A role for small RNAs in DNA double-strand break repair.

Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that ∼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells. We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, diRNAs are recruited by Argonaute 2 (AGO2) to mediate DSB repair. Knock down of Dicer or Ago2 in human cells reduces DSB repair. Our findings reveal a conserved function for small RNAs in the DSB repair pathway. We propose that diRNAs may function as guide molecules directing chromatin modifications or the recruitment of protein complexes to DSB sites to facilitate repair.

Beyond transcription: compelling open questions in plant RNA biology.

The study of RNAs has become one of the most influential research fields in contemporary biology and biomedicine. In the last few years, new sequencing technologies have produced an explosion of new and exciting discoveries in the field but have also given rise to many open questions. Defining these questions, together with old, long-standing gaps in our knowledge, is the spirit of this article. The breadth of topics within RNA biology research is vast, and every aspect of the biology of these molecules contains countless exciting open questions. Here, we asked 12 groups to discuss their most compelling question among some plant RNA biology topics. The following vignettes cover RNA alternative splicing; RNA dynamics; RNA translation; RNA structures; R-loops; epitranscriptomics; long non-coding RNAs; small RNA production and their functions in crops; small RNAs during gametogenesis and in cross-kingdom RNA interference; and RNA-directed DNA methylation. In each section, we will present the current state-of-the-art in plant RNA biology research before asking the questions that will surely motivate future discoveries in the field. We hope this article will spark a debate about the future perspective on RNA biology and provoke novel reflections in the reader.

Peptidyl-prolyl isomerase Cyclophilin71 promotes SERRATE phase separation and miRNA processing in Arabidopsis.

MicroRNAs (miRNAs) play an important role in gene regulation. In Arabidopsis, mature miRNAs are processed from primary miRNA transcripts by the Dicing complex that contains Dicer-like 1 (DCL1), SERRATE (SE), and Hyponastic Leaves 1 (HYL1). The Dicing complex can form nuclear dicing bodies (D-bodies) through SE phase separation. Here, we report that Cyclophilin71 (CYP71), a peptidyl-prolyl isomerase (PPIase), positively regulates miRNA processing. We show that CYP71 directly interacts with SE and enhances its phase separation, thereby promoting the formation of D-body and increasing the activity of the Dicing complex. We further show that the PPIase activity is important for the function of CYP71 in miRNA production. Our findings reveal orchestration of miRNA processing by a cyclophilin protein and suggest the involvement of peptidyl-prolyl cis-trans isomerization, a structural mechanism, in SE phase separation and miRNA processing.

A receptor-like protein acts as a specificity switch for the regulation of stomatal development.

Stomata are microscopic openings that allow for the exchange of gases between plants and the environment. In Arabidopsis, stomatal patterning is specified by the ERECTA family (ERf) receptor kinases (RKs), the receptor-like protein (RLP) TOO MANY MOUTHS (TMM), and EPIDERMAL PATTERNING FACTOR (EPF) peptides. Here we show that TMM and ER or ER-LIKE1 (ERL1) form constitutive complexes, which recognize EPF1 and EPF2, but the single ERfs do not. TMM interaction with ERL1 creates a binding pocket for recognition of EPF1 and EPF2, indicating that the constitutive TMM-ERf complexes function as the receptors of EPF1 and EPF2. EPFL9 competes with EPF1 and EPF2 for binding to the ERf-TMM complex. EPFL4 and EPFL6, however, are recognized by the single ERfs without the requirement of TMM. In contrast to EPF1,2, the interaction of EPFL4,6 with an ERf is greatly reduced in the presence of TMM. Taken together, our data demonstrate that TMM dictates the specificity of ERfs for the perception of different EPFs, thus functioning as a specificity switch for the regulation of the activities of ERfs.

A Dicer-Independent Route for Biogenesis of siRNAs that Direct DNA Methylation in Arabidopsis.

