The Effect Of Transforming Growth Factor ? Regulator 4 For Cell Proliferation And Apoptosis And Its Potentially Mechanism In Osteosarcoma

Osteosarcoma(OS)is the most frequent malignant primary bone tumor commonly found in children and adolescents.It is characterized by high malignancy,early metastasis,poor prognosis and low survival rate.According to the last research.The annual incidence of OS is 4.4 cases per million.And 4000-5000 new cases are diagnosed every year with only 50% of the five-year survival rate in China.Early immature treatment techniques and pulmonary metastasis lead to the low survival rate of OS.At present,with the introduction of promising chemotherapy,radiotherapy and surgery since 1970 s,the 5-year overall survival of a subset of OS patients has increased up to 70%.However,the present treatments could cause significant long-term morbidity,even lead to death.The patients with primary metastatic or relapsed disease were reported 5-year overall survival rates of less than 30%.In addition,treatments with poor prognosis,drug resistance and toxic side effects are still crucial obstacles for radical therapies.Therefore,novel treatments are required urgently.In recent years,targeted therapy has shown great potential in the treatment of OS,thus,based on the implicated pathogenesis of OS,identification of new effective prognosis markers and key molecular targets for OS will facilitate the accurate diagnosis and therapy for OS patients.The transforming growth factor ? regulatory protein 4(TBRG4),also termed cell cycle progression restoration protein2(CRP2)and Fas-activated serine/threonine kinase domain-containing protein4(FASTKD4).It is therefore conceivable that TBRG4 could be a key molecular in OS because of its regulation for TGF-?.TBRG4 is located on the7p14-p13 chromosomal region,participates in the development process of various disease,especially cancer.For instance,Sézary Syndrome is a cutaneous T-cell lymphoma leukemia,patients with Sézary Syndrome are found the abnormal replication of the TBRG4 gene region.Also,previous studies demonstrated that TBRG4 is a novel oncogene in breast cancer,with its expression level correlates with breast tumor malignancy.In addition,deficiency of TBRG4 could significantly inhibit tumor cell proliferation and migration through promoting apoptosis.In human H1299 lung cancer cells,knockdown of TBRG4 affects the role of tumorigenesis of lung cancer via deregulation of DDIT3,CAV1 and RRM2,which indicates that TBRG4 serves a crucial role in several types of human cancer.However,it is unclear whether TBRG4 could directly regulate human OS progression.Notably,Sabina Sevcikova et al collected nine samples of bone marrow plasma cells from multiple myeloma(MM)patients to evaluate the expression of 15 genes associated with the high risk signature of MM patients.The results showed that the expression of TBRG4 increased significantly in extramedullary tumors,which suggest that genetic abnormalities of TBRG4 are highly related with MM.Therefore,the hypothesis has been put forward that the altered TBRG4 expression would affect human OS growth and development.The main research contents are summarized as follows:1.Comparison of the expression of TBRG4 between OS tissues compared with para-carcinoma To investigate the expression of TBRG4 in OS,we detected the expression levels of TBRG4 in human OS tissues and cells.All specimens were emerged as OS by pathological examinations with HE staining.High TBRG4 immunoreactivity was observed in OS tissues compared with para-carcinoma.The m RNA expression levels of TBRG4 was detected in malignant human OS cells,TBRG4 was widely expressed in MG63 and U2 OS cells,rather than Saos-2 cells.These results demonstrated that TBRG4 exhibit high expression levels at both m RNA and protein levels in OS tissues and MG63 cells compared to para-carcinoma.MG63 cells would be selected for further study2.Construction of sh TBRG4 lentivirus To evaluate the efficiency of knocking down TBRG4 expression mediated by lentivirus,the MG63 cells was transfected with sh TBRG4 or sh Ctrl lentivirus.Firstly,TBRG4 gene was used as template design RNA interference target sequence and prepare specific TBRG4 interference fragments.The digested target vector was linked with