Over the past few decades, RNA sequencing has significantly progressed, becoming a paramount approach for transcriptome profiling. The revolution from bulk RNA sequencing to single-molecular, single-cell and spatial transcriptome approaches has enabled increasingly accurate, individual cell resolution incorporated with spatial information. Cancer, a major malignant and heterogeneous lethal disease, remains an enormous challenge in medical research and clinical treatment. As a vital tool, RNA sequencing has been utilized in many aspects of cancer research and therapy, including biomarker discovery and characterization of cancer heterogeneity and evolution, drug resistance, cancer immune microenvironment and immunotherapy, cancer neoantigens and so on. In this review, the latest studies on RNA sequencing technology and their applications in cancer are summarized, and future challenges and opportunities for RNA sequencing technology in cancer applications are discussed.

BackgroundThe insulin-like growth factor (IGF) signaling pathway has long been established as playing critical roles in skeletal muscle development. However, the underlying regulatory mechanism is poorly understood. Recently, a large family of small RNAs, named microRNAs (miRNAs), has been identified as key regulators for many developmental processes. Because miRNAs participate in the regulation of various signaling pathways, we hypothesized that miRNAs may be involved in the regulation of IGF signaling in skeletal myogenesis.Methodology/Principal FindingsIn the present study, we determined that the cell-surface receptor IGF-1R is directly regulated by a muscle-specific miRNA, microRNA-133 (miR-133). A conserved and functional binding site for miR-133 was identified in the 3′untranslated region (3′UTR) of IGF-1R. During differentiation of C2C12 myoblasts, IGF-1R protein, but not messenger RNA (mRNA) expression, was gradually reduced, concurrent with the upregulation of miR-133. Overexpression of miR-133 in C2C12 cells significantly suppressed IGF-1R expression at the posttranscriptional level. We also demonstrated that both overexpression of miR-133 and knockdown of IGF-1R downregulated the phosphorylation of Akt, the central mediator of the PI3K/Akt signaling pathway. Furthermore, upregulation of miR-133 during C2C12 differentiation was significantly accelerated by the addition of IGF-1. Mechanistically, we found that the expression of myogenin, a myogenic transcription factor reported to transactivate miR-133, was increased by IGF-1 stimulation.Conclusion/SignificanceOur results elucidate a negative feedback circuit in which IGF-1-stimulated miR-133 in turn represses IGF-1R expression to modulate the IGF-1R signaling pathway during skeletal myogenesis. These findings also suggest that miR-133 may be a potential therapeutic target in muscle diseases.

Skeletal muscle differentiation is controlled by multiple cell signaling pathways, however, the JNK/MAPK signaling pathway dominating this process has not been fully elucidated. Here, we report that the JNK/MAPK pathway was significantly downregulated in the late stages of myogenesis, and in contrast to P38/MAPK pathway, it negatively regulated skeletal muscle differentiation. Based on the PAR-CLIP-seq analysis, we identified six elevated miRNAs (miR-1a-3p, miR-133a-3p, miR-133b-3p, miR-206-3p, miR-128-3p, miR-351-5p), namely myogenesis-associated miRNAs (mamiRs), negatively controlled the JNK/MAPK pathway by repressing multiple factors for the phosphorylation of the JNK/MAPK pathway, including MEKK1, MEKK2, MKK7, and c-Jun but not JNK protein itself, and as a result, expression of transcriptional factor MyoD and mamiRs were further promoted. Our study revealed a novel double-negative feedback regulatory pattern of cell-specific miRNAs by targeting phosphorylation kinase signaling cascade responsible for skeletal muscle development.

Osteoarthritis is a degenerative disease that often causes patients to experience joint pain and deformity. It has been demonstrated that tumor necrosis factor (TNF)-α is associated with the progression of osteoarthritis; however, to the best of our knowledge, the mechanisms by which TNF-α simulates the progression of osteoarthritis and the signaling pathway(s) it influences remain unknown. Therefore, the aim of the present study was to investigate the therapeutic effects of TNF-α inhibitor in an iodoacetate-induced rat model of osteoarthritis and identify its potential mechanisms of action. Western blotting, ELISA and histological analyses were performed to assess the effects of the TNF-α inhibitor on osteoarthritis. The effects of TNF-α and phosphoinositide 3-kinase (PI3K) inhibition on synovial fibroblasts isolated from rats with osteoarthritis were tested in vitro. Furthermore, the expression of various inflammatory cytokines and the PI3K/protein kinase B (AKT) signaling pathway were assessed in vitro. The results indicated that the inflammatory factors TNF-α, interleukin (IL)-1β, IL-17a and IL-8 were upregulated in synovial fibroblasts taken from rats with osteoarthritis compared with normal rats. By contrast, TNF-α inhibition downregulated IL-1β, IL-17a and IL-8 expression in synovial fibroblasts in vitro. The PI3K/AKT pathway was also upregulated in synovial fibroblasts harvested from rats with osteoarthritis compared with that in normal rats. It was demonstrated that treatment with the TNF-α inhibitor downregulated the serum and protein levels of IL-1β, IL-17a and IL-8 in rats with osteoarthritis. Furthermore, treatment with the TNF-α inhibitor also decreased matrix metalloproteinase (MMP)-3, MMP-9, vascular endothelial growth factor and ADAMTS4 expression in synovial fibroblasts isolated from rats with osteoarthritis. Treatment with the TNF-α inhibitor also inhibited the PI3K/AKT pathway in synovial fibroblasts isolated from rats with osteoarthritis. Treatment with the PI3K inhibitor ameliorated TNF-α-induced increases in IL-1β, IL-17a and IL-8 expression in synovial fibroblasts isolated from rats with osteoarthritis. Furthermore, treatment with the TNF-α inhibitor decreased inflammation, as well as joint and cartilage destruction in vivo. Taken together, the results of the present study indicate that TNF-α inhibition may downregulate the expression of inflammatory factors in synovial fibroblasts, suggesting that TNF-α inhibition may be a novel method for treating osteoarthritis by downregulating the PI3K/AKT signaling pathway.