TIPE1 Participates In The Proliferation Of Osteosarcoma By Mediating PRMT1 Regulated STAT3 Arginine Methylation

?Purposes?Osteosarcoma is the most common primary malignant bone tumor,which mainly occurs in young patients with a peak incidence at 18 years.It is derived from mesenchymal stromal cells of an osteoblast origin.The pathologic appearance is characterized by the production of osteoid and new bone by spindle-shaped tumor cells.Osteosarcoma has a high degree of malignancy and poor prognosis.Although surgery,chemotherapy,targeted therapy and other comprehensive treatments have greatly improved the survival rate of patients with osteosarcoma,the 5-year survival rate of patients with recurrent osteosarcoma in China is only about 20%.Therefore,understanding the mechanisms of osteosarcoma could expand the therapeutic window and improve patient survival.The TIPE family is composed of four members(TIPE,TIPE1,TIPE2 and TIPE3),which is closely related to immune regulation and tumorigenesis.Although there are significant sequence homologies among the four members,they are involved in different processes of regulation of cell biological activities.Studies have found that TIPE1 mainly functions as a tumor suppressor gene in liver cancer,breast cancer and other tumor types.Our team has demonstrated for the first time that TIPE1 promotes the malignant biological behavior of cervical cancer and nasopharyngeal carcinoma,but its biological role in osteosarcoma is not clear.In this study,clinical specimens,cells,animal models and a variety of molecular biology techniques were used to systematically reveal how TIPE1 accurately regulates the activity of PRMT1 and how to regulate the methylation level of STAT3 arginine sites by epigenetic means,by which to participate in the process of osteosarcoma proliferation.The completion of this project clarifies a new molecular mechanism of TIPE1 in the occurrence of osteosarcoma and can provide potential tumor molecular markers for the diagnosis and prognosis of osteosarcoma.?Methods?1.The effects of TIPE1 on the proliferation of osteosarcoma cells.(1)Cell model:in order to clarify the effects of TIPE1 on the proliferation of osteosarcoma cells,we overexpressed of TIPE1in osteosarcoma cell line MNNG/HOS CL#5 which with lower expression of TIPE1 and transfected of TIPE1-si into osteosarcoma cell line MG63 which with higher expression of TIPE1.The cell proliferation was observed by CCK8 and clone formation assay,and the changes of cell cycle were detected by flow cytometry.(2)Tumor formation experiment in nude mice:4-6week-old BALB/c nude mice were selected and the osteosarcoma cell line MNNG/HOS CL#5 overexpressing TIPE1 was implanted subcutaneously in the armpit of nude mice.When the diameter of the tumor was up to 0.5cm,the changes of tumor growth rate and tumor volume were recorded and compared between the two groups.2.The molecular mechanism of TIPEl for regulating osteosarcoma proliferation by regulating PRMT1/STAT3 pathway.(1)Transcriptome microarray and classical pathway analysis based on IPA:overexpression of TIPE1 and control vector into osteosarcoma cell line MNNG/HOS CL#5.After preparing poly-A RNA controls to synthesize the first strand cDNA and synthesize the second strand cDNA,through the steps of in vitro transcription synthesis marker cRNA "cRNA purification and quantitative" cRNA fragment labeling hybridization,the chip data were obtained.Finally,the classical pathway analysis based on IPA was performed.It was found that TIPE1 regulates the STAT3 pathway in osteosarcoma.Real-time fluorescence quantitative PCR was used for further verification.Using Co-IP-MS and other techniques,the logical relationship of TIPE1 regulating downstream pathway through PRMT1 was preliminarily established:firstly,a special TIPE1 vector for Co-IP-MS was constructed and overexpressed in osteosarcoma cell lines.Finally,the exact molecular mechanism of TIPE1 regulating downstream STAT3 through PRMT1 was further verified by Co-IP mass spectrometry,quantitative proteomics-based bioinformatics analysis,and other techniques.After interfering of PRMT1,the expression of STAT3 protein was detected by Western Blot,and the reverse effect of TIPE1 on the proliferation inhibition of osteosarcoma was observed by CCK8 growth curve,so as to further clarify the regulatory logic relationship between TIPE1,PRMT1 and STAT3.(2)Construction of PRMT1 series truncated sequences and determination of interaction sites between TIPE1 and PRMT1:a series of truncated sequences were constructed according to PRMT1 protein domain,and the expression vectors of full-length or different deletion fragments of TIPE1-Flag and PRMT1-HA were cotransfected in HEK293T cells.The specific sites of the interaction between TIPE1 protein and PRMT1 were further determined by Co-IP.