Prevalence of DICER1 variants in large multinodular goiter: thyroid function, clinical and imaging characteristics

ABSTRACT Objective: Mutations in DICER1 are found in differentiated thyroid carcinoma (DTC) and in multinodular goiter (MNG) at a younger age with other tumors, which characterizes DICER1 syndrome. DICER1 is one driver to DTC; however, it is also found in benign nodules. We speculated that patients with mutations in DICER1 may present long-lasting MNG. Our aim was to investigate the frequency of DICER1 variants in patients with MNG. Subjects and methods: Patients who submitted to total thyroidectomy due to large MNG with symptoms were evaluated. DICER1 hotspots were sequenced from thyroid nodule samples. To confirm somatic mutation, DNA from peripheral blood was also analyzed. Results: Among 715 patients, 154 were evaluated with 56.2 ± 12.3 years old (28-79) and the thyroid volume was 115.7 ± 108 mL (16.2-730). We found 11% with six DICER1 variations in a homo or heterozygous state. Only rs12018992 was a somatic DICER1 variant. All remaining variants were synonymous and likely benign, according to the ClinVar database. The rs12018992 was previously described in an adolescent with DTC, measuring 13 mm. There were no significant differences according to gender, familial history of goiter, age, thyroid volume, TSH and TI-RADS classification between DICER1 carriers. Free T4 were lower in patients with DICER1 polymorphisms (13.77 ± 1.8 vs. 15.44 ± 2.4 pmol/L, p = 0.008), regardless of TSH levels. Conclusions: We conclude that germline DICER1 variants can be found in 11% of large goiters but no second-hit somatic mutation was found. DICER1 is one driver to thyroid lesion and a second-hit event seems unnecessary in the MNG development.

Pituitary blastoma, expanding the spectrum of newly recognized entities. A case report

Tumors of the pituitary gland and sellar region represent about 15% of all brain tumors, with pituitary adenoma being the commonest and pituitary carcinoma being very rare. Pituitary tumors in children are even rarer. Pituitary blastoma, a pediatric adenohypophysial tumor, is a new entity described in the 2017 WHO classification of pituitary tumors. This is a very rare tumor with only 21 cases reported so far. Hence, we are reporting this unusual case seen in a 7-month-old infant who presented with a large sellar/suprasellar mass with pressure symptoms of short duration. Typically, they present between 7–24 months of age. On histopathology, a cellular tumor was seen with primitive-looking round cells with scanty cytoplasm with few well-defined gland or rosette-like structures. The immunohistochemical stains showed diffuse strong staining for synaptophysin with a very high MIB-1 index. Other markers for common round cell tumors in this age group and hormonal markers of pituitary tumors were negative with INI-1 being intact. The initial cases described by Scheithauer presented with Cushing's disease and at least focally expressed adrenocorticotrophic hormone on immunohistochemistry. However, nonfunctioning tumors are also seen, albeit rarely. These are known to be associated with DICER 1 mutations and have a poor prognosis. Hence, morphologic recognition in the right clinical context and excluding other differential diagnoses in infants help make the correct diagnosis.

Antisense noncoding mitochondrial RNA-2 gives rise to miR-4485-3p by Dicer processing in vitro

BACKGROUND: The antisense noncoding mitochondrial RNAs (ASncmtRNAs) derive from the mitochondrial 16S gene. Knockdown of these transcripts with chemically-modified antisense oligonucleotides induces proliferative arrest, apoptosis and invasiveness reduction in tumor but not normal cells. One of these transcripts, ASncmtRNA-2, contains the complete and identical sequence of hsa-miR-4485-3p and, upon knockdown of this transcript, there is a strong increase in levels of this miRNA, suggesting ASncmtRNA-2 as a source for miR-4485-3p, which is supported by several evidences from our group and others, in the ex vivo setting. RESULTS: Here we show that incubation of in vitro-transcribed ASncmtRNA-2 with recombinant Dicer produces RNA fragments corresponding to hsa-miR-4485-3p, showing that Dicer binds to and processes ASncmtRNA-2, strongly supporting the hypothesis that ASncmtRNA-2 acts as a precursor for miR-4485-3p. CONCLUSION: The in vitro results presented here strengthen the hypothesis that miR-4485-3p is derived from ASncmtRNA-2 by Dicer processing. Since miR-4485-3p is classified as a tumor suppressor miRNA, this evidence strengthens the application of ASncmtRNA knockdown for cancer therapy.

