分子伴侣mortalin对卵巢癌细胞增殖、侵袭的影响及其相关通路的分析

发布时间：2018-09-18 09:22

【摘要】：卵巢癌是最常见的妇科恶性肿瘤之一,早期发现难,治疗效果差,预后不良,死亡率高。分子伴侣(molecular chaperone)是一类与其他蛋白的不稳定构象相结合并使之稳定的蛋白质,通过控制结合和释放来帮助被结合多肽在细胞内进行折叠、组装、转运、降解等,维持细胞的内稳态。Mortalin,又名Grp75/PBP74/mthsp70,是HSP70分子伴侣家族成员之一,其广泛分布于多个细胞器和胞浆,参与细胞增殖和胞内运输、调控应激反应、稳定细胞骨架、抑制细胞凋亡等。研究发现在许多肿瘤中,如慢性粒细胞白血病、结肠、颅脑肿瘤、乳腺癌和肝癌中mortalin表达上调。过表达mortalin可以增加乳腺癌细胞的恶性程度,参与肝转移。课题组先前通过对183例卵巢癌组织以及癌旁组织中的组织芯片检测结果显示,mortalin在卵巢癌组织中表达明显高于癌旁组织；且mortalin的表达与卵巢癌的分期有关,分期越高的卵巢癌组织中,mortalin表达量越高。提示,mortalin高表达可能与卵巢癌的发生、发展有关。本课题选取8株卵巢癌细胞系A2780/cis、COC1、HO-8910、PM-8910、 OV-90、Caov-3、SKOV-3和ES-2,分别在蛋白质水平和mRNA水平检测其mortalin的表达量。研究表明,在8株细胞系中,OV-90细胞中]mortalin的mRNA和蛋白的表达量最低,而顺铂耐药型卵巢癌细胞系A2780/cis和腹水中提取的卵巢癌细胞COC1的mortalin的mRNA和蛋白的表达量最高。课题组之前的实验结果显示,人卵巢癌顺铂敏感型细胞系A2780中mortalin表达量明显低于人卵巢癌顺铂耐药型细胞系A2780/cis。以上结果提示,motalin的表达与卵巢癌细胞的恶性程度可能有关。随后,选取人卵巢癌顺铂敏感型细胞系A2780和人卵巢癌顺铂耐药型细胞系A2780/cis,通过构建mortalin上调、下调的慢病毒载体,分别感染两株细胞,经流式筛选分别获得mortalin上调表达、下调表达稳定株。通过CCK-8(Cell Counting Kit-8)实验、克隆形成实验,细胞划痕实验和transwell细胞侵袭实验分别检测细胞活力、克隆形成率和细胞侵袭转移能力。结果显示,mortalin表达量增高可以促进卵巢癌细胞的细胞增殖、增加单个细胞克隆形成的大小和数量,并能促进细胞迁移,增加其转移侵袭能力；而降低mortalin的表达,可以有效抑制卵巢癌细胞的细胞增殖、单个细胞克隆形成的大小和数量,并能抑制细胞迁移,降低其转移侵袭能力。以上结果提示,mortalin的表达与卵巢癌细胞的发生、发展及侵袭有关,而下调mortalin表达能有效抑制卵巢癌细胞的侵袭,延缓卵巢癌的发生、发展。流式细胞仪检测mortalin表达对细胞周期的影响,Western Blot检测细胞周期蛋白的表达变化。结果显示,mortalin高表达可以促进细胞快速通过G1/S期,进入G2/M期,同时会增加细胞内Cyclin-D1和C-myc的表达量,降低细胞内Cyclin-B1的表达量；而mortalin下调表达会延迟细胞通过G1/S期,同时会增加细胞内Cyclin-D1和C-myc的表达量,降低细胞内Cyclin-B1的表达量。为了研究mortalin在卵巢癌发生发展过程中的作用机制以其可能的信号通路,通过Western Blot实验检测一系列信号通路相关蛋白表达情况,结果显示,mortalin可以特异性的促进c-Raf和Erk1/2的磷酸化,但并没有影响JNK的活化。因此推测,mortalin可以通过MAPK-ERK信号通路其促进卵巢癌细胞的增殖和侵袭迁移。综上所述,mortalin上调表达参与了卵巢癌的发生、发展,下调mortalin可以通过减慢细胞从G1期向G2/M期过渡,有效降低卵巢癌细胞的生长速度和侵袭转移率,并且这一过程与其对MAPK-ERK信号通路的激活有关。因此,mortalin可以作为治疗卵巢癌的一个潜在靶点。
[Abstract]:Ovarian cancer is one of the most common gynecological malignancies. It is difficult to detect early, poor therapeutic effect, poor prognosis and high mortality. Mortalin, also known as Grp75/PBP74/mthsp70, is a member of the HSP70 chaperone family. It is widely distributed in many organelles and cytoplasm, and participates in cell proliferation and intracellular transport, regulates stress response, stabilizes cytoskeleton and inhibits cell apoptosis. Overexpression of mortalin in granulocytic leukemia, colon, brain tumors, breast cancer and liver cancer can increase the malignant degree of breast cancer cells and participate in liver metastasis. The expression of mortalin was higher in ovarian cancer tissues than in adjacent tissues, and the higher the stage, the higher the expression of mortalin. It was suggested that the high expression of mortalin might be related to the occurrence and development of ovarian cancer. Eight ovarian cancer cell