Leucine-tRNA-synthase-2-expressing B cells contribute to colorectal cancer immunoevasion.

Immunoregulatory B cells impede antitumor immunity through unknown features and mechanisms. We report the existence of leucine-tRNA-synthase-2 (LARS2)-expressing B cell (LARS B) subset with a transforming growth factor-ß1 (TGF-ß1)-dominant regulatory feature in both mouse and human progressive colorectal cancer (CRC). Of note, LARS B cells exhibited a leucine nutrient preference and displayed active mitochondrial aminoacyl-tRNA biosynthesis. They were located outside the tertiary lymphoid structure and correlated with colorectal hyperplasia and shortened survival in CRC patients. A leucine diet induced LARS B cell generation, whereas LARS B cell deletion by Lars2 gene ablation or leucine blockage repressed CRC immunoevasion. Mechanistically, LARS2 programmed mitochondrial nicotinamide adenine dinucleotide (NAD+) regeneration and oxidative metabolism, thus determining the regulatory feature of LARS B cells in which the NAD-dependent protein deacetylase sirtuin-1 (SIRT1) was involved. We propose a leucine-dieting scheme to inhibit LARS B cells, which is safe and useful for CRC therapy.

Chimeric antigen receptor clustering via cysteines enhances T-cell efficacy against tumor.

Chimeric antigen receptor (CAR) T-cell therapy achieves great success for hematological malignancies. However, clinical trials have revealed some limitations in both improving the efficacy and reducing the relapse, which calls for innovative strategies to engineer more powerful CAR-T cells. Promoting the formation of CAR clusters provides an alternative approach and potentially improves current CAR T-cell therapy against cancers. Here, we generated CARCys-T cells using a 4-1BB-derived hinge region including 11 cysteines residues. The cysteines in the hinge were found to facilitate CARCys clustering upon antigen stimulation and promote the antitumor activity of CAR-T cells. Compared with most conventionally used CAR-T cells with CD8α-derived hinge (CARconv-T cells), CARCys-T cells exhibited larger diameter of CAR clusters and enhanced antigen-specific tumor lysis both in vitro and in vivo. In addition, the CARCys-mediated enhancement could be applied to HER2, CD19 as well as GPC3-targeted CAR-T cells. More importantly, CARCys-T cells showed potent antitumor efficacy in clinically relevant patient-derived primary tumor cells and organoids. Thus, the novel hinge containing 11 cysteines provides a promising strategy to facilitate CAR clustering and maximize anti-tumor activity of CAR-T cells, which emphasizes the importance of CAR clustering to improve CAR T-cell therapy in the clinic.

Trans-acting non-synonymous variant of FOXA1 predisposes to hepatocellular carcinoma through modulating FOXA1-ERα transcriptional program and may have undergone natural selection.

Interplay of pioneer transcription factor forkhead box A1 (FOXA1) and estrogen receptor has been implicated in sexual dimorphism in hepatocellular carcinoma (HCC), but etiological relevance of its polymorphism was unknown. In the case control study (1152 patients versus1242 controls), we observed significant increase in HCC susceptibility in hepatitis B virus carriers associated with a non-synonymous Thr83Ala variant of FOXA1 (odds ratio [OR], 1.28; 95% confidence interval [CI], 1.11-1.48, for Ala83-containing genotype, after validation in an independent population with 933 patients versus 1030 controls), a tightly linked (CGC)5/6or7 repeat polymorphism at its promoter (OR 1.32; 95% CI 1.10-1.60, for (CGC)6or7-repeat-containing genotype), and their combined haplotype (OR 1.50; 95% CI 1.24-1.81, for (CGC)6or7-Ala83 haplotype). The susceptible FOXA1-Ala83 impairs its interaction with ERα, attenuates transactivation toward some of their dual target genes, such as type 1 iodothyronine deiodinase, UDP glucuronosyltransferase 2 family, polypeptide B17 and sodium/taurocholate cotransporting polypeptide, but correlates with strengthened cellular expression of α-fetoprotein (AFP) and elevated AFP serum concentration in HCC patients (n = 1096). The susceptible FOXA1 cis-variant with (CGC)6or7 repeat strengthens the binding to transcription factor early growth response 1 and enhances promoter activity and gene expression. Evolutionary population genetics analyses with public datasets reveal significant population differentiation and unique haplotype structure of the derived protective FOXA1-Thr83 and suggest that it may have undergone positive natural selection in Chinese population. These findings epidemiologically highlight the functional significance of FOXA1-ERα transcriptional program and regulatory network in liver cancer development.

