| Background:Pelvic ring includs a cyclic structure of bilateral iliac bone, ischium, pubis and sacrum formed. A considerable number of bone tumors in this area:it is the most common site of chondrosarcoma.The proportion of chondrosarcoma in all primary bone malignancies is about 25%, and is the most common primary malignant tumors. The main feature of tumor cells is the production of hyaline cartilage case. Chondrosarcoma is the relatively low degree of malignancy,5-year survival rate is about 55%-72%. Its growth is relatively slow, but probably translates into other malignant tumors, such as osteosarcoma or fibrosarcoma. In all primary tumors of the sacrum, chondrosarcoma is the common pathology. Sacrum chondrosarcoma mostly occurs in the upper sacrum, its main clinical manifestations include neurological damage, and pain. Because the incidence of part of a special, X-ray, CT scan are a certain degree of difficulty, so early diagnosis is difficult. Mainly due to its early involvement of the nervous system, it is often misdiagnosed as back pain or lumbar disc herniation and so on. Chondrosarcoma for radiotherapy and chemotherapy are not sensitive, so surgery is the main treatment for the treatment of the sacrum chondrosarcoma. But the anatomical position of sacrum is complex, and is adjacent to important vascular, visceral and sacral nerve, which makes the sacrum chondrosarcoma resection range limited. what is more, the sacrum is connected with lumbar bone conduction, So plays the role of Conduction stress. At the same time, the sacrum is also important component of pelvic ring, which causes the sacrum spinal reconstruction and stability of the pelvic ring after resection of chondrosarcoma. The operation treatment of sacral chondrosarcoma faces a great challenge.The treatment effect for early stage of cancer is more ideal, but generally poor prognosis of advanced cancer, but tumor immunotherapy is currently an important cancer treatment, so the development of tumor markers for cancer treatment has very important significance. Epithelial-mesenchymal transition (EMT) is very important in embryonic development, tissue repair and disease. In adult, the abnormal activation of developmental programming mechanism may lead to the development of various diseases. Organ fibrosis and cancer metastasis are controlled by EMT. The control of cancer metastasis by EMT may be produced by the cancer stem cells and transplanting to other organizations to form secondary tumors. EndMT also involved in the process of fibrosis and cancer, to participate in the formation of stem-like cells, and also play an important role in the formation of heterotopic ossification and calcification of vascular progenitor cells to differentiate into bone.Epithelial cells form a polar cell layer via the adhesion of a variety of cell adhesion molecules, such as claudins and E-cadherin. The polarity of the cell layers beneath the basement membrane of epithelial cells will be anchored to the substrate surface, and maintain the top-basal polarity by connection of the fiber and hemidesmosomes. Adhesion between the base film and the the adjacent cells is very important in maintenance of epithelial morphology. In the EMT process, epithelial cells lose these features, obtained the ability to stromal invasion and metastasis, so that these cells leave the parenchyma and into the systemic circulation causing cancer metastasis. In some cases, cadherin convert to N-cadherin from E-cadherin, which is an important biological markers of EMT. In addition, increased expression of vimentin, and Snail and Slug is intercellular mesenchymal phenotype markers. Snail and Slug can change "E" to "N"-cadherin, and initiate cell EMT. Therefore, EMT pathway may be used as targeting molecules for tumor therapy and tumor metastasis suppression. Invasion, angiogenesis and metastasis of tumor cells are regulated by interaction of many overlapping molecular pathways.Chemokines and their receptors are important elements of the process, which is involved in cell movement, migration and proliferation. So far, EMT induced by SDF-1/CXCR4 has been shown to play an important role in invasion and metastasis of various cancers. CXCR4/SDF1 can mediate the proliferation and migration of tumor cells and enhanced tumor-associated angiogenesis, and thus indirectly promote tumor metastasis. Survivin is a member of the inhibitor of apoptosis protein family, which has a strong spatial and temporal specificity of expression, only expressed in embryonic and tumor tissues, and may play an important role in tumor resistance, Survivin expression is induced in most tumor tissues, and regulated by the cell cycle. Survivin has an important regulatory role in cell cycle progression. Biological effects of surviving incuding inhibition apoptosis, positioning of mitotic chromosomes, and promoting cell activity and metastasis. Important role of Survivin in cell division has now been recognized. A variety of methods and a variety of cell types mitosis deficient cells have proved this point. Survivin expresses in normal cells show a strong dependence of the