CLOCK基因参与甲状腺癌发生及进展的生物学功能及分子机制研究
发布时间：2018-08-30 20:44
【摘要】：研究背景:甲状腺癌是内分泌系统最常见的恶性肿瘤之一,已成为国际上发病率增长最快的实体瘤。临床上用于甲状腺癌早期筛查的指标极为匮乏,亟待寻找新的分子标志物。大量研究发现生物节律基因在大肠癌、乳腺癌等肿瘤的发生及进展中发挥着重要作用。其中CLOCK、BMAL1、NPAS2三种生物节律基因的蛋白产物组成正性调控元件,驱动生物节律的产生,并通过调控下游c-Myc、P53、P21等大量钟控基因参与肿瘤细胞增殖、侵袭、转移等重要功能,但生物节律基因在甲状腺癌领域的研究较少。课题组前期通过免疫组化预实验,发现甲状腺乳头状癌组织中CLOCK基因表达升高。这些提示我们CLOCK基因在甲状腺癌的发生及进展中可能发挥重要作用,但其生物学作用及分子机制尚不清楚。目的:利用CLOCK基因siRNA干涉片段,下调甲状腺癌细胞中CLOCK基因表达水平,分析干涉后甲状腺癌细胞的生物学功能变化。并检测P21、P53、CyclinD1等钟控基因的表达变化,初步探讨CLOCK基因参与甲状腺癌发生及进展的分子作用机制。方法:常规培养甲状腺癌细胞株BCPAP,常规培养。根据CLOCK基因设计特异性的siRNA,利用Lipofecter 2000试剂转染BCPAP细胞,将甲状腺癌细胞株分为空白对照组(KD组),转染试剂对照组(ZD组),单纯siRNA对照组(SD),阴性对照组(YD组)和干涉组(GS组)5组。给予不同处理,利用Western Blot检测干涉效率,CCK8实验检测细胞增殖能力,粘附实验检测细胞粘附能力,流式细胞仪检测细胞凋亡程度,Transwell侵袭实验评估侵袭与转移能力。并通过Western Blot检测P21、P53、CyclinD1等分子在干涉CLOCK基因后的表达变化。结果:1.干涉组(GS组)BCPAP细胞增殖能力较空白对照组(GD组)降低(P0.05)。ZD组、SD组、YD组与GD组比无明显改变(P0.05)。2.干涉组(GS组)BCPAP细胞凋亡较空白对照组(GD组)增多(P0.05)。ZD组、SD组、YD组与GD组比无明显改变(P0.05)。3.干涉组(GS组)BCPAP细胞粘附能力较空白对照组(GD组)减低(P0.05)。ZD组、SD组、YD组与GD组比无明显改变(P0.05)。4.干涉组(GS组)BCPAP迁移及侵袭细胞数较空白对照组(GD组)减少(P0.05)。ZD组、SD组、YD组与GD组比无明显改变(P0.05)。5.P21、P53蛋白在干涉组(GS组)较空白对照组(GD组)升高(P0.05),CyclinD1蛋白在干涉组(GS组)较空白对照组(GD组)降低(P0.05)。结论:1.下调CLOCK基因的表达可以促进甲状腺癌细胞凋亡,抑制甲状腺癌细胞增殖、浸润及转移。2.CLOCK基因可能是通过调控甲状腺癌细胞中P21、P53和CyclinD1重要基因的表达,参与甲状腺癌的发生及进展。
[Abstract]:Background: thyroid carcinoma is one of the most common malignant tumors in the endocrine system. Clinical indicators for early screening of thyroid cancer are extremely scarce, so it is urgent to find new molecular markers. A large number of studies have found that biological rhythm genes play an important role in the occurrence and progression of colorectal cancer and breast cancer. The protein products of the three biological rhythm genes of CLOCK,BMAL1,NPAS2 constitute positive regulatory elements, which drive the production of biological rhythms, and participate in the important functions of tumor cell proliferation, invasion and metastasis by regulating a large number of clock control genes such as c-Myche P53, P21 and so on. However, biological rhythm genes in the field of thyroid cancer are less studied. We found that the expression of CLOCK gene in papillary thyroid carcinoma was higher than that in thyroid papillary carcinoma by immunohistochemistry. These results suggest that our CLOCK gene may play an important role in the carcinogenesis and progression of thyroid carcinoma, but its biological role and molecular mechanism are still unclear. Aim: to down-regulate the expression of CLOCK gene in thyroid cancer cells by siRNA interference fragment of CLOCK gene and analyze the biological function of thyroid cancer cells after intervention. The expression of P21, P53, CyclinD1 and other clock controlled genes was detected, and the molecular mechanism of CLOCK gene involved in the carcinogenesis and progression of thyroid carcinoma was preliminarily investigated. Methods: thyroid cancer cell line BCPAP, was cultured routinely. Thyroid cancer cells were transfected into BCPAP cells by Lipofecter 2000 reagent according to CLOCK gene design. Thyroid cancer cells were divided into five groups: blank control group (KD group), transfection reagent control group (ZD group), siRNA control group (SD), negative control group (YD group) and intervention group (GS group). Different treatments were used to detect the ability of cell proliferation by Western Blot and CCK8, and the degree of cell apoptosis by flow cytometry and the ability of invasion and metastasis by transwell invasion assay. The expression of P21, P53, CyclinD1 and other molecules were detected by Western Blot after interfering with CLOCK gene. The result is 1: 1. The proliferative ability of BCPAP cells in the intervention group (GS group) was lower than that in the blank control group (GD group) (P0.05). There was no significant difference between the SD group (P0.05) and the GD group (P0.05). 2. The apoptosis of BCPAP cells in the intervention group (GS group) was higher than that in the blank control group (GD group) (P0.05). The adhesion ability of BCPAP cells in the intervention group (GS group) was lower than that in the blank control group (GD group) (P0.05). There was no significant difference between the SD group and the GD group (P0.05). 4. The number of BCPAP migration and invasion cells in the intervention group (GS group) was lower than that in the blank control group (GD group) (P0.05). There was no significant change in the number of BCPAP migration and invasion cells between the SD group and the GD group in the ZD group (P0.05). The expression of p53 protein in the intervention group (GS group) was higher than that in the blank control group (GD group) (P0.05). The expression of CyclinD1 protein in the intervention group (GS group) was higher than that in the control group (P0.05). ) compared with the blank control group (GD group) decreased (P0.05). Conclusion 1. Down-regulating the expression of CLOCK gene can promote the apoptosis of thyroid cancer cells, inhibit the proliferation, invasion and metastasis of thyroid cancer cells. 2. CLOCK gene may regulate the expression of P21, p53 and CyclinD1 genes in thyroid cancer cells. Participate in the development and progression of thyroid carcinoma.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R736.1