Mechanism of TWEAK promoting macrophage-derived exosomal miR-7 to epithelial ovarian cancer cell through regulating Dicer / 上海交通大学学报（医学版）

Objective • To investigate the mechanism of tumor necrosis factor like weak inducer of apoptosis (TWEAK) promoting macrophagederived exosomal miR-7 to epithelial ovarian cancer (EOC). Methods • The expression of Dicer was detected by real-time PCR and Western blotting in TWEAK-stimulated macrophages. The expression of miR-7 was detected by real-time PCR in the macrophage and macrophage-derived exosome after silencing Dicer in macrophage. While in Dicer-overexpressing macrophage, real-time PCR was used to detect the expression of miR-7. Then TWEAK was used to stimulate the macrophage, and the expression of miR-7 in the macrophage and macrophage-derived exosome was detected by real-time PCR. The expression of key proteins in the nuclear factor-κB (NF-κB) pathway was detected by Western blotting in TWEAK-stimulated macrophage. After pretreatment of NF-κB inhibitor, Western blotting was used to detect the expression of Dicer and key proteins in the NF-κB pathway in TWEAKstimulated macrophage. Results • The expression of Dicer in macrophage was down-regulated after TWEAK stimulating. The expression of miR-7 was up-regulated in Dicer-silencing macrophage and macrophage-derived exosome. While the expression of miR-7 was down-regulated in the macrophage and the macrophage-derived exosome in Dicer-overexpressing macrophage after TWEAK stimulating. The expression of key proteins in the NF-κB pathway in macrophage was also up-regulated after TWEAK stimulating. After inhibition of NF-κB signaling pathway, the expression of Dicer was not significantly changed in TWEAK-stimulated macrophage compared to the control group. Conclusion • TWEAK can active NF-κB pathway and inhibit the expression of Dicer to promote macrophage-derived exosomal miR-7 to EOC cells.

Dicer Is Down-regulated and Correlated with Drosha in Idiopathic Sudden Sensorineural Hearing Loss

Previously, we reported the expression levels of specific microRNA machinery components, DGCR8 and AGO2, and their clinical association in patients with idiopathic sudden hearing loss (SSNHL). In the present study, we investigated the other important components of microRNA machinery and their association with clinical parameters in SSNHL patients. Fifty-seven patients diagnosed with SSNHL and fifty healthy volunteers were included in this study. We evaluated mRNA expression levels of Dicer and Drosha in whole blood of patients with SSNHL and the control group, using RT & real-time PCR analysis. The Dicer mRNA expression level was down-regulated in patients with SSNHL. However, the Drosha mRNA expression level was not significantly altered in patients with SSNHL. Neither the Dicer nor Drosha mRNA expression level was not associated with any clinical parameters, including age, sex, duration of initial treatment from onset (days), initial Pure tone average, Siegel's criteria, WBC, and Erythrocyte sedimentation rate. However, mRNA expression levels of Dicer and Drosha were positively correlated to each other in patients with SSNHL. In this study, we demonstrated for the first time that the Dicer mRNA expression level was down-regulated in patients with SSNHL, suggesting its important role in pathobiology of SSNHL development.

Meta analysis for the association between a genetic variant in gene Dicer and cancer susceptibility / 国际检验医学杂志

Objective To investigate the association between a genetic variant in Dicer and cancer risk .Methods Both English and Chi-nese databases were carefully searched comprehensively .Based on the including and excluding criteria ,literatures that were eligible were carefully screened and data were retrieved .Meta analysis was performed by RevMan 5 .1 software .Pooled odds ratios(OR) and 95% confi-dential interval(CI)were used for analysis .Results Ten eligible literatures were included in the meta analysis .Meta analysis showed that the mutant allele C in 3′untranslated region polymorphism loci rs1057035 of Dicer gene might reduce the cancer risk(OR=0 .92 ,95% CI=0 .87-0 .97 ,P=0 .003) .Conclusion The variant rs1057035 T>C in Dicer gene could be related to cancer susceptibility .