cell cycle. However, in the sacral chondrosarcoma,the link between CXCR4 and Survivin is unclear, and whether the regulation of SDF-1/ CXCR4 in cell migration is dependent on Survivin needs further study.Purpose and meaning:chondrosarcoma is currently one of the most common primary malignant tumor, seriously affecting the quality of people’s lives, the early diagnosis is difficult, and treatment methods are lack of variety. Currently, the research on chondrosarcoma, especially sacral chondrosarcoma is still very scarce, and mainly focused on the clinical staging and treatment of tumors. Study of early diagnosis of sacral chondrosarcoma, biological markers, pathogenesis and tumor immunotherapy are still scarce, and these are precisely the key issues to cure cancer, and prevent recurrence of. Therefore, this study will start with the molecular mechanism of sacral chondrosarcoma, focus on the role of CXCR4 and Survivin in chondrosarcoma cell EMT and cell cycle regulation. Integrated the important signaling pathways of tumorigenesis process, including EMT, MEK/ERK and PI3K /AKT pathway and cell cycle regulation together by CXCR4 and Survivin. Preliminary study of both the occurrence and metastasis offers a possible new target for early diagnosis and immunotherapy for sacral chondrosarcoma.Methods:30 sacral chondrosarcoma samples were randomly chosen from the Department of Pathology in Qilu Hospital of Shandong University and Nanfang Hospital of Southern Medical University from January 2008 to June 2014. All samples were fixed with 10% formaldehyde and embedded with paraffin. First of al,l samples were sliced and immunohistochemical, then CXCR4 and Survivin expression in all samples were detected, including the location of protein expression in cells and its expression level. the expression of CXCR4 and Survivin and clinical characteristics of each sample were analyzed to detect the relationship between the expression of CXCR4 and Survivin and clinical characteristics; then quantitative PCR and western blot were used to detect Survivin protein and mRNA in different concentrations and time points of SDF-1 treatment; the MEK/ERK and PI3K/AKT pathway were blocked by kinase inhibitors in SW1353 cells to detect SDF-1-induced expression of survivin protein levels; Next, we use survivin siRNA transfected SW1353 cells to reduce the level of survivin mRNA and protein expression, then use flow cytometry to analyze the impact on cell cycle progression after survivin reduce; Finally, Western blot analysis of Snail and N-cadherin was done after 20 ng/ml SDF-1 treated the survivin siRNA transfected SW1353 cells.Results:CXCR4 protein mainly expresses in cell membrane and cytoplasm of cells of the sacral chondrosarcoma, while Survivin protein was mainly localized in the nucleus and cytoplasm. Both of them have some overlap in space on its subcellular localization. The CXCR4 and Survivin expression and clinical characteristics of the 30 patients were statistical analyzed, which showed that Survivin protein expression was positive in 87.6%(26/30), CXCR4 in sacral chondrosarcoma tissues The positive expression rate was 83.3%(25/30) in the sacral chondrosarcoma. In addition, in the well-differentiated sacral chondrosarcoma patients, the proportion of CXCR4 expression was 73%, while in patients with severe sacral chondrosarcoma, the proportion of CXCR4 expression was 84%; at the same time, in patients with mild sacral chondrosarcoma, positive expression of Survivin protein was 73%, and with moderate to severe for 84%, CXCR4 and Survivin protein expression was highly consistent. More importantly, Western blot showed that the expression levels of CXCR4 and Survivin was significantly higher in severe sacral chondrosarcoma tissues than that of mild tissue, indicating the severity of CXCR4 and Survivin and sacral chondrosarcoma are closely related. In severe sacral chondrosarcoma (Ⅱ, grade Ⅲ), the positive ratio of CXCR4 was 100%(16/16), while in the mild, CXCR4-positive ratio was 64.3%(9/14). As expected, Survivin in severe and mild (grade Ⅰ) sacral chondrosarcoma (Ⅱ, grade Ⅲ) were 100%(16/16)and 71.4%(10/14), respectively. Compared with mild sacral chondrosarcoma, in moderate to severe sacral chondrosarcoma tissue, CXCR4 and Survivin expression was significantly increased, (P<0.05). In addition, we also analyzed the relationship between CXCR4 and Survivin and recurrence among sacral chondrosarcoma. In all cases of recurrence, the proportion of positive expression of CXCR4 and Survivin were 100%(15/15). In the cases of recurrence not occurred, CXCR4 expression ratio was 67%(10/15), and Survivin expression was 73%(11/15). Statistics showed that there are significant differences in the expression of CXCR4 (P=0.014) and Survivin (P=0.032) recurrence and no recurrence in the proportion of cases, and had direct association with the recurrence of sacral chondrosarcoma (P<0.05).western blot results showed that with the increase of SDF-1 protein concentration, from 0 to 10,20ng/ml, the expression of Survivin protein also increased, but no significant difference in 50ng/ml and 20ng/ml SDF-1 