本文编号：2214241
[Abstract]:Background: thyroid carcinoma is one of the most common malignant tumors in the endocrine system. Clinical indicators for early screening of thyroid cancer are extremely scarce, so it is urgent to find new molecular markers. A large number of studies have found that biological rhythm genes play an important role in the occurrence and progression of colorectal cancer and breast cancer. The protein products of the three biological rhythm genes of CLOCK,BMAL1,NPAS2 constitute positive regulatory elements, which drive the production of biological rhythms, and participate in the important functions of tumor cell proliferation, invasion and metastasis by regulating a large number of clock control genes such as c-Myche P53, P21 and so on. However, biological rhythm genes in the field of thyroid cancer are less studied. We found that the expression of CLOCK gene in papillary thyroid carcinoma was higher than that in thyroid papillary carcinoma by immunohistochemistry. These results suggest that our CLOCK gene may play an important role in the carcinogenesis and progression of thyroid carcinoma, but its biological role and molecular mechanism are still unclear. Aim: to down-regulate the expression of CLOCK gene in thyroid cancer cells by siRNA interference fragment of CLOCK gene and analyze the biological function of thyroid cancer cells after intervention. The expression of P21, P53, CyclinD1 and other clock controlled genes was detected, and the molecular mechanism of CLOCK gene involved in the carcinogenesis and progression of thyroid carcinoma was preliminarily investigated. Methods: thyroid cancer cell line BCPAP, was cultured routinely. Thyroid cancer cells were transfected into BCPAP cells by Lipofecter 2000 reagent according to CLOCK gene design. Thyroid cancer cells were divided into five groups: blank control group (KD group), transfection reagent control group (ZD group), siRNA control group (SD), negative control group (YD group) and intervention group (GS group). Different treatments were used to detect the ability of cell proliferation by Western Blot and CCK8, and the degree of cell apoptosis by flow cytometry and the ability of invasion and metastasis by transwell invasion assay. The expression of P21, P53, CyclinD1 and other molecules were detected by Western Blot after interfering with CLOCK gene. The result is 1: 1. The proliferative ability of BCPAP cells in the intervention group (GS group) was lower than that in the blank control group (GD group) (P0.05). There was no significant difference between the SD group (P0.05) and the GD group (P0.05). 2. The apoptosis of BCPAP cells in the intervention group (GS group) was higher than that in the blank control group (GD group) (P0.05). The adhesion ability of BCPAP cells in the intervention group (GS group) was lower than that in the blank control group (GD group) (P0.05). There was no significant difference between the SD group and the GD group (P0.05). 4. The number of BCPAP migration and invasion cells in the intervention group (GS group) was lower than that in the blank control group (GD group) (P0.05). There was no significant change in the number of BCPAP migration and invasion cells between the SD group and the GD group in the ZD group (P0.05). The expression of p53 protein in the intervention group (GS group) was higher than that in the blank control group (GD group) (P0.05). The expression of CyclinD1 protein in the intervention group (GS group) was higher than that in the control group (P0.05). ) compared with the blank control group (GD group) decreased (P0.05). Conclusion 1. Down-regulating the expression of CLOCK gene can promote the apoptosis of thyroid cancer cells, inhibit the proliferation, invasion and metastasis of thyroid cancer cells. 2. CLOCK gene may regulate the expression of P21, p53 and CyclinD1 genes in thyroid cancer cells. Participate in the development and progression of thyroid carcinoma.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R736.1
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