外泌体miRNA29在宫颈癌转移中作用机制探讨

Mechanism of exosome miRNG29 in cervical cancer metastasis

目的miRNA-29在宫颈癌组织中表达上调，但外泌体miRNA29在宫颈癌转移中的作用目前仍未明确。本研究旨在明确外泌体miRNA29在宫颈癌转移中的作用。方法选取宫颈癌C-33A细胞和Hela细胞，以Transwell小室实验和裸鼠肺转移模型评估细胞转移能力。荧光定量PCR和荧光原位杂交技术检测C-33A和Hela细胞中miRNA29表达。收集C-33A细胞和Hela细胞外泌体，荧光定量PCR检测外泌体中miRNA29表达。以裸鼠肺转移检测Hela细胞来源外泌体对C-33A细胞转移能力的影响。结果Hela细胞组穿过基质胶细胞数[（94±11）个]高于C-33A细胞组[（46±8）个]，差异有统计学意义，t=5．841，P=0．006。Hela细胞组肿瘤体积／肺组织[（56±3．1）％]比例高于C-33A细胞组[（21±2．5）％]，差异有统计学意义，t=4．527，P=0．001。Hela细胞中miRNA29相对表达水平（1.0±0．14）高于C-33A细胞（0．16±0．18），差异有统计学意义，t=4．31，P=0．004。Hela细胞外泌体中miRNA29相对表达水平（2．8±0．15）高于C-33A细胞外泌体（1．0±0．07），差异有统计学意义，t=6．24，P=0．003。Hela细胞外泌体组穿过基质胶细胞数[（94±13）个]高于C-33A细胞对照组[（52±8）个]，差异有统计学意义，F=4．521，P=0．004。外泌体miRNA29抑制剂组穿过基质膜细胞数[（58±9）个]与对照组差畀无统计学意义，F=1．456，P=0．681。裸鼠肺转移模型中发现，Hela细胞外泌体处理组瘤体积／肺组织比例为（57±4．8）％，大于C-33A细胞对照组瘤体积／肺组织比例[（22±2．7）％]，F=3．25，P=0．002，而miR-NA29抑制剂处理后Hela细胞外泌体处理组瘤体积／肺组织比例为（24±3．5）％，小于Hela细胞外泌体组，F=4．521，P=0．003。结论高转移潜能的Hela细胞通过外泌体向低转移潜能C-33A细胞传递miRNA29，促进低转移潜能细胞提高转移潜能，证明miRNA29可促进宫颈癌细胞转移。

OBJECTIVE The enhanced miRNA29 in cervical cancer tissue was associated with the development of cervical cancer. However,the expression pattern of miRNA29 in the metastasis of cervical cancer is still unclear. The aim of this study was to evaluate the role of miRNA29 from exosomes in in the metastasis of cervical cancer. METHODS Transwell and mice pulmonary metastasis model were used to evaluate the metastatic potential of Hela and C-33 A cells. The expression of miRNA29 in Hela and C-33 A cells were detected by real-time PCR and fluorescence in situ hybridization. The exosomes were collected from Hela and C-33 A cells and the miRNA29 in exosomes were detected by real-time PCR. Mice pulmonary metastasis model was used to determine the role exosomes in the metastasis of colorectal cancer. RESUITS The results of transwell showed that the number of cells in Hela group （94± 11） was greater than that of C-33 A group （46±8）,t= 5. 841, P= 0. 006. The ration of tumor tissue/lung tissue in Hela group （56 ± 3. 1）% was greater than that of C-33 A group （21±2.5） % ,t=4. 527,P=0. 001. The expression of miRNA29 in Hela cells （1.0±0.14） was higher than C-33 A cells （0.16±0. 18）, respectively, t= 4.31, P= 0. 004. The expression of miRNA29 in Hela-exosoms （2.8 ± 0.15） was higher than C-33 A cells （ 1.0± 0.07）, respectively, t = 6.24, P = 0.003. Transwell results indicated that number of cells in Hela exosomes group （94±13） was greater than that of C-33 A group（52±8）,F=4. 521 ,P=0. 004. However,there was no difference between C-33 A group and miRNA29-inhibitor treated exosomes group （58 ± 9）,F= 1. 456,P=0. 681. The ration of tumor tissue/lung tissue of C-33 A was enhanced by Hela exosomes （57±4. 8）% vs（22±2.7） % ,F= 4. 521, P = 0. 003. The enhanced pulmonary metastasis was abolished by miRNA29 inhibitor （24 ±3.5） % vs （57±4.8） %,F=4. 521,P=0. 003. CONCLUSIONS The metastatic potential of C-33 A cells is enhanced by miRNA29 from exosomes released by Hela cells,indicating the important role of miRNA29 in the metastasis of cervical cancer cells.