Spatiotemporal Changes in Checkpoint Molecule Expression.

Immune checkpoint inhibitors (ICIs), particularly PD-1/PD-L1 blockade, have led to therapeutic breakthrough in patients with advanced malignancy, covering the lung, breast, gastrointestinal, head and neck, urinary system, lymphoma, and solid tumor harboring MSI/dMMR. In certain cancer types, the expression level of immune checkpoint molecule will be required if the immune-based approaches are considered, especially the PD-L1 expression. However, in other types, survival benefit has been proven regardless of PD-L1 expression. It raises a question of how to select patients for immune therapy and whether the expression of immune checkpoint molecules will be optimal biomarkers. Before answering this question, a comprehensive map for the expression of immune checkpoint molecules is needed. In this chapter, we describe our current knowledge on the spatiotemporal changes in the expression of checkpoint molecules. We discuss the different frequencies of expression depending on tumor types and stages, the different patterns between primary and metastatic tumors, as well as the change of expression before and after treatment. The expression of PD-L1 has been most studied, but the threshold that separate "positive" and "negative" PD-L1 expressions and the consistency of testing platform remain under debate. Better understanding on the tumor microenvironment and expression of checkpoint molecules will help to identify patients who will benefit from checkpoint blockade therapy.

Classical Monocytes Shuttling for Precise Delivery of Nanotherapeutics to Glioblastoma.

Glioblastoma (GBM) is the most aggressive brain tumor for which current therapies have limited efficacy. Immunosuppression and difficulties in accessing tumors with therapeutic agents are major obstacles for GBM treatments. Classical monocytes (CMs) possess the strongest infiltration among myeloid cells recruited into tumors during tumorigenesis. In this study, CMs are utilized to deliver the small-molecule CUDC-907 encapsulated in nanoparticles (907-NPs@CMs) for GBM therapy. Hitchhiking on CMs enables more 907-NPs to successfully penetrate the blood-brain barrier (BBB) and reach the interior of tumors. Results demonstrate that 907-NPs@CMs significantly improve the survival rates by suppressing tumor growth and reversing the immunosuppression of tumor microenvironment (TME). Furthermore, the high delivery efficiency of CMs reduces the amount of CUDC-907 required for treatments, reducing the physiological toxicity and off-target effects caused by high doses. 907-NPs@CMs is a safe and versatile therapeutic system that provides a platform for targeted drug delivery to tumors and the ability to treat GBM through a combination of chemotherapy and immunotherapy.

Inhibition of SIRT1 relieves hepatocarcinogenesis via alleviating autophagy and inflammation.

Imbalanced Sirtuin 1 (SIRT1) levels may lead to liver diseases through abnormal regulation of autophagy, but the roles of SIRT1-regulated autophagy in hepatocellular carcinoma are still controversial. In this study, we found that SIRT1 mRNA and protein levels were upregulated in hepatocellular carcinoma, and high SIRT1 expression hinted an advanced stage and a poor prognosis. The differentially expressed proteins were significantly elevated in autophagy, cellular response to stress, and immune signaling pathways. In a thioacetamide-induced hepatocellular carcinoma mouse model, we found that SIRT1 expression was highly increased with increased autophagy and excessive macrophage inflammatory response. Next, we established a Hepa 1-6 cells and macrophage co-culture system in vitro to model the alteration of tumor microenvironment, and found that the medium from CCl4-treated or SIRT1-overexpressing Hepa 1-6 cells triggered the polarization of macrophage M1, and the culture medium derived from M1 macrophage promoted Hepa 1-6 cells growth and intracellular oxidative stress. The progression of liver fibrosis in the CCl4-induced liver fibrosis mouse model showed that inhibition of SIRT1 alleviated inflammatory response and ameliorated liver fibrosis. These findings suggest that SIRT1-regulated autophagy and inflammation are oncogenic in hepatocarcinogenesis.

