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:We recently showed that the mammalian genome encodes more than a thousand large intergenic non-coding RNAs (lincRNAs) that are clearly conserved across mammals and thus functional. Gene expression patterns have implicated these lincRNAs in diverse biological processes including cell cycle regulation, immune surveillance, and embryonic stem cell pluripotency. However, the mechanism by which these lincRNAs function is unknown. Here, we expand the catalog of human lincRNAs to ~3300 by analyzing chromatin-state maps of various human cell types. Inspired by the observation that the well-characterized lincRNA HOTAIR bind the Polycomb Repressive Complex 2 (PRC2), we tested whether many lincRNAs are physically associated with PRC2. Remarkably, we observe that ~20% of lincRNAs expressed in various cell types are bound by PRC2, and that additional lincRNAs are bound by other chromatin-modifying complexes. Moreover, we show that siRNA-mediated depletion of certain lincRNAs associated with PRC2 leads to changes in gene expression and that the upregulated genes are enriched for those normally silenced by PRC2. We propose a model in which some lincRNAs guide chromatin–modifying complexes to specific genomic loci to regulate gene expression. siRNA-mediated lincRNA knockdown siRNAs targeting specific lincRNAs were transected into human fibroblasts. Non-targeting siRNAs were used as controls. Both of these experiment types were hybridized to Affymetrix gene expression arrays. PRC2 RIP-Chip The PRC2 complex was immunoprecipitated using antibodies targetting Suz12 and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for PRC2 and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 PRC2 RIP-Chip (dye swap) The PRC2 complex was immunoprecipitated using antibodies targetting Suz12 and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for PRC2 and hybridized using a Cy5 label. In parallel, RIP was performed using IGG and labelled with Cy3 PRC2 RIP-Chip (no proteins) The PRC2 complex was immunoprecipitated using antibodies targetting Suz12 and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for PRC2 and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 COREST RIP-Chip The COREST complex was immunoprecipitated using antibodies targetting COREST and the associated RNA was hybridized to Nimblegen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for COREST and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 H3K27me3 and H3K4me2 modified histones RIP-Chip The H3K27me3 and H3K4me2 modified histones were immunoprecipitated using antibodies targetting COREST and the associated RNA was hybridized to Nimbelgen tiling arrays representing lincRNAs. In parallel an IgG IP was performed and hybridized to the same NG arrays. RIP was performed for COREST and hybridized using a Cy3 label. In parallel, RIP was performed using IGG and labelled with Cy5 Human lincRNA expression Human lincRNAs were profiled in HeLa, Lung FB, and Foot FB. In addition, nuclear and cytoplasmic fractions were taken in HeLa cells Total RNA was extracted from HeLa, Foot, and Lung cells and hybridized to NG tiling array. In addition, the nucleus was fractionated and RNA was hybridized to an array.

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DEK binds to pre-mRNAs and regulated alternative splicing of Hippo signaling genes in HeLa cell [RIP-seq]

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