Rab23在皮肤鳞状细胞癌侵袭中的作用及机制研究

发布时间：2018-06-16 00:34

  本文选题：Rab23 + 鳞癌　； 参考：《第四军医大学》2012年硕士论文

【摘要】：皮肤鳞状细胞癌（cutaneous squamous cell carcinoma, cSCC）是皮肤的第二常见肿瘤，发生率仅次于基底细胞癌（basal cell carcinoma, BCC）。它是一种侵袭性癌，可早期转移。因此，关于鳞状细胞癌发生、发展及其侵袭、转移的研究一直是皮肤科学界关注的重点领域之一。 Rab23是RAS原癌基因成员，其编码的蛋白是小GTP酶超家族Rab家族成员，其突变可引起小鼠开脑综合征（open brain syndrome）。它参与囊泡转运，是sonic hedgehog信号通路的负调控因子，并在不同肿瘤中发挥不同作用。课题组曾发现Rab23在皮肤鳞癌中高表达，但目前尚未见Rab23在皮肤鳞癌侵袭中的作用及机制的研究。本研究通过免疫组化检测Rab23在光线性角化病、鲍温病、皮肤鳞癌和正常皮肤中的表达，分析其与鳞癌侵袭的相关性，，检测Rab23在鳞癌细胞系中的表达，研究Rab23对鳞癌细胞侵袭的影响，并初步探讨机制。 实验方法： ①免疫组化检测Rab23在光线性角化病、鲍温病、皮肤鳞状细胞癌及正常皮肤中的表达，统计分析Rab23在各组中的表达差异，及Rab23阳性表达与皮肤鳞癌临床病理特征的相关性；②Western blotting检测Rab23在HSC-2、HSQ-89、Sa-3、Tca四个鳞癌细胞系及Hacat中的表达；③Transwell小室检测Rab23对鳞癌细胞侵袭能力的影响；④细胞划痕试验检测Rab23对鳞癌细胞迁移能力的影响；⑤Real time RT-PCR检测干涉或过表达Rab23后鳞癌细胞中侵袭相关分子MMP2、MMP9，hedgehog信号通路中Ptch1、Gli1、Gli2的mRNA水平；⑥Western blotting检测侵袭相关分子Rac1的表达。 实验结果： ①Rab23在皮肤鳞状细胞癌、光线性角化病、鲍温病及正常皮肤中的表达：Rab23在光线性角化病、鲍温病及皮肤鳞状细胞癌组织中均有表达，在正常皮肤中不表达，非曝光部位及中、低分化鳞癌是Rab23阳性表达的危险因素； ②Rab23在HSC-2、HSQ-89、Sa-3、Tca四个鳞癌细胞系及Hacat中的表达：Rab23在四个鳞癌细胞系中均有表达，在HSC-2、HSQ-89细胞中高表达，在Sa-3及Tca中低表达，在Hacat中不表达； ③Transwell小室检测Rab23对鳞癌细胞侵袭能力的影响：HSQ-89细胞行Rab23特异的siRNA干涉后，细胞侵袭能力减弱；Sa-3细胞行质粒转染过表达Rab23后，细胞侵袭能力较对照组增强； ④细胞划痕试验检测Rab23对鳞癌细胞迁移能力的影响：HSQ-89细胞行siRNA干涉后，干涉组与对照组细胞均仅迁移1个细胞直径距离；Sa-3细胞行质粒转染后，过表达组与对照组细胞迁移能力差异无统计学意义； ⑤Real time RT-PCR检测干涉或过表达Rab23后相关分子的水平：HSQ-89细胞Rab23干涉组MMP2、MMP9、Gli2的mRNA水平较对照组低，Ptch1、Gli1的mRNA水平较对照组高；Sa-3细胞Rab23过表达组中MMP2、MMP9、Gli2的mRNA水平较对照组高，Ptch1、Gli1较对照组低； ⑥Western blotting检测干涉或过表达Rab23后Rac1的表达：HSQ-89细胞干涉Rab23表达后，Rac1水平较对照组低；Sa-3细胞过表达Rab23表达后，Rac1水平较对照组高。 主要结论： ①Rab23在光线性角化病、鲍温病及皮肤鳞癌中阳性表达，而正常皮肤中不表达，Rab23在鳞癌细胞系中表达，Hacat中不表达，说明其参与皮肤鳞癌的发生发展； ②Rab23促进鳞癌细胞的侵袭，不影响细胞迁移； ③Rab23促进鳞癌侵袭可能通过Rac1、MMP2、MMP9，可能不通过hedgehog信号通路。
[Abstract]:Cutaneous squamous cell carcinoma (cSCC) is the second common tumor of the skin, which is only inferior to basal cell carcinoma (basal cell carcinoma, BCC). It is an invasive carcinoma and can be transferred early. Therefore, the study of squamous cell carcinogenesis, development and invasion, and metastasis have always been the concern of the skin science community. One of the key areas.
Rab23 is a member of the RAS proto oncogene and its encoding protein is a member of the small GTP enzyme superfamily Rab family. Its mutation can cause open brain syndrome (open brain syndrome). It participates in vesicle transport and is a negative regulator of sonic hedgehog signaling pathway, and plays different roles in different tumors. The subject group has found Rab23 in skin squamous cell carcinoma. High expression, but there is no study of the role and mechanism of Rab23 in the invasion of skin squamous cell carcinoma. This study detected the expression of Rab23 in light keratosis, abalone disease, skin squamous cell carcinoma and normal skin by immunohistochemistry, analyzed the correlation with the invasion of squamous cell carcinoma, detected the expression of Rab23 in the squamous cell carcinoma cell lines, and studied Rab23 for squamous cell carcinoma. The effect of cell invasion and preliminary discussion of the mechanism.
Experimental methods:
