June 5438 +20201October, the research group of Wang Xiuxing of Nanjing Medical University and the research group of UCSD Jeremy Rich of the United States published an article entitled "RNA m6A Reader YTHDF2? It maintains one expression and is target-dependent in glioblastoma stem cells. " This study provides a new therapeutic opportunity for targeted therapy of glioblastoma.
Research background
Glioblastoma (GBM) is the most common primary and endogenous brain tumor, and the average survival time of patients is more than one year. In view of the role of glioblastoma stem cells (GSC) in the treatment of resistance, angiogenesis, immune escape and invasion, clinical and preclinical observations show that targeting GSC can improve tumor prognosis and precise medical research in neurooncology.
research method
research results
1.GSC upregulated oncogene transcripts are marked by RNA m6A modification.
MeRIP-seq was used to detect m6A markers of GSC and neural stem cells (NSC). The results show that the m6A distribution of GSC has changed compared with its non-tumor counterpart. GSEA analysis of RNA-seq data from 38 GSCs and 5 NSCs showed that the genes with m6A peak were highly enriched in GSC, and all the genes with m6A peak obtained in GSC were up-regulated. On the contrary, compared with NSC and GSC, the genes that lost m6A peak are usually down-regulated in GSC. In addition, in GSC, m6A peaks were obtained on important genes related to cancer stem cells, including OLIG2? And MYC.
2.？ YTHDF2？ In GSC, freedom of speech is very important to maintain GSC.
The author studied m6A YTHDF? In glioblastoma, the function of YTHDF2 was detected by CRIPR technique. Compared with the control sgRNA, YTHDF2 was knocked out? It will reduce cell viability and reduce the formation of cell spheres in GSCs. To study YTHDF2? Can exhaustion induce GSC differentiation? Orthogonal experiment found that shRNA mediated YTHDF2? Knockdown will reduce the activity of GSC and over-express YTHDF2? The activity that can save GSC shows that YTHDF2? It is a specific and effective regulator for the maintenance of glioblastoma.
3.？ YTHDF2？ M6A RNA modification supports GSCs gene expression.
The author used RNA-seq to detect YTHDF2? Downstream target, kill YTHDF2? Can cause a wide range of gene expression changes of GSCs and MYC? The target is obviously enriched, and the gene with m6A peak in GSCs is down-regulated more frequently. YTHDF2 also passed qPCR verification? Knock out MYC and VEGFA? Effect of decreased mRNA level. To predict YTHDF2? Combined with the expression data of GSCs in TCGA glioblastoma, the author found that YTHDF2? Related gene MYC? And E2F? Where are Target and G2M? Regulatory factors and oxidative phosphorylation media are highly enriched. These data show that YTHDF2? As a regulator of transcription program related to differential modification of M6 a.
4.？ YTHDF2？ By keeping? MYC？ Transcription stability shows GSC specificity dependence.
To determine YTHDF2? By mediating the specificity of MYC in GSCs, the author compares the differences between NSCs and GSCs. The effect of absence. YTHDF2 in NSCs? Knockout did not affect the level of MYC mRNA, but decreased the level of MYC mRNA in GSCs. Besides, YTHDF2? Exhaustion reduces the activity of GSC, but does not affect neural stem cells. So, YTHDF2? Represents a GSC-specific dependence, through MYC? The specific stability of this gene supports the survival of glioblastoma.
5.？ IGFBP3？ Is it in GSCs? YTHDF2-MYC？ Downstream target of axis
Because of IGFBP3? Is it YTHDF2? One of the most down-regulated genes after exhaustion, the author studied IGFBP3? Is YTHDF2-MYC regulated? Cell viability downstream of the axis. IGFBP3？ The absence of GSC decreased the activity of GSC and the formation of cell spheres. IGFBP3？ Overexpression saved the GSCs of YTHDF2? Down-regulate cell death. Finally, the author used IGFBP3? In 20 cases of glioblastoma and 20 cases of non-tumor brain tissue. The expression of IGFBP3 in GSC was confirmed. MRNA expression increased. The results showed that IGFBP3? Yhdf 2-myc in GSCs? The key downstream effector of the signal axis.
6.？ YTHDF2-MYC-IGFBP3？ Axis promotes tumor growth in vivo
In order to explore the targeting of YTHDF2 in vivo? The potential benefits of treatment, the author uses CRISPR gene knockout technology to detect mice with in situ xenografts. The results showed that YTHDF2 was knocked out compared with mice carrying GSCs of control sgRNA. The latent period of tumor is prolonged and the tumor volume is reduced. Overexpression of IGFBP3 restores YTHDF2? GSCs lack tumorigenic ability in vivo.
Research conclusion
By combining in vitro and in vivo GSCs studies, this study clarified the role of m6A medium in GSCs, and determined YTHDF2? Is GSCs-specific dependence, by stabilizing MYC? Transcripts regulate glucose metabolism in GSCs. These findings provide new therapeutic opportunities for targeted therapy of glioblastoma.
