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BACKGROUND: TNRC6B deficiency syndrome, also known as global developmental delay with speech and behavioral abnormalities (MIM 619243), is a rare autosomal dominant genetic disease mainly characterized by facial dysmorphism, developmental delay/intellectual disability (DD/ID), speech and language delay, fine and motor delay, attention deficit and hyperactivity disorder (ADHD), and variable behavioral abnormalities. It is caused by heterozygous variant in the TNRC6B gene (NM_001162501.2, MIM 610740), which encodes the trinucleotide repeat-containing adaptor 6B protein. METHODS: In this study, two Chinese patients with TNRC6B deficiency syndrome were recruited, and genomic DNA extraction from peripheral blood leukocytes of these parents and their family members was extracted for whole-exome sequencing and Sanger sequencing. RESULTS: Here, we report two unrelated Chinese patients diagnosed with TNRC6B deficiency syndrome caused by novel de novo likely pathogenic or pathogenic TNRC6B variants c.335C>T (p.Pro112Leu) and c.1632delC (p.Leu546fs*63), which expands the genetic spectrum of TNRC6B deficiency syndrome. The clinical features of the patients were DD/ID, delayed speech, ADHD, behavioral abnormalities, short stature, low body weight, café-au-lait spots, metabolic abnormalities, and facial dysmorphism including coarse facial features, sparse hair, frontal bossing, hypertelorism, amblyopia, strabismus, and downslanted palpebral fissures, which expands the phenotype spectrum associated with TNRC6B deficiency syndrome. CONCLUSION: This study expands the genotypic and phenotypic spectrum of TNRC6B deficiency syndrome. Our findings indicate that patients with TNRC6B deficiency syndrome should be monitored for growth and metabolic problems and therapeutic strategies should be developed to address these problems. Our report also suggests the clinical diversity of TNRC6B deficiency syndrome.

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Cancer microenvironment plays an important role in the proliferation and metastasis of hepatocarcinoma cancer cells (HCC). Exosomes from bone marrow-derived mesenchymal stem cells (BMSCs) are a component of the cancer microenvironment. In this study, we reveal that miRNA-652-3P from BMSC-derived exosomes promotes proliferation and metastasis in HCC. The ability of cancer proliferation, migration and invasion can be evaluated after co-culture by CCK-8, wound healing and transwell assay. Isolated exosomes were identified by transmission electron microscopy (TEM) and the biomarkers of the purified exosomes were showed in West-blotting (WB). MiR-652-3p was detected in the HepG2 and 7721 after co-culturing with exosome derived from BMSCs under different conditions. Target authentication was performed by a luciferase reporter assay to confirm the presumptive target of miR-652-3p. After overexpressing miR-652-3p, the mRNA and protein expression level of TNRC6A in HCC was examined by q-PCR and WB. Further, we observed greater miR-652-3p upregulation in hypoxic BMSCs-exosomes than in normal- exosomes. In addition, a miR-652-3p inhibitor attenuates the proliferation and metastasis of HCC cells after co-culturing with BMSCs. Our data demonstrate that hypoxic BMSCs-derived exosomal miR-652-3p promotes proliferation in HCC cells by inhibiting TNRC6A. The BMSCs-derived exosomal miR-652-3p may help find patient-targeted therapies in hepatocarcinoma cancer.

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It was revealed in our previous study that the expression of miR-30c-5p in the skeletal muscle of rabbits fed high-fat diet was highly expressed. In the present study, we further investigated the function of miR-30c-5p in proliferation and differentiation of skeletal muscle satellite cell (SMSC). The results obtained in the present study showed that the skeletal muscle fibers of the rabbits fed the standard normal diet (SND) were orderly, regular, and uniform after HE staining, however, the muscle fibers of the rabbits fed the high-fat diet (HFD) were generally atrophied, some were arranged disorderly, the intercellular space was enlarged, the nucleus was increased, and the morphology and position were abnormal. Compared with the SND group, it was observed that the weekly weight gain and fat percentage were relatively higher, and also the levels of the serum biochemical indexes such as glucose, cholesterol, and triglyceride increased significantly in the rabbits fed with HFD. In addition, the results after the transfection of miR-30c-5p mimic, mimic NC (negative control), miR-30c-5p inhibitor, and inhibitor NC into the SMSCs showed that the cell counting kit-8 (CCK-8) proliferation experiment suggested that the number of cells in the over expression group was significantly lower than that in the mimic NC group at 48, 72, 96 h of cell proliferation. At 48, 72, 120 h, the number of cells in the inhibitor group was significantly higher than that in the mimic NC group. The number of EdU positive cells decreased significantly in the over expression group compared with the mimic NC group, however, it increased significantly in the inhibitor group compared with the inhibitor NC group. Moreover, compared with the mimic NC group, the myotube area increased significantly in the miR-30c-5p mimic group, whereas it decreased significantly in the miR-30c-5p inhibitor group as compared with the inhibitor NC group. In addition, we found that trinucleotide repeat containing adaptor 6A (TNRC6A) was successfully validated as a target gene for miR-30c-5p. The expression of TNRC6A in the miR-30c-5p mimic group was significantly lower than that in the mimic NC group. It was further observed that the expression of TNRC6A increased significantly in the miR-30c-5p inhibitor group as compared to that in the inhibitor NC group. Taken together, the results obtained in this study showed that miR-30c-5p promotes the differentiation as well as inhibits the proliferation of rabbit skeletal muscle satellite cells, and TNRC6A is a target gene of miR-30c-5p.

