Yesterday, the star LncRNA hit the m6A and counterattacked today's new star.
m6A RNA methylation is currently the hottest research star after non-coding RNA such as LncRNA, circular RNA, etc. How much fire? Put the data to tell you!
Half of it was less than half in 2019, and the number of published articles has already accounted for 70% of last year. In the field of RNA methylation, there are not only a large number of articles, but also a lot of high-scoring articles. According to statistics, in the first half of 2019, there were many top journals such as Nature, Cell, Cell Stem Cell, and Nature Immunolgy. There are 18 articles with 10 or more m6A articles.
m6A RNA methylation is a natural hotspot in the country of 2018. The application topics are mainly about writers, erasers and readers.
Tired of writer, eraser? Let's take a look at the reader. The YTHDF family is the most star-studded researcher in the reader due to its involvement in protein translation and degradation. It can be used in millions of m6A reader enzymes. The genes you study may be in addition to the YTHDF family. Other readers combined .
Tired of writer, eraser? Let's take a look at the reader. The YTHDF family is the most star-studded researcher in the reader due to its involvement in protein translation and degradation. It can be used in millions of m6A reader enzymes. The genes you study may be in addition to the YTHDF family. Other readers combined .
Today, let's open our minds and talk about another reader hnRNPA2B1, and see how readers with expressions like these core kernels do research.
Article introduction
Colorectal cancer (CRC) is a type of disease that develops cancer in the intestine. At present, the molecular mechanisms related to the occurrence and metastasis of CRC have not been reported in detail. Studies have shown that LncRNA molecules play an important role in tumorigenesis, development, and even migration. Therefore, using LncRNA as the entry point, we first found a target LncRNA---RP11, which was highly expressed in CRC by LncRNA sequencing , and the phenotype was positively correlated with cell migration. Then, by RNA Pull Down-mass spectrometry , it was confirmed that the LncRNA is directly bound to the downstream protein hnRNPA2B1, which is a m6A RNA-modified star reading enzyme (Reader)! That LncRNA is bound to participate in the process of m6A RNA methylation. Next, let's follow the author's ideas and see how the LncRNA binding m6A mechanism article is done.
Published issue: Molecular Cancer
Impact factor: 7.8
Experimental methods: LncRNA sequencing , MeRIP-qPCR , RNA Pull Down , RIP , LncRNA quantitative PCR , mRNA quantitative PCR
Experimental materials: CRC clinical samples, etc., colorectal cancer cell lines, etc.
Impact factor: 7.8
Experimental methods: LncRNA sequencing , MeRIP-qPCR , RNA Pull Down , RIP , LncRNA quantitative PCR , mRNA quantitative PCR
Experimental materials: CRC clinical samples, etc., colorectal cancer cell lines, etc.
Article content
1. LncRNA RP11 is highly expressed in CRC cells and tissues
CRC tissues (cancer stage 1 and stage 3 cancer) and adjacent tissues were separately subjected to LncRNA sequencing (the cloud sequence can provide this service ) , and LncRNA-RP-RP11 which is highly differentially expressed in both stage 1 and stage 3 of cancer was screened. In the sequenced samples and 32 clinical sample tissues, it was confirmed by LncRNA quantitative PCR that it was highly expressed in the experimental group. The results of colon cancer and rectal cancer databases have also confirmed this trend of expression. RP11 is widely expressed in multiple colorectal cancer cell lines and is highly expressed in SW620 cells primary cultured in metastatic lymph node tissues of CRC patients.
CRC tissues (cancer stage 1 and stage 3 cancer) and adjacent tissues were separately subjected to LncRNA sequencing (the cloud sequence can provide this service ) , and LncRNA-RP-RP11 which is highly differentially expressed in both stage 1 and stage 3 of cancer was screened. In the sequenced samples and 32 clinical sample tissues, it was confirmed by LncRNA quantitative PCR that it was highly expressed in the experimental group. The results of colon cancer and rectal cancer databases have also confirmed this trend of expression. RP11 is widely expressed in multiple colorectal cancer cell lines and is highly expressed in SW620 cells primary cultured in metastatic lymph node tissues of CRC patients.
Figure 1. High expression of RP11 in CRC tissues and cells
2. RP11 induces CRC cell dissemination in vivo and in vitro
To further elucidate the functional mechanism of RP11 in vivo and in vitro. The authors first confirmed that the RP11 overexpressing cell line can significantly promote cell migration ability. Since this process affects the expression levels of marker proteins such as E-Cad, FN and Vim, it may be associated with EMT and cancer metastasis. Next, the authors highly expressed RP11 gene in xenografted nude mice and found that the gene can promote tumor proliferation. The authors introduced cancer cells stably expressing RP11 by tail injection in nude mice, and after 8 weeks of feeding, they were found to have metastasized to mouse liver tissues.
To further elucidate the functional mechanism of RP11 in vivo and in vitro. The authors first confirmed that the RP11 overexpressing cell line can significantly promote cell migration ability. Since this process affects the expression levels of marker proteins such as E-Cad, FN and Vim, it may be associated with EMT and cancer metastasis. Next, the authors highly expressed RP11 gene in xenografted nude mice and found that the gene can promote tumor proliferation. The authors introduced cancer cells stably expressing RP11 by tail injection in nude mice, and after 8 weeks of feeding, they were found to have metastasized to mouse liver tissues.
