tiRNA-Met/HNRNPF/NECAB1信号轴在三阴性乳腺癌生长与转移中的作用及其机制研究OA

Background and purpose:Triple-negative breast cancer(TNBC)is a particularly challenging subtype of breast cancer with a high propensity for metastasis and poor prognosis.tRNA-derived fragments(tRFs),as a new class of non-coding small RNA molecules,are involved in various physiological and pathological processes.In previous research,tiRNA-Met was identified through high-throughput sequencing of tRFs and tiRNAs from TNBC tissues.This study aimed to explore the mechanism of tiRNA-Met in the growth and metastasis of TNBC in depth.Methods:RNA Pull-Down Kit,liquid chromatography-mass spectrometry(LC-MS),and RNA immunoprecipitation(RIP)were utilized to screen and identify specific binding proteins of tiRNA-Met.The co-localization of tiRNA-Met and the specific binding protein HNRNPF was detected using cell immunofluorescence experiments.The effects of tiRNA-Met on HNRNPF mRNA and HNRNPF protein levels were assessed through real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot.RNA sequencing(RNA-seq)was performed to investigate the transcriptional profile influenced by the overexpression of tiRNA-Met in MDA-MB-231 cells,as well as the signaling pathways and target genes regulated by tiRNA-Met.The transcriptional level of the target gene NECAB1 was verified by qRT-PCR;the effect of HNRNPF knockdown on NECAB1 expression was also evaluated.The mRNA expression levels of HNRNPF and NECAB1 in TNBC tissues were analyzed using The Cancer Genome Atlas(TCGA)and GEPIA databases,while the protein expression level of HNRNPF was analyzed with the UALCAN database.Survival analysis of NECAB1 and HNRNPF was conducted using the Kaplan-Meier Plotter database.Overexpression of NECAB1 was performed in MDA-MB-231 and BT-549 cells using plasmid transfection,and the proliferation and invasion capabilities of the cells were assessed using cell counting kit-8(CCK-8)and Transwell assays,respectively.Following the knockdown of tiRNA-Met expression using a tiRNA-Met inhibitor,NECAB1 was overexpressed or HNRNPF was inhibited,and the cell proliferation and invasion capabilities were assessed using the CCK-8 assay and Transwell assay.Results:tiRNA-Met specifically binds with HNRNPF and is mainly located in the cytoplasm;neither overexpression nor knockdown of tiRNA-Met affects the levels of HNRNPF mRNA or HNRNPF protein.Both overexpression of tiRNA-Met and knockdown of HNRNPF significantly promoted the expression of NECAB1.Analysis of TCGA,GEPIA and UALCAN databases revealed that both HNRNPF mRNA and protein levels were significantly elevated,whereas NECAB1 expression was reduced in TNBC compared to adjacent normal tissues(P<0.05).Kaplan-Meier Plotter database analysis indicated a positive correlation between NECAB1 expression levels and patient survival(P<0.05),while HNRNPF protein expression level was negatively correlated with patient survival duration(P<0.05).Compared to control cells,MDA-MB-231 and BT-549 cells with NECAB1 overexpression exhibited significantly decreased proliferation(P<0.05)and invasion capabilities(P<0.05);overexpressing NECAB1 or knocking down HNRNPF on the basis of tiRNA-Met knockdown reversed the increased proliferation and invasion induced by tiRNA-Met knockdown.Conclusion:tiRNA-Met enhances NECAB1 expression by targeting HNRNPF,thereby inhibiting the malignant progression of TNBC.