treatment, indicating the inducing effect was saturated when the SDF-1 reached 20ng/ml. Using 20 ng/ml SDF-1 treated SW1353 cells in vitro, after 0,2,4,8,12, and 36 hours, the sample were harvested for western blot, respectively. The results showed that with the increase of treatment time,0,2,4,8 hours after treatment, the expression of Survivin protein gradually increased, but Survivin protein expression after treatment 8,12,36 hours, there was no significant change, indicating after 20ng/ml SDF-1 treatment for 8h, its induction effect of Survivin protein was saturated. These results suggest that SDF-1 induction of Survivin is dose and time dependent. Quantitative PCR showed that SDF-1 induced Survivin mRNA was in the same manner. In the study of where SDF-1 upregulation of survivin is dependent on MEK/ERK and PI3K/AKT pathway, we found that, compared with DMSO-treated control group, U0126 (P=0.024) and LY294002 (P=0.0293) can significantly reduce SDF-1 induced survivin protein levels in SW1353 cells. This suggests that the induction of survivin protein by SDF-1 is dependent on MEK/ERK and PI3K/AKT pathway. Then we build a survivin siRNA transfected SW1353 cells, survivin protein levels decreased significantly in transfected cells. Compared with the control group, siRNA transfection induced survivin expression level is reduced, and can inhibit the expression of cyclin D1 and CDK2, suggesting that survivin may affect cell cycle progression.We further use of flow cytometry to analyzed the cell cycle of the control group and transfected cells, and found that, compared with the SDF-1 treatment alone and the control group, survivin siRNA significantly reduced in S-phase cells proportion. After treatment 24h, compared with 23.5% in the control group, survivin siRNA transfected cells were 6.2% of the S cell count (Df=2, x2=6.73,0.01<P<0.05). To determine the role of SDF-1 in surviving-induction in the EMT, we analyzed the expression levels of Snail and N-cadherin after 20 ng/ml SDF-1 treatment in control group and of survivin siRNA transfected SW1353 cells. As shown, the expression levels Snail and N-cadherin were detected by western at different time points after the SDF-1 treatment, in the control group, the expression levels of Snail and N-cadherin significantly upregulated by SDF-1 with the extension of the time increases occur, while in survivin siRNA transfected cells, with the extension of the processing time of SDF-1, N-cadherin and Snail expression was significantly reduced, which indicating that, upregulating of Snail and N-cadherin by SDF-1 is dependent on the level of survivin protein.Conclusion:Firstly, immunohistochemical results proved CXCR4 protein was mainly localized in cytoplasmic and membrane in chondrosarcoma cells, and Survivin protein was mainly localized in the nucleus and cytoplasm in sacral chondrosarcoma cells. The positive ratio of CXCR4 and Survivin protein in the sacral chondrosarcoma was very high, in addition, expression of both has a very clear correlation with the severity and recurrence of sacral chondrosarcoma, but independent with gender and age. Therefore the high expression of CXCR4 and Survivin protein may be possible to as biological markers for the early detection of sacral chondrosarcoma. Western blot and quantitative PCR results showed that with the increase of SDF-1 protein concentration and time of treatment, the protein and mRNA expression of Survivin also increased, showed Survivin protein and mRNA levels are subject to the activity of SDF-1/CXCR4. SDF-1/CXCR4 functions upstream of Survivin, and certain components activated by SDF-1/CXCR4 can regulate Survivin expression levels at the post-transcriptional and transcription level, which will couple SDF-1/CXCR4 pathway and Survivin together, and also proved the interlinkages of the invasion and metastasis of tumor cells controlled by SDF-1/CXCR4 and cell cycle regulation by Survivin. Experiments of MEK/ERK and PI3K/AKT pathway inhibitor proved MEK/ERK and PI3K/AKT involved in regulatory role SDF-1/CXCR4 on Survivin. Our study also demonstrated that the use of siRNA inhibited Survivin of expression SW1353 cells. Our results should that inhibition of Survivin by siRNA could significantly affect the cell cycle, reduced in the proportion of SW1353 cells in G2/M phase. While Survivin siRNA transfected cells was significantly inhibited the induced expression of Snail and N-cadherin after SDF-1 treatment. As molecular markers of EMT, the reduction of Snail and N-cadherin showed decreased expression of Survivin protein can suppress EMT of sacral chondrosarcoma, mans that SDF-1 CXCR4 induced EMT is dependent on Survivin protein. Interaction between SDF-1/CXCR4 and Survivin protein showed that cell cycle and EMT can be coordinated with each other, influence each other, played an important role in the regulation of invasion, metastasis and recurrence of the sacral chondrosarcoma cells. Our study provided a possible biological maker for early diagnosis and molecular target of tumor immunotherapy for sacral chondrosarcoma. |
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