Loss of the Chr16p11.2 ASD candidate gene QPRT leads to aberrant neuronal differentiation in the SH-SY5Y neuronal cell model.

Background: Altered neuronal development is discussed as the underlying pathogenic mechanism of autism spectrum disorders (ASD). Copy number variations of 16p11.2 have recurrently been identified in individuals with ASD. Of the 29 genes within this region, quinolinate phosphoribosyltransferase (QPRT) showed the strongest regulation during neuronal differentiation of SH-SY5Y neuroblastoma cells. We hypothesized a causal relation between this tryptophan metabolism-related enzyme and neuronal differentiation. We thus analyzed the effect of QPRT on the differentiation of SH-SY5Y and specifically focused on neuronal morphology, metabolites of the tryptophan pathway, and the neurodevelopmental transcriptome. Methods: The gene dosage-dependent change of QPRT expression following Chr16p11.2 deletion was investigated in a lymphoblastoid cell line (LCL) of a deletion carrier and compared to his non-carrier parents. Expression of QPRT was tested for correlation with neuromorphology in SH-SY5Y cells. QPRT function was inhibited in SH-SY5Y neuroblastoma cells using (i) siRNA knockdown (KD), (ii) chemical mimicking of loss of QPRT, and (iii) complete CRISPR/Cas9-mediated knock out (KO). QPRT-KD cells underwent morphological analysis. Chemically inhibited and QPRT-KO cells were characterized using viability assays. Additionally, QPRT-KO cells underwent metabolite and whole transcriptome analyses. Genes differentially expressed upon KO of QPRT were tested for enrichment in biological processes and co-regulated gene-networks of the human brain. Results: QPRT expression was reduced in the LCL of the deletion carrier and significantly correlated with the neuritic complexity of SH-SY5Y. The reduction of QPRT altered neuronal morphology of differentiated SH-SY5Y cells. Chemical inhibition as well as complete KO of the gene were lethal upon induction of neuronal differentiation, but not proliferation. The QPRT-associated tryptophan pathway was not affected by KO. At the transcriptome level, genes linked to neurodevelopmental processes and synaptic structures were affected. Differentially regulated genes were enriched for ASD candidates, and co-regulated gene networks were implicated in the development of the dorsolateral prefrontal cortex, the hippocampus, and the amygdala. Conclusions: In this study, QPRT was causally related to in vitro neuronal differentiation of SH-SY5Y cells and affected the regulation of genes and gene networks previously implicated in ASD. Thus, our data suggest that QPRT may play an important role in the pathogenesis of ASD in Chr16p11.2 deletion carriers.

Co-targeting of multiple microRNAs on factor-Inhibiting hypoxia-Inducible factor gene for the pathogenesis of head and neck carcinomas.

BACKGROUND: MicroRNAs (miRNAs) are short, noncoding RNAs that inhibit the expression of target genes that play roles in tumorigenesis. MiR-21, miR-31, and miR-184 are oncogenic miRNAs for head and neck squamous cell carcinoma (HNSCC). Factor-inhibiting hypoxia (FIH)-inducible factor is known to inactivate hypoxia-induced downstream effectors and is involved in HNSCC suppression. This study investigates whether miR-21, miR-31, and miR-184 target FIH in HNSCC. METHODS: Reporter assays, Western blot analysis, quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis, and phenotypic assays were used to prove that miR-21, miR-31, and miR-184 directly target FIH. Clinicopathological implications of the gene expression were also analyzed. RESULTS: MiR-21, miR-31, and miR-184 directly bind to various sites in the 3' untranslated region (UTR) of FIH transcript, and this binding is associated with decreased FIH protein expression in HNSCC cells. Treatment with the precursors of these miRNAs increases the proliferation and migration of HNSCC cells. Concomitant treatment with precursors repressed FIH and enhanced oncogenicity most profoundly. Upregulation of miR-21, miR-31, and miR-184 expression is found in more than 80% of HNSCC tumors and 72% of tumors have concordant upregulation of these 3 oncogenic miRNAs. The highest expression of these miRNAs is present in T4b and stage IVB tumors. Downregulation of FIH mRNA expression is noted in 69% of HNSCC tumors, and in tumors exhibiting high expression of these miRNAs, the FIH mRNA expression is consistently downregulated. CONCLUSION: This study provides novel clues indicating that miR-21, miR-31, and miR-184 co-target FIH tumor suppressor during pathogenesis in the vast majority of HNSCC.

Passenger strand miRNA miR-31* regulates the phenotypes of oral cancer cells by targeting RhoA.