lines, A2780/cis, COC1, HO-8910, PM-8910, OV-90, Caov-3, SKOV-3 and ES-2, were selected in this study, respectively. The expression of]mortalin mRNA and protein was the lowest in OV-90 cells, while that in cisplatin-resistant ovarian cancer cell line A2780/cis and ascites was the highest. The results showed that the expression of mortalin in human ovarian cancer cell line A2780 was significantly lower than that in human ovarian cancer cell line A2780 / cis. These results suggest that the expression of motalin may be related to the malignancy of ovarian cancer cells. Subsequently, human ovarian cancer cell line A2780 and human ovarian cancer cell line A2780 were selected for cisplatin resistance. Drug-type cell line A2780/cis was infected with two mortalin-up-regulated and down-regulated lentiviral vectors respectively, and the stable mortalin-up-regulated and down-regulated strains were obtained by flow cytometry. Cell viability was detected by CCK-8 (Cell Counting Kit-8) assay, clone formation assay, cell scratch assay and Transwell cell invasion assay, respectively. The results showed that the increased expression of mortalin could promote the proliferation of ovarian cancer cells, increase the size and number of single cell clones, promote cell migration, increase the ability of metastasis and invasion, and decrease the expression of mortalin could effectively inhibit ovarian cancer cells. These results suggest that the expression of mortalin is related to the occurrence, development and invasion of ovarian cancer cells, and down-regulation of mortalin expression can effectively inhibit the invasion of ovarian cancer cells, delay the occurrence and development of ovarian cancer. Western Blot was used to detect the effect of mortalin expression on cell cycle, and Western Blot was used to detect the expression of cyclin. Delayed cell passage through G1/S phase increased the expression of Cyclin-D1 and C-myc, and decreased the expression of Cyclin-B1. In order to study the mechanism of mortalin in ovarian cancer development and progression, Western Blot assay was used to detect the expression of a series of signal pathway related proteins. These results suggest that mortalin can specifically promote the phosphorylation of c-Raf and Erk1/2, but does not affect the activation of JNK. Therefore, mortalin may promote the proliferation and invasion and migration of ovarian cancer cells through MAPK-ERK signaling pathway. The transition from G1 phase to G2/M phase can effectively reduce the growth rate and invasion and metastasis rate of ovarian cancer cells, and this process is related to the activation of MAPK-ERK signaling pathway.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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