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Autoimmune thyroid disease (AITD) is the most common human autoimmune disease. The two major clinical manifestations of AITD are Graves’ disease and Hashimoto’s thyroiditis (HT). AITD is characterized by lymphocytic infiltration of the thyroid gland, leading either to follicular cell damage, thyroid gland destruction, and development of hypothyroidism (in HT) or thyroid hyperplasia, induced by thyroid antibodies which activate thyrotropin receptor (TSHR) on thyrocytes, leading to hyperthyroidism. The aim of this review is to present up-to-date picture of the molecular and cellular mechanisms that underlie the pathology of AITD. Based on studies involving patients, animal AITD models, and thyroid cell lines, we discuss the key events leading to the loss of immune tolerance to thyroid autoantigens as well as the signaling cascades leading to the destruction of thyroid gland. Special focus is given on the interplay between the environmental and genetic factors, as well as ncRNAs and microbiome contributing to AITD development. In particular, we describe mechanistic models by which SNPs in genes involved in immune regulation and thyroid function, such as CD40, TSHR, FLT3, and PTPN22, underlie AITD predisposition. The clinical significance of novel diagnostic and prognostic biomarkers based on ncRNAs and microbiome composition is also underscored. Finally, we discuss the possible significance of probiotic supplementation on thyroid function in AITD.

piRNAs (PIWI-interacting RNAs) are small non-coding RNAs capable of regulation of transposon and gene expression. piRNAs utilise multiple mechanisms to affect gene expression, which makes them potentially more powerful regulators than microRNAs. The mechanisms by which piRNAs regulate transposon and gene expression include DNA methylation, histone modifications, and mRNA degradation. Genitourinary cancers (GC) are a large group of neoplasms that differ by their incidence, clinical course, biology, and prognosis for patients. Regardless of the GC type, metastatic disease remains a key therapeutic challenge, largely affecting patients’ survival rates. Recent studies indicate that piRNAs could serve as potentially useful biomarkers allowing for early cancer detection and therapeutic interventions at the stage of non-advanced tumour, improving patient’s outcomes. Furthermore, studies in prostate cancer show that piRNAs contribute to cancer progression by affecting key oncogenic pathways such as PI3K/AKT. Here, we discuss recent findings on biogenesis, mechanisms of action and the role of piRNAs and the associated PIWI proteins in GC. We also present tools that may be useful for studies on the functioning of piRNAs in cancers.

Renal cell cancer is the most frequent kidney malignancy. Most RCC cases are classified as clear cell renal cell carcinoma (ccRCC), characterized by high aggressiveness and poor prognosis for patients. ccRCC aggressiveness is defined by classification systems based on changes in morphology of nucleoli, the membraneless substructures of nuclei. The latter act as the sites of ribosome biogenesis as well as the hubs that trap and immobilize proteins, preventing their action in other cellular compartments. Thereby, nucleoli control cellular functioning and homeostasis. Nucleoli are also the sites of activity of multiple noncoding RNAs, including snoRNAs, IGS RNA, and miRNAs. Recent years have brought several remarkable discoveries regarding the role of nucleolar non-coding RNAs, in particular snoRNAs, in ccRCC. The expression of snoRNAs is largely dysregulated in ccRCC tumors. snoRNAs, such as SNHG1, SNHG4 and SNHG12, act as miRNA sponges, leading to aberrant expression of oncogenes and tumor suppressors, and directly contributing to ccRCC development and progression. snoRNAs can also act without affecting miRNA functioning, by altering the expression of key oncogenic proteins such as HIF1A. snoRNAs are also potentially useful biomarkers of ccRCC progression. Here, we comprehensively discuss the role of nucleolar proteins and non-coding RNAs in ccRCC.

