MiR-19a-3p Promotes The Progress Of Osteosarcoma By Regulating The PHLDA3/Akt/GSK-3β Signal Pathway

Background:Osteosarcoma(OS)is a primary bone tumor derived from mesenchymal cells,with high incidence and mortality in adolescents.Although some therapeutic strategies,such as surgery,adjuvant radiotherapy and chemotherapy,have appeared colossal advance in improving the survival rate of patients with OS,its 5-year survival rate is still poor.Therefore,it is urgent to explore the molecular mechanisms of osteosarcoma in order to find novel treatment targets.In recent years,gene expression profiling chip,as a highthroughput and effective technology,has been widely used in various cancers research fields.It can help reveal the disease-related genes,and provide information for the pathogenesis of cancer including osteosarcoma.Pleckstrin homology-like domain family A,member 3(PHLDA3)is one of PHLDA family.It contains the PH domain,which can compete with the PH domain of AKT to bind to membrane lipids and suppress Akt activity.Recent studies have reported that PHLDA3 as a p53-regulated tumor suppressor gene is associated with a variety of tumors,such as prostate cancer、pancreatic neuroendocrine tumors and breast cancer.However,the role of PHLDA3 in OS is still unclear.Besides,as a member of the known oncomiR-17-92 cluster,miR-19a-3p has been confirmed to play an important promoting carcinogenic effect in a variety of tumor disease development,and affect the proliferation,metastasis and chemotherapy resistance of tumors.Overexpression of miR-19a-3p was identified in OS cells,and silencing of miR-19a-3p could activate the PTEN/Akt signal to promoted its chemosensitivity.It can be seen that the role of miR-19a-3p in the progression of osteosarcoma disease is essential.Therefore,miR-19a-3p has an important research value in the molecular mechanism about osteosarcoma disease.Objective:we used bioinformatics analysis to screen differentially expressed genes of osteosarcoma by GEO database,and clarify the mechanism of osteosarcoma development in order to explore osteosarcoma treatment targets with potential research value.Methods:In the first part: by comparing and analyzing the gene expression profile of OS cells and bone mesenchymal stem cells with OS chips(GSE42352 and GSE70414),we aimed to screen differentially expressed genes of osteosarcoma.Then,the literature excavation is further screened to genes with potential value in the OS.Finally,we used GEO and USCS,the Oncomine database to further verified the most value genes as the next experimental study.In the second part: to explore the role of PHLDA3 in OS,we overexpressed PHLDA3(OE-PHLDA3)and knock down PHLD A3 expression(sh PHLDA3)in143B and U2 OS cells.Then,we used MTT 、 Colony formation assays 、 spheroid formation assays and nude mouse transplantation tumor experiment to detect the role of PHLDA3 on cell proliferation.Whereafter,the effect of PHLAD3 on OS cell migration was evaluated by wound healing and Transwell assays.Subsequently,we further assessed the effect of PHLAD3 on chemoresistance of OS cells by WB 、 flow cytometry analysis、PI straining and MTT.Finally,we used WB assay to investigate whether PHLDA3 regulates OS cell proliferation and migration through Akt/GSK-3βpathway,and treate with or without the PI3K/AKT signaling pathway inhibitor LY294002 in the OS cells to further prove it.In the third part: in order to understand the regulatory mechanism of PHLDA3 in OS,we next searched for the candidate miRNAs of PHLDA3 in three databases,including ENCORI、Target Scan Human and Tarbase.Then,we validated miR‐19a-3p was predicted miRNA of PHLDA3 in OS by literature review on Pub Med.Thus,we want to prove the relationship between miR-19a-3p and PHLDA3.Target Scan Human was used to analyze the binding site between miR-19a-3p and PHLDA3.ENCORI was used to analyze the correlation between miR‐19a-3p expression and the survival outcome of patients with Sarcoma.In the vitro study,we used WB and luciferase reporter gene analysis experiments to verify the relationship between miR‐19a-3p and PHLDA3.Finally,we used MTT、Transwell and WB to further confirm whether the effects of miR-19a-3p on OS cell proliferation 、migration and chemoresistance.Result:In the first part: to identify the differentially expressed genes(DEGs)in OS,we downloaded the expression microarray datasets,including GSE70414 and GSE42352,which