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LARP6 promotes carcinogenic features of triple negative breast cancer cell line by directly modulating alternative splicing of associated genes [RNA-Seq]

ABSTRACT: Purpose: Triple-negative breast cancer (TNBC) has the highest mortality rate of all breast cancer subtypes and currently lacks effective targeted therapies. LARP6 is an RNA-binding protein associated with cancer promotion, but its mechanism of action in TNBC remains unclear. Methods: Firstly, Western blot (WB) was used to detect the expression level of LARP6 protein in TNBC cell lines. The effects of LARP6 overexpression on TNBC cell proliferation and invasion were determined through Cell Counting Kit-8 (CCK-8) and Transwell experiments. Next, RNA sequencing (RNA-seq) and improved RNA immunoprecipitation and sequencing (iRIP-seq) were performed to analyze LARP6-bound and regulated differentially expressed genes (DEGs) and alternative splicing (AS) events in MDA-MB-231 cells. Finally, RT-qPCR and RIP-qPCR were used for confirmation. Result：Our study found that the overexpressed LARP6 TNBC cell lines significantly promoted thetumor cell proliferation and invasion of cancer (P<0.05). RNA-seq showed that LARP6 overexpression alteredaffected the expression levels of 171 genes, whileand combined with iRIP-seq, it revealedshowed that only EGR1 and LARP6 were bounddirectly bind to by the LARP6 protein, indicating. This shows that LARP6 hasis a weak ability to bind to and regulate differential expression of in regulating genes expression. However, the number of regulatedrelated alternative splicing evensgenes (RASEGs) was upwards ofreached more than 1000, and the correspondingthese genes (RASGs) were mainly enriched in important pathways such as DNA repair and cellular response to DNA damage stimulus. In addition, bassaed on binding peak calling of iRIP-seq in both IP samples,Through analysis of binding peaks, we found that there were 321 overlapping binding peaks in both IP samples between IP1 and IP2 samples awere identified, in whichnd the genes they were mainly concentrated in the post-transcriptional regulatory pathway, and it was also found that LARP6 might tend to bind motif. The top 5 binding motifs were obtained using HOMER, among which CGACGAG was the binding motifsequence that appeared simultaneously in both IP groups. The intersection of binding peak-related genes andwith RASGs suggestedrevealed that LARP6 could bind to 16 genes and regulate their alternative splicing, thus playing an important role in TNBC progression. directly bind to LARP6, most of which were related to cell proliferation, invasion and drug resistance. Conclusion: We revealed the mechanism that LARP6 promotes TNBC by directly regulating the AS of related genes, which provides a new clue for the targeted therapy of TNBC.

ORGANISM(S): Homo sapiens

PROVIDER: GSE248131 | GEO | 2025/03/13

REPOSITORIES: GEO

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Project description:Purpose: Triple-negative breast cancer (TNBC) has the highest mortality rate of all breast cancer subtypes and currently lacks effective targeted therapies. LARP6 is an RNA-binding protein associated with cancer promotion, but its mechanism of action in TNBC remains unclear. Methods: Firstly, Western blot (WB) was used to detect the expression level of LARP6 protein in TNBC cell lines. The effects of LARP6 overexpression on TNBC cell proliferation and invasion were determined through Cell Counting Kit-8 (CCK-8) and Transwell experiments. Next, RNA sequencing (RNA-seq) and improved RNA immunoprecipitation and sequencing (iRIP-seq) were performed to analyze LARP6-bound and regulated differentially expressed genes (DEGs) and alternative splicing (AS) events in MDA-MB-231 cells. Finally, RT-qPCR and RIP-qPCR were used for confirmation. Result：Our study found that the overexpressed LARP6 TNBC cell lines significantly promoted thetumor cell proliferation and invasion of cancer (P<0.05). RNA-seq showed that LARP6 overexpression alteredaffected the expression levels of 171 genes, whileand combined with iRIP-seq, it revealedshowed that only EGR1 and LARP6 were bounddirectly bind to by the LARP6 protein, indicating. This shows that LARP6 hasis a weak ability to bind to and regulate differential expression of in regulating genes expression. However, the number of regulatedrelated alternative splicing evensgenes (RASEGs) was upwards ofreached more than 1000, and the correspondingthese genes (RASGs) were mainly enriched in important pathways such as DNA repair and cellular response to DNA damage stimulus. In addition, bassaed on binding peak calling of iRIP-seq in both IP samples,Through analysis of binding peaks, we found that there were 321 overlapping binding peaks in both IP samples between IP1 and IP2 samples awere identified, in whichnd the genes they were mainly concentrated in the post-transcriptional regulatory pathway, and it was also found that LARP6 might tend to bind motif. The top 5 binding motifs were obtained using HOMER, among which CGACGAG was the binding motifsequence that appeared simultaneously in both IP groups. The intersection of binding peak-related genes andwith RASGs suggestedrevealed that LARP6 could bind to 16 genes and regulate their alternative splicing, thus playing an important role in TNBC progression. directly bind to LARP6, most of which were related to cell proliferation, invasion and drug resistance. Conclusion: We revealed the mechanism that LARP6 promotes TNBC by directly regulating the AS of related genes, which provides a new clue for the targeted therapy of TNBC.