Single-Cell Transcriptomic Analysis Reveals Macrophage-Tumor Crosstalk in Hepatocellular Carcinoma.

As one of the most malignant cancer, hepatocellular carcinoma (HCC) has a complex ecosystem featured by high heterogeneity. Cell crosstalk is demonstrated to be critical for HCC development. However, the cell communication orchestration in HCC remains largely unknown. Here, by analyzing the single-cell transcriptomes of the primary tumor tissues (n = 10) and tumor-adjacent tissues (n = 8) derived from 10 patients with HCC, we found that the proportions of plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells were reduced and that the proportion of macrophages was increased in the immune component of the primary tumor, compared with those in the tumor-adjacent tissue. Furthermore, we found widespread communication between macrophage populations and other cell types, and this communication was remarkably strengthened in the primary tumor, especially with HCC malignant cells. In addition, the SPP1-CD44 axis was identified as a unique interaction between macrophages and HCC malignant cells. Our comprehensive portrait of cell communication patterns over the HCC ecosystem reveals further insights into immune infiltration.

Data-Independent Acquisition-Based Mass Spectrometry (DIA-MS) for Quantitative Analysis of Human Intestinal Ischemia/Reperfusion.

Intestinal ischemia-reperfusion (II/R) injury is a complex pathologic process, which is of great significance to unravel the underlying mechanisms and pathophysiology. Our study represented a comprehensive proteomic analysis in the human intestine with ischemia-reperfusion injury. The proteomics analysis measured a total of 5,230 proteins, and 417 differently expressed proteins (DEPs) were identified between II/R and control samples. GO and KEGG analysis demonstrated that the 290 upregulated DEPs in II/R were significantly involved in immune-related biological process and tight junction, focal adhesion, and cAMP signaling pathway, whereas the 127 downregulated DEPs in II/R were enriched in lipid metabolic process and metabolic pathway. Furthermore, we screened out 20 hub proteins from the protein-protein interaction (PPI) network according to the degree of connectivity, and six clusters were identified. Combined with the result of KEGG analysis, 6 from the 20 hub proteins, ACTB, CAV1, FLNA, MYLK, ACTN1, and MYL9, were identified as the key proteins in the progress of II/R injury. According to the previous studies, FLNA and MYL9 were selected as the novel disease-related proteins for the first time. In conclusion, this study extended our understanding of the alteration in the human intestine during ischemia and reperfusion and highlighted the potential role of FLNA and MYL9 in the progress of II/R injury, which need to be further studied.

Integrated proteomic and transcriptomic landscape of macrophages in mouse tissues.

Macrophages are involved in tissue homeostasis and are critical for innate immune responses, yet distinct macrophage populations in different tissues exhibit diverse gene expression patterns and biological processes. While tissue-specific macrophage epigenomic and transcriptomic profiles have been reported, proteomes of different macrophage populations remain poorly characterized. Here we use mass spectrometry and bulk RNA sequencing to assess the proteomic and transcriptomic patterns, respectively, of 10 primary macrophage populations from seven mouse tissues, bone marrow-derived macrophages and the cell line RAW264.7. The results show distinct proteomic landscape and protein copy numbers between tissue-resident and recruited macrophages. Construction of a hierarchical regulatory network finds cell-type-specific transcription factors of macrophages serving as hubs for denoting tissue and functional identity of individual macrophage subsets. Finally, Il18 is validated to be essential in distinguishing molecular signatures and cellular function features between tissue-resident and recruited macrophages in the lung and liver. In summary, these deposited datasets and our open proteome server ( http://macrophage.mouseprotein.cn ) integrating all information will provide a valuable resource for future functional and mechanistic studies of mouse macrophages.