DNA methylation directed by 24-nucleotide (nt) small interfering RNAs (siRNAs) plays critical roles in gene regulation and transposon silencing in Arabidopsis. 24-nt siRNAs are known to be processed from double-stranded RNAs by Dicer-like 3 (DCL3) and loaded into the effector Argonaute 4 (AGO4). Here we report a distinct class of siRNAs independent of DCLs (sidRNAs). sidRNAs are present as ladders of ∼ 20-60 nt in length, often having the same 5' ends but differing in 3' ends by 1-nt steps. We further show that sidRNAs are associated with AGO4 and capable of directing DNA methylation. Finally we show that sidRNA production depends on distributive 3'-5' exonucleases. Our findings suggest an alternative route for siRNA biogenesis. Precursor transcripts are bound by AGO4 and subsequently subjected to 3'-5' exonucleolytic trimming for maturation. We propose that sidRNAs generated through this route are the initial triggers of de novo DNA methylation.

Exosomal PD-L1 confers chemoresistance and promotes tumorigenic properties in esophageal cancer cells via upregulating STAT3/miR-21.

Chemotherapy resistance remains a major obstacle in the treatment of esophageal cancer. Previous researches have shown that an increase in exosomal PD-L1 expression was positively associated with a more advanced clinical stage, a poorer prognosis as well as drug resistance in patients with esophageal squamous cell carcinoma (ESCC). To explore the role of exosomal PD-L1 in ESCC, we performed bioinformatics analysis as well as several in vitro/in vivo functional experiments in a parental sensitive cell line EC-9706 and its derivative, a paclitaxel-resistant subline EC-9706R, and found that the exosomal PD-L1 from EC-9706R was higher than that from EC-9706. Moreover, exosomes from EC-9706R significantly increased invasion, migration and chemoresistance of EC-9706. Anti-PD-L1 treatment in combination with chemotherapy also led to reduced tumor burden in vivo. Inhibition of the release of exosomes by GW4869 or inhibition of STAT3 phosphorylation by stattic could effectively reverse the resistance to paclitaxel mediated by exosomal PD-L1. Furthermore, we found that PD-L1, miR-21, and multidrug resistance (MDR1) gene are involved in the process of exosomal transfer. Moreover, PD-L1 could enhance miR-21 expression by increasing the enrichment of STAT3 on miR-21 promoter. Our results suggested that exosomal PD-L1 may contribute to drug resistance to paclitaxel by regulating the STAT3/miR-21/PTEN/Akt axis and promote tumorigenic phenotype. This study provides a novel potential therapeutic approach to reverse chemoresistance and tumor progression through exosomal PD-L1 in ESCC patients.

Porphyromonas gingivalis predicts local recurrence after endoscopic submucosal dissection of early esophageal squamous cell carcinoma or precancerous lesion.

BACKGROUND: Porphyromonas gingivalis plays an oncogenic role in development and progression of esophageal squamous cell carcinoma (ESCC). However, the impact of P. gingivalis on local recurrence of early ESCC or precancerous lesion after ESD treatment remains unknown. The present study aimed to evaluate the impact of P. gingivalis on local recurrence after ESD treatment of early ESCC or high-grade dysplasia (HGD). METHODS: The amount of P. gingivalis was assessed by immunohistochemistry in 205 patients with early ESCC or HGD. Univariate and multivariate Cox regression analyses were performed to determine the effect of P. gingivalis on local recurrence. Propensity score matching analysis was performed to reduce the imbalance of baseline characteristics. A nomogram integrating significant prognostic factors was built for local recurrence prediction. RESULTS: The amount of P. gingivalis increased significantly in neoplasms that invaded up to muscularis mucosa and submucosa compared with lesions confined to epithelium or lamina propria. Overabundance of P. gingivalis was positively associated with invasion depth, post-ESD stricture and local recurrence. Univariate and multivariate Cox regression analyses revealed that P. gingivalis, longitudinal length of lesion and lymphovascular invasion were independent predictors for post-ESD recurrence. A nomogram comprising P. gingivalis, lymphovascular involvement, and lesion length performed well for prediction of post-ESD local recurrence with the concordance indices of 0.72 (95%CI, 0.62 to 0.80), 0.72 (95%CI, 0.63 to 0.80), and 0.74 (95%CI, 0.65 to 0.83) in the validation cohort, the entire cohort, and the subcohort after PSM, respectively. CONCLUSION: P. gingivalis overabundance is a risk factor and a potential predictor for local recurrence of early ESCC or HGD after ESD treatment. Thus, clearance of P. gingivalis represents an attractive strategy for prognosis improvement and for prevention of ESCC.