was associated with osteosarcoma from GEO databases.Then,we screened the two datasets to obtain DEGs between OS cells and bone mesenchymal stem cells(MSC),and obtained 551 and 1113 DEGs from GSE70414 and GSE42352 dataset,including 92 common differentially expressed genes.In order to further screen genes with potential research value,we selected a total of 23 genes through literature search.Besides,we extracted those genes expression profiles from GSE42352 datasets between Osteoblast(OB)and high-grade OS pre-chemotherapy biopsy(Biopsise)and analyzed the impact of these genes on the clinical survival of patients with osteosarcoma by UCSC Xena browser.We found that the change trend of five genes(PHLDA3,FBLN5,ITGA5,LTBP2,IGFBP4)in OB and Biopisese was consistent with the results of MSC and OS and also affected the prognosis of osteosarcoma patients.The transcriptome analysis of the five genes in different types of tumors in Oncomine database displayed that PHLDA3,FBLN5,LTBP2 was low expressed in clinical samples of sarcoma.We eventually select PHLDA3 with more verification data sets as candidate genes for further experimental verification.In the second part : we overexpressed PHLDA3(OE-PHLDA3)and knock down PHLDA3 expression(sh PHLDA3)in 143 B and U2 OS cells.Through the vitro experiment,we found that OE-PHLDA3 inhibited OS Cell proliferation 、 migration and anti-chemotherapy;sh PHLDA3 enhanced OS Cell proliferation 、 migration and anti-chemotherapy.Besides,PHLDA3 silencing greatly increased the tumor weight of mice.Subsequently,to investigate whether PHLDA3 regulates OS cell proliferation and migration through Akt/GSK-3β pathway,we first assess the effect of PHLDA3 on the protein levels of phosphorylated Akt and GSK-3β in the OS cells and found that overexpression of PHLDA3 reduced the expression of p-AKT(ser 473)and p-GSK-3β(ser 9).On the contrary,PHLDA3 knockdown increased the expression of p-AKT(ser473)and p-GSK-3β(ser 9).To further prove that PHLDA3 suppressed cell growth and migration through regulating AKT pathway,the cells with or without PHLDA3 knockdown were treated with or without the PI3K/AKT signaling pathway inhibitor LY294002.The results indicated that LY294002 significantly reversed the increase of the phosphorylation of AKT and GSK-3β,and also abolished the increase of cell proliferation and migration by PHLDA3 knockdown.In the third part: in order to understand the regulatory mechanism of PHLDA3 in OS,we next searched for the candidate miRNAs of PHLDA3 in three databases,and 10 miRNAs was selected by Venn online analysis website(miR-16-5p、miR-129-2-3p、miR-19a-3p、miR-497-5p、miR-424-5p、miR-532-3p、miR-19b-3p、miR-195-5p、miR-15a-5p 和 miR-15b-5p).Then we validated the miRNAs by literature review on Pub Med.The results suggested that miR‐19a-3p,a predicted miRNA of PHLDA3,was reported to increase in OS.Subsequently,the prediction results of Target Scan Human7.1showed that there was a binding site of miR-19a-3p in the 3′UTR of PHLDA3.ENCORI was used to analyze the correlation between miR‐19a-3p expression and the survival outcome of patients with Sarcoma.The result showed that low miR‐19a-3p level might predict a better survival outcome.There was also obviously negative correlation between miR-19a-3p expression and PHLDA3 expression in Sarcoma cancer samples,and a high miR‐19a-3p level may estimate a worse survival outcome.Moreover,we also observed a negative regulation of miR-19a-3p on PHLDA3/Akt/GSK3β expression in 143B/U2 OS cell lines.The results of luciferase reporter gene analysis showed that there was a directly interaction between miR-19a-3p and the 3′UTR of PHLDA3.Finally,we transfected miR-19a-3p into OS cells with or without PHLDA3 overexpression or knockdown.The result of study revealed that restoration of PHLDA3 abolished miR-19a-3p-induced cell proliferation、 migration and chemoresistance.Conclusion:1.PHLDA3 was lowly expressed in OS cells and tissues,and also affected the prognosis of osteosarcoma patients by bioinformatics analysis,so it may become a potential therapeutic target for OS.2.We verified that PHLDA3 played a tumor suppressor role in osteosarcoma and suppressed cell proliferation,migration and enhanced cisplatin-induced cell apoptosis via regulating the Akt/ GSK-3β signaling pathway.3.PHLDA3 is a target mRNA of miR-19a-3p;miR-19a-3p may exert its protumorigenic functions through inhibiting PHLDA3 expression in OS.