G-quadruplex formation within the promoter region of HSPB2 and its effect on transcription.

G-rich sequences in DNA and RNA tend to fold into stable secondary structures called G-quadruplexes. Except for the telomere region, G-quadruplex-forming sequences are widely present in gene promoters and have been implicated in transcriptional regulation. Single nucleotide polymorphisms (SNPs) can disrupt the G-quadruplex structure of a gene promoter. In this study, we confirmed the promoter of HSPB2, a cancer-related gene, tends to form an unusual DNA secondary structure. The dual luciferase assay revealed that the SNP rs2234704 in the HSPB2 promoter with a single G > A mutation increased the transcriptional activity of the HSPB2 promoter. Circular dichroism and native PAGE revealed that the G-rich strand of the DNA in this promoter preferred to form a parallel G-quadruplex, which could be destabilized by the SNP rs2234704 (G > A) mutation. Furthermore, we found that the SNP rs2234704 (G > A) greatly increased and influenced the overexpression of HSPB2 in breast cancer samples. These results suggest SNP rs2234704 (G > A) may play a role in the occurrence of breast cancer by destroying the G-quadruplex structure and promoting the expression of HSPB2.

A novel copper chelator for the suppression of colorectal cancer.

Copper ions play a crucial role in the progression of cancers. Tumor tissue is rich in copper ions, and copper chelators could potentially scavenge these copper ions and thus exert an antitumor effect. In this study, we report the synthesis of a novel thieno[3,2-c]pyridine compound we have called "JYFY-001" that can act as the copper chelator thanks to the inclusion of an N-(pyridin-2-yl)acetamide moiety that targets copper ions. JYFY-001 potently inhibited cancer proliferation, inducing cell apoptosis and impairing the extracellular acidification rate and oxygen consumption rate of colorectal cancer (CRC) cells. JYFY-001 also inhibited the growth of a CRC-transplanted tumor in a dose-dependent manner, inducing apoptosis of the tumor cells and promoting the infiltration of lymphocytes in the CRC-transplanted tumor tissues. JYFY-001 also enhanced the antitumor effects of the programmed cell death protein 1 (PD-1) inhibitor. The relatively benign nature of JYFY-001 was demonstrated by the effect on normal cell viability and acute toxicity tests in mice. Our findings suggest that JYFY-001 is a prospective copper chelator to be used as a targeted drug and a synergist of immunotherapy for CRC treatments.

Tumor cell-secreted exosomal miR-22-3p inhibits transgelin and induces vascular abnormalization to promote tumor budding.

Tumor budding (TB) is considered a histomorphological marker of poor prognosis in patients with breast cancer (BC). Tumor vasculature is disordered and unstable in BC, which causes restricted drug delivery, hypoxia, and tumor metastasis. Traditional anti-angiogenic treatments cause extreme hypoxia, increased invasion, metastasis, and drug resistance due to blood vessel rarefaction or regression. Therefore, the application of anti-angiogenic strategies for vascular normalization in tumors is crucial to improve therapeutic efficacy in BC. In the present study, we found that transgelin (TAGLN) promoted the normalization of tumor vessels by regulating the structure and function of endothelial cells, and knockout of TAGLN in tumor-bearing mice resulted in tumor vessel abnormalization, an increase in epithelial-mesenchymal transition characteristics of tumor cells, and promotion of TB. Moreover, BC cells secrete exosomal miR-22-3p that mediates tumor vessel abnormalization by inhibiting TAGLN. We demonstrated for the first time that TAGLN plays an essential role in tumor vessel normalization, and thus it impairs TB and metastasis. Additionally, the findings of this study indicate that exosomal miR-22-3p is a potential therapeutic target for BC.

Two receptor-like protein kinases, MUSTACHES and MUSTACHES-LIKE, regulate lateral root development in Arabidopsis thaliana.

Receptor-like protein kinases (RLKs) play key roles in regulating plant growth, development and stress adaptations. There are at least 610 RLKs (including receptor-like cytoplasmic kinases) in Arabidopsis. The functions of the majority of RLKs have not yet been determined. We previously generated promoter::GUS transgenic plants for all leucine-rich repeat (LRR)-RLKs in Arabidopsis and analyzed their expression patterns during various developmental stages. We found the expression of two LRR-RLKs, MUSTACHES (MUS) and MUSTACHES-LIKE (MUL), are overlapped in lateral root primordia. Independent mutants, mus-3 mul-1 and mus-4 mul-2, show a significantly decreased emerged lateral root phenotype. Our analyses indicate that the defects of the double mutant occur mainly at stage I of lateral root development. Exogenous application of auxin can dramatically enhance the transcription of MUS, which is largely dependent on AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19. MUS and MUL are inactive kinases in vitro but are phosphorylated in planta, possibly by an unknown kinase. The kinase activity of MUS is dispensable for its function in lateral root development. Many cell wall related genes are down regulated in mus-3 mul-1. In conclusion, we identified MUS and MUL, two kinase-inactive RLKs, in controlling the early development of lateral root primordia likely via regulating cell wall synthesis and remodeling.

TCP Transcription Factors Associate with PHYTOCHROME INTERACTING FACTOR 4 and CRYPTOCHROME 1 to Regulate Thermomorphogenesis in Arabidopsis thaliana.

