目的 探讨长链非编码RNA（lncRNA）抗分化非编码RNA（ANCR）对结直肠癌（CRC）细胞生物学行为的影响及其作用机制。方法 收集50例CRC患者的癌组织和癌旁组织，通过RT-qPCR检测组织中ANCR和叉头框蛋白O1（FOXO1）的表达水平并进行Pearson相关性分析。利用RNA 拉下和RNA免疫沉淀实验（RIP）确认ANCR是否可与FOXO1相互作用。将经过慢病毒包装的ANCR和FOXO1慢病毒质粒转染至人CRC细胞系SW480细胞中，细胞分为6组：对照组（未转染），NC组（转染慢病毒质粒空载），ANCR组（转染 pHRi-ANCR），sh-ANCR组（转染pHRi-sh-ANCR），FOXO1组（pHRi-FOXO1），sh-ANCR + sh-FOXO1组（同时转染pHRi-sh-ANCR和pHRi-sh-FOXO1）。RT-qPCR检测过表达和抑制ANCR后FOXO1的表达水平，5-乙炔基-2′脱氧尿嘧啶核苷（EdU）标记技术和流式细胞术分别检测各组细胞增殖、细胞周期和细胞凋亡情况。Western blot检测B淋巴细胞瘤-2（BCL-2）、BCL2相关蛋白X（BAX）、细胞周期蛋白D1（cyclin D1）及人P27蛋白（P27）的表达情况。结果 与癌旁组织相比，CRC组织中ANCR表达明显增加，FOXO1表达明显减少（P<0.01），二者表达水平负相关（P<0.01）。ANCR能够结合并抑制FOXO1表达。与NC组相比，sh-ANCR组和FOXO1组细胞增殖数减少，细胞阻滞于G0/G1期，细胞凋亡数增多，BAX和P27增多，BCL-2和cyclin D1减少（P<0.05）。与sh-ANCR组相比，sh-ANCR + sh-FOXO1组上述指标得到逆转（P<0.05）。结论 lncRNA ANCR在CRC中表达升高并通过调控FOXO1的表达调控CRC细胞的细胞增殖和凋亡。
Abstract
Objective To investigate the effect of long chain noncodingRNA (lncRNA) on the biological behavior of colorectal cancer (CRC) cells and its mechanism. Methods Cancer and adjacent tissues of 50 CRC patients were collected.The expression levels of ANCR and forkhead box protein O1 (FOXO1) in tissues were detected by RT-qPCR and Pearson correlation analysis was performed. RNA pull-down and RNA immunoprecipitation (RIP) experiments were used to confirm whether ANCR could interact with FOXO1. ANCR and FOXO1 lentivirus were transfected into CRC cell line SW480 cells. The cells were divided into the control group (untransfected), NC group (transfected with lentiviral vector), ANCR group (transfected with ANCR over-expressed lentivirus), sh-ANCR group (transfected with ANCR knockdown lentivirus), FOXO1group (transfected with FOXO1 overexpressing lentivirus), sh-ANCR + sh-FOXO1group (transfected with ANCR and FOXO1 simultaneously inhibited lentivirus). RT-qPCR was used to detect the expression of FOXO1 after over-expression and inhibition of ANCR. 5-ethynyl-2′-deoxyuridine (EdU) labeling technology and flow cytometry were used to detect cell proliferation, cell cycle and apoptosis in each group. Western blot was used to detect the expression of B-cell lymphoma-2 (BCL-2), BCL2-Associated X Protein (BAX), cycle proteins cyclin D1 (cyclin D1) and Human P27 protein (P27 protein). Results Compared with adjacent tissues, the expression of ANCR in CRC tissues increased significantly, while the expression of FOXO1 decreased significantly (P<0.01). The expression levels of the two were negatively correlated (P<0.01). ANCR is able to bind and inhibit FOXO1 expression. Compared with the NC group, the number of cells in the sh-ANCR group and FOXO1group was reduced, the cells were arrested in the G0 / G1 phase, the number of apoptosis was increased, BAX and P27 were increased, and BCL-2 and cyclin D1 were reduced (P<0.05). Compared with the sh-ANCR group, the above indexes of the sh-ANCR + sh-FOXO1 group were reversed (P<0.05). Conclusion LncRNA ANCR expression is increased in CRC and regulates cell proliferation and apoptosis of CRC cells by regulating FOXO1 expression.
Key words
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Long noncoding RNA ANCR｜Forkhead box protein O1｜Colorectal cancer｜Cell proliferation｜Cell cycle｜Apoptosis
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基金
绵阳市卫生健康委医学科研课题（201922）
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