Porphyromonas gingivalis promotes progression of esophageal squamous cell cancer via TGFß-dependent Smad/YAP/TAZ signaling.

Microbial dysbiosis in the upper digestive tract is linked to an increased risk of esophageal squamous cell carcinoma (ESCC). Overabundance of Porphyromonas gingivalis is associated with shorter survival of ESCC patients. We investigated the molecular mechanisms driving aggressive progression of ESCC by P. gingivalis. Intracellular invasion of P. gingivalis potentiated proliferation, migration, invasion, and metastasis abilities of ESCC cells via transforming growth factor-ß (TGFß)-dependent Drosophila mothers against decapentaplegic homologs (Smads)/Yes-associated protein (YAP)/Transcriptional coactivator with PDZ-binding motif (TAZ) activation. Smads/YAP/TAZ/TEA domain transcription factor1 (TEAD1) complex formation was essential to initiate downstream target gene expression, inducing an epithelial-mesenchymal transition (EMT) and stemness features. Furthermore, P. gingivalis augmented secretion and bioactivity of TGFß through glycoprotein A repetitions predominant (GARP) up-regulation. Accordingly, disruption of either the GARP/TGFß axis or its activated Smads/YAP/TAZ complex abrogated the tumor-promoting role of P. gingivalis. P. gingivalis signature genes based on its activated effector molecules can efficiently distinguish ESCC patients into low- and high-risk groups. Targeting P. gingivalis or its activated effectors may provide novel insights into clinical management of ESCC.

Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal nucleotide.

Argonaute (AGO) proteins recruit small RNAs to form the core of RNAi effector complexes. Arabidopsis encodes ten AGO proteins and a large network of small RNAs. How these small RNAs are sorted into specific AGO complexes remains largely unknown. We have cataloged small RNAs resident in four AGO complexes. We found that AGO2 and AGO4 preferentially recruit small RNAs with a 5' terminal adenosine, whereas AGO1 harbors microRNAs (miRNAs) that favor a 5' terminal uridine. AGO5 predominantly binds small RNAs that initiate with cytosine. Changing the 5' terminal nucleotide of an miRNA predictably redirected it into a different AGO complex and alters its biological activity. These results reveal a role for small RNA sequences in assorting among AGO complexes. This suggests that specialization of AGO complexes might involve remodeling the 5' end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine.

Evolution of lmiRNAs and their targets from MITEs for rice adaptation.

Twenty-four nucleotide long microRNAs (lmiRNAs) direct DNA methylation at target genes and regulate their transcription. The evolutionary origin of lmiRNAs and the range of lmiRNA-mediated regulation remain obscure. Here, we reannotated lmiRNAs and their targets in rice by applying stringent criteria. We found that the majority of lmiRNAs are derived from Miniature Inverted-repeat Transposable Elements (MITEs) and most sites targeted by MITE-derived lmiRNAs reside within MITEs, suggesting co-evolution of lmiRNAs and their targets through MITE amplification. lmiRNAs undergo dynamically changes under stress conditions and the genes targeted by lmiRNAs show an enrichment for stress-responsive genes, suggesting that lmiRNAs are widely involved in plant responses to stresses. We constructed the evolutionary histories of lmiRNAs and their targets. Nearly half of lmiRNAs emerged before or when the AA genome was diverged, while the emergence of lmiRNA targets coincided with or followed the emergence of lmiRNAs. Furthermore, we found that the sequences of a lmiRNA target site underwent variations, coincident with the divergence of rice accessions and the distribution of rice accessions in different geographical locations and climatic conditions. Our findings highlight MITEs as an important origin of lmiRNAs and suggest that the evolution of lmiRNA-target regulatory modules may contribute to rice adaptation to environmental changes.

