| OBJECTIVE:This study is designed to gain more insight into the function of synovial sarcoma (SS) fusion gene. Screening part of genes on downstream of SYT-SSX, the aim was to explain the relationship of SYT-SSX and other pathways. These results would be hoped to elicit a novel path to discovery the function of the fusion genes in SS.METHODS:Two distinct siRNA duplexes for SYT-SSX carried by vector were synthesized, transfected by LipofectamineTM 2000. Total cellular RNAs were isolated using Trizol reagent. Then quantitative real-time PCR (qRT-PCR) was performed using SYBR Green Premix Ex TaqTM in order to manifest the inhibiting effect of SYT-SSX. The study evaluated the whole genome expression in SYO-1 cells inhibited as a result of specific small interfering RNA for SYT-SSX. All microarray data were submitted to the Database of Annotation, Visualization and Integrated Discovery (DAVID) 2008. Some down-stream genes of SYT-SSX chosen from the microarray analysis and involved in cell proliferation were further verified. Their expression was compared in SYT-SSX-blocked SYO-1 and control SYO-1 cells by qRT-PCR. The protein expression of some related down-stream genes was detected by western blot. Cell proliferation was measured using the methyl thiazolyl blue tetrazolium bromide colorimetric dye assay. Cell cycle analysis was conducted by fluorescence-activated cell sorting with cells stained by propidiumiodide. Apoptotic cells were measured with an Annexin V/FITC kit. and analyzed by flow cytometer after transfection. Seventy-four cases were selected into the study, including 54 SYT-SSX positive SSs tested by RT-PCR,4 SYT-SSX negative SSs (diagnosed as SS according to typical clinical context, histologic aspect and immunohistochemical profile), and 16 non-SSs (4 malignant melanoma,4 Ewing's sarcomas,4 malignant peripheral nerve sheath tumor and 4 hemangio-peritheliomas). A tissue microarray (TMA) was constructed. Visualization of DNA fragmentation, a marker of apoptosis, was performed by the TUNEL method using the In Situ Apoptosis Detection Kit. Immunohistochemistry staining was performed on TMA. The expressions of Cyclin D1, CDK4, and p-ERK were analyzed in considering the staining intensity and area extent. Statistical comparisons were made by using ANOVA with subsequent application or t test where appropriate. P value of less than 0.05 was considered statistically significant.RESULTS:1 Compared with the cells only treated with lipofectamineTM, the expression of SYT-SSX reduced about 93.7%in the cells transfected with SYT-SSX-specific siRNA1.2. Compared with gene expression in cells transfected with negative control siRNA, the expression of 375 genes was up-regulated and of 418 genes was down-regulated in cells transfected with SYT-SSX-specific siRNAl. Among the 793 genes with significant change,653 genes could be assigned into different functional categories with DAVID gene functional category analysis. Subsequently,5 pathways hit by biocarta pathway analysis were indicated, including ERK1/2 pathway (P=0.043).3.The expression of ERK mRNA was confirmed to be significantly deceased after blocking SYT-SSX. Moreover, protein expression of ERK and p-ERK detected by western blot was equal to the results of qRT-PCR.4. The survival rate of SYO-1 cells transfected with specific siRNAl was most significantly reduced compared with that of SYO-1 cells only treated with lipofectamineTM.5. In comparison with control SYO-1 cells treated with lipofectamineTM, SYT-SSX-specific siRNAl and siRNA2 caused an increase in the percentage of G1/G0 phase cells, accompanied by a significant decrease in the percentage of S phase cells, cell cycle related proteins, Cyclin D1 and CDK4 were detected in SYO-1 cells after transfection with specific siRNAl and negative control siRNA. The expression of CyclinDl in siRNA1 transfected SYO-1 cells was significantly lower than that in negative control siRNA transcripted cells and cells untreated. But there was no notable change of CDK4 expression in SYO-1 cells treated with different siRNAs. It was shown the apoptotic rate in SYT-SSX-specific siRNAs transfected cells obviously increased.6. Cases positive for SYT-SSX showed higher ki-67 LI than SYT-SSX negative ones. In comparison of cases not detected SYT-SSX, expression of CyclinDl, CDK4 and p-ERK showed a trend to be more expressed in cases with SYT-SSX. Although the AI means in SYT-SSX positive SSs was higher than that in SYT-SSX negative cases, there were not significant difference between TUNEL AI according to expression of SYT-SSX.7. Higher ki-67 LI were shown in p-ERK positive group. Moreover, CyclinD1 and CDK4 were more expressed in p-ERK positive group than those in negative group.CONCLUSIONS:1. vector-based siRNA to block the expression of SYT-SSX had high specificity and efficiency.2. All genes changed were involved in phosphoprotein, transcription regulation, alternative splicing, direct protein sequencing, nucleotide-binding, transport, cell cycle, kinase and so on. Then pathway analysis indicated the ERK pathway were included in. SYT-SSX might play an important role via ERK pathway. Therefore activation of ERK may affect a broad array of cellular functions, including proliferation, survival, apoptosis, motility, transcription, metabolism and differentiation, and is in part responsible for oncogenesis.3. The results in vivo also suggested that the fusion gene SYT-SSX should be considered to play important role on SS cell growth maybe via ERK pathway. |
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