OBJECTIVES: MicroRNAs (miRNAs) are endogenous small non-coding RNAs that negatively regular target gene expression by RNA interference. The processing of the pre-miRNA hairpin generates a miRNA duplex, which consists of a miRNA (guide strand) and a miRNA(*) (passenger strand). miR-31 is an oncogenic miRNA and is up-regulated in oral squamous cell carcinoma (OSCC). miR-31(*) shows a high level of conservation across species and, based on this, this study hypothesized that miR-31(*) is a functional miRNA. MATERIALS AND METHODS: The expression of miR-31 and miR-31* in OSCC tissues and oral cells were analyzed. Functional studies were performed on OSCC cells. RESULTS: miR-31(*) is up-regulated in OSCC tissues, but its expression is less abundant than miR-31. miR-31(*) decreases the proliferation and migration of both SAS and Fadu cells. Furthermore, miR-31(*) targets the 3'UTR of RhoA and is able to down-regulate RhoA expression. Knockdown of RhoA expression is known to decrease the proliferation and migration of OSCC cells. However, up-regulation of both miR-31 and miR-31(*) by delivery of pre-mir-31 does still enhance OSCC oncogenicity. CONCLUSION: miR-31(*) is a functional miRNA involving in regulating RhoA, and the activity of miR-31(*)'s activity seems to counteract the functions of miR-31 during OSCC tumorigenesis.

miR-196a overexpression and miR-196a2 gene polymorphism are prognostic predictors of oral carcinomas.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is prevalent worldwide, and survival in OSCC has not improved significantly in the last few decades. MicroRNAs have an important regulatory role in oral carcinogenesis. This study investigated the prognostic implications of miR-196 expression and the rs11614913 genotype of the miR-196a2 gene in OSCC. METHODS: The clinicopathologic implications of miR-196 in OSCC were investigated using expression assays and genotyping, and the functional role of miR-196 in OSCC pathogenesis was investigated using exogenous expression and knockdown. RESULTS: miR-196 was up-regulated in OSCC tissue relative to control mucosa. High expression of miR-196a, but not miR-196b, was associated with tumor recurrence, nodal metastasis, and mortality. Plasma miR-196a levels could be used to distinguish patients from controls with a separating power of 0.75. Multivariate analysis showed that both high miR-196a expression and TT genotype were independent predictors for poor survival in OSCC. The risk of mortality was greatest for patients with high miR-196a level and positive node status. Expression of miR-196 enhanced oncogenesis of OSCC cells, while inhibition of miR-196 expression attenuated such effects. CONCLUSIONS: High miR-196a expression in tumor tissue and the presence of the TT variant of miR-196a2 gene indicate worse survival in OSCC.

miR-221 and miR-222 expression increased the growth and tumorigenesis of oral carcinoma cells.

BACKGROUNDS: MicroRNAs are small noncoding RNAs involved in posttranscriptional gene regulation, which play an important role in both physiological functioning and pathological progression. The miR-221/miR-222 microRNA family has been shown to be related to the neoplastic process in a number of different types of cancers; nevertheless, its function in oral squamous cell carcinoma (OSCC) remained uncertain. MATERIALS AND METHODS: Paired OSCC and matched noncancerous oral mucosa were examined for miR-221/miR-222 expression using quantitative reverse-transcription PCR. Ectopic expression of miR-221/miR-222 by lentiviral infection was investigated to explore its in vitro and in vivo impact on the oncogenic phenotype and the expression of various target genes. The expression of Cip/Kip cell cycle regulator p27 in tumors was analyzed with immunohistochemistry. RESULTS: The expression levels of miR-221 and miR-222 were highly correlated in OSCC. Increased miR-221/miR-222 expression was found in 40% of OSCC tissues. The ectopic expression of miR-221 or of miR-222 increased growth and anchorage-independent colony formation of OSCC cell lines. It also resulted in an increase in the tumorigenesis of an OSCC cell line in nude mice. Western blot analysis suggested that p27 and p57 might be the targets of miR-221/miR-222. p27 expression was reversely associated with the miR-221 and miR-222 expression level in OSCC tissues. CONCLUSIONS: Our findings suggested that increased miR-221/miR-222 expression was associated with the OSCC cell growth.

miR-31 ablates expression of the HIF regulatory factor FIH to activate the HIF pathway in head and neck carcinoma.

MicroRNAs (miRNA) are endogenously expressed noncoding RNAs with important biological and pathological functions that are yet to be fully defined. This study investigated alterations in miRNA expression in head and neck squamous cell carcinoma (HNSCC), the incidence of which is rising throughout the world. Initial screening and subsequent analysis identified a panel of aberrantly expressed miRNAs in HNSCC tissues, with miR-31 among the most markedly upregulated. Ectopic expression of miR-31 increased the oncogenic potential of HNSCC cells under normoxic conditions in cell culture or tumor xenografts. Conversely, blocking miR-31 expression reduced the growth of tumor xenografts. The in silico analysis suggested that miR-31 may target the 3' untranslated region (UTR) of factor-inhibiting hypoxia-inducible factor (FIH), a hypoxia-inducible factor (HIF) regulatory factor that inhibits the ability of HIF to act as a transcriptional regulator under normoxic conditions. In support of this likelihood, miR-31 expression repressed FIH expression and mutations within the predictive miR-31 target site in the FIH 3' UTR abrogated FIH repression. Furthermore, miR-31 expression increased HIF transactivation activity. We found that FIH suppressed oncogenic phenotypes under normoxic conditions and that this activity was abrogated by functional mutations. Lastly, increased miR-31 expression was correlated with decreased levels of FIH in tumor tissues. Our findings suggest that miR-31 contributes to the development of HNSCC by impeding FIH to activate HIF under normoxic conditions.