| BACKGROUNDS AND OBJECTIVES:The tumorgenesis and progression is a complex biologic process in which various factors and numerous genes are involved, including many signal molecules. The role and the possible mechanisms of these signaling molecules in this process, and the interactions between these signaling molecules have not yet been clarified.S-100 protein is a family of low molecular weight protein found in vertebrates characterized by two EF-hand types. There are at least 25 different types of S100 proteins. They can combine with calcium and play a variety of physiological roles, including cell proliferation and differentiation, calcium homeostasis, protein phosphorylation, enzyme activity and transcription factor regulation. It was found that there are abnormal expression of some S100 proteins in tumor cells and that these S100 proteins are involved in the tumor cell proliferation, apoptosis and migration. S100A6 is one of them and shows high expression in most of osteosarcoma, ovarian cancer and other tumors. Based on the facts above, we took this subject into further studies about effects of S100A6 in human osteosarcoma cells both in vitro and in vivo.Wnt/β-catenin signaling pathway is a classic pathway, which is closely related to tumors. Overexpression ofβ-catenin, which is a central molecule of the Wnt/β-catenin signaling pathway, can cause the activation of this pathway and promote the occurrence and development of tumor. Osteosarcoma is no exception.Our previous study found: 1) that hS100A6 plays an inhibitive role in human osteosarcoma cell lines MG63 and U2OS; 2) that hS100A6 increases the level of Wnt signal pathway in MG63 and U2OS; 3) that the direct interaction between S100A6 and some effector molecules of Wnt signaling pathway(includingβ-catenin, GSK-3βand DVL) was found by Pulldown assay. And then, the overexpressions of hS100A6 andβ-catenin were found simultaneously in osteoasarcoma. So, we supposed that hS100A6 may be involved in regulating Wnt/β-catenin signaling activity, and that hS100A6 may play its biological roles on osteosarcoma cells via Wnt/β-catenin signaling pathway.So, we investigated the effects of S100A6 onβ-catenin level in human osteosarcoma 143B cell line and further confirmed the direct interaction between S100A6 and the main components of Wnt signaling pathway, includingβ-catenin, GSK-3β, DVL and Axin by co-immunoprecipitation (Co-IP). At the same time, we intend to look for the interactive molecules with S100A6 in 143B cell line by yeast two-hybrid technology (Y2H) and to provide more data for understanding the effects and mechanisms of S100A6 on human osteosarcoma.In this study, recombinant adenoviruses with the genes of hS100A6, siRNA- hS100A6 were used to treat human osteosarcoma cell line 143B and the effects of S100A6 on the cell proliferation, migration, apoptosis andβ-catenin level of 143B cells were studied. Then the direct interactions between S100A6 and the main components of Wnt signaling pathway, including S100A6 andβ-catenin, S100A6 and GSK-3β, S100A6 and DVL, S100A6 and Axin, were detected by co-immunoprecipitation (Co-IP); At last, the interactive molecules with S100A6 in 143B cell line were investigated by yeast two-hybrid technology (Y2H).METHODS:1. The recombinant adenoviruses with hS100A6 gene (AdS100A6), hS100A6-siRNA gene (AdsiS100A6) ,β-catenin gene(Adβ-catenin) and control adenoviruses (AdGFP and AdRFP) were amplified in human embryonic kidney cell 293 (HEK293 cell).2. After up-regulating and down-regulating S100A6 expression in human osteosarcoma cell line 143B by AdS100A6 and AdsiS100A6, respectively, MTT assay and Trypan blue staining were used to check the cell proliferation; Hoechst staining was used to check the cell apoptosis; Transwell room experiment was used to check the cell migration. Animal tumor model were used to verify the effects of hS100A6 on human osteosarcoma in vivo.3. After up-regulating and down-regulating S100A6 expression in human osteosarcoma cell line 143B by AdS100A6 and AdsiS100A6, respectively,β-catenin level was determined by Western Blot.4. The direct interaction between S100A6 and the main components of Wnt signaling pathway (β-catenin, GSK-3β, DVL and Axin) were detected by co-immunoprecipitation(Co-IP).5. Build cDNA library of 143B cells by using Clontech kit, and then look for the interactive molecules with S100A6 in 143B cell line by yeast two-hybrid technology (Y2H).RESULTS:1. The green or red fluorescence protein was expressed in 293 cells infected by adenovirus. The infection rate was about 90% after been infected for 72 h and the titer of virus solution was about 108-109 pfu / ml.2. There was endogenous expression of S100A6 in 143B cells( Immunocytochemistry). 3. In the 143B cells infected with AdS100A6, S100A6 level was1.19 times that of the AdGFP group; In the 143B cells infected with AdsiS100A6, S100A6 level was 61.2% of the AdGFP group's S100A6 level. These results showed that S100A6 gene and siRNA for S100A6 gene carried by recombinant adenoviruses AdS100A6 and AdsiS100A6 could express well in target cells.4. Exogenous hS100A6 inhibited cell proliferation and migration and induced apoptosis in human osteosarcoma cell line 143B.