(3)The effect of TIPE1 on the activity of arginine methyltransferase of PRMT1:A.The quantitative proteomics technique of arginine monomethylation modification was used to identify the degree of arginine monomethylation modification in osteosarcoma cell line by protein extraction-arginine monomethylation-MMA(arginine monomethylation)enrichment-mass spectrometry-information analysis and other processes.B.TIPE1 inhibits the methylation level of STAT3 by PRMT1.Due to the lack of STAT3 methylation antibody,all groups were first transfected with STAT3-His,and enriched STAT3 protein by immunoprecipitation with His antibody.Finally,the level of STAT3 methylation was detected by universal methylation antibody.First,TIPE1-Flag was transferred into HEK293T cells to detect whether TIPE1 regulates the methylation ability of endogenous PRMT1 to its substrate STAT3 in a dose-dependent manner under normal physiological conditions.TIPE1-sh was transfected into HEK293T cells by siRNA technique,and the effect of endogenous PRMT1 on the methylation level of STAT3 was detected after suppressing the expression of endogenous TIPE1,and the methylation level of PRMT1 substrate was detected after suppressing the expression of endogenous PRMT1 in HEK293T cells after overexpressing TIPE1.(4)The ability of TIPElfor inhibiting the transcriptional activation of STAT3:HEK293T cells were co-transfected with pGL3-promoter vector or APRE luciferase reportor gene,and PRMT1-HA,TIPE1-Flag and STAT3-His vectors.Under the stimulation of IL-6,the transcriptional activation ability of TIPE1 to the downstream gene STAT3 regulated by PRMT1 was detected by double luciferase activity assay.3.Clinical specimens verify the phenomenon for TIPE1 participates in the process of osteosarcoma through PRMT1/STAT3 pathway.Using tissue microarray of osteosarcoma patients to verify the relationship between the expression level of TIPE1 in tumor tissue and tumor stage,and to analyze the correlations between the expression of TIPE1 and STAT3 in tumor tissues.?Results?1.TIPE1 inhibits the proliferation of osteosarcoma cells.The results of in vitro experiments showed that TIPE1 significantly inhibited the proliferation rate of osteosarcoma cells;the results of cell cycle showed that TIPE1 could significantly reduce the ratio of S phase in the cell cycle;in addition,TIPE1 could significantly weaken the clone formation ability of osteosarcoma cells.The results of in vivo experiment showed that TIPE1 could significantly inhibit the tumorigenesis ability of nude mice,and the immunohistochemical Ki67 results showed that TIPE1 significantly inhibited the expression of Ki67,an index of proliferation.2.TIPE1-mediated PRMT1 regulates STAT3 arginine methylation and participates in the process of osteosarcoma proliferation.(1)The results of transcriptome microarray and IPA-based classical pathway analysis showed that TIPE1 mainly inhibited STAT3 pathway in osteosarcoma cell line MNNG/HOS CL#5.qPCR verification also showed that the expression of STAT3 was significantly decreased after overexpression of TIPE1.Using Co-IP-MS and other techniques,it was found that TIPE1 could interact with PRMT1,and after interfering with PRMT1,the inhibition of STAT3 caused by overexpression of TIPE1 could be reversed,and the inhibition of osteosarcoma cell proliferation was also reversed.(2)A series of truncated PRMT1 sequences were constructed,and it was found that TIPE1 protein mainly binds to the catalytic region of PRMT1,and TIPE1 may inhibit the methylase activity of PRMT1 by binding to the catalytic region of PRMT1.(3)The quantitative proteomic technique of arginine monomethylation modification showed that TIPE1 could down-regulate the methylation level of STAT3 arginine site.Further analysis showed that overexpression of TIPE1 inhibited endogenous PRMT1-mediated STAT3 methylation in a concentration-dependent manner,while interfering with TIPE1 promoted endogenous PRMT 1-mediated STAT3 methylation.(4)The ability of TIPE1 to inhibit the transcriptional activation of downstream gene STAT3 regulated by PRMT1 was found by double luciferase activity assay.The results showed that TIPE1 could significantly inhibit the activation of STAT3 mediated by PRMT1,which further suggested that TIPE1 inhibited the down-regulation of STAT3 methylation level of PRMT1 substrate,which would eventually lead to the down-regulation of STAT3 transcription level.3.Clinical specimens confirmed that TIPE1 was involved in the process of osteosarcoma by inhibiting PRMT1/STAT3 pathway.Using tissue microarray of osteosarcoma patients,it was found that the expression level of TIPE1 in tumor tissue was negatively correlated with tumor stage,and the expression of TIPE1 in tumor tissues were also negatively correlated with the expression of STAT3.?Conclusion?.Although the important biological functions of PRMT1 and STAT3 in tumors have been reported,and the related mechanisms of PRMT1 regulating STAT3 have been preliminarily described,how TIPE1 regulates PRMT1 and related downstream signal pathways is not clear.The completion of this project clarifies the way that TIPE1 accurately regulates the activity of PRMT1,and clarifies the exact molecular mechanism that TIPE1 regulates the activity of PRMT 1,and then affects the expression of downstream molecules STAT3,and finally has a significant inhibitory effect on the proliferation of osteosarcoma cells.The completion of this project provides a new target and treatment strategy for the clinical diagnosis and treatment of osteosarcoma,and provides theoretical and practical possibility for the prediction of prognosis and the discovery of early diagnostic markers of osteosarcoma.