CRY2激活Wnt/β-catenin通路促进卵巢癌侵袭的研究

Mechanism of CRY2 promoting invasion of ovarian cancer cells by activating Wnt/β-Catenin pathway

目的探讨生物钟基因CRY2通过Wnt/β-catenin信号通路调控卵巢癌细胞侵袭性的作用,并阐明其对细胞外基质可能的作用机理。方法利用GEPIA数据库下载符合本研究需要的卵巢癌组织426例和正常卵巢组织88例的基因表达谱,并通过R语言分析两组的CRY2基因表达。将TCGA数据库中下载到的的353例卵巢癌患者以CRY2基因表达水平中位值为中心分为CRY2低表达组和CRY2高表达两组,并行差异分析,随后筛选差异基因富集的京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)信号通路,通过STRING和cBioPortal分析蛋白互作。采用实时荧光定量聚合酶链反应(RT-qPCR)及免疫组织化学检测组织中CRY2 mRNA和蛋白表达水平。采用脂质体转染法将shCRY2质粒转染卵巢癌细胞,过表达CRY2,利用Transwell实验研究卵巢癌细胞的侵袭性。采用荧光素酶报告实验检测过表达CRY2组和对照组两组的TOP/Flash荧光素酶比率,Western-blot检测β-catenin蛋白表达水平。结果CRY2在卵巢癌中的表达水平较正常卵巢组织低(P<0.05),CRY2的差异表达基因主要和细胞外基质受体相互作用等信号通路相关。CRY2主要与生物钟基因蛋白相互作用,并与Wnt信号通路密切相关。RT-qPCR及免疫组织化学染色法检测CRY2在卵巢癌中的基因表达水平低于正常卵巢组织(P<0.05),shCRY2组侵袭细胞数较shNC组多,并具有统计学差异(P<0.05)。过表达CRY2后卵巢癌HO8910细胞株TOP/Flash荧光素酶的比率降低(P<0.05),β-catenin蛋白的表达量减少(P<0.05)。结论CRY2在卵巢癌组织中低表达,可能通过调控Wnt/β-catenin信号通路促进细胞侵袭,并影响细胞外基质受体相互作用。

Objective To investigate the effect of the biological clock gene CRY2 on ovarian cancer cell invasiveness and its regulation of the Wnt/β-catenin signal pathway,and to elucidate its possible mechanism of action on the extracellular matrix.Methods Gene expression profiles of 426 ovarian cancer tissues and 88 normal ovarian tissues that met the requirements of this study were downloaded from the GEPIA database,and CRY2 gene expression in both groups was analyzed using R.According to the median value of CRY2 gene expression level,353 ovarian cancer patients in the TCGA ovarian cancer database were divided into low and high CRY2 expression groups,which were subjected to differential gene analysis and screened for differential gene-enriched Kyoto Encyclopedia of Genes and Genomes(KEGG)signalling pathway,and CRY2 interacting proteins were analyzed by STRING and cBioPortal.Real-time PCR(RT-qPCR)and immunohistochemistry were used to detect CRY2 expression levels in two groups.Liposomal transfection was used to transfect shCRY2 and shNC plasmids into ovarian cancer cells HO8910,overexpressing CRY2 in ovarian cancer cells,and cell invasion was observed using Transwell assay.The luciferase reporter assay was used to detect the TOP/Flash luciferase ratio in the overexpression of CRY2 group and the control group,and Western-blot was used to detect the expression level ofβ-catenin protein in the two groups.Results Bioinformatics analysis showed that the expression level of CRY2 in ovarian cancer was lower than that in normal ovarian tissues(P<0.05).The differentially expressed genes of CRY2 were mainly related to signaling pathways such as extracellular matrix receptor interactions.CRY2 mainly interacts with clock gene proteins and was closely related to the Wnt pathway.CRY2 gene expression in ovarian cancer was lower than that in normal ovarian tissues by RT-qPCR and immunohistochemical staining(P<0.05),the number of invasive cells in shCRY2 group was higher than that in shNC group,and the difference was statistically significant(P<0.05).The ratio of TOP/Flash luciferase in ovarian cancer HO8910 cell line decreased after overexpression of CRY2(P<0.05),the expression ofβ-catenin protein decreased(P<0.05).Conclusion CRY2 showed low expression in ovarian cancer tissues,which may promote cell invasion and affect extracellular matrix receptor interactions by regulating the Wnt/β-catenin signal pathway.