Dicer knockdown enhance the proliferation, migration and invasion of cholangiocarcinoma cell line TFK-1 / 中华肝胆外科杂志

Objective To investigate the effect of Dicer knockdown on the proliferation,migration and invasion of cholangiocarcinoma cell line TFK-1.Methods The study used liposome-mediated method to transfect Dicer-siRNA into TFK-1 cells.The mRNA and protein expressions of Dicer after transfection were detected by real-time PCR and western blot.CCK8 assay,scratch test and transwell assays were performed to analyze the influence on the proliferation,migration and invasion of cells,respectively.Results Compared with the control,Dicer mRNA level at 48 h and Dicer protein level at 96 h were both significantly reduced after transfection with Dicer-siRNA (P ＜ 0.01).CCK8 assay revealed that the absorbance value significantly increased at 72 h,96 h and 120 h after transfection (P ＜0.01).Scratch test and transwell assay showed that the migration ability and the number of invasive cells significantly increased (P ＜ 0.05).Conclusion Dicer knockdown enhanced the proliferation,migration and invasion of cholangiocarcinoma cell line TFK-1.

Significance and expression of PIWIL1 protein and DICER enzyme in hepatocellular carcinoma / 中华肝胆外科杂志

Objective To explore the expression of PIWIL1 protein and DICER enzyme in hepa tocellular carcinoma (HCC) and their significance.Methods Immunohistochemical method was used to detect the expression of PIWIL1 and DICER in 47 cases of HCC and the adjacent HCC tissues.Western blot method was used to detect the expression of PIWIL1 and DICER in 31 cases of fresh HCC tissues and their adjacent HCC tissues.The relationship between PIWIL1 and DICER and their relationships were analysed with clinical features.12 cases of normal liver tissues were used as control group.Results The expression of PIWIL1 was high in HCC but low in normal liver tissues (P＜ 0.05).The expression of DICER was high in normal liver tissues but low in HCC (P＜0.05).The expression of PIWIL1 was positively correlated with invasion to adjacent tissues and histological differentiation (P＜0.05).The expression of DICER was negatively correlated with invasion to the adjacent tissues and histological differentiation (P＜0.05).There was a negative correlation between PIWIL1 and DICER (P＜ 0.05).Conclusions High expression of PIWIL1 and low/missing expression of DICER was related to pathological differentiation and invasion of adjacent tissues.

Conditional deletion of the human ortholog gene Dicer1 in Pax2-Cre expression domain impairs orofacial development.

Background: Orofacial clefts are common worldwide and result from insufficient growth and/or fusion during the genesis of the derivatives of the first pharyngeal arch and the frontonasal prominence. Recent studies in mice carrying conditional and tissue-specific deletions of the human ortholog Dicer1, an RNAse III family member, have highlighted its importance in cell survival, differentiation, proliferation, and morphogenesis. Nevertheless, information regarding Dicer1 and its dependent microRNAs (miRNAs) in mammalian palatogenesis and orofacial development is limited. Aims: To describe the craniofacial phenotype, gain insight into potential mechanisms underlying the orofacial defects in the Pax2-Cre/Dicer1 CKO mouse, and shed light on the role of Dicer1 in mammalian palatogenesis. Materials And Methods: Histological and molecular assays of wild type (WT) and Pax2-Cre/Dicer1 loxP/loxP (Dicer1 CKO) mice dissected tissues have been performed to characterize and analyze the orofacial dysmorphism in Pax2-Cre/Dicer1 loxP/loxP mouse. Results: Dicer1 CKO mice exhibit late embryonic lethality and severe craniofacial dysmorphism, including a secondary palatal cleft. Further analysis suggest that Dicer1 deletion neither impacts primary palatal development nor the initial stages of secondary palatal formation. Instead, Dicer1 is implicated in growth, differentiation, mineralization, and survival of cells in the lateral palatal shelves. Histological and molecular analysis demonstrates that secondary palatal development becomes morphologically arrested prior to mineralization around E13.5 with a significant increase in the expression levels of apoptotic markers (P < 0.01). Conclusions: Pax2-Cre-mediated Dicer1 deletion disrupts lateral palatal outgrowth and bone mineralization during palatal shelf development, therefore providing a mammalian model for investigating the role of miRNA-mediated signaling pathways during palatogenesis.

