辐射/乏氧诱导的HIF-1-miR-1290-GSK-3β促进宫颈癌细胞侵袭和转移的研究

The mechanisms underlying metastasis of survival cervical cancer after radiotherapy remain to be elucidated. Our study supported by a grant from National Natural Science Foundation of China revealed that hypoxia and radiation induced HIF-1 expression in human cervical cancer cells and promoted radioresistance of cervical cancer cells. We further demonstrated that miR-1290 was up-regulated in radioresistant cervical cancer cells，hypoxia and radiation induced miR-1290 expression in human cervical cancer cells in a HIF-1-dependent manner. In vitro experiment shows that miR-1290 mimics promoted metastasis of cervical cancer cells.Bioinformatic analysis suggesting that GSK-3β could be a possible target for miR-1290, GSK-3β was reported as inhibitor of tumor metastasis, our pilot experiments displayed that GSK-3β expression level in cervical cancer tissues was significantly lower than that in normal cervical tissus. Based on these data, we hypothesized that HIF-1 mediated hypoxia and radiation induced miR-1290 expression and miR-1290 in turn promoted metastasis of cervical cancer cells via down-regulating GSK-3β. In the current study, reporter gene assay, EMSA and Chip assay will be employed to uncover how HIF-1 regulate miR-1290 expression. Gain-of-function and loss-of-function will be used to verify GSK-3β is a functional target of miR-1290. Transwell migration and invasion assays and tail vein injection metastasis assay will be performed to observe the effects of HIF-1-miR-1290-GSK-3β on metastasis of cervical cancer. The study will deepen our understanding of tumor metastasis and help us to find novel therapeutic targets for tumor metastasis inhibition reagents.

放疗后宫颈癌残癌转移的机制仍未完全阐明。申请者在国科金资助下发现，乏氧和辐射可上调HIF-1并增强癌细胞辐射抗性。进一步发现，miR-1290在辐射抗性细胞中表达增强，HIF-1介导了乏氧和辐射对miR-1290的诱导表达，miR-1290模拟物促进宫颈癌细胞的侵袭和转移；生物信息学分析提示miR-1290基因调控区含有HIF-1结合位点，GSK-3β是miR-1290的候选靶基因。免疫组化表明，GSK-3β在宫颈癌中表达下降。提示：辐射/乏氧诱导的HIF-1-miR-1290-GSK-3β能够促进宫颈癌细胞转移。本项目将借助报告基因实验、EMSA、Chip明确HIF-1调控miR-1290表达的机制，通过干预和拮抗实验确证GSK-3β是miR-1290的功能靶分子，利用体内外转移实验阐明HIF-1-miR-1290-GSK-3β对宫颈癌转移的调节。研究结果有助于加深对残癌转移机制的理解。

我们根据临床宫颈癌的放射治疗方式，模拟其分割疗法，建立具有辐射抗性的宫颈癌HeLa细胞株。利用慢病毒载体调变miR-1290在宫颈癌HeLa细胞中的表达。随后通过离体细胞实验明确miR-1290诱导宫颈癌HeLa细胞发生EMT。采用蛋白印迹实验、双荧光素酶报告基因实验初步解析miR-1290的靶基因。取得的研究结果有：1.将HeLa细胞经不同剂量γ 射线单次照射后，qRT-PCR结果提示，照射后HeLa细胞中miR-1290的表达上调。通过连续11次60Co γ 射线照射HeLa细胞，获得细胞株HeLa-R11。qRT-PCR结果显示，HeLa-R11中miR-1290的表达明显上调。上述实验结果证明，单次或多次60Co γ 射线辐照可诱导HeLa细胞中miR-1290表达。2..qRT-PCR证实成功构建稳定调变miR-1290表达的HeLa细胞株。显微镜下观察发现，HeLa-miR -1290细胞呈现出明显的间质细胞样外观，表现为胞间隙增宽、细胞尾足拉长、细胞极性消失出现梭形细胞形态。3.Western blot结果显示，上调HeLa细胞中miR-1290的表达，可降低E-cadherin的表达，同时增高N-cadherin的表达。裸鼠移植瘤石蜡切片免疫组织化学染色结果显示，miR-1290的表达与间质标志物N-cadherin的表达呈正相关，与上皮标志物E-cadherin的表达呈负相关。4.划痕实验结果证明，上调miR-1290的表达可增加HeLa细胞的迁移能力；Transwell侵袭实验结果证明，上调miR-1290的表达可增加HeLa细胞的侵袭能力。5.在线数据库分析及双荧光素酶报告基因实验证实SMAD4和GSK3B是miR-1290的靶基因。6.上调HeLa细胞中miR-1290的表达，可降低Smad4和GSK-3β的蛋白表达水平。裸鼠移植瘤石蜡切片免疫组织化学染色结果显示，miR-1290的表达与Smad4和GSK-3β的表达呈负相关。此结果提示，miR-1290可抑制Smad4和GSK-3β的表达。7..Western blot结果显示，下调HeLa细胞中Smad4的表达，可降低E-cadherin的表达，同时升高N-cadherin的表达；下调HeLa细胞中GSK-3β的表达，可升高N-cadherin的表达。此结果表明，Smad4和GSK-3β