(1) immunohistochemistry was used to detect the expression of Rab23 in light keratosis, abalone disease, skin squamous cell carcinoma and normal skin. The difference of expression of Rab23 in each group was statistically analyzed, and the correlation between the positive expression of Rab23 and the clinicopathological features of skin squamous cell carcinoma; and Western blotting was used to detect four squamous cell carcinoma cell lines and H in HSC-2, HSQ-89, Sa-3, Tca. The expression in ACAT; (3) the effect of Rab23 on the invasiveness of squamous cell carcinoma cells by Transwell; (4) the effect of Rab23 on the migration ability of squamous cell carcinoma cells; (5) Real time RT-PCR detection of interference or overexpression of Rab23 in squamous cell carcinoma cells, MMP2, MMP9, Hedgehog signaling pathway The expression of invasion related molecule Rac1 was detected by Western blotting.
Experimental results:
(1) the expression of Rab23 in skin squamous cell carcinoma, ray keratosis, bauwen disease and normal skin: Rab23 is expressed in light keratosis, bauwen disease and skin squamous cell carcinoma, and is not expressed in normal skin, unexposed parts and medium, and low differentiated squamous cell carcinoma is a risk factor for Rab23 positive expression.
(2) the expression of Rab23 in four squamous cell carcinoma cell lines and Hacat in HSC-2, HSQ-89, Sa-3, Tca: Rab23 is expressed in four squamous cell carcinoma cell lines, expressed in HSC-2, HSQ-89 cells, low in Sa-3 and Tca, and not expressed in Hacat.
(3) the effect of Rab23 on the invasive ability of squamous cell carcinoma cells in Transwell cells: the invasion ability of HSQ-89 cells was weakened after Rab23 specific siRNA interference, and the invasion ability of Sa-3 cells was enhanced after transfection of Rab23.
The effect of Rab23 on the migration ability of squamous cell carcinoma cells was detected by cell scratch test: after siRNA interference in HSQ-89 cells, only 1 cell diameter distances were migrated between the interference group and the control group. After the transfection of Sa-3 cells, there was no statistical significance between the over expression group and the control group.
Real time RT-PCR detected the level of related molecules after interference or overexpression of Rab23: the Rab23 interference group of HSQ-89 cells MMP2, MMP9, Gli2 was lower than the control group, Ptch1, and the level of Gli1 was higher than that of the control group;
(6) Western blotting detected the expression of Rac1 after interference or overexpression of Rab23: after HSQ-89 cells interfered with Rab23 expression, the level of Rac1 was lower than that of the control group, and the level of Rac1 was higher than that of the control group after the expression of Rab23 in Sa-3 cells.
The main conclusions are as follows:
(1) Rab23 is positive in light keratosis, bauwen disease and skin squamous cell carcinoma, but not in normal skin. Rab23 is expressed in squamous cell carcinoma cell lines and Hacat is not expressed, indicating that it participates in the development of squamous cell carcinoma of the skin.
(2) Rab23 promotes the invasion of squamous cell carcinoma and does not affect cell migration.
(3) Rab23 promotes the invasion of squamous cell carcinoma possibly through Rac1, MMP2 and MMP9, probably not through the hedgehog signaling pathway.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.5

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