Effect of transfer RNA-derived molecule fragment 770 on proliferation of breast cancer cells by targeting cytoskeletal-associated protein 2 / 肿瘤研究与临床

ABSTRACT

Objective: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.