Serine and arginine rich splicing factor 2(SRSF2) belongs to the serine/arginine (SR)-rich family of proteins that regulate alternative splicing. Previous studies suggested that SRSF2 can contribute to carcinogenic processes. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, highly aggressive and difficult to treat, mainly due to resistance to apoptosis. In this study we hypothesized that SRSF2 contributes to the regulation of apoptosis in ccRCC. Using tissue samples obtained from ccRCC patients, as well as independent validation on The Cancer Genome Atlas (TCGA) data, we demonstrate for the first time that expression of SRSF2 is decreased in ccRCC tumours when compared to non-tumorous control tissues. Furthermore, by employing a panel of ccRCC-derived cell lines with silenced SRSF2 expression and qPCR arrays we show that SRSF2 contributes not only to splicing patterns but also to expression of multiple apoptotic genes, including new SRSF2 targets

Type 1 iodothyronine deiodinase (DIO1) regulates peripheral metabolism of thyroid hormones that control cellular proliferation, differentiation and metabolism. The significance of DIO1 in cancer is unknown. In this study we hypothesized that diminished expression of DIO1, observed in renal cancer, contributes to the carcinogenic process in the kidney. Here, we demonstrate that ectopic expression of DIO1 in renal cancer cells changes the expression of genes controlling cell cycle, including cyclin E1 and E2F5, and results in inhibition of proliferation. The expression of genes encoding collagens (COL1A1, COL4A2, COL5A1), integrins (ITGA4, ITGA5, ITGB3) and transforming growth factor-β-induced (TGFBI) is significantly altered in renal cancer cells with induced expression of DIO1. Finally, we show that overexpression of DIO1 inhibits migration of renal cancer cells. In conclusion, we demonstrate for the first time that loss of DIO1 contributes to renal carcinogenesis and that its induced expression protects cells against cancerous proliferation and migration.

Metabolic reprogramming is one of the hallmarks of renal cell cancer (RCC). We hypothesized that altered metabolism of RCC cells results from dysregulation of microRNAs targeting metabolically relevant genes. Combined large-scale transcriptomic and metabolic analysis of RCC patients tissue samples revealed a group of microRNAs that contribute to metabolic reprogramming in RCC. miRNAs expressions correlated with their predicted target genes and with gas chromatography-mass spectrometry (GC-MS) metabolome profiles of RCC tumors. Assays performed in RCC-derived cell lines showed that miR-146a-5p and miR-155-5p targeted genes of PPP (the pentose phosphate pathway) (G6PD and TKT), the TCA (tricarboxylic acid cycle) cycle (SUCLG2), and arginine metabolism (GATM), respectively. miR-106b-5p and miR-122-5p regulated the NFAT5 osmoregulatory transcription factor. Altered expressions of G6PD, TKT, SUCLG2, GATM, miR-106b-5p, miR-155-5p, and miR-342-3p correlated with poor survival of RCC patients. miR-106b-5p, miR-146a-5p, and miR-342-3p stimulated proliferation of RCC cells. The analysis involving &gt, 6000 patients revealed that miR-34a-5p, miR-106b-5p, miR-146a-5p, and miR-155-5p are PanCancer metabomiRs possibly involved in global regulation of cancer metabolism. In conclusion, we found that microRNAs upregulated in renal cancer contribute to disturbed expression of key genes involved in the regulation of RCC metabolome. miR-146a-5p and miR-155-5p emerge as a key &ldquo, metabomiRs&rdquo, that target genes of crucial metabolic pathways (PPP (the pentose phosphate pathway), TCA cycle, and arginine metabolism).

To assess the occurrence of antibiotic-resistant Lactic Acid Bacteria (LAB) in Polish raw milk and fermented artisanal products, a collection comprising 500 isolates from these products was screened. Among these isolates, six strains (IBB28, IBB160, IBB161, IBB224, IBB477 and IBB487) resistant to tetracycline were identified. The strains showing atypical tetracycline resistance were classified as Lactococcus lactis: three of them were identified as L. lactis subsp. cremoris (IBB224, IBB477 and IBB487) and the other three (IBB28, IBB160, IBB161) were identified as L. lactis subsp. lactis. The mechanism involving Ribosomal Protection Proteins (RPP) was identified as responsible for tetracycline resistance. Three of the tested strains (IBB28, IBB160 and IBB224) had genes encoding the TetS protein, whereas the remaining three (IBB161, IBB477 and IBB487) expressed TetM. The results also demonstrated that the genes encoding these proteins were located on genetic mobile elements. The tet(S) gene was found to be located on plasmids, whereas tet(M) was found within the Tn916 transposon.

