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摘要:
目的 探讨importin5（IPO5）基因的表达对食管癌（esophageal cancer, EC）进展的影响以及相关作用机制。 方法 收集2020年9 月至2022 年4 月50 例于锦州医科大学附属第一医院确诊为EC的患者的资料及手术标本，免疫组化验证IPO5在癌组织与癌旁组织中的表达情况，评估IPO5与患者临床信息的相关性；通过慢病毒转染构建IPO5基因沉默EC细胞模型；通过 CCK-8实验、Transwell侵袭实验、细胞划痕实验检测细胞增殖、侵袭及迁移能力；应用流式细胞术检测细胞周期，Western blot实验检测细胞周期相关蛋白的表达及RAS通路相关蛋白的表达。构建裸鼠皮下成瘤模型，免疫组化验证Ki-67表达。 结果 临床实验中，IPO5在癌组织的表达明显高于癌旁组织（P<0.01），并与肿瘤大小、分期及分化程度呈正相关（P<0.05）。IPO5在EC细胞中的表达高于正常食管细胞，尤其是ECA109及OE33细胞（P<0.01）。与阴性对照组相比，IPO5干扰组细胞增殖能力减弱（P<0.05）、侵袭能力减弱（P<0.01）。IPO5基因沉默后EC细胞生长周期阻滞于G2期，细胞周期相关蛋白表达减少。sh-IPO5干扰后RAS-ERK通路下游蛋白表达水平下降（P<0.05）。裸鼠成瘤实验证实敲除IPO5后肿瘤缩小（P<0.05），Ki-67表达减少（P<0.01）。 结论 IPO5在EC组织中高表达，抑制IPO5的表达对于EC细胞的生长和迁移存在显著影响，IPO5通过激活RAS-ERK信号通路促进EC的进展。 Abstract:Objective To investigate the effect and mechanism of importin5 (IPO5) gene expression on esophageal cancer (EC) progression. Methods The data and surgical specimens of 50 patients diagnosed with EC in The First Affiliated Hospital of Jinzhou Medical University from September 2020 to April 2022 were collected. The expression of IPO5 in cancer tissues and adjacent tissues was determined using immunohistochemistry. The correlation between IPO5 expression and patient clinical information was evaluated. Lentivirus transfection was used to generate an IPO5-silenced EC cell model. Cell proliferation, invasion, and migration were measured using CCK-8, Transwell invasion, and cell scratch assays. Flow cytometry and Western blot were used to detect cell cycle-related and RAS pathway-related proteins. A subcutaneous tumorigenesis nude mouse model was established, and Ki-67 expression was determined using immunohistochemistry. Results In clinical samples, IPO5 expression was significantly higher in cancer tissues than that in adjacent tissues (P<0.01) and was positively correlated with tumor size, stage, and degree of differentiation (P<0.05). EC cells expressed higher IPO5 levels than normal esophageal cells, especially ECA109 and OE33 cells (P<0.01). The proliferation and invasion abilities of the IPO5 gene silencing group were decreased (P<0.05 and P<0.01, respectively) compared with that of the negative control group. After IPO5 silencing, the EC cells were arrested in the G2 phase of the cell cycle, and the expression of cell cycle-related proteins decreased. The level of activated RAS-ERK pathway proteins decreased after sh-IPO5 knockdown (P<0.05). Tumorigenesis experiments in nude mice confirmed that tumor shrank ( P <0.05) and Ki-67 expression decreased (P<0.01) after IPO5 gene silending. Conclusions IPO5 is highly expressed in EC tissues, and inhibition of IPO5 has a significant inhibitory effect on the growth and migration of EC cells. IPO5 activates the RAS-ERK signaling pathway and promotes EC development. -
Key words:
- importin5 (IPO5) gene /
- RAS-ERK pathway /
- esophageal cancer (EC) /
- proliferation
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图 1 EC中IPO5的表达情况
A～C：EC组织与正常食管组织之间差异表达基因的火山图，IPO5在EC组织中呈现高表达。D：EC组织中IPO5高表达；E：EC癌旁组织中IPO5低表达，F：依据免疫组化评分计算出IPO5在癌组织中表达升高。G：qRT-PCR分析IPO5在ECA109和OE33细胞中表达高于HEEC细胞；H：Western blot实验显示sh-IPO5可以明显下调EC细胞中IPO5的表达。注：*：P<0.05；**：P<0.01
图 2 IPO5影响EC细胞的侵袭、迁移、增殖能力