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The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its targets through RNA-supported interaction with TNRC6/GW182, a core component of the miRNA-induced silencing complex (miRISC). We demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that the loss of AGO proteins or of AGO binding to TNRC6 enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that the use of TNRC6 as a central hub provides a means to integrate their activities.

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The potential for microRNAs (miRNAs) to regulate gene expression remains incompletely understood. DROSHA initiates the biogenesis of miRNAs while variants of Argonaute (AGO) and trinucleotide repeat containing six (TNRC6) family proteins form complexes with miRNAs to facilitate RNA recognition and gene regulation. Here we investigate the fate of miRNAs in the absence of these critical RNAi protein factors. Knockout of DROSHA expression reduces levels of some miRNAs annotated in miRBase but not others. The identity of miRNAs with reduced expression matches the identity of miRNAs previously identified by experimental approaches. The MirGeneDB resource offers the closest alignment with experimental results. In contrast, the loss of TNRC6 proteins had much smaller effects on miRNA levels. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of the miRNAs most strongly associated with AGO2 as determined from enhanced crosslinking immunoprecipitation (AGO2-eCLIP). Evaluation of miRNA binding to endogenously expressed AGO proteins revealed that miRNA:AGO association was similar for AGO1, AGO2, AGO3, and AGO4. Our data emphasize the need to evaluate annotated miRNAs based on approximate cellular abundance, DROSHA-dependence, and physical association with AGO when forming hypotheses related to their function.

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Previous studies have uncovered the key role of circular RNAs (circRNAs) in various diseases, including cancer. However, the growth-inhibitory effects of circRNAs on esophageal squamous cell carcinoma (ESCC) have not been completely elucidated. This study characterized a newly identified circRNA derived from exons 9-13 of TNRC6B (named circ-TNRC6B). The expression of circ-TNRC6B in ESCC tissues was markedly downregulated when compared to that in non-tumor tissues. In 53 ESCC cases, circ-TNRC6B expression was negatively correlated with the T stage. Multivariate Cox regression analysis showed that circ-TNRC6B upregulation was an independent protective factor for ESCC patients' prognosis. Overexpression and knockdown functional experiments demonstrated that circ-TNRC6B inhibited ESCC cell proliferation, migration, and invasion. RNA immunoprecipitation and dual-luciferase reporter assays demonstrated that circ-TNRC6B sponges oncogenic miR-452-5p to upregulate the expression and activity of DAG1. Treatment with miR-452-5p inhibitor partially reversed the circ-TNRC6B-induced changes in the biological behavior of ESCC cells. These findings demonstrated that circ-TNRC6B exerts a tumor-suppressing effect in ESCC through the miR-452-5p/DAG1 axis. Thus, circ-TNRC6B is a potential prognostic biomarker for the clinical management of ESCC.

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Non-small cell lung cancer (NSCLC) accounts for over 80% of lung cancer cases and have poor clinical outcomes. Increasing number of lncRNAs are reported to be implicated in the carcinogenesis of NSCLC. Previous lncRNA-seq results showed that LINC01082 was under-expressed in several cancer types. In the current study, we focused on the role of LINC01082 in NSCLC development. An online bioinformatics tool was utilized to assess the expression profile of LINC01082, miR-543, and TNRC6A in NSCLC samples. RT-qPCR analysis was performed for evaluating LINC01082, TNRC6A and miR-543 expression in cells (NSCLC cells vs. normal lung cells). Impact of LINC01082 upregulation on cell proliferation in vitro was investigated by MTT and EdU experiments. Transwell assay was applied to analyze the migration and invasion of NSCLC cells. The cell apoptosis after plasmid transfection was detected by flow cytometry. The interactions among LINC01082, miR-543 and TNRC6A were measured by RNA pulldown and luciferase reporter assays. We showed that LINC01082 levels were downregulated in NSCLC samples and NSCLC cells. Overexpression of LINC01082 inhibited NSCLC cell proliferation, migration and invasion and strengthened cell apoptosis. LINC01082 directly bound to miR-543, and miR-543 targeted TNRC6A. TNRC6A was downregulated and miR-543 was overexpressed in NSCLC cells. miR-543 inhibition suppressed malignant cellular behaviors. TNRC6A knockdown reversed the effects of LINC01082 on the malignant character of NSCLC cells. In conclusion, LINC01082 exerts an antioncogenic role in NSCLC via interaction with miR-543 to regulate TNRC6A expression.

