Human Positive Coactivator 4 Is Involved In The Malignancy Of Osteosarcoma

Objective: Osteosarcoma is the most common primary malignant bone tumor with an extraordinarily poor prognosis and early pulmonary metastasis formation as a frequent occurrence.Transcriptional positive coactivator 4(PC4)is a transcriptional coactivator which plays multiple roles in DNA transcription,replication,repair and chromatin organization,even in tumorigenesis.However,the precise function of PC4 in osteosarcoma is still unclear.Our purpose is to uncover the effect of PC4 on osteosarcoma and find the possible mechanism.Based on the previous research,we sought to evaluate the expression level of PC4 in human osteosarcoma and explore the relationship between PC4 and tumor stages or prognosis.To characterize the functional relevance of increased PC4 expression in osteosarcoma,we performed further experiments in vitro in seven osteosarcoma cell lines.To further investigate the role of PC4 in the malignancy of 143 B cells,PC4 was silenced in 143 B cells utilizing lentivirus sh RNA.RNA-seq analysis was used to investigate the molecular alterations after PC4 interference in 143 B cells.Analysis the relevance of PC4 and MMP-9,in order to explain the pulmonary metastasis malignancy change after PC4 silence.Analysis the possible molecular mechanism of PC4 involved in the bone resorptioin of osteosarcoma.Method:1.First,we evaluated the expression level of PC4 in human osteosarcoma tissues to investigate whether PC4 level is correlated with osteosarcoma progression and prognosis.1.1 IHC was used to analysis the expression of PC4 in 198 osteosarcoma tissues and 44 normal tissues.And reveal the relevance of PC4 in Enneking stages or tumor size or sex or age.1.2 WB was used to analyze 5 osteosarcoma tissues and adjacent normal counterparts.1.3 Total 59 cases were followed up to evaluate whether there is correlation between PC4 expression and osteosarcoma survival rate.2.Evaluate the expression of PC4 in various osteosarcoma cell lines,find the possible malignancy controlled by PC4.2.1 Detect the expression of PC4 in 7 osteosarcoma cell lines.2.1.1 Immunofluoresence staining of PC4 in 7 osteosarcoma cell lines to detect the expression level and molecular location.2.1.2 Western blotting of PC4 in 7 osteosarcoma cell lines to detect the expression level.2.2 Soft Agar Colony Formation assay to examine the potential of colony formation in 7 osteosarcoma cell lines.2.3 Tumorigenicity assays were carried out to test the tumorigenicity in various osteosarcoma cell lines.2.4 RT-PCR was performed to test several malignant phenotype related genes in 7 osteosarcoma cell lines.2.5 Lv-sh RNA treatment for PC4 gene silencing in 143 B and stable transfected clone selection.2.5.1 The establishment of lentivirus of PC4 silencing.2.5.2 Immunofluoresence staining to evaluate the transfection rate,and limited dilution assay for stable transfection subclone selection.2.5.3 Western blotting to test the transfection efficiency of PC4 silencing.2.5.4 CCK-8 assay to check the alteration of proliferation after PC4 knocking down.2.5.5 Flow cytometry to check the alteration of cell cycle distribution after PC4 knocking down.2.5.6 Soft agar assay to check the alteration of colony formation ability after PC4 knocking down.2.5.7 Scarification test to check the alteration of mobility after PC4 knocking down.2.5.8 Transwell chamber assay to check the alteration of invasion after PC4 knocking down.2.5.9 CCK-8 assay to check the alteration of adherence ability after PC4 knocking down.2.5.10 Tumorigenicity assays to check the alteration of tumorigenicity ability and pulmonary metastasis ability after PC4 knocking down.3.RNA-seq