Suppression of RNA Interference Pathway in vitro by Grass Carp Reovirus / 中国病毒学·英文版

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined.The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK).The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay.GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells,no GCRV-specific siRNA could be detected.Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene.These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway,unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.

Expression and clinical significance of dicer in hilar cholangiocarcinoma tissues and cells / 中华肝胆外科杂志

ObjectiveTo investigate the relationship between Dicer expression and clinicopathological characteristics and prognosis by detecting the expression of Dicer in hilar cholangiocarcinoma tissues and cells.MethodsThe expression of Dicer in tissues was detected using immunohistochemistry.Western blotting and RT-PCR were used to investigate Dicer expression in QBC939 and HIBEpic cells.The relationship between Dicer expression and clinicopathological characteristics was analyzed.A Kaplan-Maier analysis was performed to analyze the disease-free survival (DFS) and overall survival (OS) after radical surgical resection of hilar cholangiocarcinoma.ResultsWhen compared to control,Dicer was significantly down-regulated in hilar cholangiocarcinoma tissues (P＜0.05) and in QBC939 (P＜0.05).The expression of Dicer was higher in well differentiated adenocarcinoma than poorly and moderately differentiated tumours. Univariate analysis showed low expression of Dicer protein was significantly correlated with short disease-free survival and overall survival of patients with hilar cholangiocarcinoma after radical surgical resection (P＜0.01). Multivariate analysis revealed that the expression of Dicer was the most important factor for predicting prognosis after radical surgical resection of hilar cholangiocarcinoma (P＜0.05).ConclusionsDicer could be used as a prognostic marker for hilar cholangiocarcinoma.

The expression and significance of Dicer in gastric cell lines and gastric cancer tissue / 重庆医科大学学报

Objective:To detect the expression and significance of Dicer in gastric cell lines and gastric cancer tissue.Methods:Western-Blot and reverse transcriptase polymerase chain reaction(RT-PCR) were used to detect the expression of Dicer in normal gastric mucosa cell line GES-1,human gastric cancer cell line SGC-7901,25 surgical removed gastric cancer tissue and paired para-tumor tissue.Results:1.The expression of Dicer in human gastric cancer cell line SGC-7901 was lower than that in normal gastric mucosa cell line GES-1(P

Real time fluorescent quantitative RT-PCR in determination of Dicer mRNA in primary hepatocellular carcinoma / 第二军医大学学报

Objective:To develop a real time fluorescent quantitative reverse transcriptase polymerase chain reaction(RT-PCR) system for determining the expression of Dicer mRNA in human hepatoma cell lines and 20 samples of primary hepatocellular carcinoma(PHC)tissues.Methods: The specific primers,designed according to the complete sequence of Dicer mRNA,and the fluorescence dye SYBR Green Ⅰwere used for RT-PCR amplification.The fluorescence was monitored in a real time manner.The expression levels of Dicer mRNA in samples were calculated according to the standard curve and the nonspecific amplifications were excluded by melting curve analysis.The mRNA levels of Dicer were presented as the ratios of Dicer mRNA to 18S rRNA.Results: The linear detection range of this system was from 5?10~(2)to 5?10~(9)copies/?l and the coefficient of variation values for intra-experimental and inter-experimental reproducibility ranged from 4.13% to 19.72% and from 6.25% to(18.76%,) respectively.The expression levels of Dicer mRNA in HBV positive hepatoma cell line HepG2.2.15 or in HBV negative hepatoma cell line HepG2 were significantly lower(P

Research progresses of RNA interference / 中国药理学通报

RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Although it is a short time since the discovery of the phenomenon, the technology develops fastly for the character of easy operation, low investment, specificity and potentiality, and researchers have made some progress on the mechanism of RNA interference and application in functional genomics and disease treatment. This article reviews the history, mechanism, biological significance and application of RNA interference.