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RNA interference is almost always associated with post-transcriptional silencing in the cytoplasm. MicroRNAs (miRNAs) and critical RNAi protein factors like argonaute (AGO) and trinucleotide repeat binding containing 6 protein (TNRC6), however, are also found in cell nuclei, suggesting that nuclear miRNAs may be targets for gene regulation. Designed small duplex RNAs (dsRNAs) can modulate nuclear processes such as transcription and splicing, suggesting that they can also provide leads for therapeutic discovery. The goal of this Perspective is to provide the background on nuclear RNAi necessary to guide discussions on whether nuclear RNAi can play a role in therapeutic development programs.

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BACKGROUND: Hepatocellular carcinoma (HCC) was one of the most prevalent life-threatening cancers. Metastasis is the leading cause of cancer-related death in HCC. MiRNAs play essential roles in cancer metastasis. METHODS: Expression of miR-652-3p in HCC was assessed. Function experiments of miR-652-3p and trinucleotide repeat-containing gene 6A protein (TNRC6A) were performed both in vitro and in vivo. mRNA sequencing, PCR, and western blot were performed to verify the target genes and pathway of miR-652-3p. The lung metastasis and xenograft cancer model in nude mice was established to investigate the effects of the miR-652-3p and TRNC6A on tumor metastasis in vivo. The relationship between the expression of the miR-652-3p, TNRC6A and the prognosis of HCC patients was analyzed. RESULTS: Upregulated miR-652-3p was found in the tumor tissues of HCC, especially in metastatic HCC patients. Overexpression of miR-652-3p promoted and knockdown of miR-652-3p suppressed HCC metastasis both in vitro and in vivo. MiR-652-3p promoted HCC metastasis via regulating the EMT pathway. TNRC6A was identified as a direct target of miR-652-3p, and the knockdown of TNRC6A restored repressed EMT and HCC metastasis caused by the inhibition of miR-652-3p. Clinical results revealed that high expression of miR-652-3p and low expression of TNRC6A were positively correlated to shortened overall survival and disease-free survival in HCC patients. CONCLUSIONS: MiR-652-3p promotes EMT and HCC metastasis by inhibiting TNRC6A expression in HCC. MiR-652-3p and TNRC6A may serve as potential biomarkers to predict prognosis in HCC patients with metastasis.

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GW182 family proteins are a key component of microRNA-protein complex eliciting translational repression and/or degradation of microRNA-targets. The microRNAs in complex with Argonaute proteins bind to target mRNAs, and GW182 proteins are recruited by association with Argonaute proteins. The GW182 protein acts as a scaffold that links the Argonaute protein to silencing machineries including the CCR4-NOT complex which accelerates deadenylation and inhibits translation. The carboxyl-terminal effector domain of GW182 protein, also called the silencing domain, has been shown to bind to the subunits of the CCR4-NOT complex, the CNOT1 and the CNOT9. Here we show that a small region within the amino-terminal Argonaute-binding domain of human GW182/TNRC6A can associate with the CCR4-NOT complex. This region resides between the two Argonaute-binding sites and contains reiterated GW/WG-motifs. Alanine mutation experiments showed that multiple tryptophan residues are required for the association with the CCR4-NOT complex. Furthermore, co-expression and immunoprecipitation assays suggested that the CNOT9 subunit of the CCR4-NOT complex is a possible binding partner of this region. Our work, taken together with previous studies, indicates that the human GW182 protein contains multiple binding interfaces to the CCR4-NOT complex.

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The Argonaute (AGO) and the Trinucleotide Repeat Containing 6 (TNRC6) family proteins are the core components of the mammalian microRNA-induced silencing complex (miRISC), the machinery that mediates microRNA function in the cytoplasm. The cytoplasmic miRISC-mediated post-transcriptional gene repression has been established as the canonical mechanism through which AGO and TNRC6 proteins operate. However, growing evidence points towards an additional mechanism through which AGO and TNRC6 regulate gene expression in the nucleus. While several mechanisms through which miRISC components function in the nucleus have been described, in this review we aim to summarize the major findings that have shed light on the role of AGO and TNRC6 in mammalian chromatin biology and on the implications these novel mechanisms may have in our understanding of regulating gene expression.