(1) MTT assay: The OD values of AdS100A6 group were 94.35%,88.66% and 86.61% of those of AdGFP group at d 3, d 4 and d5(P<0.05)and significantly decreased in time-dependent manner; the OD values of AdsiS100A6 group were was 109%,106%,126% and 120% of those of AdGFP group from d2(P<0.05)and significantly increased in time-dependent fashion. There were no significant difference between the OD vaules of AdGFP group and blank group. The consistent results have been found in Trypan blue staining.(2) Hoechst stainning: Compared with AdGFP group, the apoptosis rate of AdS100A6 group increased by 25.3%,37.2% and 45.8% at 48, 72 and 96 h (P<0.05); The apoptosis rate of AdsiS100A6 group decreased by 25.9%,36.4% and 39.3% at 48, 72 and 96 h(P<0.05). There was no significant difference between the AdGFP group and blank group.(3) Transwell experiment: Compared with AdGFP group, the transmembrane cell number of AdS100A6 group decreased by 41.2% at 72 h (P<0.05) while the number of AdsiS100A6 group increased by 60.3%(P< 0.05). There were no significant difference between the AdGFP group and blank group.(4) After injection for 30 days, the mean tumor volumes of blank control, AdGFP group, AdS100A6 group and AdsiS100A6 group were 2796±154 mm3, 2876±263 mm3, 1716±120 mm3 and 4520±400 mm3, respectively. There were no significant difference between the mean tumor volumes of blank group and AdGFP group(p>0.05)while there were significant difference between the mean tumor volumes of AdGFP group and experiment groups(p<0.01). A metastatic tumor was found in the liver of one mice of AdsiS100A6 group while none was found in other groups.These results suggested that exogenous hS100A6 could inhibit cell proliferation and migration and induce cell apoptosis in human osteosarcoma cell line 143B, that exogenous hS100A6 could inhibit the growth of human osteosarcoma in vivo.5. Exogenous hS100A6 increasedβ-catenin expression in human osteosarcoma cell line 143B.The 143B cells were infected with AdS100A6 and AdsiS100A6 for 48 h, respectively. Compared with AdGFP group,β-catenin levels of the two cell groups increased by 1.64 times and decreased by 79.8% (P<0.05), respectively. There was no significant difference betweenβ-catenin levels of AdGFP group and blank group. These results suggested that exogenous hS100A6 increaseβ-catenin expression in human osteosarcoma cell line 143B.6. In Co-IP/Western blot, the interactions between hS100A6 andβ-catenin, hS100A6 and GSK-3β, and hS100A6 and DVL were verified. No direct interaction between S100A6 and Axin was found.7. The cDNA library of 143B cells for yeast two-hybrid was successfully constructed.(1) Total RNA of 143B cell was successfully extracted and the mRNA was purified. UV spectrophotometer showed that the concentration of mRNA was high enough and its OD260/OD280 ratio was in the 1.8-2.0 range and suitable for next test.(2) The cDNA library of 143B cells for yeast two-hybrid was constructed. It was showed that double-stranded cDNA appears in a diffused state among the size of 200-10000bp, and that the second chain was slightly larger than the first chain.(3) The library titer was 7.6×10~7 CFU / ml.(4) The inserts were amplified from 9 of the 10 colonies by colony PCR. It suggested that recombinant rate of cDNA library was 90%.(5) The sizes of the inserts were between 500-1000 bp. The polymorphism of the library was good enough.8. pGBKT7-S100A6, yeast two-hybrid bait vector, was successfully constructed. Using primers designed from the pGST-S100A6, S100A6 gene fragment was amplified and subcloned into pGBKT7 vector. pGBKT7-S100A6 was sequenced and the result suggested that pGBKT7-S100A6 was successfully constructed. We tested the expression product of pGBKT7-S100A6 in AH109 yeast cells and didn't find toxic effects, leakage or self-activation.9. The interactive molecules with S100A6 in 143B cell line were detected by yeast two-hybrid technology (Y2H)The library transformed into Y187 cells and the bait plasmids were co-transfected into yeast AH109 cells, grew in nutritional deficiencies selective agar plate. After four rigorous screening, only two positive insert paragraph clones were found. After sequencing and BLAST analysis, the nucleotides of the 2 clones were similar to S100A6 gene and pGADT7 sequence.CONCLUSION:1. Exogenous hS100A6 inhibits cell proliferation and migration and induces cell apoptosis in human osteosarcoma cell line 143B in vitro and the growth of human osteosarcoma in vivo.2. hS100A6 increasesβ-catenin expression of human osteosarcoma cell line 143B.3. The direct interactions between hS100A6 andβ-catenin, hS100A6 and GSK-3β, hS100A6 and DVL were verified by Co-IP/Western blot. No direct interaction between hS100A6 and Axin was found.4. The cDNA library construction of 143B cell line for yeast two-hybrid is successful; the construction of pGBKT7-S100A6, yeast two-hybrid bait vector, is successful.5. Two positive colonies are found by the yeast two-hybrid. Their sequences have higher homology with S100A6 gene and pGADT7 sequence, respectively. They need further identification.6. Based on the results above, we suppose that the inhibitory effect of hS100A6 on human osteosarcoma may be mediated by other signaling pathways and that there may be antagonistic effects between these signaling pathways and the Wnt /β-catenin signaling pathway upregulated by hS100A6. They play together to realize the fine adjustment on cell proliferation, migration and apoptosis of human osteosarcoma.
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