Molecular Cancer : Hepatocellular carcinoma continues to make progress! Cai Xiujun/Liang Wei reveals a new mechanism for cyclic RNA-mediated sorafyni resistance

Hepatocellular carcinoma is the most common primary liver cancer, and as a large country of liver cancer, more than 50% of new liver cancer occurs in China.
is the first FDA-approved drug for targeted treatment of advanced hepatocellular carcinoma.
However, due to the complexity and heterogeneity of the development mechanism of liver cancer, after 6 months of treatment with soraphinium, most patients develop resistance to soraphinium, which makes the disease worse and makes treatment extremely difficult.
November 23, 2020, Cai Xiujun and Liang Wei of Yifu Hospital, affiliated with Zhejiang University School of Medicine, published an online newsletter entitled "N6-methynosine-modified circRNA-SORERERES in he Patocellular carcinoma by regulatoring β-catenin signaling" research paper, which describes the mechanism of circRNA-SORE (a circular RNA upregulated in sorafenib-resistant HCC cells) to mediate sorafinib-resistant drug resistance, and found the mechanism of oscular genetics that regulates the expression level of circRNA-SORE.
In summary, this study shows a new mechanism for maintaining sorafinist resistance and is a concept validation study targeting circRNA-SORE in HCC patients treated with sorafinie as a new drug intervention for late-stage HCC.
addition, on November 13, 2020, Zhejiang University Wang Xiaojian and Cai Xiujun published a research paper entitled "IRF3 prevents coloration tumorigenesis via resing the nuclear translocation of β-catenin" online, which found that IRF3 defective mice were highly sensitive to the development of intestinal tumors in AOM/DSS and Apcmin/ plus models.
study found that cytostic IRF3 in a stationary state inhibits the occurrence of colorectal tumors by blocking the transmission of the Wnt/β-catenin signal.
Therefore, the study identified IRF3 as a negative regulator of the Wnt/β-catenin pathway and a potential prognostic marker for the occurrence of Wnt-related tumors, and described the link between the gut microbiome and CRC via the IRF3-β-catenin axis (click to read).
study data show that circRNA-SORE can act as ceRNA (competitive endoen endo-RNA) to induce sorafonine resistance by adsorbing miR-103a-2-5p and β miR-660-3p competitively.
more importantly, the team found that elevated levels of Adenosine-specific Adenosine (m6A) in circRNA-SORE helped maintain the stability of their RNA, which in turn increased the level of circRNA-SORE and promoted drug resistance.
This study is the first time in the world that m6A modification of specific locations on circRNA can affect its expression level through RNA stability, is an innovative exploration of the molecular mechanism of the ostogenesis of liver cancer, expounds the new mechanism of drug resistance in targeted treatment of liver cancer, and provides new clues for the search for new therapeutic targets for patients with advanced liver cancer.
1. The mechanism showed that the team first established the sorafinist liver cancer cell line in an earlier study and found circRNA-SORE, which plays an important role in the formation of drug resistance.
consistent with expectations, circRNA-SORE showed a significant increase in expression in both Solafinib-resistant cells and animal tumors and was critical to maintaining Sorafinib resistance.
in drug-resistant cells, silent circRNA-SORE resulted in a significant decrease in cell proliferation, a significant increase in apoptosis, and a significant decrease in cloning capacity.
team believes that silent circRNA-SORE improves the efficacy of sorafinist by inducing apoptosis of tumor-resistant cells.
Figure 2. CircRNA-SORE significantly increases in soraphine-resistant hepatocellular liver cancer, and plays an important role in the maintenance of drug resistance CircRNA the most common mechanism of action is as ceRNA, adsorption of a variety of miRNAs, resulting in the original combination of mRNA mRNA translation can not be blocked by miRNA, forming the competitive activation of mRNA, resulting in an increase in downstream protein expression levels and produce corresponding physiological pathological manifestations.
is the circRNA-SORE also miRNA sponge characteristic? Figure 3. circRNA-SORE as ceRNA adsorption miR-103a-2-5p and miR-660-3p, competitive activation of wnt/β-catenin pathway induces solafneni resistance first, the research team through target-based Scan andamp; MiRanda's miRNA, in combination with prediction software, obtained 12 miRNAs that predicted the binding of circRNA-SORE, and laboratory analysis of RNA-pulldown for circRNA-SORE, and found that 8 miRNAs were significantly rich in circRNA-SORE, proving that circRNA-SORE could indeed play the role of ceRNA.
further functional experiments showed that the sensitivity of drug-resistant cells to sorafinie increased significantly after the expression of miR-103a-2-5p and miR-660-3p.
to explore the specific mechanisms by which circRNA-SORE regulates miR-103a-2-5p and miR-660-3p, the team first used fluorescent in-place hybridization techniques to find that circRNA-SORE and miR-2-5p and miR-660-3p have co-location in cells that conform to the characteristics of the combination of the two.
RNA immunoassecipitation shows further indications that miR-103a-2-5p and miR-660-3p can be combined with circRNA-SORE.
combination of circRNA-SORE and Ago2 is easier to detect in drug-resistant strains, further suggesting the properties of circRNA-SORE as a ceRNA function.
reporting test of double luciferase for mutations at specific bits on circRNA-SORE further suggests the binding of circRNA-SORE with miR-103a-2-5p and miR-660-3p.