A Role for MINIYO and QUATRE-QUART2 in the Assembly of RNA Polymerases II, IV, and V in Arabidopsis.

RNA polymerases IV and V (Pol IV and Pol V) are required for the generation of noncoding RNAs in RNA-directed DNA methylation (RdDM). Their subunit compositions resemble that of Pol II. The mechanism and accessory factors involved in their assembly remain largely unknown. In this study, we identified mutant alleles of MINIYO (IYO), QUATRE-QUART2 (QQT2), and NUCLEAR RNA POLYMERASE B11/D11/E11 (NRPB/D/E11) that cause defects in RdDM in Arabidopsis thaliana We found that Pol IV-dependent small interfering RNAs and Pol V-dependent transcripts were greatly reduced in the mutants. NRPE1, the largest subunit of Pol V, failed to associate with other Pol V subunits in the iyo and qqt2 mutants, suggesting the involvement of IYO and QQT2 in Pol V assembly. In addition, we found that IYO and QQT2 were mutually dependent for their association with the NRPE3 subassembly prior to the assembly of Pol V holoenzyme. Finally, we show that IYO and QQT2 are similarly required for the assembly of Pol II and Pol IV. Our findings reveal IYO and QQT2 as cofactors for the assembly of Pol II, Pol IV, and Pol V and provide mechanistic insights into how RNA polymerases are assembled in plants.

Using a machine learning approach to identify key prognostic molecules for esophageal squamous cell carcinoma.

BACKGROUND: A plethora of prognostic biomarkers for esophageal squamous cell carcinoma (ESCC) that have hitherto been reported are challenged with low reproducibility due to high molecular heterogeneity of ESCC. The purpose of this study was to identify the optimal biomarkers for ESCC using machine learning algorithms. METHODS: Biomarkers related to clinical survival, recurrence or therapeutic response of patients with ESCC were determined through literature database searching. Forty-eight biomarkers linked to recurrence or prognosis of ESCC were used to construct a molecular interaction network based on NetBox and then to identify the functional modules. Publicably available mRNA transcriptome data of ESCC downloaded from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets included GSE53625 and TCGA-ESCC. Five machine learning algorithms, including logical regression (LR), support vector machine (SVM), artificial neural network (ANN), random forest (RF) and XGBoost, were used to develop classifiers for prognostic classification for feature selection. The area under ROC curve (AUC) was used to evaluate the performance of the prognostic classifiers. The importances of identified molecules were ranked by their occurrence frequencies in the prognostic classifiers. Kaplan-Meier survival analysis and log-rank test were performed to determine the statistical significance of overall survival. RESULTS: A total of 48 clinically proven molecules associated with ESCC progression were used to construct a molecular interaction network with 3 functional modules comprising 17 component molecules. The 131,071 prognostic classifiers using these 17 molecules were built for each machine learning algorithm. Using the occurrence frequencies in the prognostic classifiers with AUCs greater than the mean value of all 131,071 AUCs to rank importances of these 17 molecules, stratifin encoded by SFN was identified as the optimal prognostic biomarker for ESCC, whose performance was further validated in another 2 independent cohorts. CONCLUSION: The occurrence frequencies across various feature selection approaches reflect the degree of clinical importance and stratifin is an optimal prognostic biomarker for ESCC.

Geminiviral V2 Protein Suppresses Transcriptional Gene Silencing through Interaction with AGO4.