Project description:Background: Angiogenesis is an essential step for tissue development and its dysregulation is associated with various diseases, including moyamoya disease. Among the regulators of angiogenesis, galectin-1 encoded by LGALS1, plays essential roles, but the underlying mechanisms need to be further expounded. In this study, we systematically investigated the potential targets of galectin-1 by silencing LGALS1 (siLGALS1) in human umbilical vein endothelial cells (HUVECs) and performing whole transcriptome sequencing (RNA-seq) experiments. We also integrated galectin-1-interacting RNA data to explore how galectin-1 regulates gene expression and alternative splicing. Results: By systematical analysis, we found siLGALS1 treatment significantly regulated 1451 differentially expressed genes (DEGs), including 604 up-regulated DEGs and 847 down-regulated DEGs. The down-regulated DEGs were significantly enriched in angiogenesis and inflammatory response pathways. The involved genes included CCL2, GJA5, CALCRL, ACKR3, HEY1, AQP1, CD34, ECM1, RAMP2, and SELP, which were also validated by RT-qPCR experiment. We also analyzed the dysregulated alternative splicing (AS) profile by siLGALS1, and found siLGALS1 had obvious bias for dysregulated AS types, preferring to promote exon skipping and intron retention, and inhibit cassette exon events. Interestingly, we found the regulated AS genes (RASGs) were enriched in focal adhesion and VEGF signaling pathway, which were highly related to angiogenesis. Finally, we found hundreds of RASGs were also bound by galectin-1 using the published iRIP-seq data of galectin-1, including RASGs from angiogenesis pathway. Conclusions: In summary, our results demonstrated that galectin-1 could regulate angiogenesis-related genes at transcriptional and post-transcriptional levels, which is associated its RNA binding ability in HUVECs. The discovery extends our understanding on the functions and molecular mechanisms of galectin-1 during the development of angiogenesis, which could be served as a therapeutic option in anti-angiogenic treatment in future.

Project description:Accumulating evidence indicates that RNA-binding proteins (RBPs) are involved in tumorigenesis. As a member of DEAD-box RBPs, DDX17 has a significant role in various contexts, such as mRNA translation, pre-mRNA splicing, ribosome biogenesis, and DNA repair. However, whether DDX17 is involved in the regulation of gene expression and alternative splicing in lung adenocarcinoma (LUAD) remains unclear. Methods: CCK-8 assay was used to evaluate cells proliferation. Cell apoptosis was evaluated by an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit. We used high-throughput RNA sequencing (RNA-seq) to determine differentially expressed genes (DEGs) and alternative splicing (AS) events in DDX17 knockdown LUAD cells. Functional and pathway enrichment analysis was also performed. Results: Knockdown of DDX17 by siRNA significantly inhibited the proliferation and induced apoptosis in A549 cells. 5243 DEGs were identified. Functional enrichment analyses indicated that these genes were mainly enriched in biological processes including regulation of transcription, regulation of cell growth, DNA repair. DDX17 knockdown increased the level of pro-apoptotic gene expression and decreased the expression of pro-proliferation genes. We also showed that DDX17-regulated AS events revealed by computational analysis using ABLas software in A549 cells were highly validated by RT-qPCR, and also validated by TCGA-LUAD dataset. Functional enrichment analyses showed regulated alternatively spliced genes (RASGs) were mainly enriched in biological processes related to apoptotic process, DNA repair, positive regulation of translation, positive regulation of apoptotic process. Conclusion: The present findings suggest that DDX17 may function as an oncogene by regulating both the expression and the alternative splicing of genes associated with proliferation, invasion and apoptosis in LUAD cells. Our findings may bring new insights into understanding the molecular mechanisms of LUAD and provide a new direction for LUAD treatment.

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LARP6 promotes carcinogenic features of triple negative breast cancer cell line by directly modulating alternative splicing of associated genes [RNA-Seq]

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