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The androgen receptor (AR) signaling pathway plays an important role in the initiation and progression of prostate cancer. Circular RNAs (circRNAs), the novel noncoding RNAs without 5' to 3' polarity or 3' poly (A), play an important role in multiple diseases. However, the potential roles of androgen-responsive circRNAs in prostate cancer remain unclear. In this study, we identified 3237 androgen-responsive circRNAs and 1954 androgen-responsive mRNAs after dihydrotestosterone (DHT) stimulation using microarray. Among them, the expression of 1296 androgen-responsive circRNAs was consistent with that of their parent genes, and we thought AR might regulate the expression of these circRNAs at the transcriptional level. In addition, 1941 circRNAs expression was not consistent with their parent genes, and we speculated that AR may regulate the expression of those circRNAs at the posttranscriptional level through affecting alternative splicing. Analyzing the androgen-responsive circRNAs regulated at the posttranscriptional level, we identified two key RNA binding proteins (RBPs), WTAP and TNRC6, using the circInteractome database, which may play important role in the biogenesis of androgen-responsive circRNAs. Furthermore, we explored the potential biological functions and predicted the molecular mechanisms of two dysregulated circRNAs (circNFIA and circZNF561) in prostate cancer. In this study, we revealed that circNFIA was upregulated in prostate cancer tissues and plasma samples from patients with prostate cancer; circNFIA may play an oncogenic role in prostate cancer. In contrast, circZNF561 was downregulated and may act as a tumor suppressor in prostate cancer. Our results suggest that androgen-responsive circRNAs might regulate the progression of prostate cancer and could be novel diagnostic biomarkers.

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BACKGROUND: Considering the combined role of long non-coding RNA (lncRNAs)-microRNA (miRNA)-mRNA in tumorigenesis, the purpose of this study was to investigate how TNRC6C-AS1 regulates the expression of lysophosphatidic acid receptor 5 (LPAR5) by modulating miR-513c-5p, thus influencing the progression of thyroid cancer (THCA). METHODS: qRT-PCR and Western blotting were performed to detect the expression levels of TNRC6C-AS1, miR-513c-5p, and LPAR5 in THCA tissues and cell lines. The viability, proliferation, migration, and invasion were assessed using CCK-8, BrdU, wound healing, and transwell migration assays, respectively. Dual-luciferase reporter assay, RIP assay, and RNA pull-down assay were used to evaluate the relationship between TNRC6C-AS1, miR-513c-5p, and LPAR5. RESULTS: TNRC6C-AS1 was highly expressed in THCA tissues, and knockout of TNRC6C-AS1 reduced the viability, proliferation, migration, and invasion of THCA cells. TNRC6C-AS1 competitively adsorbed miR-513c-5p. In addition, the biological function of TNRC6C-AS1 was blocked by knocking down the thyroid cell line TNRC6C-AS1 with miR-513c-5p inhibitor transfection. LPAR5 is the target gene for miR-513c-5p, which has the ability to eliminate the influence of miR-513c-5p on THCA cells. CONCLUSION: The TNRC6C-AS1/miR-513c-5p/LPAR5 axis is a novel signaling pathway that modulates THCA progression and may be a potential target for cancer therapy.

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Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss-of-function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.

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Precise development of the dendritic architecture is a critical determinant of mature neuronal circuitry. MicroRNA (miRNA)-mediated regulation of protein synthesis plays a crucial role in dendritic morphogenesis, but the role of miRNA-induced silencing complex (miRISC) protein components in this process is less studied. Here, we show an important role of a key miRISC protein, the GW182 paralog TNRC6A, in the regulation of dendritic growth. We identified a distinct brain region-specific spatiotemporal expression pattern of GW182 during rat postnatal development. We found that the window of peak GW182 expression coincides with the period of extensive dendritic growth, both in the hippocampus and cerebellum. Perturbation of GW182 function during a specific temporal window resulted in reduced dendritic growth of cultured hippocampal neurons. Mechanistically, we show that GW182 modulates dendritic growth by regulating global somatodendritic translation and actin cytoskeletal dynamics of developing neurons. Furthermore, we found that GW182 affects dendritic architecture by regulating the expression of actin modulator LIMK1. Taken together, our data reveal a previously undescribed neurodevelopmental expression pattern of GW182 and its role in dendritic morphogenesis, which involves both translational control and actin cytoskeletal rearrangement. This article has an associated First Person interview with the first author of the paper.

