TRPS1Is Associated With The Multidrug Resistance Of Osteosarcoma By Regulating MDR1Gene Expression

[Background]Osteosarcoma is the most prevalent primary malignant bone tumor. The prognosis was very poor prior to the discovery of chemotherapy. However, the development of multidrug resistance (MDR), in which tumor cells become resistant to a wide spectrum of anti-cancer agents with different structures or different target sites, severely limits the success of chemotherapy in osteosarcoma.The human MDR gene1(MDR1), which encodes the membrane-located efflux pump P-glycoprotein (P-gp), plays an important role in the drug resistance process. Overexpression of MDR1/P-gp resulted in an active efflux of anti-cancer agents from cells, thus lowering intracellular drug concentrations and inducing cancer cell resistance to chemotherapeutic drugs, such as doxorubicin and paclitaxel etc.Tricho-rhino-phalangeal syndrome1(TRPS1) is a gene involved in Tricho-rhino-phalangeal syndrome (TRPS), which is characterized by craniofacial and skeletal malformations. It encodes a transcription factor that contains nine zinc-finger domains, including a GATA-type zinc finger through which it binds DNA. Trpsl is found in malignant tumor cells and has been suggested as a promising cytologic tumor marker in breast cancer and prostatic carcinoma. Furthermore, Trpsl enhanced chondrogenesis and apopotosis in ATDC5cells. In our previous study,we have found that TRPS1suppress TGF-β signaling and the expression of TGF-J31, which has been reported to inhibit MDR1in human glioma cells. Thus TRPS1may be associated with multidrug resistance by regulating MDR1expression.Whether Trpsl regulates the MDR in osteosarcoma through MDR1gene expression is quite a significant question for improving the poor prognosis of drug-resistance osteosarcoma. We have occasionally observed a positive correlation between MDR1/P-gp and TRPS1in our clinical samples of osteosarcoma. The putative promoter region of MDR1has three GATA box-like sequences to which Trpsl may bind. We therefore hypothesized that the silencing of TRPS1by plasmid-mediated expression of small interfering RNA (siRNA) results in the downregulation of MDR1/P-gp. If so, MDR in clinical chemotherapy could be partly reversed through downregulating Trpsl.In this study, to elucidate the regulating effect of Trpsl on MDR in osteosarcoma, we examined the functional interactions between Trpsl and MDRl/P-gp in three typical osteosarcoma cell lines by both downregulation and upregulation of Trpsl. Furthermore, we performed immunohistochemistry on the tissues of osteosarcoma patients to analyze the expression of both proteins and to evaluate the predictive significance of Trpsl for MDR1/P-gp..[Methods]1. The expression of TRPS1in the osteosarcoma cell lines U2-OS, MNNG/HOS Cl#5and Saos-2were evaluated by Real-time Quantitative PCR and Western blot.2. Clinical undecalcified osteosarcoma samples were collected and Immunohistochemical Analysis of TRPS1and MDR1were used to find the possible relationship between them.3. Small hairpin siRNA sequences targeted at TRPS1and non-targeting siRNAs (as negative controls) sequences were designed were selected and synthesized as64oligonucleotides, annealed, and then cloned into the pSUPER. neo+GFP expression vector respectively. Plasmids of full-length of TRPS1gene were purchased from open biosystem, and then the target gene were subcloned into a mammalian expression vector, pcDNA3.1(+)(Invitrogen), negative control vector was build similar to prior control plasmid.After comfirmed by DNA sequencing, the recombinant plasmids were extracted.4. Three groups of osteosarcoma cells were transduced with the above plasmids respectively. After another48h culture, Q-PCR was adopted to analysis the expression of TRPS1and MDR1mRNA, Western blot was used to detect the TRPS1and P-gp protein expression. MTT assay was used to assess the effect of TRPS1silencing on the chemosensitivity of MDR cells to anticancer drugs as doxorubicin, cisplatin ect..Rh123efflux assays were performed to evaluate the function of P-gp.5. ChIP analysis was performed in U2-OS cell and chromatin samples were immunoprecipitated using an anti-Trpsl antibody. Bindings of Trpsl to the MDR1promoter were analyzed by PCR using specific primers.6. TGFβ-1mRNA and protein levels were analyzed in different groups of affected U2-OS cell sublines to find the possible effect of TRPS1on TGFβ signal pathway.[Results]1. TRPS1was found to have higher mRNA and protein levels in relation to P-gp expression in U2-OS and MNNG/HOS C1#5, compared with the Saos-2.In addition, P-gp expression in Saos-2are hard to detect.2. Twenty four of the74osteosarcoma cases showed Trpsl-positive expression in the central portion of tumors(32.4%),while The MDR1/P-gp expression was observed in12patients out of74(16.2%).Significant correlations were found between Trpsl and MDRl/P-gp expression in all osteosarcoma patients (r=0.478, P<0.01, Fisher’s Exact Test). We observed no immunohistochemical staining of the two markers in9osteoid osteoma samples.3. Both mRNA and protein level of TRPS1and MDR1were with the same tendency of changes in the TRPS1affected osteosarcoma cell lines groups compared with the negative control groups.4. The P-gp efflux function was changed a lot after48h of transfection in different cell groups. MTT assay showed that IC50values of the four anti-cancer drugs all decreased remarkably after TRPS1interference and the over-expression groups were on the contrary. Rh123fluorescence intensity was examined to compare MDRl/P-gp function among the three cell lines. The retained fluorescence intensity in each negative control group was set as the background. After1h of incubation in Rh123-free culture medium, a rightward shift in the fluorescence peak associated with cellular inhibition of TRPS1and a rapid reduction in intracellular Rh123was observed in all TRPS1-transfected cells.5. We examined the MDR1gene sequence and identified a putative Trpsl binding site (ACTATCTTGT) in the-932～-925bp of the MDR1gene promoter. There was a reduction of TGFβ-1mRNA and protein levels in TRPS1over-expressed U2-OS cell lines compared with negative controls.[Conclusions]TRPS1and MDRl/P-gp was expressed to some degree and positive correlation between MDR1/P-gp and TRPS1was also found in the osteosarcoma cell lines and clinical tissues. The silencing of TRPS1decreased MDR1expression and the over-expression was on the contrary, which indicate that TRPS1may be possible to be identified as a new molecular marker to predict multidrug resistance and target for MDR reversing in osteosarcoma. We confined a single Trpsl binding site in the MDR1/P-gp promoter in U2-OS cell lines.TGFβ-1was also inhibited by Trpsl in the context of human osteosarcoma.