Proteogenomic insights into the biology and treatment of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor prognosis. Proteogenomic characterization and integrative proteomic analysis provide a functional context to annotate genomic abnormalities with prognostic value. METHODS: We performed an integrated multi-omics analysis, including whole-exome sequencing, RNA-seq, proteomic, and phosphoproteomic analysis of 217 PDAC tumors with paired non-tumor adjacent tissues. In vivo functional experiments were performed to further illustrate the biological events related to PDAC tumorigenesis and progression. RESULTS: A comprehensive proteogenomic landscape revealed that TP53 mutations upregulated the CDK4-mediated cell proliferation process and led to poor prognosis in younger patients. Integrative multi-omics analysis illustrated the proteomic and phosphoproteomic alteration led by genomic alterations such as KRAS mutations and ADAM9 amplification of PDAC tumorigenesis. Proteogenomic analysis combined with in vivo experiments revealed that the higher amplification frequency of ADAM9 (8p11.22) could drive PDAC metastasis, though downregulating adhesion junction and upregulating WNT signaling pathway. Proteome-based stratification of PDAC revealed three subtypes (S-I, S-II, and S-III) related to different clinical and molecular features. Immune clustering defined a metabolic tumor subset that harbored FH amplicons led to better prognosis. Functional experiments revealed the role of FH in altering tumor glycolysis and in impacting PDAC tumor microenvironments. Experiments utilizing both in vivo and in vitro assay proved that loss of HOGA1 promoted the tumor growth via activating LARP7-CDK1 pathway. CONCLUSIONS: This proteogenomic dataset provided a valuable resource for researchers and clinicians seeking for better understanding and treatment of PDAC.

Proteomic analysis reveals key differences between squamous cell carcinomas and adenocarcinomas across multiple tissues.

Squamous cell carcinoma (SCC) and adenocarcinoma (AC) are two main histological subtypes of solid cancer; however, SCCs are derived from different organs with similar morphologies, and it is challenging to distinguish the origin of metastatic SCCs. Here we report a deep proteomic analysis of 333 SCCs of 17 organs and 69 ACs of 7 organs. Proteomic comparison between SCCs and ACs identifies distinguishable pivotal pathways and molecules in those pathways play consistent adverse or opposite prognostic roles in ACs and SCCs. A comparison between common and rare SCCs highlights lipid metabolism may reinforce the malignancy of rare SCCs. Proteomic clusters reveal anatomical features, and kinase-transcription factor networks indicate differential SCC characteristics, while immune subtyping reveals diverse tumor microenvironments across and within diagnoses and identified potential druggable targets. Furthermore, tumor-specific proteins provide candidates with differentially diagnostic values. This proteomics architecture represents a public resource for researchers seeking a better understanding of SCCs and ACs.

Integrated MicroRNA Expression Profile Reveals Dysregulated miR-20a-5p and miR-200a-3p in Liver Fibrosis.

MicroRNAs (miRNAs) have been demonstrated to involve in liver fibrogenesis. However, the miRNA-gene regulation in liver fibrosis is still unclear. Herein, the miRNA expression profile GSE40744 was obtained to analyze the dysregulated miRNAs between liver fibrosis and normal samples. Then, we predicted the target genes of screened miRNAs by miRTarBase, followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Then, the protein-protein interaction (PPI) network was constructed to identify the functional miRNA-gene regulatory modules. Furthermore, we verified the hub gene expression using the gene expression profile GSE14323. Finally, 89 DEMs were identified in fibrotic liver samples compared to normal liver samples. The top 3 upregulated DEMs (miR-200b-3p, miR-200a-3p, and miR-182-5p) and downregulated DEMs (miR-20a-5p, miR-194-3p, and miR-148a-3p) were further studied. 516 and 1416 target genes were predicted, respectively. KEGG analysis demonstrated that the predicted genes were enriched in the p53 signaling pathway and hepatitis B, etc. Through constructing a PPI network, the genes with the highest connectivity were identified as hub genes. Of note, most of the hub genes were potentially targeted by miR-20a-5p and miR-200a-3p. Based on the data from GSE14323, the expression of EGFR, STAT3, CTNNB1, and TP53 targeted by miR-200a-3p was significantly downregulated in fibrotic liver samples. Oppositely, the expression of PTEN, MYC, MAPK1, UBC, and CCND1 potentially targeted by miR-20a-5p was significantly upregulated. In conclusion, it is demonstrated that miR-20a-5p and miR-200a-3p were identified as the novel liver fibrosis-associated miRNAs, which may play critical roles in liver fibrogenesis.

