BPNT1 Regulates GSK3β Phosphorylation To Stabilize β-catenin To Promote The Proliferation,invasion And Migration Of Human Osteosarcoma

Objective: Osteosarcoma(Osteosarcoma,OS)is a bone-like matrix derived from the malignant proliferation of primitive mesenchymal cells.It is the most common malignant primary bone tumor,which mostly occurs in children and adolescents and may be associated with sudden growth during adolescence,is second only to the third highest incidence of lymphoma and brain tumors of adolescent malignant tumor,male patients more than women.The second peak of osteosarcoma onset is around age 65 years and usually presents as a secondary tumor.With the development of neoadjuvant chemotherapy as a treatment modality,the prognosis of patients has improved,but the therapeutic effect of osteosarcoma is still unsatisfactory due to the high rate of metastasis or recurrence.With the development of molecular targeted therapy technology,scholars’ research on osteosarcoma is also focused more on the molecular mechanism,trying to improve the clinical diagnosis and treatment effect from the molecular mechanism,and reduce the mental and economic burden of patients,their families and the society.BPNT1 is a novel mammalian lithium-sensitive enzyme with phosphatase and nucleotiase activities and belongs to the family of inositol monophosphatases.The activity of the BPNT1 phosphatase allows it to hydrolyze 3’(2’)-phosphate adenosine 5’-phosphate(PAP)into adenosine monophosphate(AMP).Previous studies found that mice with BPNT 1 gene deletion accumulated excessive PAP in vivo,causing liver damage and even failure,and mice developed hypoproteinemia,edema and even death.BPNT1 inhibition also leads to selective neuronal dysfunction,which may participate in the development of head and neck squamous cell carcinoma and the glycolytic process of gastric cancer,but its function and action mechanism in osteosarcoma are still unclear.Therefore,this study sought to investigate the role and the underlying mechanism of BPNT1 in osteosarcoma.Methods: 1.The analysis of BPNT 1 expression in osteosarcoma by online bioinformatics databases including TCGA,GEO and CCLE,Western blot(WB)and real-time PCR(RT-q PCR)were used to detect BPNT1 expression in osteosarcoma cell lines and normal cell,and determine the localization of BPNT1 in osteosarcoma cells by cellular immunofluorescence experiments.The expression of BPNT 1 in the osteosarcoma tissue microarray was detected by immunohistochemical staining.2.The constructed BPNT1 overexpression plasmid and small interfering RNA(si RNA)were transferred into osteosarcoma cells by liposome transfection(plasmid transfection),and the transfection efficiency was measured by WB for downstream experiments.3.The effects of BPNT 1 overexpression and knockdown on osteosarcoma cell proliferation capacity in vitro was tested by CCK-8 cell proliferation assay,colony formation assay and Ed U cell proliferation assay,and the effects of upregulated BPNT 1 expression in osteosarcoma in vivo was evaluated by xenograft experiments in nude mice.4.The effect of BPNT 1 overexpression and knockdown on the invasive capacity of osteosarcoma cells was evaluated by Transwell invasion assay,and the effect of BPNT 1 overexpression and knockdown on the migratory capacity of osteosarcoma cells was evaluated by Transwell migration assay and wound healing assay.5.Potential mechanism of BPNT1 function in osteosarcoma as predicted by GSEA and KEGG enrichment analysis.The results of the database predictions were validated by WB,ubiquitination experiments,and cellular immunofluorescence experiments.6.Rescue experiments were designed by addition of Wnt signaling pathway inhibitor XAV-939 and co-transduced si-BPNT1 and overexpressed β-catenin plasmid.7.We detected proteins interacting with BPNT1 by coimmunoprecipitation assay(Co-IP),plasmids overexpressing BPNT1 and GSK3β were also transfected to design the rescue experiments of BPNT 1 acting on GSK3β.8.Transfection of a plasmid with a BPNT1 domain deletion and its phosphorylation point mutation were used to determine that BPNT1 acts by affecting the phosphorylation of GSK3β.9.Statistical analysis: All experiments in this study were repeated three times under the same conditions,and Graph Pad prism 9.0.0 was used for statistical analysis of data.The data of numerical variables meet the normal distribution and the homogeneity test of variance,the T-test was used to compare the two groups,One-way ANOVA was used for comparing the three groups;When the data satisfied a normal distribution but not the test of