Figure 2. In vitro: RP11 promotes cancer cell migration in vivo: RP11 promotes cancer tissue proliferation and migration
3. The transcription factor Zeb1 mediates RP11 regulation of CRC cell spread
Studies have shown that LncRNA can influence the expression of transcripts around it through homeopathic regulation. Therefore, six transcripts were found around LncRNA RP11, namely NUDT12, C5orf30, PPIP5K2, GIN1, RP11-6 N13.1 and CTD-2374C24. It was confirmed by mRNA quantitative PCR that the above transcripts were not differentially expressed in the RP11 high expression group and the SW620 cell line with the highest RP11 expression. Therefore, the functional mechanism of RP11 is not affected by the mode of homeopathic regulation.
Studies have shown that LncRNA can influence the expression of transcripts around it through homeopathic regulation. Therefore, six transcripts were found around LncRNA RP11, namely NUDT12, C5orf30, PPIP5K2, GIN1, RP11-6 N13.1 and CTD-2374C24. It was confirmed by mRNA quantitative PCR that the above transcripts were not differentially expressed in the RP11 high expression group and the SW620 cell line with the highest RP11 expression. Therefore, the functional mechanism of RP11 is not affected by the mode of homeopathic regulation.
Since it is not caused by homeopathic regulation, what will it be?
Is it a downstream binding protein?
In order to confirm the above conjecture, the authors examined the expression levels of multiple EMT-related factors in mRNA over-expressed by over-expressing cells. The results showed that Zeb1 was positively correlated with the expression pattern of RP11. And in the Zeb1 overexpressing cell line, it was confirmed that the expression levels of the FN and Vim markers were affected. Therefore, the downstream of RP11 may be Zeb1.
In order to verify the previous conjecture, the authors confirmed that Zeb1 and RP11 are not directly combined at the mRNA and protein levels by Zeb1 RIP-LncRNA PCR (the cloud sequence can provide this service) and RP11 Pull Down-mass spectrometry (the cloud sequence can provide this service) . Relationship.
In order to confirm the above conjecture, the authors examined the expression levels of multiple EMT-related factors in mRNA over-expressed by over-expressing cells. The results showed that Zeb1 was positively correlated with the expression pattern of RP11. And in the Zeb1 overexpressing cell line, it was confirmed that the expression levels of the FN and Vim markers were affected. Therefore, the downstream of RP11 may be Zeb1.
In order to verify the previous conjecture, the authors confirmed that Zeb1 and RP11 are not directly combined at the mRNA and protein levels by Zeb1 RIP-LncRNA PCR (the cloud sequence can provide this service) and RP11 Pull Down-mass spectrometry (the cloud sequence can provide this service) . Relationship.
Figure 3. Zeb1 may be an indirect downstream of RP11
4.RP11-hnRNPA2B1-mRNA complex participates in RP11 regulation of Siah1 and Fbxo45
Similarly, the authors speculate that the downstream proteins may be Siah1 and Fbxo45. At the mRNA level, both of them regulated the expression of RP11 and Zeb1 by RP11 RIP-mRNA PCR . At the protein level, it was confirmed by RP11 Pull Down-mass spectrometry that the three were not directly bound.
Similarly, the authors speculate that the downstream proteins may be Siah1 and Fbxo45. At the mRNA level, both of them regulated the expression of RP11 and Zeb1 by RP11 RIP-mRNA PCR . At the protein level, it was confirmed by RP11 Pull Down-mass spectrometry that the three were not directly bound.
So what is the protein that connects the three?
To demonstrate how RP11 regulates downstream mRNAs Siah1 and Fbxo45, the authors found a hnRNPA2B1 protein that binds directly to RP11 in a previous RP11 pull down-mass spectrometry . hnRNPA2B1 RIP-mRNA PCR experiments confirmed that RP11, Siah1 and Fbxo45 are directly bound at the mRNA level. Studies have shown that hnRNP2B1 is an RNA-binding protein widely distributed in the nucleus and cytoplasm. The structural analysis of the LncRNA structure confirmed that it was able to bind to the CDS region of Siah1 and the 3'UTR region of Fbxo45. In general, RP11 regulates the expression of downstream mRNA Siah1 and Fbxo45 by forming the RP11-hnRNPA2B1-mRNA complex.
Figure 4. RP11 forms RP11-hnRNPA2B1-Siah1-Fbxo45 complex involved in cell migration
5. m6A modification is involved in the up-regulation of RP11 expression in CRC cells
First, the authors suspected that high expression of RP11 was associated with DNA methylation. After treatment of cells with DNA methylation inhibitory enzymes, the expression of RP11 was found to be unchanged, confirming that RP11 expression in CRC cells was not associated with DNA methylation. At the same time, experiments have also confirmed that it is not related to histone acetylation.