SRSF1, SRSF2 and hnRNP A1 are splicing factors that regulate the expression of oncogenes and tumor suppressors. SRSF1 and SRSF2 contribute to the carcinogenesis in the kidney. Despite their importance, the mechanisms regulating their expression in cancer are not entirely understood. Here, we investigated the microRNA-mediated regulation of SRSF1, SRSF2 and hnRNP A1 in renal cancer. The expression of microRNAs predicted to target SRSF1, SRSF2 and hnRNP A1 was disturbed in renal tumors compared with controls. Using qPCR, Western blot/ICC and luciferase reporter system assays we identified microRNAs that contribute to the regulation of expression of SRSF1 (miR-10b-5p, miR-203a-3p), SRSF2 (miR-183-5p, miR-200c-3p), and hnRNP A1 (miR-135a-5p, miR-149-5p). Silencing of SRSF1 and SRSF2 enhanced the expression of their targeting microRNAs. miR-183-5p and miR-200c-3p affected the expression of SRSF2-target genes, TNFRSF1B, TNFRSF9, CRADD and TP53. 3'UTR variants of SRSF1 and SRSF2 differed by the presence of miRNA-binding sites. In conclusion, we identified a group of microRNAs that contribute to the regulation of expression of SRSF1, SRSF2 and hnRNP A1. The microRNAs targeting SRSF1 and SRSF2 are involved in a regulatory feedback loop. microRNAs miR-183-5p and miR-200c-3p that target SRSF2, affect the expression of genes involved in apoptotic regulation.

In our previous study we found altered expression of 19 adhesion-related genes in renal tumors. In this study we hypothesized that disturbed expression of adhesion-related genes could be caused by microRNAs: short, non-coding RNAs that regulate gene expression. Here, we found that expression of 24 microRNAs predicted to target adhesion-related genes was disturbed in renal tumors and correlated with expression of their predicted targets. miR-25-3p, miR-30a-5p, miR-328 and miR-363-3p directly targeted adhesion-related genes, including COL5A1, COL11A1, ITGA5, MMP16 and THBS2. miR-363-3p and miR-328 inhibited proliferation of renal cancer cells, while miR-25-3p inhibited adhesion, promoted proliferation and migration of renal cancer cells. TGF-β1 influenced the expression of miR-25-3p, miR-30a-5p, and miR-328. The analyzed microRNAs, their target genes and TGF-β1 formed a network of strong correlations in tissue samples from renal cancer patients. The expression signature of microRNAs linked with TGF-β1 levels correlated with poor survival of renal cancer patients. The results of our study suggest that TGF-β1 coordinates the expression of microRNA network that regulates cellular adhesion in cancer.

SRSF1 jest wielofunkcyjnym białkiem biorącym udział w procesach związanych z metabolizmem RNA. Następstwem zaburzeń ekspresji SRSF1, obserwowanych w wielu typach nowotworów, są nieprawidłowości w składaniu pre-mRNA, zmiany stabilności transkryptów i poziomu translacji onkogenów oraz genów supresorowych. Regulując różnicowe składanie transkryptów genów CCND1, RAC1, KLF6, BCL2L1, MCL1 oraz CASP9, SRSF1 indukuje zmiany w cyklu komórkowym, proliferacji i apoptozie. Czynnik SRSF1 wpływa także na angiogenezę nowotworową i przerzutowanie, m.in. promując powstawanie proangiogennych wariantów VEGF oraz wariantu splicingowego genu RON, który aktywuje proces przejścia nabłonkowo-mezenchymalnego. Ze względu na istotną rolę SRSF1 w rozwoju i progresji nowotworów, białko to jest obiecującym celem terapii przeciwnowotworowych wykorzystujących związki hamujące jego aktywność.W artykule przedstawiono najnowsze informacje o wpływie SRSF1 na nowotworzenie oraz jego potencjalne znaczenie w opracowaniu nowych strategii w leczeniu chorych z nowotworami.

Purpose Cellular metabolism of renal cell carcinoma (RCC) tumors is disturbed. The clinical significance of these alterations is weakly understood. We aimed to find if changes in metabolic pathways contribute to survival of RCC patients. Material and methods 35 RCC tumors and matched controls were used for metabolite profiling using gas chromatography-mass spectrometry and transcriptomic analysis with qPCR-arrays targeting the expression of 93 metabolic genes. The clinical significance of obtained data was validated on independent cohort of 468 RCC patients with median follow-up of 43.22 months. Results The levels of 31 metabolites were statistically significantly changed in RCC tumors compared with controls. The top altered metabolites included beta-alanine (+ 4.2-fold), glucose (+ 3.4-fold), succinate (− 11.0-fold), myo-inositol (− 4.6-fold), adenine (− 4.2-fold), uracil (− 3.7-fold), and hypoxanthine (− 3.0-fold). These disturbances were associated with altered expression of 53 metabolic genes. ROC curve analysis revealed that the top metabolites discriminating between tumor and control samples included succinate (AUC = 0.91), adenine (AUC = 0.89), myo-inositol (AUC = 0.87), hypoxanthine (AUC = 0.85), urea (AUC = 0.85), and beta-alanine (AUC = 0.85). Poor survival of RCC patients correlated (p

