Introduction/Background*RNA modifications play a key role in the regulation of gene expression. Among them, N6-methyladenosine (m6A) is the most prevalent mRNA modification. m6A methylation modulates the gene expression by influencing numerous aspects of mRNA metabolism, including pre-mRNA processing, nuclear export, and translation. Many studies have demonstrated the importance of m6A-mediated post-transcriptional gene regulation in numerous physiological and pathophysiological processes, especially in tumorigenesis. This modification is dynamically modulated by proteins such as methyltransferases (writers), demethylases (erasers) and, recruitment of m6A-binding proteins (readers). In this study we investigated the different m6A methylation patterns between HeLa, SiHa and C33A cervical cancer (CC) cell lines and normal HCK1T cervical keratinocyte cells.
Methodology Gene expression levels of writers (METTL3, METTL14 and WTAP), erasers (ALKBH5 and FTO), and readers (IGF2BP-I, YTHDF1 and HNRNPA2B) was determined at mRNA and protein level by qPCR and Western blot, respectively. The quantification of RNA methylation was performed by colorimetric assay using the EpiQuick® methylation kit.
Result(s)*The expression of METTL3 and METTL14 methyltransferases in C33A cells was significantly higher (p=0,0274 and p=0,0343, respectively) while a statistically significant increase was observed for both SiHa and HeLa for WTAP compared to HCK1T cells (p=0,0153 and p=0,0312, respectively). In the case of demethylases, FTO was statistically increased in C33A and SiHa (p=0,0015 and p=0,0342, respectively), while ALKBH5 was expressed at higher levels only in C33A (p=0,0054) in comparison with HCK1T cells. The expression of readers, YTHDF1 and IGF2BP-1, was statistically increased in C33A (p=0,0058 and p=0,0031, respectively) and SiHa (p=0,0123 and p=0,0161, respectively). Moreover, the methylation rate of all three CC cell lines was higher (HeLa 0.25%, SiHa 0.17% and C33A 0.20%) compared to HCK1T normal cervical cells (0.14%).
Conclusion*Overall, the expression of writers, erasers and readers was enhanced in CC cells, highlighting a deregulation in this critical process. Ongoing experiments focus on the differential expression of the genes of the three groups in samples from cervical cancer with different degrees of dysplasia, to investigate their individual role in cervical cancer progression.
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