First, the authors suspected that high expression of RP11 was associated with DNA methylation. After treatment of cells with DNA methylation inhibitory enzymes, the expression of RP11 was found to be unchanged, confirming that RP11 expression in CRC cells was not associated with DNA methylation. At the same time, experiments have also confirmed that it is not related to histone acetylation.
What is the modification that regulates the expression of RP11?
Studies have shown that m6A modification regulates the expression and function of LncRNA. The authors confirmed the m6A RNA methylation modification on RP11 in three cell lines by MeRIP-qPCR technology (the cloud sequence provides this service) . Moreover, overexpression of methyltransferase METTL3 and demethyltransferase ALKBH5, respectively, can affect the expression of RP11. Next, the authors aimed to investigate how m6A modification regulates RP11 expression in CRC cells. First, the authors used Act-D to inhibit the transcription process, and found that the expression level of RP11 was not affected in the Mettl3 overexpression group. The reason may be that Mettl3 promoted the accumulation of RP11 in chromatin, which stabilized the expression of RP11. The m6A reader protein hnRNPA2B1 is involved in this process.
Figure 5. RP11 has m6A modification and is regulated by Mettl3 and AlKBH5
6. Relationship between m6A/RP11/Zeb1 and progression in CRC cells in vivo
Analysis of the expression of a large number of different stages of clinical samples showed that m6A modification first regulated RP11, and RP11 affected Siah1-Fbxo45/Zeb1. With the KM curve, the authors found that the survival of patients with high expression of RP11 rectal cancer is not optimistic. At the same time, the relationship between downstream genes and other clinical indicators of patients was also analyzed. Overall, it was confirmed that m6A/RP11/Zeb1 promoted the development of CRC.
Analysis of the expression of a large number of different stages of clinical samples showed that m6A modification first regulated RP11, and RP11 affected Siah1-Fbxo45/Zeb1. With the KM curve, the authors found that the survival of patients with high expression of RP11 rectal cancer is not optimistic. At the same time, the relationship between downstream genes and other clinical indicators of patients was also analyzed. Overall, it was confirmed that m6A/RP11/Zeb1 promoted the development of CRC.
to sum up
In the present study, the authors first screened high-expression LncRNA-RP11 in the CRC disease group by LncRNA high-throughput sequencing and confirmed that it can promote tumor migration.
Subsequently, the authors aimed to investigate what is the downstream binding protein of RP11 and how it regulates the migration phenotype.
On the one hand: with LncRNA Pull Down , the protein that binds rapidly to RP11 is the m6A reader hnRNPA2B. By RIP-PCR , the RNA that quickly finds binding to this protein is RP11. On the other hand, by MeRIP-qPCR , it was confirmed that m6A modification was present on RP11 in the three disease-related cell lines. At the same time, Mettl3 promotes RP11 stability as its upstream, thereby promoting the development of CRC.
In the present study, the authors first screened high-expression LncRNA-RP11 in the CRC disease group by LncRNA high-throughput sequencing and confirmed that it can promote tumor migration.
Subsequently, the authors aimed to investigate what is the downstream binding protein of RP11 and how it regulates the migration phenotype.
On the one hand: with LncRNA Pull Down , the protein that binds rapidly to RP11 is the m6A reader hnRNPA2B. By RIP-PCR , the RNA that quickly finds binding to this protein is RP11. On the other hand, by MeRIP-qPCR , it was confirmed that m6A modification was present on RP11 in the three disease-related cell lines. At the same time, Mettl3 promotes RP11 stability as its upstream, thereby promoting the development of CRC.
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Cloud Biological Sequence provides a detection level of the overall methylation modification m6A colorimetric method, RNA methylation sequencing, MeRIP-qPCR verification, RIP, and RNA Pull Down Mechanism service. RNA methylation sequencing technology is the real implementation of m6A, m5C and m1A modification, detection of molecules in addition to mRNA, but also detection of circular RNA, LncRNA and other non-coding RNA. Since 2016, the number of samples has accumulated more than 5000+, and the integrated power of MeRIP has reached more than 98%.
Now, in order to solve the problem of small sample size of customers, ultra-micro-MeRIP sequencing technology is introduced, and 500 ng of total RNA can be used for sequencing experiments. Special samples can also be sequenced for RNA methylation, such as serum, plasma, exosomes, and paraffin samples.
Cloud Biological Sequence provides a detection level of the overall methylation modification m6A colorimetric method, RNA methylation sequencing, MeRIP-qPCR verification, RIP, and RNA Pull Down Mechanism service. RNA methylation sequencing technology is the real implementation of m6A, m5C and m1A modification, detection of molecules in addition to mRNA, but also detection of circular RNA, LncRNA and other non-coding RNA. Since 2016, the number of samples has accumulated more than 5000+, and the integrated power of MeRIP has reached more than 98%.
Now, in order to solve the problem of small sample size of customers, ultra-micro-MeRIP sequencing technology is introduced, and 500 ng of total RNA can be used for sequencing experiments. Special samples can also be sequenced for RNA methylation, such as serum, plasma, exosomes, and paraffin samples.
Full text link:
Https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-019-1014-2
Https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-019-1014-2
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