In April 2020, Hong Jie team of Renji Hospital affiliated to Shanghai Jiaotong University School of Medicine published a research paper entitled "M6 a-dependent glycolysis promotes the progress of colored cancer" in Molecular Cancer. Studies have shown that targeting METTL3? Its pathway provides another reasonable therapeutic target for colorectal cancer patients with high glucose metabolism.
Research background
Colorectal cancer (CRC) is the fourth most common malignant tumor and the third most common cause of cancer death in the world, and lactic acid, as the final product of glycolysis, is considered as a promising method to treat cancer. The change of m6A regulatory gene plays an important role in the pathogenesis of many human diseases, but it is not clear whether m6A modification plays a role in the glucose metabolism of CRC.
research method
research results
1.？ METTL3 is closely related to glycolysis of colorectal cancer.
In order to explore the correlation between m6A modification and glycolysis metabolism of colorectal cancer, the author analyzed 47 patients with colorectal cancer by RT-PCR, and found that FDG uptake and METTL3? There is the most significant correlation between expressions. Further analysis found that FDG uptake and METTL3? Immunohistochemical staining showed significant correlation. Finally, the gene expression profiles of METTL 3 knockout and wild-type (WT) HCT 1 16 CRC cells were compared by RNA-seq, and it was found that METTL3 knockout cells showed higher expression of METTL3. These results show that METTL3? It may mediate glycolysis metabolism and carcinogenesis in patients with colorectal cancer.
2.METTL3？ Promote glycolysis metabolism in colorectal cancer
To get to know METTL3 Does the change directly affect glycolytic metabolism, and it is found that METTTL 3 is knocked out? The extracellular acidification rate (ECAR) of HCT 1 16 and SW480 cells can be significantly reduced, and the overexpression of METTTL 3 can significantly increase the lactic acid production, glucose absorption and ECAR of DLD 1 cells. Level. Clarify Mettl3? Does the induced glycolysis of CRC depend on its methyltransferase function? The study of wild type and mutant found that Mettl3? The deletion of MTase domain blocks METTTL 3? Inducing glycolysis process. These data indicate that METTTL 3 regulates glycolytic metabolism of colorectal cancer through its methyltransferase domain.
3. In colorectal cancer, METLC3? Induced proliferation depends on the activation of glycolysis.
METLC3？ Knocking out HCT 1 16 cells eliminated cell proliferation and colony formation, and reduced the growth of HCT 1 16 tumor and the tumor weight of xenotransplantation mouse model. In functional analysis, METTL3? Overexpression increased cell proliferation, colony formation, tumor growth and tumor weight. Treatment with 2-DG (an inhibitor of glycolytic pathway) significantly blocked METTL3? Inducing cell proliferation and colony formation, these results indicate that METTTL 3 promotes CRC progress by regulating glucose metabolism in colorectal cancer.
4.METTL3？ Potential target of colorectal cancer
In order to determine the potential goal of METTTL 3, the author chose METTTL 3? MeRIP-seq and RNA-seq were performed on knockout and wild-type HCT 1 16 cells. The most common motif "GGAC" is significantly enriched in m6a peak, and most of them are METTL3? The binding site is located in the CDS region which is highly enriched in 5'UTR and 3'UTR, and m6A is hypomethylated at the transcription level. Combined with RNA-seq data, 429 hypomethylated m6A genes were identified, with down-regulated mRNA transcription and 595 hypomethylated m6A genes up-regulated. Based on the decrease of methylation level and mRNA expression level, the target gene HK2 closely related to glycolysis was found. And SLC2A 1(GLUT 1).
6.？ HK2？ And then what? SLC2A 1？ what's up METTL3？ Important functional target genes in colorectal cancer
Through HCT 1 16 WT and mettl3? With control and HK2? Or SLC2A 1? Over-expression experiment found that HK2? Or SLC2A 1? The ectopic expression of metll 3 partially recovered. Cell proliferation, colony forming ability and tumor growth can also restore HCT 1 16 metlt 3? Decrease in lactic acid production in knockout cells. At the same time, SLC2A 1 was overexpressed in vitro and in vivo. Significant recovery of HCT 1 16 metl 3? The trend of decreasing glucose uptake in knockout cells. So, HK2? And SLC2A 1 mediate the regulatory function of METLL3 in CRC cells.
Research conclusion
METTL3？ It is a functional and clinical oncogene of colorectal cancer. METTL3？ M6a-IGF-2bp2/3-/3 Dependence Mechanism Stabilizes Colorectal Cancer HK2? And SLC2A 1. Aim for METTL3? Its pathway provides another reasonable therapeutic target for colorectal cancer patients with high glucose metabolism.
refer to
[1] dixit d, praeger b, gipsyn r, et al. RNA m6A reader YTHDF2? Maintaining oncogene expression is a targeted dependence of glioblastoma stem cells [J]. Cancer discovery, 2020.
[2] Shen C, Xuan B, Yan T, et al. M6A-dependent glycolysis promotes the progress of colorectal cancer [J]. Molecular Cancer, 2020, 19( 1).