microRNAs, short non-coding RNAs, influence key physiological processes, including hormonal regulation, by affecting the expression of genes. In this study we hypothesised that the expression of microRNAs targeting thyroid hormone pathway genes may be in turn regulated by thyroid hormone signalling. It is known that the expression of DIO1, a gene contributing to triiodothyronine (T3) signalling, is regulated by miR-224. Thus, we analysed mutual regulation between triiodothyronine pathway and miR-224/miR-452/GABRE cluster. Firstly, we found that miR-452 directly regulates the expression of thyroid hormone receptor TRβ1 in renal cancer cells. In turn, the expression of miR-224/452/GABRE cluster and other microRNAs targeting TRβ1 was influenced by T3 treatment and/or TR silencing. miR-452 expression correlated with intracellular T3 concentrations in renal tumours. In conclusion, we propose a new mechanism of feedback regulation, by which in renal cancer microRNAs regulate the expression of T3 pathway genes, while T3 in turn regulates expression of microRNAs.

Genitourinary cancers (GCs) include a large group of different types of tumors localizing to the kidney, bladder, prostate, testis, and penis. Despite highly divergent molecular patterns, most GCs share commonly disturbed signaling pathways that involve the activity of TGF-β (transforming growth factor beta). TGF-β is a pleiotropic cytokine that regulates key cancer-related molecular and cellular processes, including proliferation, migration, invasion, apoptosis, and chemoresistance. The understanding of the mechanisms of TGF-β actions in cancer is hindered by the “TGF-β paradox” in which early stages of cancerogenic process are suppressed by TGF-β while advanced stages are stimulated by its activity. A growing body of evidence suggests that these paradoxical TGF-β actions could result from the interplay with microRNAs: Short, non-coding RNAs that regulate gene expression by binding to target transcripts and inducing mRNA degradation or inhibition of translation. Here, we discuss the current knowledge of TGF-β signaling in GCs. Importantly, TGF-β signaling and microRNA-mediated regulation of gene expression often act in complicated feedback circuits that involve other crucial regulators of cancer progression (e.g., androgen receptor). Furthermore, recently published in vitro and in vivo studies clearly indicate that the interplay between microRNAs and the TGF-β signaling pathway offers new potential treatment options for GC patients.

Serine and arginine rich splicing factor 2(SRSF2) belongs to the serine/arginine (SR)-rich family of proteins that regulate alternative splicing. Previous studies suggested that SRSF2 can contribute to carcinogenic processes. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, highly aggressive and difficult to treat, mainly due to resistance to apoptosis. In this study we hypothesized that SRSF2 contributes to the regulation of apoptosis in ccRCC. Using tissue samples obtained from ccRCC patients, as well as independent validation on The Cancer Genome Atlas (TCGA) data, we demonstrate for the first time that expression of SRSF2 is decreased in ccRCC tumours when compared to non-tumorous control tissues. Furthermore, by employing a panel of ccRCC-derived cell lines with silenced SRSF2 expression and qPCR arrays we show that SRSF2 contributes not only to splicing patterns but also to expression of multiple apoptotic genes, including new SRSF2 targets: DIABLO, BIRC5/survivin, TRAIL, BIM, MCL1, TNFRSF9, TNFRSF1B, CRADD, BCL2L2, BCL2A1, and TP53. We also identified a new splice variant of CFLAR, an inhibitor of caspase activity. These changes culminate in diminished caspase-9 activity and inhibition of apoptosis. In summary, we show for the first time that decreased expression of SRSF2 in ccRCC contributes to protection of cancer cells viability.

