N6-methyladenosine (m6A) is the most abundant epitranscriptome modification in mammalian mRNA. Recent years have seen substantial progress in m6A epitranscriptomics, indicating its crucial roles in the initiation and progression of cancer through regulation of RNA stabilities, mRNA splicing, microRNA processing and mRNA translation. However, by what means m6A is dynamically regulated or written by enzymatic components represented by methyltransferase-like 3 (METTL3) and how m6A is significant for each of the numerous genes remain unclear. We focused on METTL3 in pancreatic cancer, the prognosis of which is not satisfactory despite the development of multidisciplinary therapies. We established METTL3-knockdown pancreatic cancer cell line using short hairpin RNA. Although morphologic and proliferative changes were unaffected, METTL3-depleted cells showed higher sensitivity to anticancer reagents such as gemcitabine, 5-fluorouracil, cisplatin and irradiation. Our data suggest that METTL3 is a potent target for enhancing therapeutic efficacy in patients with pancreatic cancer. In addition, we performed cDNA expression analysis followed by gene ontology and protein-protein interaction analysis using the Database for Annotation, Visualization, and Integrated Discovery and Search Tool for the Retrieval of Interacting Genes/Proteins databases, respectively. The results demonstrate that METTL3 was associated with mitogen-activated protein kinase cascades, ubiquitin-dependent process and RNA splicing and regulation of cellular process, suggesting functional roles and targets of METTL3.
Recent studies that have emerged on the diversity of RNA modification in tumors suggest their eligibility as bona fide targets in diagnosis and drug discovery. N6-methyladenosine (m6A) was first reported and is most common in epitranscriptome modification of various RNAs. The YT521-B homology (YTH) domain family are representative m6A-binding proteins, but how the YTH domain family is involved in cancer remains to be clearly understood. Given that clinical sequence data in colorectal cancer indicate that overexpression of YTHDF1 is outstanding among other family members, we studied the role of Ythdf1 and the transcriptional control of YTHDF1. Immunostaining of Ythdf1 showed that its expression was associated with various malignant tumor behaviors, such as depth, lymph node metastasis, and poorer cancer stages. The study of patient survival indicated that patients with high Ythdf1 expression had significantly poorer overall survival. The results indicated that Ythdf1 expression is an independent prognostic factor of patients. The in vitro study showed that the knockdown of YTHDF1 resulted in the suppression of cancer proliferation and sensitization to the exposure of anticancer drugs such as fluorouracil and oxaliplatin. Importantly, the study upstream of the YTHDF1 gene indicated that an oncogenic transcription factor c-Myc was associated with YTHDF1 in both expression and chromatin immunoprecipitation data. Moreover, the knockdown experiments of c-Myc showed the inhibition of YTHDF1, supporting a notion of c-Myc-driven YTHDF1 axis significance. These data suggest that m6A reader Ythdf1 plays a significant role in colorectal cancer progression.
Pyruvate kinase M2 (PKM2) is an alternatively spliced variant of the pyruvate kinase gene that is preferentially expressed during embryonic development and in cancer cells. PKM2 alters the final rate-limiting step of glycolysis, resulting in the cancer-specific Warburg effect (also referred to as aerobic glycolysis). Although previous reports suggest that PKM2 functions in nonmetabolic transcriptional regulation, its significance in cancer biology remains elusive. Here we report that stimulation of epithelial-mesenchymal transition (EMT) results in the nuclear translocation of PKM2 in colon cancer cells, which is pivotal in promoting EMT. Immunoprecipitation and LC-electrospray ionized TOF MS analyses revealed that EMT stimulation causes direct interaction of PKM2 in the nucleus with TGF-β-induced factor homeobox 2 (TGIF2), a transcriptional cofactor repressor of TGF-β signaling. The binding of PKM2 with TGIF2 recruits histone deacetylase 3 to the E-cadherin promoter sequence, with subsequent deacetylation of histone H3 and suppression of E-cadherin transcription. This previously unidentified finding of the molecular interaction of PKM2 in the nucleus sheds light on the significance of PKM2 expression in cancer cells.pyruvate kinase M2 | epithelial-mesenchymal transition | colorectal cancer | invasion | transforming growth factor-β-induced factor homeobox 2 C olorectal cancer (CRC) is the second most common cancer in the world, with more than 1.2 million new cases and about 600,000 deaths annually (1). Cancerous cells exploit a cancerspecific glycolytic system known as the Warburg effect (also referred to as aerobic glycolysis), which involves rapid glucose uptake and preferential conversion to lactate, despite an abundance of oxygen (2, 3). The precise mechanism underpinning aerobic glycolysis was unclear for a long time. However, in 2008, pyruvate kinase M2 (PKM2) gained attention when its expression was shown to be required for the maintenance of aerobic glycolysis (4). PKM2 is an alternatively spliced variant of the PKM gene that regulates the final rate-limiting step of glycolysis. PKM2 is expressed during embryonic development, but it is generally not expressed in most adult tissues. However, its counterpart, PKM1, is exclusively expressed in adult tissues. PKM2 has been shown to be reactivated in tumor development (5, 6). In cancer cells, PKM2 expression allows the diversion of glycolytic flux into the pentose phosphate pathway associated with attenuated pyruvate kinase activity, thereby meeting the biosynthetic demands for rapid proliferation (3).Investigations about the nuclear function of PKM2 arose after elucidation of the PKM2 metabolic function. It was identified that in cancer cells, PKM2 can translocate into the nucleus and function as a transcriptional cofactor in response to several extracellular signals, including EGF and hypoxia, subsequently activating CYCLIN D1, C-MYC, or hypoxia-inducible factor 1α (HIF-1α) (7,8). Particularly in the hypoxic condition, PKM2 interacts with HIF-1α and partici...
The biological significance of micro (mi)RNAs has traditionally been evaluated according to their RNA expression levels based on the assumption that miRNAs recognize and regulate their targets in an unvarying fashion. Here we show that a fraction of mature miRNAs including miR-17-5p, -21-5p, and -200c-3p and let-7a-5p harbor methyl marks that potentially alter their stability and target recognition. Importantly, methylation of these miRNAs was significantly increased in cancer tissues as compared to paired normal tissues. Furthermore, miR-17-5p methylation level in serum samples distinguished early pancreatic cancer patients from healthy controls with extremely high sensitivity and specificity. These findings provide a basis for diagnostic strategies for early-stage cancer and add a dimension to our understanding of miRNA biology.
Background:Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance.Methods:We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining.Results:The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients.Conclusions:miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.
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