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:Papillary thyroid cancer (PTC) is an ever-increasing cancer type worldwide, and greatly decreases the life quality and affects survival time of patients during its development and progression, but the underlying mechanisms and key factors for PTC progression are not clear. Recent studies demonstrated the potassium channel protein SK4, also named as KCNN4, participates in the progression of many cancers, while it lacks the molecular mechanism study for SK4 function. In this study, we performed functional and molecular explorations for SK4 by overexpressing its level in thyroid cancer BHT101 cells. Cellular proliferation and invasion experiments were performed to assess the influences of SK4 on cell behaviors. Further, whole transcriptome sequencing (RNA-seq) analysis helped us systematically investigated the down stream targets of SK4, including differentially expressed genes (DEGs) and regulated alternative splicing events (RASEs). We finally validated several DEGs and RASEs associated with the functions of SK4 by RT-qPCR experiment. In thyroid cancer patients, SK4 expression was completely lost in normal tissues and significantly increased in every stage of tumor tissues compared with normal tissues, the reason of which probably results from the low DNA methylation level at its promoter region. Consistent with previous study, SK4 overexpression (SK4-OE) promoted proliferation level and invasion ability of BHT10 cells compared with negative control (NC). By analyzing the RNA-seq data, we detected dozens of DEGs and found that up DEGs were enriched in negative regulation of apoptotic progress, including VTCN1, MSX1, FATE1, TEK, and PRAMEF2. More importantly, we found SK4-OE globally changed the alternative splicing pattern and identified 1639 RASEs. The genes of RASEs were enriched in DNA damage/repair, viral process, translation, and mRNA splicing pathways, which were tightly associated with the pathogenesis and progression of cancers. The splicing regulatory genes from RASGs could partly explain the reason of global AS dysregulation by SK4-OE in BHT101 cells. Finally, we validated several DEGs and RASEs associated with PTC progression by RT-qPCR. We found the expression of VTCN1, EDN1, SLC29A4, RP11-473M20.16, and CH507-513H4.4 were validated by RT-qPCR, as well as the AS pattern of TMEM116, RBM39, and RBM6. In summary, our results deeply explored the molecular targets of SK4 associated them with SK4-induced progression of PTC. We highlight that SE4-regulated AS pattern probably is a novel regulatory mechanism for SK4 in PTC. The identified DEGs and RASEs, as well as SK4 itself, could be used as potential therapeutic targets for PTC treatment in future.

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SF3B6 promotes the cellular proliferation by extensively interacting with and regulating transcripts expression and alternative splicing in MDA-MB-231 cells [RIP-seq]

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