analysis of PC4 knocking down in 143 B cell lines.3.1 Analysis of different expressed genes after PC4 knocking down.3.2 Gene ontology analysis of different expressed genes after PC4 knocking down.3.3 KEGG pathway analysis of different expressed genes after PC4 knocking down.4.To explore the molecular mechanism of PC4 in MMP-9 regulation in 143 B.4.1 Lv-sh RNA treatment for PC4 gene silencing in 7 osteosarcoma cell lines.RT-PCR to check the alteration of MMP-2,MMP-9,FN after PC4 knocking down.4.2 Test the function of human FN protein and FNsi RNA in the regulation of MMP-2 and MMP-9 in 143 B cell model and MG63 cell model.4.3 Chromatin Immunoprecipitation assay to analyze the bind of PC4 protein and MMP-9 promoter region.4.4 Luciferase reporter Assay to test the regulation of PC4 and SP1 on MMP-9.4.5 Co-immunoprecipitation to test the combination of PC4 and SP1 protein in 143 B.5.To evaluate the effect of PC4 on bone remolding regulation in osteosarcoma.5.1 To test the alteration of bone resorption after PC4 knocking down in vivo.5.1.1 Micro CT to evaluate the bone fracture,bone volume,trabecular number,trabecular thickness.5.1.2 Using transwell co-culture cell model and conditional medium cell model,to test the effect of PC4 knocking down on RAW264.7 osteoclast differentiation.Trap staining and scanning electron microscopy were used to evaluate the osteoclast differentiation.5.1.3 Analyze the RNA-seq data,screen the osteoclast related genes,and confirm these genes by RT-PCR.5.1.4 Western blotting to evaluate the expression alteration of IGFBP5 after PC4 knocking down.5.1.5 To evaluate the effect of IGFBP5 on osteoclast differentiation using IGFBP5 protein.5.1.6 To evaluate the rescue effect of IGFBP5 on osteoclast differentiation in PC4 knocking down conditional medium cell model using IGFBP5 protein.Results:1.PC4 expression level was increased in osteosarcoma.1.1 PC4 expression level was increased in osteosarcoma comparing with the normal tissues.And its expression level is correlated with osteosarcoma stages and tumor size.1.2 WB showed us PC4 is upregulated in osteosarcoma comparing to the adjacent normal tissues.1.3 PC4 positive rate is correlated with osteosarcoma patient survival rate.2.PC4 and malignancy phenotype in vivo and vitro.2.1 PC4 expression in 7 osteosarcoma cell lines.2.1.1 Immunofluorescent staining for PC4 again demonstrated prominent nuclear localization and exhibited greatest intensity in 143 B and MNNG-HOS cells.2.1.2 Western-blotting demonstrated highest PC4 expression in 143 B and MNNG-HOS cells,moderate PC4 levels in MG63 and U2 OS cells,and lowest PC4 expression in HOS,SAOS2,and 9901 cells.2.2 Clonogenicity assays demonstrated greatest sphere formation in 143 B cells,followed by moderate formation in MNNG-HOS,MG63,and 9901 cells,and lowest formation in HOS,U2 OS,and SAOS2 cells.2.3 143 B and MNNG-HOS cells exhibited greatest tumorigenicity in xenografted mouse models,while MG63 and 9901 cells had lower tumorigenicity;HOS xenografts did not develop visible tumors in our experiment.2.4 RT-PCR demonstrated that highest PC4 expression in 143 B and MNNG-HOS cells,143 B cells had the highest expression of MMP-9,m RNA levels of P53 were extraordinary low in 143 B cells.2.5 Establishment of Lv-sh RNA for PC4 gene silencing in 143 B,to explore the malignancy phenotype changed.2.5.1 Establishment of Lv-sh RNA for PC4 gene silencing.2.5.2 IF staining showed us 90% transfection rate for PC4 Lv-sh RNA.2.5.3 WB showed us stably transfected 143BPC4-cells were obtained with effective downregulation of PC4.After 10 passages,PC4 expression was maintained at 30% comparing with parental cells.2.5.4 Cell proliferation was decreased after PC4 knockdown,utilizing