SRSF7 is a SR splicing factor involved in the regulation of splicing and mRNA export of cancer-related genes. The mechanisms regulating the expression of SRSF7 are unknown. This study shows that SRSF7 expression in cancer cells is regulated by microRNAs: short, non-coding RNAs that bind to 3'UTR of target genes and downregulate their expression. We show that microRNAs miR-30a-5p and miR-181a-5p together with SRSF7 form regulatory feedback loop in which the expression of microRNAs is recurrently regulated by its target. Finally, we demonstrate that silencing of SRSF7 affects the expression of osteopontin splice variants and decreases proliferation rate of renal cancer cells.

Thyroid hormone receptor β1 (TRβ1) is a hormone-dependent transcription factor activated by 3,5,3'- l -triiodothyronine (T3). TRβ1 functions as a tumor suppressor and disturbances of the THRB gene are frequent findings in cancer. Translational control mediated by untranslated regions (UTRs) regulates cell proliferation, metabolism and responses to cellular stress, processes that are involved in carcinogenesis. We hypothesized that reduced TRβ1 expression in clear cell renal cell cancer (ccRCC) results from regulatory effects of TRβ1 5′ and 3′UTRs on protein translation. We determined TRβ1 expression and alternative splicing of TRβ1 5′ and 3′UTRs in ccRCC and control tissue together with expression of the type 1 deiodinase enzyme (coded by DIO1 , a TRβ1 target gene). Tissue concentrations of T3 (which are generated in part by D1) and expression of miRNA-204 (an mRNA inhibitor for which a putative interaction site was identified in the TRβ1 3′UTR) were also determined. TRβ1 mRNA and protein levels were reduced by 70% and 91% in ccRCC and accompanied by absent D1 protein, a 58% reduction in tissue T3 concentration and 2-fold increase in miRNA-204. Structural analysis of TRβ1 UTR variants indicated that reduced TRβ1 expression may be maintained in ccRCC by posttranscriptional mechanisms involving 5′UTRs and miRNA-204. The tumor suppressor activity of TRβ1 indicates that reduced TRβ1 expression and tissue hypothyroidism in ccRCC tumors is likely to be involved in the process of carcinogenesis or in maintaining a proliferative advantage to malignant cells.

Tetracycline-resistant Lactococcus lactis strains originally isolated from Polish raw milk were analyzed for the ability to transfer their antibiotic resistance genes in vitro, using filter mating experiments, and in vivo, using germfree rats. Four of six analyzed L. lactis isolates were able to transfer tetracycline resistance determinants in vitro to L. lactis Bu2-60, at frequencies ranging from 10 −5 to 10 −7 transconjugants per recipient. Three of these four strains could also transfer resistance in vitro to Enterococcus faecalis JH2-2, whereas no transfer to Bacillus subtilis YBE01, Pseudomonas putida KT2442, Agrobacterium tumefaciens UBAPF2, or Escherichia coli JE2571 was observed. Rats were initially inoculated with the recipient E. faecalis strain JH2-2, and after a week, the L. lactis IBB477 and IBB487 donor strains were introduced. The first transconjugants were detected in fecal samples 3 days after introduction of the donors. A subtherapeutic concentration of tetracycline did not have any significant effect on the number of transconjugants, but transconjugants were observed earlier in animals dosed with this antibiotic. Molecular analysis of in vivo transconjugants containing the tet (M) gene showed that this gene was identical to tet (M) localized on the conjugative transposon Tn 916 . Primer-specific PCR confirmed that the Tn 916 transposon was complete in all analyzed transconjugants and donors. This is the first study showing in vivo transfer of a Tn 916 -like antibiotic resistance transposon from L. lactis to E. faecalis . These data suggest that in certain cases food lactococci might be involved in the spread of antibiotic resistance genes to other lactic acid bacteria.