SPRY4-AS1, A Novel Enhancer RNA, Is a Potential Novel Prognostic Biomarker and Therapeutic Target for Hepatocellular Carcinoma.

A growing number of evidence have demonstrated the involvement of enhancer RNAs (eRNAs) in tumor progression. However, the possible functions of eRNAs in hepatocellular carcinoma (HCC) remain largely unclear. Our present research aimed to screen critical eRNAs and to further delve into the clinical significance of eRNAs in HCC patients. In this study, we identified 124 prognosis-related eRNAs by analyzing The Cancer Genome Atlas (TCGA) datasets. Among them, SPRY4 antisense RNA 1 (SPRY4-AS1) may be a key eRNA involved in HCC progression. SPRY4 was a regulatory target of SPRY4-AS1. High SPRY4-AS1 expression was associated with poor prognosis of HCC patients. Kyoto Encyclopedia of Genes and Genomes (KEGG) assays revealed that the mainly enriched biological process included Human papillomavirus infection, Hippo signaling pathway, and Proteoglycans in cancer. Besides, RT-PCR and immunohistochemical staining confirmed SPRY4-AS1 as an overexpressed eRNA in HCC specimens. The pan-cancer assays revealed that SPRY4-AS1 was associated with glioblastoma multiforme (GBM), adrenocortical carcinoma (ACC), brain lower grade glioma (LGG) and mesothelioma(MESO). Positive associations were observed between SPRY4-AS1 and SPRY4 (its target gene) in 16 tumor types. Collectively, our findings reveal a novel eRNA SPRY4-AS1 for HCC progression and suggest that SPRY4-AS1 may be a potential biomarker and therapeutic target for HCC.

Comparative Proteomics Demonstrates Altered Metabolism Pathways in Cotrimoxazole- Resistant and Amikacin-Resistant Klebsiella pneumoniae Isolates.

Antibiotic resistance (AMR) has always been a hot topic all over the world and its mechanisms are varied and complicated. Previous evidence revealed the metabolic slowdown in resistant bacteria, suggesting the important role of metabolism in antibiotic resistance. However, the molecular mechanism of reduced metabolism remains poorly understood, which inspires us to explore the global proteome change during antibiotic resistance. Here, the sensitive, cotrimoxazole-resistant, amikacin-resistant, and amikacin/cotrimoxazole -both-resistant KPN clinical isolates were collected and subjected to proteome analysis through liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A deep coverage of 2,266 proteins were successfully identified and quantified in total, representing the most comprehensive protein quantification data by now. Further bioinformatic analysis showed down-regulation of tricarboxylic acid cycle (TCA) pathway and up-regulation of alcohol metabolic or glutathione metabolism processes, which may contribute to ROS clearance and cell survival, in drug-resistant isolates. These results indicated that metabolic pathway alteration was directly correlated with antibiotic resistance, which could promote the development of antibacterial drugs from "target" to "network." Moreover, combined with minimum inhibitory concentration (MIC) of cotrimoxazole and amikacin on different KPN isolates, we identified nine proteins, including garK, uxaC, exuT, hpaB, fhuA, KPN_01492, fumA, hisC, and aroE, which might contribute mostly to the survival of KPN under drug pressure. In sum, our findings provided novel, non-antibiotic-based therapeutics against resistant KPN.

TNF-α-Induced miR-21-3p Promotes Intestinal Barrier Dysfunction by Inhibiting MTDH Expression.