Figure 4. How does circRNA-SORE's relationship with m6A, and the role of circRNA-SORE in animal models, in combination with miR-103a-2-5p and miR-660-3p, regulate the esotypes of sorafinist resistance? In liver cancer, the wnt/β-catenin path line plays a key role, and the team found that the protein levels of β-catenin in drug-resistant cells were significantly higher than in wild cells, and that their expression was positively related to the expression levels of circRNA-SORE.
further experiments have shown that the protein levels of β-catenin and its upstream wnt2b in drug-resistant cells are affected by circRNA-SORE and miR-103a-2-5p and miR-The regulation of 660-3p, and the corresponding esolysed experimental results are consistent with the direction of regulation, indicating that the downstream of these two miRNAs regulated by circRNA-SORE is likely to be the wnt/β-catenin signal path.
based on this hypothesis, the team predicted the binding point between mRNA of wnt2b and miR-103a-2-5p and miR-660-3p, and conducted a double fluorinase reporting experiment before and after the bit mutation.
results show that miR-103a-2-5p and miR-660-3p can be combined with the mRNA 3'UTR region of wnt2b, promoting the degradation of mRNA, thereby suppressing the wnt2b/β-catenin signal path.
to this point, the team demonstrated that circRNA-SORE isolated miR-103a-2-5p and miR-660-3p in the role of ceRNA, competitively activating the wnt/β-catenin signaling path, inducing and maintaining resistance to sorafinib from liver cancer.
in the study of non-coding RNA, the cause of abnormal RNA expression has always been one of the problems that has plagued many researchers.
m6A is one of the most abundant RNA modifications, and researchers continue to find that RNA m6A modification plays an important role in tumor progression, while in recent years researchers have found that circRNA m6A modification (often in RRm6ACH molds, R? =? G or A; H? =? A, C or U) regulates its functionality.
is there an m6A modification on the circRNA-SORE? What role does m6A retouch play in it? With cutting-edge questions, the team first predicted using the SRAMP and RMBase v2.0 databases that there was a potentially m6A-modified site near the action site of the circRNA-SORE sequence.
RNA Pulldown test showed that circRNA-SORE could be combined with m6A-related enzyme proteins such as YTHDF1, YTHDF2, METTL3, and FFO, suggesting that circRNA-SORE may be regulated by m6A.
methylated RNA immunoprecipitation (meRIP) experiments have shown that the level of m6A modification of crcRNA-SORE in drug-resistant cells is higher than in wild cells.
team went on to silence the m6A modification-related proteins and found that the m6A modification level of circRNA-SORE decreased significantly after knocking down METTL3 and METTL14, while the modification level increased significantly after knocking down FFO, suggesting the presence of m6A modification points on circRNA-SORE.
series of experiments show that circRNA-SORE is not only modified by m6A, but also its expression level is regulated by m6A modification level.
, however, it is still uncertain whether this regulatory role is at the overall m6A level or at the m6A level at a particular location. To answer this question, the team further explored and designed a set of sequence specificity for m6A bits in circRNA-SORE.
, the m6A level decreased significantly in HepG2-SR cells treated with MAO-targeted circRNA-SORE (MAO-SORE) compared to cells treated with control MAO (MAO-NC).
even more exciting, the team used the latest CRISPR-Cas9-ALKBH5 complex to target the m6A site for de-methylation, further confirming the presence of the predicted m6A site on circRNA-SORE, and the m6A modification level of circRNA-SORE decreased significantly after the m6A site mutation.
studies have shown that m6A modification involves all aspects of RNA metabolism after transcription, including mRNA positioning, shearing, translation and degradation, which in turn regulate important biological processes.
team found that silent adenosine methyl transferase METTL3 / 14 lowers the level of circRNA-SORE.
it is important to note that MAO-SORE can reduce the level of circRNA-SORE in drug-resistant cells, but not in wild cells, further suggesting that m6A modification may promote the upward of circRNA-SORE in drug-resistant cells.
interestingly, RNA stability tests using linen D have shown that MAO-SORE reduces the stability of circRNA-SORE in drug-resistant cells, while wild circRNA-SORE is more stable than circRNA-SORE with m6A bit mutations.
these results suggest that m6A modification may regulate its expression by increasing the stability of circRNA-SORE.
finally, the team used the built CDX mouse liver cancer tumor model and its corresponding soraphine resistance model to confirm at in vivo levels that circRNA-SORE can induce soraphine resistance through the wnt/β-catenin pathway.
, the team found that circRNA-SORE played an important role in the formation of sorafneid resistance.
study is well designed and novel in thinking, which provides a new way of thinking for the clinical diagnosis and treatment of hepatocellular liver cancer.
Xiujun and Liang Wei are the correspondents of the paper, and Dr. Xu Junjie is the first author of the paper.
the study was supported by major national scientific research projects, the National Natural Science Foundation of China, the Zhejiang Provincial Key Research and Development Program, the China Postdoctoral Science Foundation Project, and the Zhejiang Natural Science Foundation.
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