Calcyclin在卵巢上皮性癌中的表达及其临床意义

发布时间：2018-09-14 15:02

【摘要】：目的卵巢上皮性癌是常见的女性恶性肿瘤之一，在女性生殖系统恶性肿瘤中，其死亡率也是最高的。卵巢癌患者的早期症状不典型，目前尚无有效的早期筛查和特异性诊断方法，超过70%的患者已是中晚期。目前标准治疗方案为理想的肿瘤减灭术和以铂类为基础的化疗，但近年来卵巢癌患者五年生存率仅为30%-40%左右[42]。寻找卵巢癌有效的基因治疗的分子靶点是卵巢癌目前亟待解决的临床问题。钙周期蛋白(Calcyclin，Cacy)属于S100蛋白家族。文献报道，Calcyclin在胰腺癌、肺癌、乳腺癌、卵巢癌、肝癌、结直肠癌等多种类型的恶性肿瘤中均出现表达明显增加[3,27-32]。有研究证实Calcyclin与上皮性癌发生发展相关[36,37]。还有研究表明Calcyclin高表达与胰腺癌、骨肉瘤、胃癌、肺腺癌、黑色素瘤等多种恶性肿瘤的转移和不良预后相关[28,38-40]。这些研究均提示，Calcyclin可能是肿瘤基因治疗的良好候选靶点。目前Calcyclin蛋白在卵巢上皮性癌中的表达及其临床意义尚不清楚。本研究旨在从组织水平、血清水平统计分析Calcyclin蛋白表达与卵巢癌的相关性，并初步探讨Calcyclin作为肿瘤基因治疗的可行性。方法（1）本研究首先用免疫组化技术检测60例卵巢癌病理组织标本中Calcyclin的蛋白表达情况，统计分析Calcyclin与卵巢癌的发生发展的关系，探讨Calcyclin与卵巢上皮性癌临床病理因素之间的相关性；（2）用ELISA法检测正常血清、良性卵巢肿瘤和卵巢癌患者Calcyclin血清浓度，统计分析Calcyclin血清浓度与卵巢癌的相关性，初步验证Calcyclin作为检测卵巢癌病情发展的分子标志物的可行性；（3）我们利用RNA干扰技术特异性降低Calcyclin表达水平后，观察卵巢癌细胞细胞周期、侵袭能力、顺铂敏感性等生物学行为的变化，探讨Calcyclin作为卵巢癌靶向治疗候选基因的可行性。结果（1）免疫组化检测卵巢癌组织中Calcyclin表达情况，结果表明低分化的卵巢癌患者中Calcyclin高表达比例（55.0%）显著高于高分化的卵巢癌患者中Calcyclin高表达比例（15.4%），有统计学差异(P＜0.05)。Calcyclin表达水平与手术分期有关，随着手术分期的增加，Calcyclin表达水平也随之明显上升，有统计学差异(P＜0.05)。在出现腹水和淋巴结转移的卵巢癌患者中，Calcyclin高表达的比例明显增加，有统计学意义(P＜0.001)。（2）ELISA法检测Calcyclin血清浓度，结果表明卵巢癌患者Calcyclin血清平均浓度为1242.47+178.17pg/ml，显著高于正常血清（489.73+37.86pg/ml）和良性卵巢肿瘤（700.75+43.14pg/ml），差异有统计学意义（P0.01）。然后我们统计分析了不同手术分期和细胞学分级的卵巢癌患者Calcyclin血清浓度。结果显示Ⅲ期、Ⅳ期卵巢癌患者Calcyclin血清浓度分别为644.79+67.89pg/ml和1087.44+104.66pg/ml，显著高于Ⅰ期、Ⅱ期卵巢癌患者（359.35+37.82pg/ml和499.44+42.56pg/ml），差异有统计学意义（P0.001）。同时我们还发现低分化的卵巢癌患者Calcyclin血清浓度显著升高，为1320.08+102.59pg/ml，与高分化（573.58+37.28pg/ml）和中分化（832.91+75.62pg/ml）相比，差异有统计学意义（P0.05）。（3）利用RNA干扰技术特异性降低Calcyclin表达水平后，细胞周期检测结果表明，Calcyclin表达降低后A2780细胞出现了G0/G1期比例升高，，S期比例降低。转染6.25、12.5nmol/LCalcyclinsiRNA后A2780细胞增殖指数分别为39.4和31.0，较空白对照组和阴性对照siRNA组显著降低。（4）Transwell试验结果表明，CalcyclinsiRNA分别转染A2780细胞后穿膜细胞数目显著减少，有统计学意义（p＜0.05）。这一结果提示随着Calcyclin表达降低，A2780细胞侵袭能力也随之明显降低。（5）MTT法检测CalcyclinsiRNA转染后A2780细胞对顺铂敏感性的影响，结果显示CalcyclinsiRNA引起顺铂对A2780细胞的抑制率增加，即一定程度提高了A2780细胞对顺铂的敏感性。对顺铂半数致死剂量（IC50）的计算结果显示，转染6.25、12.5nmol/LCalcyclinsiRNA的A2780细胞对顺铂IC50值分别为13.42μM和11.32μM，和阴性对照siRNA组IC50值（18.12μM）均显著降低。结论（1）免疫组化结果显示Calcyclin表达水平与卵巢癌细胞分化程度、手术分期、腹水和淋巴结转移有关，提示Calcyclin表达和与卵巢癌的发生发展、侵袭转移和不良预后密切相关，有可能成为临床评价病情进展和随访的参考指标。（2）Calcyclin的血清浓度在卵巢癌患者中显著升高，并与手术分期和细胞学分级相关，提示Calcyclin的血清浓度与卵巢癌病情发展相关。但本研究由于时间有限，病例样本量较小，Calcyclin能否成为监测肿瘤病情发展的分子标志物还需要进一步大样本研究验证。（3）CalcyclinsiRNA转染卵巢癌A2780细胞后，引起A2780细胞G0/G1期阻滞，侵袭能力降低，同时对顺铂的敏感性有所增强。Calcyclin蛋白高表达对卵巢癌A2780细胞的恶性生物学行为有促进作用，有可能成为卵巢癌靶向治疗的新候选靶标。
[Abstract]:objective
Ovarian epithelial carcinoma is one of the most common malignant tumors in women, and its mortality rate is the highest among the malignant tumors of the female reproductive system. The early symptoms of ovarian cancer patients are atypical, there is no effective early screening and specific diagnosis method, more than 70% of the patients are in the middle and advanced stage. In recent years, the 5-year survival rate of ovarian cancer patients is only about 30%-40%[42]. It is an urgent clinical problem to find effective gene therapy for ovarian cancer.
