| Dermatofibrosarcoma protuberans (DFSP) is a rare, slow-growing, infiltrating soft tissue neoplasm of intermediate malignancy, which means that this tumor has a propensity for local recurrence after tumor resection but rarely metastasizes. DFSP is characterized for its particular histological features, cytogenetics and molecular rearrangements.With the development of histology and molecular biology, DFSP is gradually familiar with more and more pathologists and clinicians. However, there are still a variety of questions in the diagnosis of DFSPs. It is common to mistake DFSPs with numbers of other binign and malignant soft tissue tumors, but the treatment and prognosis are absolutely different among these tumors. A number of immunohistochemical markers are thought to be really helpful in the differential diagnosis between DFSPs and other tumors, though a high degree of specificity is insufficientCurrently, most of studies about DFSPs focus on the applications of diagnosis and treatment, while the researches of its molecular mechanism mainly focus on the characteristic COL1A1-PDGFB gene fusion; few studys aim at the functions of other molecular markers and drugs, however. There are approximately 10% of DFSPs reported with fibrosarcomatous areas. It has been hypothesized that these areas in DFSP increase the risk of recurrence and shorten the interval to recurrence. However, the molecular relationship between classical DFSP and DFSP with fibrosarcomatous areas (FS-DFSP) has not been clarified.With the help of gene microarray technique, some scientists successfully separated differentially expressed genes in DFSPs. Our study aimed at some of the highly expressed genes, Apo D, RAR?and RAR?, identified their expressions in DFSP tumor tissues and cell lines and studied the relationships between gene expression patterns and clinical data. Then we regulated the expression of Apo D in human DFSP cell line CRL-7043 by RNAi and drugs, to investigate the influences of changed Apo D expression level on RAR?and RAR?and on cell proliferation.Methods:1. A tissue microarray was constructed, containing 25 cases of DFSPs, 30 cases of cutaneous fibrous histiocytoma, 10 cases of malignant fibrous histiocytoma, 4 cases of fibrosarcoma, 10 cases of neurofibroma and 5 cases of nodular fasciitis. Immunohistochemical markers, including apolipoprotein D and CD34 were used on these tissue microarray sections by the SP immunohistochemical staining method. In situ hybridization(ISH) using apolipoprotein D oligonucleotide probe was performed on the tissue microarray sections, too;2. Anohter tissue microarray containing 53 cases of DFSPs was constructed. Immunohistochemistry was performed to identify the expressions of RAR?,RAR?and COX-2 in human DFSP tumor tissues. The correlations of expression patterns between RAR?,RAR?and COX-2 as well as clinicopathologic variables were analyzed;3. The expressions of Apo D, RAR?,RAR?in CRL-7043 cell line were identified by RT-PCR and Western Blot;4. siRNA sequences of Apo D gene were synthesized, and transiently transfected into CRL-7043 cell line by liposomes. The efficiency of transfection was analyzed by RT-PCR, Western Blot of Apo D and immunofluorescence;5. The expression changes of RAR?, RAR?proteins after transient transfection in CRL-7043 cell line were observed by RT-PCR and Western Blot; PI staining by flow cytometry was performed to detect the change of cell cycles in CRL-7043 cells after transient transfection;6. CRL-7043 cell line was treated with different combinations of drugs including all-trans retinoic acid(all-trans RA, ATRA) , RAR?selective antagonist Ro41-5253 and RAR?selective antagonist LE-135. The corresponding expression changes of Apo D, RAR??RAR?proteins in CRL-7043 cell line were observed by Western Blot. The different cell proliferation activities were measured by MTT assay. The following changes of cell cycles were analysed by PI staining.Results:1. Immunohistochemically , positive staining of Apo D was seen in 23 of 25(92.0%) cases of DFSPs and 2 of 30(6.7%) cases of cutaneous fibrous histiocytoma, whereas by ISH, positive staining of Apo D was seen in 20 of 22(90.9%) cases of DFSPs and 2 of 26(7.7%) cases of cutaneous fibrous histiocytoma. The protein and mRNA expression patterns of Apo D between DFSPs and cutaneous fibrous histiocytoma had statistical differences(?2=36.79,p?0.05;?2=33.24,p?0.05, respectively);2. 52 DFSPs (98.11%) showed immunohistochemical positive staining for RAR?. 30 showed strong staining (grade 3), 16 showed moderate staining (grade 2) and 7 showed negative or weak staining (grade 1) .The expression patterns between RAR?and RAR?,and between RAR?and COX-2 were of no significant differences;3. 48 DFSPs(90.58%) showed immunohistochemical positive staining for RAR?. 10 showed strong staining (grade 3), 13 showed moderate staining (grade 2) and 30 showed negative or weak staining (grade 1);4. Immunohistochemically, 32 DFSPs (60.38%) stained positive for COX-2. 1 showed strong staining (grade 3), 32 showed moderate staining (grade 2) and 20 showed negative or weak staining (grade 1) .RAR?staining was significantly inversely correlated with COX-2 staining (p<0.001; r=-0.668);5. RT-PCR and Western Blot proved the expressions of Apo D, RAR?and RAR?in CRL-7043 cell line;6. siRNA sequences of Apo D gene were synthesized, and transiently transfected into CRL-7043 cell line by liposomes successfully. Apo D mRNA and protein expressions were down-regulated after transfection;7. The cell cycles of CRL-7043 cell line were varied after transfection, showing a decreased percentage of cells at G0/G1 phase, while RAR?and RAR?protein showed no change after transfection;8. Apo D and RAR?proteins were up-regulated after treated with all-trans RA in CRL-7043, but RAR?showed no alteration at protein level;9. In CRL-7043 cell line, RAR?selective antagonist Ro41-5253 down-regulated the induction of all-trans RA on Apo D while RAR?selective antagonist LE-135 did not, but both Ro41-5253 and LE-135 repressed the induction of all-trans RA on RAR?, which was proved by Western Blot;11. The results of MTT assay showed that after 72h, the inhibition rate of cell proliferation activity of all-trans RA treated group was up to 67.4%±3.5%, while those of ATRA+Ro41-5253 group, ATRA+LE-135 group were 41.2%±5.1%, 44.6%±2.8%, respectively. The data between all-trans RA treated group and ATRA+Ro41-5253 group, and between all-trans RA treated group and ATRA+LE-135 group were significantly different respectively(p<0.05, respectively). PI staining demonstrated the changes in cell cycles after treated with all-trans RA, showing an increased percentage of cells at G0/G1 phase.Conclusions:1. Apo D was overexpressed in DFSP tumor tissues, indicating that it may be a useful immunohistochemical marker in the differential diagnosis between DFSPs and cutaneous fibrous histiocytoma;2. RAR?, RAR?and COX-2 expressed in DFSP tumor tissues and the expression of RAR?was inversely correlated with the expression of COX-2. RARs may be potential therapeutic targets for unresectable DFSP cases;3. The transient transfection of Apo D gene siRNA sequences into human DFSP CRL-7043 cell line could repress Apo D expression, with shortened cell cycles, but could not influence the expressions of RAR?and RAR?;4. RAR?selective antagonist significantly down-regulated the induction of all-trans RA on Apo D, indicating a RAR?pathway in the regulation of all-trans RA on Apo D in the DFSP cell line;5. All-trans RA repressed the cell proliferation of CRL-7043 through both RAR?and RAR?pathways, with retarded cell cycles showing an increased percentage of cells at G0/G1 phase. |
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