Background N6-methyladenosine (m6A) is the most abundant reversible methylation modification of eukaryotic mRNA, and it plays vital roles in tumourigenesis. This study aimed to explore the role of the m6A demethylase ALKBH5 in pancreatic cancer (PC). Methods The expression of ALKBH5 and its clinicopathological impact were evaluated in PC cohorts. The effects of ALKBH5 on the biological characteristics of PC cells were investigated on the basis of gain-of-function and loss-of-function analyses. Subcutaneous and orthotopic models further uncovered the role of ALKBH5 in tumour growth. mRNA and m6A sequencing and assays of m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the targeted effect of ALKBH5 on PER1. P53-binding sites in the ALKBH5 promoter were investigated by ChIP and luciferase assays to reveal the interplay between ALKBH5 and PER1-activated ATM-CHK2-P53/CDC25C signalling. Results ALKBH5 loss characterized the occurrence and poor clinicopathological manifestations in patients with PC. Overexpression of ALKBH5 reduced tumoural proliferative, migrative, invasive activities in vitro and ameliorated tumour growth in vivo, whereas ALKBH5 knockdown facilitated PC progression. Mechanistically, ALKBH5 posttranscriptionally activated PER1 by m6A demethylation in an m6A-YTHDF2-dependent manner. PER1 upregulation led to the reactivation of ATM-CHK2-P53/CDC25C signalling, which inhibited cell growth. P53-induced activation of ALKBH5 transcription acted as a feedback loop regulating the m6A modifications in PC. Conclusion ALKBH5 serves as a PC suppressor by regulating the posttranscriptional activation of PER1 through m6A abolishment, which may highlight a demethylation-based approach for PC diagnosis and therapy.
BackgroundTumor metastasis is one of the leading causes of poor prognosis for colorectal cancer (CRC) patients. Loss of Smad4 contributes to aggression process in many human cancers. However, the underlying precise mechanism of aberrant Smad4 expression in CRC development is still little known.ResultsmiR-20a-5p negatively regulated Smad4 by directly targeting its 3′UTR in human colorectal cancer cells. miR-20a-5p not only promoted CRC cells aggression capacity in vitro and liver metastasis in vivo, but also promoted the epithelial-to-mesenchymal transition process by downregulating Smad4 expression. In addition, tissue microarray analysis obtained from 544 CRC patients’ clinical characters showed that miR-20a-5p was upregulated in human CRC tissues, especially in the tissues with metastasis. High level of miR-20a-5p predicted poor prognosis in CRC patients.MethodsFive miRNA target prediction programs were applied to identify potential miRNA(s) that target(s) Smad4 in CRC. Luciferase reporter assay and transfection technique were used to validate the correlation between miR-20a-5p and Smad4 in CRC. Wound healing, transwell and tumorigenesis assays were used to explore the function of miR-20a-5p and Smad4 in CRC progression in vitro and in vivo. The association between miR-20a-5p expression and the prognosis of CRC patients was evaluated by Kaplan–Meier analysis and multivariate cox proportional hazard analyses based on tissue microarray data.ConclusionsmiR-20a-5p, as an onco-miRNA, promoted the invasion and metastasis ability by suppressing Smad4 expression in CRC cells, and high miR-20a-5p predicted poor prognosis for CRC patients, providing a novel and promising therapeutic target in human colorectal cancer.
We previously discovered that Ras association domain family member 6 (RASSF6) was downregulated and predicted poor prognosis in GC patients. However, the mechanisms of the down regulation of RASSF6 in GC remained unclear. Increasing evidence indicates that dysregulation of microRNAs promotes the progression of cancer through the repression of tumour suppressors. Here, we identified miR-181a-5p as a novel regulator of RASSF6 in GC. Functionally, ectopic expression or silencing of miR-181a-5p, respectively, promoted or inhibited GC cell proliferation, colony formation and cell cycle transition, as well as enhanced or prevented the invasion, metastasis of GC cells and epithelial to mesenchymal transition of GC cells in vitro and in vivo. Molecularly, miR-181a-5p functioned as an onco-miRNA by activating the RASSF6-regulated MAKP pathway. Overexpression or silencing of RASSF6 could partially reverse the effects of the overexpression or repression of miR-181a-5p on GC progress caused by activation of the MAKP pathway in vitro and in vivo. Clinically, high miR-181a-5p expression predicted poor survival in GC patients, especially combined with low RASSF6 expression. Collectively, we identified miR-181a-5p as an onco-miRNA, which acts by directly repressing RASSF6 in GC.
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