Cell Du Peng's group reveals plant immune proteins by rescuing miRNA deficiency.

Cancer, as a disease of abnormal cell proliferation, is a major global public health problem

Excessive activation of the cell cycle is necessary for abnormal proliferation of cancer cells, so cell cycle genes are an important target for

Unlike miRNAs, siRNAs are derived from double-stranded RNA substrates

On May 26, 2022, The Du Peng Research Group of Peking University and the Center for Life published a research paper

In this study, the authors mainly made the following findings:

1.

To perform plant RDR1-based bioengineering in mammalian cells, the authors cloneDR1 genes from Arabidopsis thaliana (At) and rice (Os) onto Dox-induced lentiviral vectors

In the ensuing study, to the authors' surprise and surprise, AtRDR1 and OsRDR1 were able to significantly inhibit proliferation in all 10 cancer cell lines in vitro, but had no effect on the other 5 non-cancer cells (Figure 1

Figure 1 AtRDR1 and OsRDR1 broad-spectrum and specific inhibit cancer cell proliferation without affecting non-cancer cell lines

Figure 2 RDR1 can specifically target and interfere with the cell cycle of cancer cells, but does not work on non-cancer cells

2.

Since RDR1 is involved in small RNA pathways in plants, the authors performed small RNA sequencing

To further investigate the mechanism by which RDR1 specifically targets cancer cells, the authors systematically analyzed published data on the sequencing of small RNA, including data from 9980 groups of cancer patients in the TCGGA database and data from cancer patients, cancer cell lines, and normal tissues

Figure 3 Abnormal 3' terminal short 1-nt miRNA isomers accumulate extensively in different human tumors

3.

Plant RDR6 has been reported to have both RNA polymerase and nucleotide transferase activity[7], while in subsequent small RNA sequencing data analysis, the authors found that mature miRNAs with 3'-terminal single nucleotide tailing were specifically enriched in
cancer cells with RDR1 ectopic expression.
Interestingly, this single nucleotide tailing occurs mainly on miRNAs with a short 1-nt end of 3' but rarely on the cleavage ends of the
annotation.
The authors subsequently purified the recombinant rAtRDR1 and 3DA mutant rAtRDR1 mutant rAtRDR1
mutant in Escherichia coli.
Through in vitro biochemical experiments, the authors directly demonstrated that rAtRDR1 is capable of performing 3'-terminal single nucleotide modifications on single-stranded miRNAs and miRNA double strands with 1-nt or 2-nt overhang, but not miRNA double strands
with flat ends.

Combining the above findings, the authors hypothesize that RDR1 tends to recognize as substrates the free miRNA double strands with 1-nt overhang in AGO2, which accumulate particularly in cancer cells but not in non-cancer cells, and modify these problematic miRNAs
with single nucleotide tailing.
To verify this, the authors first performed an AGO2 knockdown experiment in lung cancer A549 cells expressing RDR1, followed by small RNA sequence analysis
.
As expected, AGO2 deletion did further enhance RDR1-mediated miRNA modification, with a significantly higher proportion of single nucleotide tailing on miRNA isomers with a short 1-nt in cancer cells (Figure 4
).
After that, rAGO2 was pre-incubated with miRNA double-stranded with 1-nt/2-nt overhang in vitro and then rRDR1 was added to the tailing experiment of free miRNA double stranding, and the results showed that miRNA double strands with lower affinity with AGO2 1-nt overhang were more likely to be modified by RDR1 after pre-incubation with rAGO2 protein (Figure 5
).

