Cloud order customer published RIP & RNA pull down article : Full text link: https://doi.org/10.1172/JCI124508 Cloud order related product recommendation: RIP sequencing RNA pull down Whole transcriptome sequencing miRNA sequencing miRNA quantitative PCR
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Article introduction
Vascular smooth muscle cells (VSMC) are in a dedifferentiated phenotype under healthy conditions. When they are damaged, platelets in the circulatory system are rapidly activated, and hemostasis is stopped by the coagulation reaction, and the biological activities such as PDGF and thromboxane are released to the wound site. The medium switches the VSMC to a highly differentiated phenotype while the cells are in a proliferative state. Intimal hyperplasia type diabetes is caused by dysregulation of VSMC differentiation and dedifferentiation state switching mechanisms. However, the molecular mechanisms of such diseases are still unclear. 
Impact factor: 13.3
Experimental method: AGO2 RIP experiment , miRNA sequencing
Experimental materials: VSMC and platelet co-culture group (experimental group), VSMC untreated group (blank group); model mice, etc. 
miRNA quantitative PCR showed that the three were highly expressed in co-cultured cells. 
Figure 3. AGO2 RIP-miRNA quantitative PCR confirms that the above three miRNAs are involved in the sponge mechanism 
Figure 5. Fluorescence quantification confirms a negative correlation between miRNA and target gene expression 
Figure 5. Fluorescence quantification confirms that miRNAs are underexpressed in the diabetic group, both human and murine; inversely proportional to the expression of the target gene;
Low expression of miRNA promotes intimal hyperplasia in diabetic patients 
Figure 6. Platelets in normal and diabetic groups participate in VSMC function by transporting miRNAs
The author of this article first co-cultured platelets with VSMC and found that the cell differentiation is contrary to common sense. It is thus speculated that miRNAs in platelets are at work. The basis for the inference is: 1. It was observed that platelets were internalized by VSMC. 2. Previous studies have found that platelets contain a large number of miRNAs. To confirm the speculation results, the authors quickly identified miR-223, which may function by miRNA high-throughput sequencing technology, and confirmed that miR-223 participates in the sponge mechanism through AGO2 RIP experiments. The target gene PDGFRβ was identified by biomarker prediction, validation and luciferase experiments. Finally, in the model of VSMC injury repair and the diabetes model, the authors found the opposite mechanism, which has brought great help to future clinical research. Cloud order advantage summary
Summary of advantages:
It is worth mentioning that the cloud sequence biology not only solves the sequencing problem for the majority of scientific research users, but also provides a mechanism solution. So far, the RIP and RNA pull down experiments have been completed to reach 100+ cases, helping the majority of researchers to sprint high-scoring articles.
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[JCI 13.3] Reveals the involvement of diabetic platelet-derived miRNAs in the mechanism of sponge-mediated arterial injury repair
Cloud order customer 13 points top story, revealing that diabetic platelet-derived miRNA participates in sponge mechanism to mediate arterial injury repair
Yunxu Bio and the research team of Guangzhou Women and Children Medical Center first revealed that miR-223 in platelets regulates the process of wound repair by regulating target gene expression, and established a molecular mechanism model of diabetes, which provides a reference for future cell damage repair process.
Published journal: Journal of Clinical Investigation
research content
1. Activated platelets promote differentiation and proliferation of human VSMC <br> First, the authors of this article used the cells co-cultured with human VSMC and activated platelets as the experimental group (AP), and the untreated cells were used as the blank control group (RP) for qPCR. Compared with WB assay, the results showed that the marker associated with differentiation in the co-culture group was significantly expressed, and the cell proliferation ability was weakened, which is contrary to common sense. Next, the authors observed that VSMC secreted platelets from both in vitro and in vivo perspectives by confocal microscopy. So the question is, will platelets carry other molecules dynamically involved in the differentiation process?
Figure 1. WB results of differentiation-related proteins; confocal microscopy observation of VSMC capable of autonomously secreting platelets
2. Platelets carry miRNAs into VSMC and participate in the differentiation process <br> With the previous problems, the authors have reviewed the literature and learned that predecessors have reported that platelets are rich in miRNAs. Is that because miRNAs in platelets are involved in cell differentiation? The next experiment became simpler, using high-throughput miRNA sequencing ( cloud sequencing bio-miRNA sequencing services ) to compare differentially expressed miRNAs in co-cultured and untreated cells. Three miRNAs significantly associated with the study were detected by sequencing: miR-223 and miR-143/145, respectively. miRNA quantitative PCR confirmed that all three were highly expressed in co-cultured cells, in which miR-223 was highly expressed at 8 h of co-culture, and miR-143/145 was highly expressed at 24 h of co-culture. Then I know the expression patterns of the three on the timeline. What kind of mechanism do they play? Therefore, the authors used the AGO2 RIP experiment (the cloud sequence organism provided this service ) to pull the miRNA directly bound to the AGO2 protein in the experimental group, and confirmed that the above three miRNAs can directly bind to the AGO2 protein and participate in the sponge by miRNA quantitative PCR. Mechanism, and also confirmed that miRNA secreted by platelets is indeed active.
Figure 2. miRNA high-throughput sequencing clustering;
3. The target gene of miR-223 is PDGFRβ
The target genes with the strongest binding to miRNA were predicted by biosignal analysis: KLF4, KLF5 and PDGFRβ. Of course, all of the above speculations, the author confirmed by luciferase experiments that the two are directly related. Next, the authors mainly studied the relationship between miR-223 and PDGFRβ. After quantitative PCR of the target gene PDGFRβ, it was found to be highly expressed in the injured femoral artery, demonstrating that the wound can change its expression level. Moreover, it is inversely proportional to the amount of expression of miR-223. In summary, it is indicated that PDGFRβ is indeed a miR-223 target gene.
Figure 4. Biosignal analysis predicts binding of miRNA to target gene (3'UTR region)
4.miR-223 is low in diabetic mice and promotes intimal hyperplasia
The VSMC differentiation disorder mainly causes people to develop vascular proliferative diabetes, so the author hopes to study the mechanism of miR-223 in diabetes by molecular means. miRNA quantitative PCR results showed that miR-223 is a low expression pattern in both human and mouse diabetic groups. How is the location of miR-223 in the cell? By fluorescence in situ hybridization, the authors found that platelets in diabetic patients were also internalized into VSMC. Therefore, it is speculated that after internalization of platelets to cells, it causes low expression of miR-223 in platelets, thereby promoting high expression of miRNA target genes in vivo, and ultimately leading to phenotype of vascular smooth muscle cell proliferation and intimal hyperplasia.
5. Construction of VSMC injury repair model <br> The authors first proposed the VSMC injury repair mechanism model. The participating members of the mechanism mainly include VSMC, endothelial tissue and platelets, as shown in the following figure. In uninjured individuals, endothelial tissue is intact and there is no interaction between VSMC and platelets. When damaged, the endothelial barrier is impaired, platelet activation, on the one hand release PDGF, TXA2 and other substances, on the other hand carrying miRNA into VSMC, improve the expression of target genes, thereby promoting cell differentiation and inhibiting intimal hyperplasia. However, when the diabetic patient is injured, the platelet is consistent with the former in releasing the prosthetic substance, but the molecular mechanism is completely opposite to the ordinary damage mechanism.
to sum up:
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