In plants, RNA-directed DNA methylation (RdDM)-mediated transcriptional gene silencing (TGS) is a natural antiviral defense against geminiviruses. Several geminiviral proteins have been shown to target the enzymes related to the methyl cycle or histone modification; however, it remains largely unknown whether and by which mechanism geminiviruses directly inhibit RdDM-mediated TGS. In this study, we showed that Cotton leaf curl Multan virus (CLCuMuV) V2 directly interacts with Nicotiana benthamiana AGO4 (NbAGO4) and that the L76S mutation in V2 (V2L76S) abolishes such interaction. We further showed that V2, but not V2L76S, can suppresses RdDM and TGS. Silencing of NbAGO4 inhibits TGS, reduces the viral methylation level, and enhances CLCuMuV DNA accumulation. In contrast, the V2L76S substitution mutant attenuates CLCuMuV infection and enhances the viral methylation level. These findings reveal that CLCuMuV V2 contributes to viral infection by interaction with NbAGO4 to suppress RdDM-mediated TGS in plants.IMPORTANCE In plants, the RNA-directed DNA methylation (RdDM) pathway is a natural antiviral defense mechanism against geminiviruses. However, how geminiviruses counter RdDM-mediated defense is largely unknown. Our findings reveal that Cotton leaf curl Multan virus V2 contributes to viral infection by interaction with NbAGO4 to suppress RNA-directed DNA methylation-mediated transcriptional gene silencing in plants. Our work provides the first evidence that a geminiviral protein is able to directly target core RdDM components to counter RdDM-mediated TGS antiviral defense in plants, which extends our current understanding of viral counters to host antiviral defense.

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair.

Cytoplasmic assembly and selective nuclear import of Arabidopsis Argonaute4/siRNA complexes.

In plants, DNA methylation can be mediated by a class of Argonaute4 (AGO4)-associated heterochromatic siRNAs (hc-siRNAs), through a pathway termed RNA-directed DNA methylation (RdDM). It has been thought that RdDM is solely a nuclear process, as both the biogenesis and functioning of hc-siRNAs take place in the nucleus. In this study, we unexpectedly found that hc-siRNAs are predominantly present in the cytoplasm. We demonstrated that AGO4 is loaded with hc-siRNAs in the cytoplasm and the formation of mature AGO4/siRNA complexes requires HSP90 and the cleavage activity of AGO4. Intriguingly, siRNA binding facilitates the redistribution of AGO4 into the nucleus, likely through inducing conformational change that leads to the exposure of the nuclear localization signal (NLS). Our findings reveal an unsuspected cytoplasmic step in the RdDM pathway. We propose that selective nuclear import of mature AGO4/siRNA complexes is a key regulatory point prior to the effector stage of RdDM.

Derivation of adult canine intestinal organoids for translational research in gastroenterology.

BACKGROUND: Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures. RESULTS: Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research. CONCLUSIONS: We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.

Stress-responsive regulation of long non-coding RNA polyadenylation in Oryza sativa.

Recently, long non-coding RNAs (lncRNAs) have been demonstrated to be involved in many biological processes of plants; however, a systematic study on transcriptional and, in particular, post-transcriptional regulation of stress-responsive lncRNAs in Oryza sativa (rice) is lacking. We sequenced three types of RNA libraries (poly(A)+, poly(A)- and nuclear RNAs) under four abiotic stresses (cold, heat, drought and salt). Based on an integrative bioinformatics approach and ~200 high-throughput data sets, ~170 of which have been published, we revealed over 7000 lncRNAs, nearly half of which were identified for the first time. Notably, we found that the majority of the ~500 poly(A) lncRNAs that were differentially expressed under stress were significantly downregulated, but approximately 25% were found to have upregulated non-poly(A) forms. Moreover, hundreds of lncRNAs with downregulated polyadenylation (DPA) tend to be highly conserved, show significant nuclear retention and are co-expressed with protein-coding genes that function under stress. Remarkably, these DPA lncRNAs are significantly enriched in quantitative trait loci (QTLs) for stress tolerance or development, suggesting their potential important roles in rice growth under various stresses. In particular, we observed substantially accumulated DPA lncRNAs in plants exposed to drought and salt, which is consistent with the severe reduction of RNA 3'-end processing factors under these conditions. Taken together, the results of this study reveal that polyadenylation and subcellular localization of many rice lncRNAs are likely to be regulated at the post-transcriptional level. Our findings strongly suggest that many upregulated/downregulated lncRNAs previously identified by traditional RNA-seq analyses need to be carefully reviewed to assess the influence of post-transcriptional modification.

