MicroRNA-34a mediates the autocrine signaling of PAR2-activating proteinase and its role in colonic cancer cell proliferation.

The tumor microenvironment is replete with proteinases. As a sensor of proteinases, proteinase activated receptor 2 (PAR2) plays critical roles in tumorigenesis. We showed that PAR2 and its activating proteinase were coexpressed in different colon cancer cell lines, including HT29. Inactivating proteinase or knockdown of PAR2 significantly not only reduced cell proliferation in vitro but also inhibited tumorigenicity of HT29 in vivo. In addition, activation of PAR2 promoted DNA synthesis and upregulated Cyclin D1 activity at both transcriptional and post-transcriptional levels. Further studies showed that miRNA-34a mediated PAR2-induced Cyclin D1 upregulation. Inhibition of miR-34a partially abolished the suppression of Cyclin D1 induced by PAR2 deficiency. In addition, we showed that TGF-β contributed to the regulation of miR-34a by PAR2. Finally, in colorectal carcinoma samples, upregulation of PAR2 and downregulation of miR-34a were significantly correlated with grade and lymphomatic metastasis. Our findings provide the first evidence that miRNA mediates autocrine proteinase signaling-mediated cancer cell proliferation

PAR₂ regulates Cyclin D1 expression at transcriptional and post-transcriptional levels.

<p>A549 cells were pretreated with actinomycin D (2 µg/ml) 30 min before challenge with PAR₂-AP (50 µM) for 24 h. (A) mRNA and (B) protein levels of Cyclin D1 were detected by real time PCR and Western Blot. siRNA targeting beta-catenin was transfected into A549 cells 24 h before treatment with PAR₂-AP. (C) mRNA and (D) protein levels of Cyclin D1 were detected by real time PCR and Western Blot, respectively.</p

Knockdown of PAR₂ blocked cell proliferation in HT29 cells.

<p>A. mRNA expression of PAR₂, trypsinogen, KLK14 was measured with regular PCR in colon cancer cell lines. B. Colonic cancer cells were pretreated with the inhibitors of trypsin (T9128 and T6522) or serine proteinases (proteinase inhibitor cocktail, PIC) and cell proliferation was measured by MTT assay. C. The mRNA expression of PAR₂ in stable transfectant cells (PAR₂-1 and PAR₂-2) was measured by real time PCR. HT29 RS represents the control cells stably transfected with scrambled control RNA. Protein level of PAR₂ was measured by Western Blot and shown as insert. The effect of PAR₂ knockdown on cell proliferation was measured by (D) MTT assay and (E) colony formation in HT29 cells. F. Cells (10⁶) were injected subcutaneously into nude mice, tumor volumes were measured as indicated and tumor weights were determined at sacrifice. *<i>p</i><0.05, **<i>p</i><0.01 All data are shown as mean±SEM. N = 3–6. All experiments have been repeated at least 3 times independently.</p

TGF-β mediated PAR₂-related miRNA-34a expression.

<p>(A) HT-29-RS or HT-29-PAR-2 cells were treated with conditional medium from HT-29-RS cells (CM-RS) for 3 days. (B) HT-29-PAR-2 cells were treated with TGF-β (5 ng/ml) for 12 hours. (C) A549 cells were treated with TGF-β (5 ng/ml) for 6 hours. (D) Conditional medium from HT-29-RS (CM-RS) was incubated with anti-TGF β antibody and protein A/G agarose at 4°C for 2 hours. After centrifuge, the supernatant (CM-RS-anti-TGF β) was used to treat HT-29-PAR-2 cell for 3 days. After the different treatments as mentioned above, the cells were collected for the assay of miR-34a with real time PCR. *<i>p</i><0.05, **<i>p</i><0.01 All data are shown as mean±SEM. N = 4–8.</p

The overall pathway by which the activation of PAR₂ regulates cell proliferation.

<p>The overall pathway by which the activation of PAR₂ regulates cell proliferation.</p

Activation of PAR₂ increases cell proliferation through Cyclin D1-E2F pathway.

<p>A549 cells were treated with PAR₂ activating peptide (PAR₂-AP, 50 µM) or reverse peptide (PAR₂-RP) for 24 h. A. DNA synthesis was measured by BrdU labeling assay.B. Levels of Cyclin D1 and Cyclin E mRNA were detected with PCR after treatment with or without PAR₂-AP. C. Protein level of Cyclin D1 was determined by Western blot. D. A549 cells were treated with PAR₂-AP (50 µM) 24 h after transient transfection with E2F-luciferase (1 µg/well). The data were normalized with tk-RL and shown as the percentage of the control group. E. HT29 cells were treated with PIC (proteinase inhibitor cocktail) or T9128 (trypsin inhibitor). DNA synthesis was measured by BrdU labeling assay. F. Protein (left) and mRNA (right) levels of Cyclin D1 in PAR₂ knockout cells were measured. *<i>p</i><0.05, ***<i>p</i><0.001 All data are shown as mean±SEM. N = 3–6. All experiments have been repeated at least 3 times independently.</p

Wheat genomic study for genetic improvement of traits in China

Bread wheat (Triticum aestivum L.) is a major crop that feeds 40% of the world’s population. Over the past several decades, advances in genomics have led to tremendous achievements in understanding the origin and domestication of wheat, and the genetic basis of agronomically important traits, which promote the breeding of elite varieties. In this review, we focus on progress that has been made in genomic research and genetic improvement of traits such as grain yield, end-use traits, flowering regulation, nutrient use efficiency, and biotic and abiotic stress responses, and various breeding strategies that contributed mainly by Chinese scientists. Functional genomic research in wheat is entering a new era with the availability of multiple reference wheat genome assemblies and the development of cutting-edge technologies such as precise genome editing tools, high-throughput phenotyping platforms, sequencing-based cloning strategies, high-efficiency genetic transformation systems, and speed-breeding facilities. These insights will further extend our understanding of the molecular mechanisms and regulatory networks underlying agronomic traits and facilitate the breeding process, ultimately contributing to more sustainable agriculture in China and throughout the world