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N1, N12-Diacetylspermine Is Elevated in Colorectal Cancer and Promotes Proliferation through the miR-559/CBS Axis in Cancer Cell Lines.

ABSTRACT: N1, N12-Diacetylspermine (DiAcSpm) has been reported to be upregulated in the urine of cancer patients. Mass spectrometry has shown elevated DiAcSpm expressions in colorectal cancer (CRC) tissues. However, the diagnostic application of DiAcSpm is not available due to a lack of diagnostic grade antibodies. Also, its biological roles in CRC cells remain unexplored. In the present study, we developed an antibody that directly detected DiAcSpm expression in paraffin-embedded tissues. We also characterized its biological characteristics and underlying mechanisms. Polyclonal antibodies were generated by immunizing animals with a synthetic product of DiAcSpm. Antibody DAS AB016 showed strong sensitivity against DiAcSpm in CRC tissues. Immunohistochemistry results showed that DiAcSpm expression was significantly elevated in CRC tissues. High levels of DiAcSpm correlated with the clinical stage and Ki67 index. DiAcSpm treatment increased levels of proliferation, cell cycle progression, and cyclin D1 and cyclin E proteins in CRC cell lines, SW480 and Caco-2. DiAcSpm also upregulated ATP production in these two cell lines. RNA-sequencing showed that DiAcSpm downregulated miR-559, which was confirmed using RT-qPCR. The luciferase reporter assay, western blotting, and RT-qPCR showed that cystathionine β-synthase (CBS) was the target of miR-559. miR-559 inhibited, while CBS accelerated, CRC proliferation. In addition, CBS siRNA knockdown blocked the biological effects of DiAcSpm on CRC cells. In conclusion, DiAcSpm was found to be increased in CRC tissues using a newly developed antibody. DiAcSpm accelerated CRC proliferation by regulating the miR-559/CBS axis.

SUBMITTER: Mu T

PROVIDER: S-EPMC8486517 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOsteosarcoma (OS) is one of the most common malignant bone tumors in children and adolescents. Circular RNAs (circRNAs) are critical regulators involved in multiple physiological and pathological processes. However, the underlying regulatory mechanisms of circRNA in OS are still not fully understood.MethodsThe circRNA expression profiles were downloaded from the Gene Expression Omnibus (GEO) database and analyzed by GEO2R. Bioinformatics analysis was performed to predict the potential target miRNAs of hsa_circ_0069117 and its downstream mRNAs. The co-expression of hsa_circ_0069117/miR-875-3p/PF4V1 axis was further validated in OS tissue samples via quantitative real-time PCR (qRT-PCR). Luciferase reporter gene plasmids containing the sequence of PF4V1 and hsa_circ_0069117 were constructed to verify the putative sites of miR-875-3p. Gain/loss-of-function assays were performed to verify the effect of hsa_circ_0069117 on miR-875-3p/PF4V1 expression and related pathways via qRT-PCR and Western blot. Cell counting kit-8 (CCK-8) and wound-healing assays were performed to evaluate the effect of hsa_circ_0069117 on cell proliferation and migration of MG63 and U2OS, respectively.ResultsWe identified hsa_circ_0069117 as the most markedly dysregulated circRNA in OS cell lines. Bioinformatics analysis indicated that hsa_circ_0069117 might inhibit the expression of miR-875-3p, thereby promoting the expression of platelet factor 4 variant 1 (PF4V1). The expression of miR-875-3p was negatively correlated to hsa_circ_0069117 and PF4V1 in clinical samples. Luciferase reporter gene assays confirmed the binding sites of miR-875-3p on hsa_circ_0069117 and PF4V1. Gain/loss-of-function and rescue assays further indicated that hsa_circ_0069117 could significantly promote the expression of PF4V1 by sponging miR-875-3p, thereby inhibiting the proliferation and migration of OS cells by suppressing ERK1 and AKT.ConclusionOur study revealed that hsa_circ_0069117 is an anti-OS molecule that could substantially attenuate cell proliferation and migration of OS, which may provide a novel and reliable molecular target for the treatment of OS patients.

Project description:Several theories on the origin of adenomyosis (ADS) have been proposed, of which the most widely accepted is the fundamental pathogenic role of uterine eutopic endometrium. Emerging evidence suggests that circular RNAs participate in the multiple tumorgenesis. The vital importance of circular RNA PVT1 (circPVT1) in the pathological progress like malignancies has been well documented. Nevertheless, its underlying correlation with ADS remains elusive yet. The purpose of this study was to investigate the expression pattern, regulatory effect, and internal mechanism of circPVT1 in ADS. qRT-PCR was performed to detect the relative mRNA expression of circPVT1, miR-145, and Talin1 in ADS endometrial tissue and cells. The protein level of Talin1 was measured by Western blot and immunochemistry. Immunofluorescence was used to identify the primary endometrial epithelial and stromal cells. circPVT1 knockdown in vitro was achieved by transfecting with specific lentivirus vector CCK-8, and colony formation assays were utilized to assess cell proliferation; meanwhile, the transwell assay was employed for evaluating cell invasion ability. By conducting bioinformatics, dual-luciferase reporter assay, or RNA immunoprecipitation (RIP) experiment, the interaction between miR-145 and circPVT1 or Talin1 was verified. Rescue experiments further determined the regulatory effect of circPVT1/miR-145/Talin1 axis. We found both circPVT1 and Talin1 were markedly upregulated in ADS endometrial tissue and cells, whereas miR-145 was decreased. Elevated expression of circPVT1 was closely related to the severity of dysmenorrhea, menorrhagia, and uterine enlargement of patients with ADS. Knockdown of circPVT1 inhibited adenomyotic epithelial and stromal cell proliferation and invasion. Further mechanistic experiments revealed that circPVT1 negatively regulated miR-145 through serving as a molecular sponge. And the facilitating effect of circPVT1 was partially reversed by miR-145. Talin1 was demonstrated to be a down target of miR-145 and indirectly affected by circPVT1. Our findings unveiled that enhanced circPVT1 may be involved in the pathogenesis of ADS via stimulating endometrial cell proliferation and invasion. The establishment of circPVT1/miR-145/Talin1 pathway might present a novel therapeutic insight for ADS.

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N1, N12-Diacetylspermine Is Elevated in Colorectal Cancer and Promotes Proliferation through the miR-559/CBS Axis in Cancer Cell Lines.

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