Intestinal barrier dysfunction is characterized by increased intestinal permeability to lumen endotoxin, showing remarkable predisposition to immune enteropathy, and colorectal cancer tumor necrosis factor (TNF)-α is associated with this pathological process, while the mechanism remains unknown. In this study, different doses of TNF-α were used for Caco-2 cell treatment. We discovered that miR-21-3p expression was obviously increased by TNF-α in a dose-dependent manner. Further study demonstrated that TNF-α could upregulate miR-21-3p expression through the NF-κB signaling pathway. Then, TargetScan and miRWalk miRNA-mRNA interaction prediction online tools were introduced, and metadherin (MTDH) was screened out as a potential target of miR-21-3p. We subsequently found that miR-21-3p could directly target the 3'-untranslated region (UTR) of MTDH mRNA and inhibit its expression. Furthermore, it was demonstrated that miR-21-3p could regulate the Wnt signaling pathway by targeting MTDH mRNA, suggesting the effect of miR-21-3p/MTDH/Wnt axis on intestinal barrier dysfunction. Our findings provide a novel potential biomarker and therapeutic target for intestinal barrier dysfunction and related diseases.

FOXA1 Promotes Cell Proliferation and Suppresses Apoptosis in HCC by Directly Regulating miR-212-3p/FOXA1/AGR2 Signaling Pathway.

BACKGROUND: Forkhead box protein A1 (FOXA1), acting as a transcriptional activator for liver-specific transcripts, plays a vital part in proliferation, apoptosis and cell cycle. METHODS: The mRNA expression of FOXA1 in 90 HCC tissues and matched adjacent non-tumor tissues was determined by qRT-PCR. The downstream and upstream regulators of FOXA1 were identified by bioinformatics analysis and experimental confirmation. RESULTS: We found out that the expression of FOXA1 was obviously higher in hepatocellular carcinoma (HCC) tissues than that in matched non-tumor tissues. Similarly, FOXA1 is also highly expressed in HCC cell lines as compared with normal human hepatic cell line L02. Clinical association analysis indicated that high expression of FOXA1 was prominently correlated with high HBV level, large tumor size, high venous infiltration, high Edmondson-Steiner grading, and advanced tumor-node-metastasis tumor stage. Furthermore, the in vitro tests showed that ectopic expression of FOXA1 promoted HepG2 cell proliferation and suppressed apoptosis. In contrast, the downregulation of FOXA1 inhibited cell proliferation, and induced apoptosis in Hep3B cells. To investigate the functional mechanism of FOXA1, anterior gradient 2 (AGR2), an executor in proliferation and apoptosis, was identified as the direct target gene of FOXA1. Meanwhile, we also found the expression of FOXA1 could be inhibited by miR-212-3p, which working as a tumor suppressor downregulated in HCC. CONCLUSION: We revealed that FOXA1 exerted its biological function by regulating AGR2 expression, and its ectopic expression may be blamed for low expression of miR-212-3p.

DCAF13 promotes triple-negative breast cancer metastasis by mediating DTX3 mRNA degradation.

DCAF13 is firstly identified as a substrate receptor of CUL4-DDB1 E3 ligase complex. This study disclosed that DCAF13 acted as a novel RNA binding protein (RBP) that contributed to triple-negative breast cancer (TNBC) metastasis. Clinical data obtained from TCGA and our collection showed that DCAF13 was closely correlated with poor clinicopathological characteristics and overall survival, which indicated DCAF13 may serve as a diagnostic marker for TNBC metastasis. Functionally, DCAF13 overexpression or suppression was sufficient to enhance or decrease breast cancer cell migration and invasion. Mechanistically, DCAF13 functioned as an RBP by binding with the AU-rich element (ARE) of DTX3 mRNA 3'UTR to accelerate its degradation. Moreover, we identified that DTX3 promoted the ubiquitination and degradation of NOTCH4. Finally, increased DCAF13 expression led to post-transcriptional decay of DTX3 mRNA and consequently activated of NOTCH4 signaling pathway in TNBC. In conclusion, these results identified that DCAF13 as a diagnostic marker and therapeutic target for TNBC treatment. Abbreviation: DCAF13: DDB1 and CUL4-associated factor 13; DDB1: DNA-binding protein 1; CUL4: Cullin 4; CRL4, Cullin-ring finger ligase 4; RBP: RNA binding protein; TNBC: triple-negative breast cancer; ARE: AU-rich element; DTX3: Deltex E3 ubiquitin ligase 3; HER2: human epidermal growth factor receptor 2; ER: estrogen receptor; PR: progesterone receptor; PTEN: phosphatase and tensin homolog deleted on chromosome 10; EMT: epithelial-mesenchymal transition.