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TNRC6 is a scaffolding protein that bridges interactions between small RNAs, argonaute (AGO) protein, and effector proteins to control gene expression. There are three paralogs in mammalian cells, TNRC6A, TNRC6B, and TNRC6C These paralogs have ∼40% amino acid sequence identity and the extent of their unique or redundant functions is unclear. Here, we use knockout cell lines, enhanced crosslinking immunoprecipitation (eCLIP), and high-throughput RNA sequencing (RNA-seq) to explore the roles of TNRC6 paralogs in RNA-mediated control of gene expression. We find that the paralogs are largely functionally redundant and changes in levels of gene expression are well-correlated with those observed in AGO knockout cell lines. Splicing changes observed in AGO knockout cell lines are also observed in TNRC6 knockout cells. These data further define the roles of the TNRC6 isoforms as part of the RNA interference (RNAi) machinery.

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Human GW182 family proteins have Argonaute (AGO)-binding domains in their N-terminal regions and silencing domains, which interact with RNA silencing-related proteins, in their C-terminal regions. Thus, they function as scaffold proteins between the AGO protein and RNA silencing-related proteins, such as carbon catabolite repressor4-negative on TATA (CCR4-NOT) or poly(A)-binding protein (PABP). Our mass spectrometry analysis and the phosphorylation data registered in PhosphoSitePlus, a post-translational modification database, suggested that the C-terminal region of a human GW182 family protein, TNRC6A, has at least four possible phosphorylation sites, which are located near the region interacting with the CCR4-NOT complex. Among them, two serine residues at amino acid positions 1332 and 1346 (S1332 and S1346) were certainly phosphorylated in human HeLa cells, but other two serine residues (S1616 and S1691) were not phosphorylated. Furthermore, it was revealed that the phosphorylation patterns of TNRC6A affect the interaction with the CCR4-NOT complex. When S1332 and S1346 were dephosphorylated, the interactions of TNRC6A with the CCR4-NOT complex were enhanced, and when S1616 and S1691 were phosphorylated, such interaction was suppressed. Thus, phosphorylation of TNRC6A was considered to regulate the interaction with RNA silencing-related factors that may affect RNA silencing activity.

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In cells, closely spaced microRNA (miRNA) target sites within a messenger RNA (mRNA) can act cooperatively, leading to more repression of the target mRNA than expected by independent action at each site. Using purified miRNA-Argonaute (AGO2) complexes, synthetic target RNAs, and a purified domain of TNRC6B (GW182 in flies) that is able to simultaneously bind multiple AGO proteins, we examined both the occupancies and binding affinities of miRNA-AGO2 complexes and target RNAs with either one site or two cooperatively spaced sites. On their own, miRNA-AGO2 complexes displayed little if any cooperative binding to dual sites. In contrast, in the presence of the AGO-binding region of TNRC6B, we observed strong cooperative binding to dual sites, with almost no singly bound target RNAs and substantially increased binding affinities and Hill coefficients. Cooperative binding was retained when the two sites were for two different miRNAs or when the two sites were bound to miRNAs loaded into two different AGO paralogs, AGO1 and AGO2. The improved binding affinity was attributable primarily to a reduced rate of dissociation between miRNA-AGO complexes and their dual-site targets. Thus, the multivalent binding of TNRC6 enables cooperative binding of miRNA-AGO complexes to target RNAs, thereby explaining the basis of cooperative action.

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Head and neck squamous cell carcinoma (HNSCC) is a common cancer in China, which was mainly caused by smoking and HPV infection. With the advancement of molecular research, it is meaningful to explore the biomarkers of HNSCC. LINC01207 (small integral membrane protein 31, also known as SMIM31) is a verified oncogene in colorectal adenocarcinoma. Present study aimed to explore the function of LINC01207 in HNSCC cells. Function assays including EdU, colony formation, TUNEL and JC-1 assay revealed that LINC01207 was an oncogene in HNSCC cells. Next, by some mechanism assays including RIP assay and luciferase reporter assay, miR-5047 was identified as the downstream gene of LINC01207. Subsequently, trinucleotide repeat containing adaptor 6B (TNRC6B) was verified as the target of miR-5047. LINC01207 boosted HNSCC cell proliferation and stemness characteristics via acting as a ceRNA of TNRC6B to bind miR-5047. Then, we identified that transcription of both LINC01207 and TNRC6B was induced by FOXA1, which played a tumor facilitator role in HNSCC cells. In a word, present study uncovered a novel ceRNA mechanism of LINC01207/miR-5047/TNRC6B in HNSCC cells, which might contribute to HNSCC treatment.

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