and metastasis of osteosarcoma by activating the classic Wnt signaling pathway.URG4 may be a new biomarker and potential therapeutic target for osteosarcoma. |
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