As a widely used anticancer and immunosuppressive agent, methotrexate (MTX) can induce multiple adverse drug reactions (ADRs), such as gastrointestinal toxicity, the mechanisms are poorly understood. Gut microbiota has been widely reported to be associated with the onset of multiple diseases as well as treatment outcomes of different drugs. In this study, mucosal injury was observed in MTX-treated mice, leading to significant changes in macrophages (i.e., M1/M2 ratio, P < 0.05) but not in dendritic cells. Moreover, the population, diversity and principal components of the gut microbiota in mice were dramatically altered after MTX treatment in a time-dependent manner, and Bacteroidales exhibited the most distinct variation among all the taxa (P < 0.05). Bacteroides fragilis was significantly decreased with MTX treatment (P < 0.01) and tended to decrease proportionately with increasing macrophage density. Gavage of mice with B. fragilis ameliorated MTX-induced inflammatory reactions and modulate macrophage polarization. In conclusion, our results delineate a strong impact of the gut microbiota on MTX-induced intestinal mucositis and provide a potential method for the prevention of such ADRs.

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MicroRNAs (miRNAs) have been demonstrated to modulate cellular processes in the liver. However, the role of miRNAs in liver fibrosis is poorly understood. Because the activation of hepatic stellate cells (HSCs) is a pivotal event in the initiation and progression of hepatic fibrosis, we investigate the differential expression of miRNAs in activated and quiescent rat HSCs by microarray analysis and find that miR-214 (miR-214-3p) is significantly upregulated during HSC activation. Moreover, the robust induction of miR-214 is correlated with liver fibrogenesis in carbon tetrachloride (CCl4)-treated rats and mice, high-fat diet-induced non-alcoholic steatohepatitis in mice, and cirrhosis in humans. We identify that miR-214 expression is driven by the helix–loop–helix transcription factor Twist1 via the E-box element. The increased miR-214 inhibits the expression of suppressor-of-fused homolog (Sufu), a negative regulator of the Hedgehog signaling pathway, thereby contributing to HSC activation to promote the accumulation of fibrous extracellular matrix and the expression of profibrotic genes in HSCs and LX2 cells. Furthermore, miR-214 expression is inversely correlated with the expression of Sufu in clinical cirrhosis samples. To explore the clinical potential of miR-214, we inject antagomiR-214 oligos into mice to induce hepatic fibrosis. The knockdown of miR-214 in vivo enhances Sufu expression and reduces fibrosis marker expression, which ameliorates liver fibrosis in mice. In conclusions, the Twist1-regulated miR-214 promotes the activation of HSC cells through targeting Sufu involved in the Hedgehog pathway and participates in the development of hepatic fibrosis. Hence, the knockdown of miR-214 expression may be a promising therapeutic strategy for liver fibrosis.

Plant phloem-based defence (PBD) against phloem-feeding insects is characteristic of the sieve occlusion by phloem lectins and β-1,3-glucan callose, both of which are produced under regulation by ethylene and MYB transcription factors. Wheat PBD requires β-1,3-glucan synthase-like proteins GSL2, GSL10, and GSL12, and may also require insect-resistant mannose-binding lectins Hfr-1 and Wci-1, which can accumulate in the phloem upon aphid feeding. This study elucidates whether any of the 73 MYB genes identified previously in the common wheat Triticum aestivum genome plays a role in wheat PBD activation with regard to the GSLs and lectins. Wheat MYB genes TaMYB19, TaMYB29, and TaMYB44 are highly activated in response to infestation of English grain aphid, and their silencing facilitates aphid feeding on wheat phloem and represses wheat PBD responses. Repressed PBD is shown to decrease aphid-induced callose deposition in wheat leaf epidermis and decrease aphid-induced expression of genes GSL2, GSL10, GSL12, Hfr-1, and Wci-1 in wheat leaf tissues. Based on single gene silencing effects, TaMYB19, TaMYB29, and TaMYB44 contribute 55-82% of PBD responses. However, the contributions of TaMYB genes to PBD are eliminated by ethylene signalling inhibitors, while simultaneous silencing of the three TaMYB genes cancels the tested PBD responses. Therefore, TaMYB19, TaMYB29, and TaMYB44 are co-regulators of wheat PBD and execute this function through crosstalk with the ethylene signalling pathway.

The advent of single-cell RNA sequencing (scRNA-seq) has provided insight into the tumour immune microenvironment (TIME). This review focuses on the application of scRNA-seq in investigation of the TIME. Over time, scRNA-seq methods have evolved, and components of the TIME have been deciphered with high resolution. In this review, we first introduced the principle of scRNA-seq and compared different sequencing approaches. Novel cell types in the TIME, a continuous transitional state, and mutual intercommunication among TIME components present potential targets for prognosis prediction and treatment in cancer. Thus, we concluded novel cell clusters of cancer-associated fibroblasts (CAFs), T cells, tumour-associated macrophages (TAMs) and dendritic cells (DCs) discovered after the application of scRNA-seq in TIME. We also proposed the development of TAMs and exhausted T cells, as well as the possible targets to interrupt the process. In addition, the therapeutic interventions based on cellular interactions in TIME were also summarized. For decades, quantification of the TIME components has been adopted in clinical practice to predict patient survival and response to therapy and is expected to play an important role in the precise treatment of cancer. Summarizing the current findings, we believe that advances in technology and wide application of single-cell analysis can lead to the discovery of novel perspectives on cancer therapy, which can subsequently be implemented in the clinic. Finally, we propose some future directions in the field of TIME studies that can be aided by scRNA-seq technology.