miR-20a-5p/TGFBR2 Axis Affects Pro-inflammatory Macrophages and Aggravates Liver Fibrosis.

Combined inhibition of programmed death-ligand 1 (PD-L1) and transforming growth factor-ß (TGF-ß) displayed additive anti-tumor response in a subgroup of cancer patients, highlighting the importance of understanding the multifaceted roles of TGF-ß in immunity and fibrosis. In the present research, we show that TGF-ß signaling pathway, controlled by miR-20a-5p and transforming growth factor-ß receptor 2 (TGFBR2), alters the inflammation and fibrosis processes in liver. We performed integrated analysis of differently expressed miRNA (DEM) associated with liver fibrosis and screened miR-20a-5p out as a key regulator in inflammation-driven liver fibrosis. We subsequently conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the genes targeted by miR-20a-5p. And the result showed that 12 target genes were significantly enriched in TGF-ß signaling pathway. Further study showed that miR-20a-5p was down-regulated and involved in inflammation during liver fibrosis in human and mouse samples, indicating that miR-20a-5p and inflammation are functionally linked during liver fibrosis progression. To uncover the underlying pro-inflammatory mechanism of miR-20a-5p in liver fibrosis, we selected and verified TGFBR2, which is a key functional receptor in TGF-ß signaling pathway, as a direct target gene of miR-20a-5p. The downregulation of miR-20a-5p in liver fibrosis resulted in TGFBR2-activated TGF-ß signaling pathway, followed by the activation of macrophage and extracellular matrix (ECM) production by hepatic stellate cell (HSC). Our results identify the miR-20a-5p/TGFBR2 axis as a key regulator of TGF-ß signaling, and highlight the critical role of miR-20a-5p in the development of liver fibrosis.

Genetic polymorphism of SLC31A1 is associated with clinical outcomes of platinum-based chemotherapy in non-small-cell lung cancer patients through modulating microRNA-mediated regulation.

SLC31A1 is the major transporter for platinum drug intake, its expression correlates with drug disposition and response. In 1004 Chinese NSCLC patients with platinum-based chemotherapy, we investigated the association between SLC31A1 polymorphisms and clinical outcomes. Heterozygotes of rs10759637 at 3'UTR was associated with severe thrombocytopenia (odds ratio [OR]: 2.69; P = 0.012) and shorter overall survival (hazard ratio [HR]: 1.24; P = 0.005). Variant homozygote of rs2233914 was correlated with longer overall survival (hazard ratio [HR]: 0.73; P = 0.008). Haplotype and diplotype of these linked SNPs were associated with hematologic toxicities. In stratification analyses, rs10759637 and rs2233914 consistently correlated with overall survival in specific subgroups such as men, smoker, patients older than 58 years, or with ECOG PS 0-1, or with squamous cell carcinoma. rs10759637 could change the local structure of 3'UTR harboring putative binding sites for hsa-miR-29, whose transfection into 16HBE cells resulted in remarkable suppression of gene expression. The rs10759637 variant significantly correlated with lowered luciferase activity in reporter assays and decreased expression of SLC31A1 transcript in tumorous tissues. The study thereby identified functional polymorphism of SLC31A1 that modulates miRNA-3'UTR interaction and gene expression as potential pharmacogenetic biomarker for clinical outcomes of platinum-based chemotherapy in NSCLC patients.