摘要目的:探讨转运RNA来源的小分子片段770（tRF-770）调控细胞骨架相关蛋白2（CKAP2）对乳腺癌细胞增殖的影响。方法:使用MINTbase数据库v2.0序列与基因组中成熟的tRNA进行比对，确认tRF-770的染色体定位；TA克隆实验鉴定tRF-770；利用TargetScan和miRBase数据库分析预测tRF-770的靶基因；利用癌症基因组图谱（TCGA）数据库数据分析CKAP2在乳腺癌中的表达情况。选择乳腺癌细胞株MDB-MA-231和MCF7，分别分为空白对照组（不予任何处理）、tRF-770过表达组（转染tRF-770过表达序列）及阴性对照组（转染阴性对照序列）；另外设立tRF-770过表达+CKAP2-HA组（共转染tRF-770过表达序列与CKAP2过表达序列）。采用实时荧光定量聚合酶链反应（qRT-PCR）检测乳腺癌细胞中tRF-770的相对表达量；CCK-8法检测乳腺癌细胞增殖能力并进行挽救实验；双荧光素酶报告基因实验验证tRF-770的靶基因；蛋白印迹法检测CKAP2-ERK2信号通路相关蛋白的表达情况。结果:tRF-770与9类tRNA剪切修饰的5'端完全匹配，TA克隆测序验证结果表明产物大小以及碱基与预期tRF-770序列一致。基于TCGA数据库数据分析发现CKAP2在乳腺癌组织中高表达（ t=7.21， P<0.05）。qRT-PCR检测结果显示，在MDA-MB-231细胞中，空白对照组、阴性对照组、tRF-770过表达组tRF-770相对表达量分别为1.00±0.00、2.42±0.11、3.75±0.01，差异有统计学意义（ F=1 395.00， P<0.001）；在MCF7细胞中，3组tRF-770相对表达量分别为1.00±0.00、2.45±0.21、3.26±0.16，差异有统计学意义（ F=169.30， P<0.001）；两种细胞中，与空白对照组及阴性对照组比较，tRF-770过表达组中tRF-770相对表达量均升高（均 P<0.05）。双荧光素酶报告基因实验结果表明，tRF-770与CKAP2 mRNA 3'UTR结合。CCK-8法检测结果显示，在MDA-MB-231、MCF7细胞中，第3天和第4天tRF-770过表达组细胞增殖能力均低于空白对照组和阴性对照组（均 P<0.05）；第3天和第4天阴性对照组与tRF-770过表达组细胞增殖能力比较，差异均有统计学意义（均 P<0.05）。CCK-8法挽救实验结果显示，在两株乳腺癌细胞中，tRF-770过表达+CKAP2-HA组自转染第2天起，细胞增殖能力均高于tRF-770过表达组（均 P<0.05）。蛋白质印迹法检测结果显示，两种乳腺癌细胞中，tRF-770过表达组与阴性对照组比较，CKAP2、p-ERK2、PCNA蛋白表达均下降（均 P<0.05）；ERK2蛋白在MDA-MB-231细胞中表达量变化较小，而在MCF7细胞中tRF-770过表达组蛋白表达下降。 结论:tRF-770可能通过CKAP2-ERK2信号通路抑制乳腺癌细胞增殖。

abstractsObjective:To explore the effect of transfer RNA-derived small molecule fragment 770(tRF-770) on proliferation of breast cancer cells through regulating cytoskeletal-associated protein 2 (CKAP2).Methods:Chromosome localization of tRF-770 was identified using the MINTbase database v2.0 sequence alignment with mature tRNA in the genome. TA cloning assay was used to identify tRF-770; TargetScan and miRBase database were used to analyze and predict the target genes of tRF-770. The expression of CKAP2 in breast cancer was analyzed by using the data from The Cancer Genome Atlas (TCGA) database. Breast cancer cell lines MDB-MA-231 and MCF7 were selected and divided into three groups: blank control group (without any treatment), tRF-770 overexpression group (transfected with tRF-770 overexpression sequence) and negative control group (transfected with negative control sequence). In addition, tRF-770 overexpression+CKAP2-HA group was established (co-transfected with tRF-770 overexpression sequence and CKAP2 overexpression sequence). Real-time quantitative fluorescence polymerase chain reaction (qRT-PCR) was used to detect the relative expression of tRF-770 in breast cancer cells. CCK-8 assay was used to detect the proliferation of breast cancer cells and perform rescue experiment. Dual luciferase reporter gene assay was used to verify the target genes of tRF-770. The protein expression of CKAP2-ERK2 signaling pathway was detected by Western blotting.Results:tRF-770 completely matched 5' UTR spliced and modified by 9 kinds of tRNA. TA clone sequencing verification results showed that the product size and bases were consistent with the expected tRF-770 sequences. CKAP2 was highly expressed in breast cancer tissues based on analysis of the data from TCGA database ( t = 7.21, P < 0.05). qRT-PCR showed that the relative expressions of tRF-770 in MDA-MB-231 cells of blank control group, negative control group and tRF-770 overexpression group were 1.00±0.00, 2.42±0.11 and 3.75±0.01, respectively, and the difference was statistically significant ( F = 1 395.00, P < 0.001). The relative expressions of tRF-770 in MCF7 cells of 3 groups were 1.00±0.00, 2.45±0.21 and 3.26±0.16, respectively, and the difference was statistically significant ( F = 169.30, P < 0.001). Compared with blank control group and negative control group, the relative expression of tRF-770 in tRF-770 overexpression group in 2 cell lines was increased (all P < 0.05). Dual luciferase reporter assay showed that tRF-770 bound to CKAP2 mRNA 3'UTR. CCK-8 assay showed that in MDA-MB-231 and MCF7 cells, the cell proliferation ability of tRF-770 overexpression group on the 3rd and 4th day was lower than that of blank control group and negative control group (both P < 0.05); there were significant differences in cell proliferation ability between the negative control group and tRF-770 overexpression group on the 3rd and 4th day (all P < 0.05). CCK-8 assay showed that in 2 breast cancer cell lines, the cell proliferation ability of tRF-770 overexpression+ CKAP2-HA group was higher than that of tRF-770 overexpression group since the second day after transfection (all P < 0.05). Western blotting showed that the expressions of CKAP2, p-ERK2 and PCNA proteins in tRF-770 overexpression group were decreased compared with the negative control group (all P < 0.05). The change of ERK2 protein expression was small in MDA-MB-231 cells, but the expression of protein in tRF-770 overexpression group in MCF7 cells was decreased. Conclusions:tRF-770 may inhibit the proliferation of breast cancer cells through CKAP2-ERK2 signaling pathway.