Renal cell carcinoma is the most common highly metastatic kidney malignancy. Adhesion has a crucial role in the metastatic process. TGF (transforming growth factor)-β1 is a pleiotropic cytokine that influences cancerous transformation. We hypothesized that 1) changes in the expression of adhesion related genes may influence survival rate of patients with renal cell carcinoma and 2) TGF-β1 may contribute to changed expression of adhesion related genes.Two-step quantitative real-time polymerase chain reaction arrays were used to analyze the expression of adhesion related genes in 77 tumors and matched pair controls. The prognostic significance of genes was evaluated in TCGA (The Cancer Genome Atlas) data on 468 patients with renal cell carcinoma. Quantitative real-time polymerase chain reaction and Western blot were applied for TGF-β1 analysis. TGF-β1 mediated regulation of gene expression was analyzed by TGF-β1 supplementation of Caki-2 cells and quantitative real-time polymerase chain reaction.The expression of 19 genes related to adhesion and extracellular matrix remodeling was statistically significantly disturbed in renal cell carcinoma compared with controls. The 10-gene expression signature (COL1A1, COL5A1, COL11A1, FN1, ICAM1, ITGAL, ITGAM, ITGB2, THBS2 and TIMP1) correlated with poor survival (HR 2.85, p = 5.7e-10). TGF-β1 expression was 22 times higher in renal cell carcinoma than in controls (p0.0001). TGF-β1 induced expression of TGFBI, COL1A1, COL5A1, COL8A1, FN1, ITGA5, ITGAM and TIMP1 in a renal cell carcinoma derived cell line.Disturbed expression of genes involved in adhesion and extracellular matrix remodeling develops early during renal cell carcinoma carcinogenesis and correlates with poor survival. TGF-β1 contributes to changed expression of extracellular matrix and adhesion related genes. Bioinformatic analysis performed on a broad panel of cancers of nonkidney origin suggests that disturbed expression of genes related to extracellular matrix and adhesion may be a universal feature of cancerous progression.

Thyroid hormone receptor beta (THRB) gene is commonly deregulated in cancers and, as strengthened by animal models, postulated to play a tumor-suppressive role. Our previous studies revealed downregulation of THRB in clear cell renal cell carcinoma (ccRCC), but the culpable mechanisms have not been fully elucidated. Since epigenetic regulation is a common mechanism influencing the expression of tumor suppressors, we hypothesized that downregulation of THRB in renal cancer results from epigenetic aberrances, including CpG methylation and microRNA-dependent silencing. Our study revealed that ccRCC tumors exhibited a 56% decrease in THRB and a 37% increase in DNA methyltransferase 1 (DNMT1) expression when compared with paired non-neoplastic control samples. However, THRB CpG methylation analysis performed using BSP, SNaPshot and MSP-PCR consistently revealed no changes in methylation patterns between matched tumor and control samples. In silico analysis resulted in identification of four microRNAs (miR-155, miR-425, miR-592, and miR-599) as potentially targeting THRB transcript. Luciferase assay showed direct binding of miR-155 and miR-425 to 3′UTR of THRB, and subsequent in vivo analyses revealed that transfection of UOK171 cell line with synthetic miR-155 or miR-425 resulted in decreased expression of endogenous TRHB by 22% and 64%, respectively. Finally, real-time PCR analysis showed significant upregulation of miR-155 (354%) and miR-425 (162%) in ccRCC when compared with matched controls. Moreover, microRNA levels were negatively correlated with the amount of THRB transcript in tissue samples. We conclude that CpG methylation is not the major mechanism contributing to decreased THRB expression in ccRCC. In contrast, THRB is targeted by microRNAs miR-155 and miR-425, whose increased expression may be responsible for downregulation of THRB in ccRCC tumors.

Context:Loss of the thyroid hormone receptor is common in tumors. In mouse models, a truncated THRB gene leads to thyroid cancer. Previously, we observed up-regulation of the expression of eight microRNAs (miRs) in papillary thyroid carcinoma (PTC) tumors.Objective:Our objective was to determine whether THRB might be inhibited by miRs up-regulated in PTC.Design:The potential binding of miR to the 3′-untranslated region of THRB was analyzed in silico. Direct inhibition by miRs binding to the cloned 3′-untranslated region of THRB was evaluated using luciferase assays. Inhibition of endogenous THRB and its target genes (DIO1 and APP) was examined in cell lines transfected by pre-miRs. The impact on thyroid hormone response element (TRE) was evaluated in promoter assays. Correlations between the expression of THRB and miRs was evaluated in 13 PTC tumor/normal tissue pairs.Results:THRB contains binding sites for the top seven miRs up-regulated in PTC (P = 0.0000002). Direct interaction with THRB was shown for miR-21 and miR-146a. We observed lower levels of THRB transcripts in cell lines transfected with miR-21, -146a, and -221 (down-regulation of 37–48%; P < 0.0001), but not with miR-181a. THRB protein was suppressed down to 10–28% by each of four miRs. Concomitant expression of DIO1 and APP was affected (down-regulation of 32–66%, P < 0.0034 and up-regulation of 48–57%, P < 0.0002, respectively). All four miRs affected TRE activity in promoter assays. Down-regulation of luciferase occurred after transfection with pTRE-TK-Luc construct and each of four miRs. The analysis of tumor/normal tissue pairs revealed down-regulation of THRB in 11 of 13 pairs (1.3- to 9.1-fold), and up-regulation of miR-21, -146a, -181a, and -221 in almost all pairs.Conclusions:MiRs up-regulated in PTC tumors directly inhibit the expression of THRB, an important tumor suppressor gene.