Temperature, one of the most critical environmental cues, greatly affects plant growth, development, and reproduction. PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), a key transcription factor in light signaling pathway, plays a central role in temperature-mediated growth responses. How higher temperature regulates the function of PIF4, however, is not well understood. Here we demonstrate that three phylogenetically related TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) transcription factors, TCP5, TCP13, and TCP17, play fundamental roles in promoting thermoresponsive hypocotyl growth by positively regulating the activity of PIF4. TCP17 was found to interact with a blue light receptor, CRYPTOCHROME 1 (CRY1), at lower temperature, leading to reduced activity of TCP17. Higher temperature can increase the stability of TCP17, and release TCP17 from the CRY1-TCP17 complex, allowing it to upregulate the expression of PIF4 and enhance the transcriptional activity of PIF4. This study revealed the important roles of TCPs in regulating the activity of PIF4 in thermomorphogenesis.

RGF1 INSENSITIVE 1 to 5, a group of LRR receptor-like kinases, are essential for the perception of root meristem growth factor 1 in Arabidopsis thaliana.

RGF1, a secreted peptide hormone, plays key roles in root meristem development in Arabidopsis. Previous studies indicated that a functional RGF1 needs to be sulfated at a tyrosine residue by a tyrosylprotein sulfotransferase and that RGF1 regulates the root meristem activity mainly via two downstream transcription factors, PLETHORA 1 (PLT1) and PLT2. How extracellular RGF1 is perceived by a plant cell, however, is unclear. Using genetic approaches, we discovered a clade of leucine-rich repeat receptor-like kinases, designated as RGF1 INSENSITIVE 1 (RGI1) to RGI5, serving as receptors of RGF1. Two independent rgi1 rgi2 rgi3 rgi4 rgi5 quintuple mutants display a consistent short primary root phenotype with a small size of meristem. An rgi1 rgi2 rgi3 rgi4 quadruple mutant shows a significantly reduced sensitivity to RGF1, and the quintuple mutant is completely insensitive to RGF1. The expression of PLT1 and PLT2 is almost undetectable in the quintuple mutant. Ectopic expression of PLT2 driven by an RGI2 promoter in the quintuple mutant greatly rescued its root meristem defects. One of the RGIs, RGI1, was subsequently analyzed biochemically in detail. In vitro dot blotting and pull-down analyses indicated that RGI1 can physically interact with RGF1. Exogenous application of RGF1 can quickly and simultaneously induce the phosphorylation and ubiquitination of RGI1, indicating that RGI1 can perceive and transduce the RGF1 peptide signal. Yet, the activated RGI1 is likely turned over rapidly. These results demonstrate that RGIs, acting as the receptors of RGF1, play essential roles in RGF1-PLT-mediated root meristem development in Arabidopsis thaliana.

Genome-Wide Expression Pattern Analyses of the Arabidopsis Leucine-Rich Repeat Receptor-Like Kinases.

Receptor-like protein kinases (RLKs) are a large group of transmembrane proteins playing critical roles in cell-cell and cell-environment communications. Based on extracellular domain structures, RLKs were classified into more than 21 subfamilies, among which leucine-rich repeat RLKs (LRR-RLKs) belong to the largest subfamily in plants such as Arabidopsis and rice. In Arabidopsis, there are approximately 223 LRR-RLKs, but only about 60 of which have been functionally described to date. To systematically investigate the roles of LRR-RLKs in regulating plant growth, development, and stress adaptations, we generated promoter::GUS transgenic plants for all 223 LRR-RLK genes in Arabidopsis and analyzed their detailed expression patterns at various developmental stages. The results provide valuable resources for functionally elucidating this large and essential signaling protein subfamily.

PAG1, a cotton brassinosteroid catabolism gene, modulates fiber elongation.

Cotton (Gossypium hirsutum) is the major source of natural textile fibers. Brassinosteroids (BRs) play crucial roles in regulating fiber development. The molecular mechanisms of BRs in regulating fiber elongation, however, are poorly understood. pagoda1 (pag1) was identified via an activation tagging genetic screen and characterized by genome walking and brassinolide (BL) supplementation. RNA-Seq analysis was employed to elucidate the mechanisms of PAG1 in regulating fiber development. pag1 exhibited dwarfism and reduced fiber length due to significant inhibition of cell elongation and expansion. BL treatment rescued its growth and fiber elongation. PAG1 encodes a homolog of Arabidopsis CYP734A1 that inactivates BRs via C-26 hydroxylation. RNA-Seq analyses showed that the constitutive expression of PAG1 downregulated the expression of genes involved in very-long-chain fatty acids (VLCFA) biosynthesis, ethylene-mediated signaling, response to cadmium, cell wall development, cytoskeleton organization and cell growth. Our results demonstrate that PAG1 plays crucial roles in regulating fiber development via controlling the level of endogenous bioactive BRs, which may affect ethylene signaling cascade by mediating VLCFA. Therefore, BR may be a critical regulator of fiber elongation, a role which may in turn be linked to effects on VLCFA biosynthesis, ethylene and cadmium signaling, cell wall- and cytoskeleton-related gene expression.