A：细胞划痕实验显示在24h时sh-IPO5组闭合率低于sh-NC组；B：Transwell侵袭实验证明干扰IPO5表达可使细胞侵袭能力减弱；C：CCK-8分析sh-IPO5可以明显减低ECA109和OE33细胞活力。注：*：P<0.05；**：P<0.01
图 3 IPO5影响食管癌的细胞周期及肿瘤大小
A、B：流式细胞术检测ECA109细胞转染sh-IPO5后G2期细胞比例增多；C、D：流式细胞术检测OE33细胞转染sh-IPO5后G2期细胞比例增多。E：Western blot分析显示敲低IPO5基因可使CDK4、CDK6、CyclinD1表达减少。 F：裸鼠成瘤实验显示sh-IPO5组裸鼠生长速度明显小于对照组；G：免疫组化实验显示阳性组与阴性对照组相比Ki67表达明显降低。*：P<0.05；**：P<0.01
表 1 临床数据与IPO5表达之间的关系 例
项目 例数 IPO5低表达 IPO5高表达 χ2 P 性别 0.109 0.741 男 27 14 13 女 23 13 10 年龄(岁) 0.216 0.642 ≤60 22 14 8 >60 28 16 12 肿瘤大小(cm) 5.547 0.019 ≤3.9 28 17 11 >3.9 22 6 16 TNM分期 4.276 0.039 Ⅰ～Ⅱ 36 22 14 Ⅲ～Ⅳ 14 4 10 淋巴结转移 2.803 0.094 有 12 3 9 无 38 20 18 分化程度 6.388 0.041 低 8 1 7 中 29 14 15 高 13 9 4 
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[1] Uhlenhopp DJ, Then EO, Sunkara T, et al. Epidemiology of esophageal cancer: update in global trends, etiology and risk factors[J]. Clin J Gastroenterol, 2020, 13(6):1010-1021. doi: 10.1007/s12328-020-01237-x [2] Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015[J]. CA Cancer J Clin, 2016, 66(2):115-132. doi: 10.3322/caac.21338 [3] Huang FL, Yu SJ. Esophageal cancer: risk factors, genetic association, and treatment[J]. Asian J Surg, 2018, 41(3):210-215. doi: 10.1016/j.asjsur.2016.10.005 [4] Domper Arnal MJ, Ferrández Arenas Á, Lanas Arbeloa Á. Esophageal cancer: risk factors, screening and endoscopic treatment in Western and Eastern countries[J]. World J Gastroenterol, 2015, 21(26):7933-43. doi: 10.3748/wjg.v21.i26.7933 [5] Patouret R. The nuclear transport protein importin-5: a promising target in oncology and virology[J]. Chimia (Aarau), 2021, 75(4):319-322. doi: 10.2533/chimia.2021.319 [6] van der Watt PJ, Okpara MO, Wishart A, et al. Nuclear transport proteins are secreted by cancer cells and identified as potential novel cancer biomarkers[J]. Int J Cancer, 2022, 150(2):347-361. doi: 10.1002/ijc.33832 [7] Quan L, Qiu T, Liang J, et al. Identification of target genes regulated by KSHV miRNAs in KSHV-infected lymphoma cells[J]. Pathol Oncol Res, 2015, 21(4):875-880. doi: 10.1007/s12253-015-9902-2 [8] Sung H, Ferlay J, Siegel RL, et al. Global Cancer statistics 2020: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3):209-249. doi: 10.3322/caac.21660 [9] Pumroy RA, Cingolani G. Diversification of importin-α isoforms in cellular trafficking and disease states[J]. Biochem J, 2015, 466(1):13-28. doi: 10.1042/BJ20141186 [10] Vaidyanathan S, Thangavelu PU, Duijf PHG. Overexpression of ran GTPase components regulating nuclear export, but not mitotic spindle assembly, marks chromosome instability and poor prognosis in breast cancer[J]. Target Oncol, 2016, 11(5):677-686. doi: 10.1007/s11523-016-0432-y [11] Mehmood R, Jibiki K, Shibazaki N, et al. Molecular profiling of nucleocytoplasmic transport factor genes in breast cancer[J]. Heliyon, 2021, 7(1):e06039. doi: 10.1016/j.heliyon.2021.e06039 [12] Nagashima S, Maruyama J, Honda K, et al. CSE1L promotes nuclear accumulation of transcriptional coactivator TAZ and enhances invasiveness of human cancer cells[J]. J Biol Chem, 2021, 297(1):100803. doi: 10.1016/j.jbc.2021.100803 [13] Khan M, Khan Z, Uddin Y, et al. Evaluating the oncogenic and tumor suppressor role of XPO5 in different