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions.Methodology/principal findingsUsing real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1.Conclusions/significanceWe found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis.

Alternative splicing, one of the sources of protein diversity, is often disturbed in cancer. Type 1 iodothyronine deiodinase (DIO1) catalyzes deiodination of thyroxine generating triiodothyronine, an important regulator of cell proliferation and differentiation. The expression of DIO1 is disturbed in different types of cancer. The aim of the study was to analyze the alternative splicing of DIO1 and its possible disturbance in renal cancer.Using real-time PCR, we analyzed 19 tissue samples (T) of renal cancer and 19 matched control samples (C) of the opposite pole of the kidney, not infiltrated by tumor, and 6 control samples (N) (nonneoplastic kidney abnormalities).Cloning of DIO1 mRNA isoforms revealed 11 different transcripts, among them 7 new splice variants, not previously reported. The expression of all variants of DIO1 was dramatically (90%) and significantly (por = 0.0003) lowered in samples T compared to control samples C. The ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center was lowered in samples T compared with control samples C, suggesting disturbed alternative splicing of DIO1. The expression of mRNA of splicing factors SF2/ASF (splicing factor-2/alternative-splicing factor) and hnRNPA1 (heterogeneous ribonucleoprotein A1), regulating 5'-splice site selection, was significantly but not proportionally lowered in samples T compared to samples C. The mRNA ratio of splicing factors SF2/ASF and hnRNPA1 correlated with the ratio of mRNA isoforms encoding DIO1 protein variants possessing or lacking the active center in controls C but not in samples T.Our results show that the expression and alternative splicing of DIO1 mRNA is disturbed in renal cancer, possibly due to changes in expression of splicing factors SF2/ASF and hnRNPA1.

Context: Loss of the thyroid hormone receptor is common in tumors. In mouse models, a truncated THRB gene leads to thyroid cancer. Previously, we observed up-regulation of the expression of eight micro-RNAs (miR) in papillary thyroid carcinoma (PTC) tumors. Objective: Our objective was to determine whether THRB might be inhibited by miR up-regulated in PTC. Design: The potential binding of miR to the 3′-untranslated region of THRB was analyzed in silico. Direct inhibition by miR binding to the cloned 3′-untranslated region of THRB was evaluated using luciferase assays. Inhibition of endogenous THRB and its target genes (DIO1 and APP) was examined in cell lines transfected by pre-miR. The impact on thyroid hormone response elements (TRE) was evaluated in promoter assays. Correlations between the expression of THRB and miR was evaluated in 13 PTC tumor/normal tissue pairs. Results: THRB contains binding sites for the top seven miR up-regulated in PTC (P = 0.0000002). Direct interaction with THRB was shown for miR-21 and miR-146a. We observed lower levels of THRB transcripts in cell lines transfected with miR-21, −146a, and −221 (down-regulation of 37–48%; P < 0.0001), but not with miR-181a. THRB protein was suppressed down to 10–28% by each of four miR. Concomitant expression of DIO1 and APP was affected (down-regulation of 32–66%, P < 0.0034 and up-regulation of 48–57%, P < 0.0002, respectively). All four miR affected TRE activity in promoter assays. Down-regulation of luciferase occurred after transfection with pTRE-TK-Luc construct and each of four miR. The analysis of tumor/normal tissue pairs revealed down-regulation of THRB in 11 of 13 pairs (1.3- to 9.1-fold), and up-regulation of miR-21, −146a, −181a, and −221 in almost all pairs. Conclusions: MiR up-regulated in PTC tumors directly inhibit the expression of THRB, an important tumor suppressor gene.