the prepared interference fragment and transferred to the competent cells of E.coli for positive clone and sequencing.The cloning results were identical with the target sequence.The plasmid can be used in the next transfection experiment.Lentiviral vectors(including TBRG4 vector,Helper 1.0 and Helper 2.0)were prepared by co-transfection protocol.Three plasmids were co-transfected into 293 T cells.After concentration and purification,the lentiviral particles were detected in physical condition,sterility and viral titer.After transfection with lentivirus for 72 h,the proportions of infected MG63 cells expressing GFP more than 80%,which was confirmed a high infection efficiency and could be used for further analysis.TBRG4 m RNA expression and protein level was determined by RT-q PCR and Western blot assay respectively.OS cells transfected with sh TBRG4 lentivirus showed an 50.3% decrease in TBRG4 m RNA expression. Consistent with qPCR data,the protein expression level of the shTBRG4 lentivirus transfected group showed the similar trend compared to control group.Therefore,these results demonstrated that TBRG4 expression was downregulated by sh TBRG4 lentivirus infection successfully at both m RNA and protein levels.3.Effect of TBRG4 on the function of OS cells The lentiviral expression vectors sh TBRG4 and sh Ctrl constructed in previous experiments were used to transfect MG63 cells.MG63 cells was determined by both CCK8 assay,High content screening analysis,cell viability experiment,tumor sphere formation assay,cloning formation assay and cell apoptosis assay.In the high content screening analysis,the number of cells in control group increased in a time-dependent manner,while only a slight increase was observed in the TBRG4-si RNA group,and the difference was statistically significant.Cell viability test by CCK8 assay showed that TBRG4 knockdown inhibited cell proliferation significantly compared with the control group.All these results showed that TBRG4 knockdown could inhibit MG63 cells proliferation significantly.,indicating that TBRG4 knockdown could inhibit MG63 cells proliferation significantly.In the tumor sphere formation assay,TBRG4 significantly reduced the size and number of spheres than that formed by the control lentivirus cells,demonstrated that the tumorigenicity of MG63 cells could be inhibited after TBRG4 knockdown.In cloning formation assay,the average number of colony in sh TBRG4 group was significantly lower than that in sh Ctrl group after 14 days of culture,indicating that depletion of TBRG4 can inhibit ability of tumor proliferation of MG63 cells. The apoptotic rate of MG63 cells transfected with sh TBRG4 and sh Ctrl was detected by Annexin V-APC staining.The results showed that 1.88% of the apoptotic rate in cells transfected sh Ctrl lentivirus,while,in OS cells infected with sh TBRG4,apoptotic cell rate reached 12.95%.The difference was statistically significant.The results revealed that TBRG4 deficiency could lead to induction of apoptosis in MG63 cells.4.Genome-wide effect of TBRG4 knockdown The genome-wide effect of TBRG4 knockdown was analyzed by the Prime View Human Gene Expression Array.A total of 2268 differentially expressed genes were identified,in which 1134 were upregulated and 1134 were downregulated.To verify the accuracy of the gene chip,we selected 30 genes with significant differences to be checked using RT-q PCR.The results showed that all the 30 genes expression was consistent with the results of gene expression array.IPA program was performed to analyze the regulator effect,diseases and functions,canonical pathway,upstream analysis,and molecular network in the 2268 differently regulated genes.The enrichment analysis of differential genes using IPA database revealed 13 significantly altered ingenuity canonical pathways,in which 7 pathways were significantly inhibited(Z-score?-2)and 6 were activated(Z-score?2).3-phosphoinositide Biosynthesis pathway was significantly inhibited with Z-score of-2.401 and Superpathway of Cholesterol pathway was significantly activated with Z-score of 2.449.Among these networks,32 focus molecules were involved with 16 upregulated and 16 downregulated.These results provide foundation for the subsequent molecular mechanism study of TBRG4 in MG63 cells.