Finally, by experiments and analyses such as reconjugation of 1-nt suspended miRNA double strands and in vitro transfection of 1-nt/2-nt exogenous miR-34c double strands, the authors also demonstrated that RDR1 has nucleotide transferase activity and can perform single nucleotide modification of short 1-nt miRNA double-stranded isomers free of AGO2 to restore the loading efficiency of these isomers to AGO2, and ultimately repair defective miRNA pathways in cancer
。

Figure 4 AGO2 deletion significantly enhances RDR1-mediated miRNA tailing events in cancer cells

Figure 5 RDR1 is more inclined to identify free miRNA double-stranded isomers with 1-nt overhang as substrates

4.
RDR1 inhibits the progression of a variety of mouse solid tumors and leukemia

Next, the authors wanted to validate the antitumor effects
of the plant RDR1 in mice.
First, the authors injected the RDR1-induced cancer cell line into immunodeficient mice for in vivo tumor-bearing experiments and achieved ectopic expression
of RDR1 by Dox feeding water.
The results showed that wild-type rather than mutant RDR1 significantly inhibited the size, volume, and weight
of the tumors.
The authors also found that in tumors formed by A549, H1299 and PC-3 cells, plant RDR1 was able to significantly improve the expression of miRNAs in cancer cells, thereby inhibiting the cell cycle
of cancer cells.

Similarly, the authors also evaluated the antitumor effects
of RDR1 in mouse models of leukemia in vivo.
Compared with vector-controlled or mutated RDR1, wild-type RDR1 was able to significantly inhibit the proliferation of three leukemia cell lines (Jurkat, K562, and NALM6) in the peripheral blood of immunodeficient mice, and ultimately extend the lifespan
of xenografted mice.
Similarly, RDR1 can significantly increase miRNA expression in leukemia cells in vivo to inhibit key cell cycle components in leukemia cells, including CDK1, CCNE2, PLK1, CDK6, etc.
, thereby inhibiting the cell cycle
.

Finally, direct delivery and tumor suppression of RDR1 at the in vitro cell and solid tumor levels were achieved by nano-vesicle-wrapped purified RDR1 protein and AAV-wrapped RDR1 at the in vitro cell and in vivo solid tumor levels, respectively (Figure 6
).

Figure 6 AAV-delivered RDR1 inhibits solid tumor growth in mice

Taken together, this study reveals for the first time the widespread accumulation of abnormal 3' terminal short 1-nt miRNA isomers in a variety of human primary tumors, which provides new insights
into the global miRNA dose reduction during tumorigenesis.
Using the plant immune protein RDR1, we achieved a broad-spectrum anti-tumor response by rescuing miRNA defects in cancer cells and developed a new strategy to edit and manipulate miRNAs as a powerful weapon
against human diseases such as cancer.

The pattern plot represents the broad-spectrum anti-tumor response of plant RDR1 by salvaging microRNA defects in cancer

Peng Du, a researcher at the School of Life Sciences/Joint Center for Life Sciences at Peking University, is the corresponding author
of the paper.
Qi Ye, a doctoral student at the Institute of Frontier Interdisciplinarity at Peking University, and Ding Li, a doctoral candidate at the School of Life Sciences, are the tie-in first authors
of this paper.
Zhang Siwen, a doctoral student at the Institute of Frontier Interdisciplinarity at Peking University, participated in part of the work, and Yao Shengze (who graduated) and Weng Jianli, a postdoctoral fellow at Peking University, also made important contributions
to this paper.
Peking University Yi Li and Professor Wu Hong cooperated to complete this work and gave strong support
.
The project was funded
by the National Natural Science Foundation of China, the Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education of Peking University, the Joint Center for Life Sciences, and the Peking University-Qidong Innovation Fund.

Link to the original article: https://doi.
org/10.
1016/j.
cell.
2022.
04.
030

Postdoctoral Recruitment:

Relying on the School of Life Sciences and the Joint Center for Life Sciences of Peking University, The Du Peng Laboratory of Peking University is engaged in research
on RNA regulation, stem cells and tumor biology.
His research focuses on the analysis and identification of unknown RNA regulatory pathways, and the study of the function
of related RNA regulatory pathways in the differentiation fate of embryonic stem cells and early embryonic development.
At the same time, we also try to recombinant RNA regulatory pathways specific to plants or microorganisms in animal cells and investigate their potential applications
in translational medicine.
Now we are recruiting 2-3 postdoctoral fellows
.
For more information, see the link:  

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