RNAi in Plants: An Argonaute-Centered View.

Argonaute (AGO) family proteins are effectors of RNAi in eukaryotes. AGOs bind small RNAs and use them as guides to silence target genes or transposable elements at the transcriptional or posttranscriptional level. Eukaryotic AGO proteins share common structural and biochemical properties and function through conserved core mechanisms in RNAi pathways, yet plant AGOs have evolved specialized and diversified functions. This Review covers the general features of AGO proteins and highlights recent progress toward our understanding of the mechanisms and functions of plant AGOs.

TRANSPORTIN1 Promotes the Association of MicroRNA with ARGONAUTE1 in Arabidopsis.

In Arabidopsis thaliana, microRNAs (miRNAs) are mainly loaded into ARGONAUTE1 (AGO1) to posttranscriptionally regulate gene expression. We previously found that ENHANCED MiRNA ACTIVITY1 (EMA1), an importin ß family protein, negatively regulates miRNA loading into AGO1. In this study, through a suppressor screening of ema1, we identified another importin ß protein, TRANSPORTIN1 (TRN1), as a regulatory component in the miRNA pathway. Mutation of TRN1 did not reduce miRNA accumulation, but it impaired miRNA activity. We found that TRN1 interacted with AGO1. Mutation of the three conserved residues required for cargo recognition of TRN1 reduced its interaction with AGO1 and compromised its function in regulating miRNA activity. Intriguingly, TRN1 dysfunction did not change the cytoplasmic-nuclear distribution of miRNAs and AGO1 but reduced the amount of miRNAs associated with AGO1. These results indicate that TRN1 positively regulates miRNA activity by promoting the association of miRNAs with AGO1, and they reveal opposing roles of two importin ß family proteins in miRNA loading.

SERPINH1 overexpression in clear cell renal cell carcinoma: association with poor clinical outcome and its potential as a novel prognostic marker.

Precision therapy for clear cell renal cell carcinoma (ccRCC) requires molecular biomarkers ascertaining disease prognosis. In this study, we performed integrated proteomic and transcriptomic screening in all four tumour-node-metastasis stages of ccRCC and adjacent normal tissues (n = 18) to investigate differentially expressed genes. Most identified differentially expressed genes revealed a strong association with transforming growth factor-ß level and the epithelial-to-mesenchymal transition process. Of them, Serpin peptidase inhibitor clade H member 1 (SERPINH1) revealed the strongest association with poor prognosis and regulation on the expression levels of epithelial-to-mesenchymal transition markers. Subsequently, two independent sets (n = 532 and 105) verified the high level of SERPINH1 in ccRCC tissues and its association with reduced overall survival and disease-free survival in all tumour-node-metastasis stages and patients with von Hippel-Lindau wild-type (VHL-WT). SERPINH1 was an independent predictor of poor overall survival (hazard ratio 0.696 for all patients) and disease-free survival (hazard ratio 0.433 for all patients and 0.362 for patients with VHL-WT) in ccRCC. We have thus shown for the first time that SERPINH1 is an independent precision predictor for unfavourable prognosis in ccRCC. This could assist in identifying patients who need early aggressive management and deepen our understanding of the pathogenesis of VHL-WT ccRCC.