tissue tumor types[J]. Asian Pac J Cancer Prev, 2018, 19(4):1119-1125. [14] Shigeyasu K, Okugawa Y, Toden S, et al. Exportin-5 functions as an oncogene and a potential therapeutic target in colorectal cancer[J]. Clin Cancer Res, 2017, 23(5):1312-1322. doi: 10.1158/1078-0432.CCR-16-1023 [15] Li D, Fu S, Wu Z, et al. DDX56 inhibits type I interferon by disrupting assembly of IRF3-IPO5 to inhibit IRF3 nucleus import[J]. Cell Sci, 2019, 133(5):jcs230409. [16] Goto T, Sato A, Adachi S, et al. IQGAP1 protein regulates nuclear localization of β-catenin via importin-β5 protein in Wnt signaling[J]. Biol Chem, 2013, 288(51):36351-36360. doi: 10.1074/jbc.M113.520528 [17] Zhang W, Lu Y, Li X, et al. IPO5 promotes the proliferation and tumourigenicity of colorectal cancer cells by mediating RASAL2 nuclear transportation[J]. Exp Clin Cancer Res, 2019, 38(1):296. doi: 10.1186/s13046-019-1290-0 [18] Chen S, Li F, Xu D, et al. The function of RAS mutation in cancer and advances in its drug research[J]. Curr Pharm Des, 2019, 25(10):1105-1114. doi: 10.2174/1381612825666190506122228 [19] Pang X, Liu M. Defeat mutant KRAS with synthetic lethality[J]. Small GTPases, 2017, 8(4):212-219. doi: 10.1080/21541248.2016.1213783 [20] Zinatizadeh MR, Momeni SA, Zarandi PK, et al. The role and function of ras-association domain family in cancer: a review[J]. Genes Dis, 2019, 6(4):378-384. doi: 10.1016/j.gendis.2019.07.008 [21] Osaka N, Hirota Y, Ito D, et al. Divergent mechanisms activating RAS and small GTPases through post-translational modification[J]. Front Mol Biosci, 2021, 8:707439. doi: 10.3389/fmolb.2021.707439 [22] Degirmenci U, Wang M, Hu J. Targeting aberrant RAS/RAF/MEK/ERK signaling for cancer therapy[J]. Cells, 2020, 9(1):198. doi: 10.3390/cells9010198 [23] Choi S, Anderson RA. And Akt-ion! IQGAP1 in control of signaling pathways[J]. EMBO J, 2017, 36(8):967-969. doi: 10.15252/embj.201796827 [24] Wei T, Lambert PF. Role of IQGAP1 in Carcinogenesis[J]. Cancers (Basel), 2021, 13(16):3940. doi: 10.3390/cancers13163940 [25] Zhou D, Wei Z, Deng S, et al. SASH1 regulates melanocyte transepithelial migration through a novel Gαs-SASH1-IQGAP1-E-Cadherin dependent pathway[J]. Cell Signal, 2013, 25(6):1526-1538. doi: 10.1016/j.cellsig.2012.12.025 [26] Johnson MA, Sharma M, Mok MT, et al. Stimulation of in vivo nuclear transport dynamics of actin and its co-factors IQGAP1 and Rac1 in response to DNA replication stress[J]. Biochim Biophys Acta, 2013, 1833(10):2334-2347. doi: 10.1016/j.bbamcr.2013.06.002 [27] Erdemir HH, Li Z, Sacks DB. IQGAP1 binds to estrogen receptor-α and modulates its function[J]. J Biol Chem, 2014, 289(13):9100-9112. doi: 10.1074/jbc.M114.553511 [28] Zeng F, Jiang W, Zhao W, et al. Ras GTPase-activating-like protein IQGAP1 (IQGAP1) promotes breast cancer proliferation and invasion and correlates with poor clinical outcomes[J]. Med Sci Monit, 2018, 24:3315-3323. doi: 10.12659/MSM.909916 [29] Goto T, Sato A, Adachi S, et al. IQGAP1 protein regulates nuclear localization of β-catenin via importin-β5 protein in Wnt signaling[J]. Biol Chem, 2013, 288(51):36351-36360. doi: 10.1074/jbc.M113.520528 -
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