homogeneity of variance,The comparison method between the two groups is the Welch test,The comparison method of the three groups was Welch one-way ANOVA;Not satisfying the normal distribution,The comparison of the two groups was the Wilcoxon-test,The method for comparing the three groups was the Kruskal-Wallis test,P <0.05 was considered statistically significant,*P <0.05,**P<0.01,***P<0.001,****P<0.0001.Results: 1.Pan-cancer analysis of TCGA database showed that BPNT1 was highly expressed in most tumors.Since there was no separate osteosarcoma grouping in TCGA database,we found GSE42352 and GSE33383 osteosarcoma data sets in GEO database.Statistical analysis found that BPNT1 expression was higher in osteosarcoma than normal cells or tissues.After differential analysis in GSE12856 osteosarcoma data set,BPNT1 was found in the up-regulated differential gene set(P <0.05,∣log FC∣> 1.5).BPNT1 expression was found to be relatively high in osteosarcoma cell lines in the CCLE database.To validate the results of the database,we examined the expression of BPNT1 in osteosarcoma cell lines and normal human osteoblasts,and found that the expression levels of protein and m RNA in osteosarcoma cell lines were higher than those in normal human osteoblasts(all P <0.05).To understand the localization of BPNT1 in cells,we found that BPNT1 is localized to the cytoplasm and nucleus by cellular immunofluorescence experiments.2.To make the study closer to the clinic,we examined the expression of BPNT1 in the osteosarcoma tissue chip,and found that it was localized in the cytoplasm and nucleus,consistent with the immunofluorescence results.Its expression was higher in osteosarcoma tissue than in normal bone tissue,and was related to the grade of osteosarcoma patients and whether tumors occurred(all P <0.05),but not to the age,sex and tumor site.3.The results of cell proliferation experiment showed that the overexpression of BPNT1 could promote the proliferation of osteosarcoma in vitro,the results of cell invasion experiment showed that the overexpression of BPNT1 could promote the invasion ability of osteosarcoma in vitro,and the results of cell migration experiment showed that the overexpression of BPNT1 could promote the migration ability of osteosarcoma in vitro;knock out BPNT1 and get the opposite result(all P <0.01).4.KEGG enrichment analysis found that BPNT1 was associated with Wnt signaling pathway,and after overexpressed BPNT1,overexpression of BPNT1 can promote the nuclear entry of β-catenin,a key protein of the Wnt signaling pathway,and reduce its ubiquitination degradation;the opposite result was obtained by knockdown of BPNT1 expression.The use of the Wnt signaling pathway inhibitor XAV-939 can rescue the activation effect of BPNT1 on the Wnt signaling pathway,and also rescue the promotion effect of BPNT1 on the proliferation and migration of osteosarcoma.We believe that BPNT1 does affect the malignant progression of osteosarcoma through the Wnt signaling pathway.In order to further support the above results,we cotransferred the plasmid of si-BPNT1 and β-catenin,and obtained consistent results(all P<0.05).5.Considering the phosphatase action of BPNT 1 and the mechanism of β-catenin entry into the nucleus,we examined the effect of BPNT1 on β-catenin phosphorylation and found that BPNT1 could inhibit the expression level of p-β-catenin(S33/S37/Th41),but did not interact with it(all P <0.05).6.Based on p-β-catenin(S33/S37/Th41)being directly regulated by GSK3β,we found that BPNT1 could inhibit the expression of GSK3β and its activated form p-GSK3β(Y216).Co-IP experiments showed that BPNT1 interacted with GSK3β and p-GSK3β(Y216),and cellular immunofluorescence results confirmed the co-localization of BPNT1 with p-GSK3β(Y216).Cotransversion of BPNT1 and GSK3β overexpression plasmid can restore BPNT1 to the activation of Wnt signaling pathway and its promotion on the malignant progression of osteosarcoma(all P <0.05).7.Compared with the BPNT1 wild-type plasmid,the BPNT1 domain knockout and phosphorylation site mutants lost the regulation of GSK3β and p-GSK3β(Y216),and also promoted Wnt signaling(all P <0.05).Conclusion: 1.BPNT1 is highly expressed in OS and is associated with its grade and metastasis.2.BPNT1 promotes the proliferation,invasion,and migration of OS.3.BPNT1 promotes the malignant progression of OS through the Wnt signaling pathway4.BPNT1 inhibited the activity of GSK3β by phosphatase acting on p-GSK3β(Y216)and stabilizes β-catenin into the nucleus to activate Wnt signaling pathway and then plays a role in promoting the malignant progression of OS.