m6A-seq (Micro MeRIP-seq) Service
m6A is a common RNA methylation modification that has been shown to play important roles in regulating gene expression, splicing, RNA editing, RNA stability, mRNA decay, and mediating translation of circular RNAs. CD Genomics has developed a new technology called Micro MeRIP-seq, which utilizes a novel rRNA depletion method. This technology enables the simultaneous detection of methylation profiles in mRNA, lncRNA, and circRNA, allowing researchers to study the post-transcriptional methylation landscape of RNA, making it a crucial technique in the field of epitranscriptomics.
It is worth noting that the Micro MeRIP-seq technology has significantly reduced the sample input requirement by optimizing the technique. Only 1-20 μg of total RNA is now sufficient to meet the experimental requirements.
Technical Principle
Applications
Tumor heterogeneity: Revealing the RNA methylation landscape of cancer cells.
Cell differentiation: Mapping the epigenetic regulation of cell differentiation.
Immune microenvironment: Investigating the epigenetic regulatory mechanisms of immune cell activation and differentiation.
Neuroscience: Exploring the molecular mechanisms of neuro-related diseases.
Advantages
Low RNA input requirement. Rich experience in sample processing. Authoritative algorithms and workflows. Flexible customization of analysis content.
Species Information: Human, Rat, Mouse.
Sample Requirements
Tissue quality of 10-20mg, Cell count of 5×106~107
Sequencing: Illumina HiSeq PE150
Recommended Data Output: ≥6Gb per sample.
Data Analysis
| Basic Analysis | Advanced Analysis |
|---|---|
| 1. Raw data quality assessment | 1. Joint analysis with RNA-seq |
| 2. Raw data preprocessing | 2. prediction of transcription factor network interaction diagram |
| 3. Data alignment to reference genome | |
| 4. Peaks identification | |
| 5. Heatmap of peak centers | |
| 6. Average signal distribution and heatmap near all genes | |
| 7. Chromosome and peak length distribution of peaks | |
| 8. Gene position annotation of peaks | |
| 9. Distribution of peaks in various gene elements | |
| 10. Differential peaks analysis and gene correlation | |
| 11. Motif analysis | |
| 12. KEGG and GO analysis |
Study of m6A in Endometrial Cancer
Abstract
Endometrial cancer refers to cancer originating from the endometrium, the inner lining of the uterus. Its pathogenesis is characterized by abnormal cell growth with the ability to invade or spread to other parts of the body. The current treatment primarily involves surgical removal of the uterus, often accompanied by bilateral salpingo-oophorectomy (removal of both fallopian tubes and ovaries). In more severe cases, radiation therapy, chemotherapy, and hormone therapy may also be considered. Early detection and timely treatment of endometrial cancer lead to better prognosis. In the United States, the five-year survival rate for patients exceeds 80%.
To investigate the role of m6A mRNA methylation in cell proliferation and tumor development, the authors studied endometrial cancer cases with the R298P hotspot mutation in the methyltransferase complex METTL14. They found that approximately 70% of endometrial tumor patients showed a decrease in m6A methylation, which may be attributed to the mutation in METTL14 (writer of m6A) or reduced expression of METTL3 (writer of m6A). These changes led to increased proliferation and tumor development in endometrial cancer cells through the activation of the AKT pathway. The reduced m6A methylation resulted in decreased expression of PHLPP2, a negative regulator in the AKT pathway, and increased expression of mTORC2, a positive regulator of the AKT pathway. In conclusion, these findings reveal that decreased m6A mRNA methylation is a carcinogenic mechanism in endometrial cancer and identify m6A methylation as a regulator of the AKT signaling pathway.
Results
Figure 1 shows the relative low m6A methylation in tumor tissue compared to normal tissue in AKT pathway-related genes.
Figure 2 illustrates the mechanism by which decreased m6A methylation mediates the promotion of cancer development in the AKT pathway. m6A methylation typically weakens AKT pathway activity by promoting the translation of m6A-dependent PHLPP2 and degradation of m6A-dependent mTORC2 subunit transcripts.
The upregulation of PHLPP2 phosphatase and downregulation of mTORC2 kinase both inhibit AKT activity by maintaining its dephosphorylation state. The reduction in m6A methylation disrupts the regulation of these transcripts, resulting in decreased PHLPP2 expression, increased mTORC2 expression, and enhanced AKT activity.
Reference
- Liu J, Eckert MA, Harada BT, Liu SM, Lu Z, Yu K, Tienda SM, Chryplewicz A, Zhu AC, Yang Y, Huang JT, Chen SM, Xu ZG, Leng XH, Yu XC, Cao J, Zhang Z, Liu J, Lengyel E, He C. m6A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer. Nat Cell Biol. 2018 Sep;20(9):1074-1083.