CCK8 test analysis.2.5.5 After PC4 knockdown,the percentage of cells in G1 phase increased(p<0.05)while those in S phase decreased but there was no significant difference.2.5.6 143BPC4-2.5.7 The migration distances of 143 B stably transfected subclones showed decreased colony formation ability compared to 143 B parental cell.PC4-2.5.8 Crystal violet staining showed that the number of 143 B cells crossing the basement membrane of the transwell was decreased in the 143 B cells were decreased comparing with the control group.PC4-2.5.9 In the adherence ability test,the 143 B group.PC4-2.5.10 Downregulation of PC4 in 143 B cells supresses tumor growth in vivo and development of pulmonary metastases.group showed slow adherent speeds at 30 min,60min and 120 min,but no obvious differences at 24 h.3.RNA-seq analysis to investigate the molecular alterations after PC4 interference in 143 B cells.3.1 In comparison with 143 B parental cells,572 genes were upregulated and 513 genes were downregulated in 143BPC4-3.2 Gene ontology functional classification of differentially expressed genes demonstrated that PC4 might be involved in protein bind,cell junction,extracellular matrix,locomotion and anatomical structure development.cells.3.3 KEGG pathway of enrichment analysis of differentially expressed genes demonstrated that p53 and other 20 pathway were enriched in 143BPC4-4.Downregulation of PC4 inhibits the transcription of MMP9 and affect the pulmonary metastasis ability of 143 B.cells.4.1 In 143 B,MNNG-HOS,MG63,Sa OS2,and 9901 cells,PC4 knockdown downregulated MMP9 m RNA levels,compared to parental cells respectively.PC4 knockdown also reduced m RNA levels of MMP2 in all osteosarcoma cell lines except MNNG-HOS.4.2 After exposure to 35ug/ml fibronectin protein for 4h,or FN si RNA,the m RNA levels of both MMP2 and MMP9 showed no obvious changes in either 143 B or 143BPC4-4.3 Chi P analysis suggested that PC4 protein bound to the promoter region of MMP9.cells but dramatical changes in MG63.4.4 Sp1 si RNA and PC4 si RNA can both reduce the luciferase activity of MMP9 promoter,and this effect was more obvious under the PMA stimulation[1],SP1 si RNA and PC4 si RNA seemed to have synergistic effect.4.5 SP1 could be detected in the samples immunoprecipitated using PC4 antibody,the combination between PC4 and SP1 was found.5.PC4 and bone resorption in osteosarcoma.5.1 PC4 knocking down reduced the bone resorption of 143 B cell in vivo and in vitro.5.1.1 Micro CT showed that PC4 knocking down decreased the bone resorption in nude mice,and reduced the TRAP positive cells in tumor related bone resorption region.5.1.2 TRAP staining images of representative groups.143 B increased the TRAP-positive cells,and this effect was attenuated by PC4 knocking down.5.1.3 RNA-seq showed CXCL2,IGFBP5,MCP1,IL1 A,IL1B were reduced by PC4 knocking down,and confirmed by RT-PCR.5.1.4 IGFBP5 increase the TRAP positive cell in a dose dependent manner.5.1.5 143 B conditional medium can increase the TRAP positive cells,PC4 interference can attenuate this effect,while 2n M IGFBP5 can intensify this in 143BPC4-Conclusion: CM group.1.PC4 expression level was increased in osteosarcoma.2.Upregulated PC4 correlates with poor patient prognosis and tumor progression.3.PC4 was involved in proliferation,pulmonary metastases and migration,invasion,adherence,colony formation pathological process in osteosarcoma.4.Transcriptional level regulation is the predominant access of PC4 affects the MMP-9 of 143 B by the combination to SP1.5.PC4 is involved in the bone resorption of 143 B cell lines.6.Suppression of PC4 in 143 B can reduce the secretion of CXCL2,CCL2,IGFBP5,IL1 A,IL1B.And lead to the restrain of bone remolding.