Calcyclin (Cacy) belongs to the S100 protein family. The expression of Calcyclin in pancreatic cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, colorectal cancer and other types of malignant tumors was significantly increased [3,27-32]. Overexpression is associated with metastasis and poor prognosis in pancreatic cancer, osteosarcoma, gastric cancer, lung adenocarcinoma, melanoma and other malignant tumors [28,38-40].
At present, the expression of Calcyclin protein in ovarian epithelial carcinoma and its clinical significance are still unclear. The purpose of this study is to analyze the correlation between the expression of Calcyclin protein and ovarian cancer at tissue level and serum level, and to explore the feasibility of Calcyclin as a gene therapy for ovarian cancer.
Method
(1) Immunohistochemical technique was used to detect the expression of Calcyclin in 60 ovarian cancer specimens. The relationship between Calcyclin and the development of ovarian cancer was analyzed statistically.
(2) Serum Calcyclin levels in normal serum, benign ovarian tumors and ovarian cancer patients were detected by ELISA, and the correlation between serum Calcyclin levels and ovarian cancer was statistically analyzed.
(3) After specific reduction of Calcyclin expression by RNA interference, we observed the changes of cell cycle, invasiveness and cisplatin sensitivity of ovarian cancer cells, and explored the feasibility of Calcyclin as a candidate gene for targeted therapy of ovarian cancer.
Result
(1) The expression of Calcyclin in ovarian cancer tissues was detected by immunohistochemistry. The results showed that the expression of Calcyclin in poorly differentiated ovarian cancer patients (55.0%) was significantly higher than that in well differentiated ovarian cancer patients (15.4%). The expression of Calcyclin was related to the stage of operation (P < 0.05). The expression of Calcyclin increased significantly with the increase of lymph node metastasis and ascites (P < 0.05). In ovarian cancer patients with ascites and lymph node metastasis, the expression of Calcyclin increased significantly (P < 0.001).
(2) Calcyclin serum concentration was detected by ELISA. The results showed that the average serum concentration of Calcyclin in ovarian cancer patients was 1242.47+178.17 pg/ml, which was significantly higher than that in normal serum (489.73+37.86 pg/ml) and benign ovarian tumor (700.75+43.14 pg/ml), and the difference was statistically significant (P 0.01). The results showed that the serum concentration of Calcyclin in patients with stage III and stage IV ovarian cancer was 644.79+67.89pg/ml and 1087.44+104.66 pg/ml, respectively, which was significantly higher than that in patients with stage I and stage II ovarian cancer (359.35+37.82 pg/ml and 499.44+42.56 pg/ml), and the difference was statistically significant (P 0.001). The serum concentration of Calcyclin in cancer patients was 1320.08+102.59 pg/ml, which was significantly higher than that in well-differentiated (573.58+37.28 pg/ml) and moderately differentiated (832.91+75.62 pg/ml) patients (P 0.05).
(3) Cell cycle analysis showed that the proportion of G0/G1 phase in A2780 cells increased and that of S phase decreased after specific reduction of Calcyclin expression by RNA interference technique. Group decreased significantly.
(4) Transwell assay showed that the number of transfected A2780 cells significantly decreased (p < 0.05). The results suggested that the invasive ability of A2780 cells decreased with the decrease of Calcyclin expression.
(5) MTT assay showed that the inhibition rate of cisplatin on A2780 cells was increased after transfection of CalcyclinsiRNA, that is to say, the sensitivity of A2780 cells to cisplatin was increased to a certain extent. The IC50 values of 2780 cells to cisplatin were 13.42 and 11.32 mu M, respectively. The IC50 values (18.12 mu M) of the negative control siRNA group were significantly decreased.
conclusion
(1) Immunohistochemistry showed that the expression of Calcyclin was correlated with the differentiation of ovarian cancer cells, surgical stage, ascites and lymph node metastasis, suggesting that the expression of Calcyclin was closely related to the occurrence and development of ovarian cancer, invasion and metastasis and poor prognosis, which might be a reference index for clinical evaluation of disease progression and follow-up.
(2) The serum concentration of Calcyclin was significantly increased in ovarian cancer patients and correlated with surgical stage and cytological grade, suggesting that the serum concentration of Calcyclin was associated with the development of ovarian cancer. Validation of large sample studies.
(3) After transfection of CalcyclinsiRNA into ovarian cancer A2780 cells, the G0/G1 phase arrest of A2780 cells was induced, and the invasiveness of A2780 cells was decreased, and the sensitivity to cisplatin was enhanced. The high expression of Calcyclin protein could promote the malignant biological behavior of ovarian cancer A2780 cells and might be a new candidate target for targeted therapy of ovarian cancer.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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