Using CRISPR/Cas9 to Edit a Thyroid Cancer Cell Line.

Thyroid cancer is the most prevalent endocrine malignancy, comprising multiple types of cancer, with distinct clinical-pathological characteristics. The oncogenesis of thyroid cancer is related to genetic alterations in MAPK signaling that induce proliferation and modulate noncoding genes, such as microRNAs and long noncoding RNAs. In this context, CRISPR/Cas9 emerges as a potential tool to modify gene sequence and modulate gene expression in thyroid cancer cell lines. In this chapter, we explore some of the current studies in which researchers have applied CRISPR/Cas9 in vitro to investigate thyroid cancer biology (Fig. 5.1).

Modulation of EZH2 Activity Induces an Antitumoral Effect and Cell Redifferentiation in Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is a rare and lethal form of thyroid cancer that requires urgent investigation of new molecular targets involved in its aggressive biology. In this context, the overactivation of Polycomb Repressive Complex 2/EZH2, which induces chromatin compaction, is frequently observed in aggressive solid tumors, making the EZH2 methyltransferase a potential target for treatment. However, the deregulation of chromatin accessibility is yet not fully investigated in thyroid cancer. In this study, EZH2 expression was modulated by CRISPR/Cas9-mediated gene editing and pharmacologically inhibited with EZH2 inhibitor EPZ6438 alone or in combination with the MAPK inhibitor U0126. The results showed that CRISPR/Cas9-induced EZH2 gene editing reduced cell growth, migration and invasion in vitro and resulted in a 90% reduction in tumor growth when EZH2-edited cells were injected into an immunocompromised mouse model. Immunohistochemistry analysis of the tumors revealed reduced tumor cell proliferation and less recruitment of cancer-associated fibroblasts in the EZH2-edited tumors compared to the control tumors. Moreover, EZH2 inhibition induced thyroid-differentiation genes' expression and mesenchymal-to-epithelial transition (MET) in ATC cells. Thus, this study shows that targeting EZH2 could be a promising neoadjuvant treatment for ATC, as it promotes antitumoral effects in vitro and in vivo and induces cell differentiation.

Age-related increase of let-7 family microRNA in rat retina and vitreous.

Vitreous alterations occur from early stages and continue through the normal aging, with gradual lamellae formation and the appearance of liquefied spaces, which eventually leads to complications, such as retinal tear, retinal detachment, and intravitreal hemorrhage. The aim of the present study was to investigate the expression of let-7 miRNA family in the vitreous and retina in newborn (1-3- day-old), young adult (2-month-old), and aging (12-month-old) rats, as well as their role as regulators of vitreous components. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression. Our results showed detection of all investigated let-7 isoforms (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f and let-7i) in the retina and vitreous. Although most let-7 members were significantly upregulated in the vitreous during development, only let-7b, let-7c, and let-7e followed this same expression pattern in the retina. Let-7b and -7c increased in aging vitreous as well, and were expressed in vitro by Müller glial cells and their extracellular vesicles. Moreover, let-7 targeted hyaluronan synthase 2 (Has2) mRNA, a synthesizing enzyme of hyaluronan. These observations indicate that let-7 function is important during retina and vitreous development, and that isoforms of let-7 increased with aging, potentially modulating hyaluronan content.

Targeting the Highly Expressed microRNA miR-146b with CRISPR/Cas9n Gene Editing System in Thyroid Cancer.

Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the pre-mir-146b structure to target miR-146b-5p and miR-146b-3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-miR-146b-GuideA-puromycin and pSp-Cas9n-miR-146b-GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.

Nc886 is epigenetically repressed in prostate cancer and acts as a tumor suppressor through the inhibition of cell growth.

BACKGROUND: Nc886 is a 102 bp non-coding RNA transcript initially classified as a microRNA precursor (Pre-miR-886), later as a divergent homologue of the vault RNAs (vtRNA 2-1) and more recently as a novel type of RNA (nc886). Although nc886/vtRNA2-1/Pre-miR-886 identity is still controversial, it was shown to be epigenetically controlled, presenting both tumor suppressor and oncogenic function in different cancers. Here, we study for the first time the role of nc886 in prostate cancer. METHODS: Nc886 promoter methylation status and its correlation with patient clinical parameters or DNMTs levels were evaluated in TCGA and specific GEO prostate tissue datasets. Nc886 level was measured by RT-qPCR to compare normal/neoplastic prostate cells from radical prostatectomies and cell lines, and to assess nc886 response to demethylating agents. The effect of nc886 recovery in cell proliferation (in vitro and in vivo) and invasion (in vitro) was evaluated using lentiviral transduced DU145 and LNCaP cell lines. The association between the expression of nc886 and selected genes was analyzed in the TCGA-PRAD cohort. RESULTS: Nc886 promoter methylation increases in tumor vs. normal prostate tissue, as well as in metastatic vs. normal prostate tissue. Additionally, nc886 promoter methylation correlates with prostate cancer clinical staging, including biochemical recurrence, Clinical T-value and Gleason score. Nc886 transcript is downregulated in tumor vs. normal tissue -in agreement with its promoter methylation status- and increases upon demethylating treatment. In functional studies, the overexpression of nc886 in the LNCaP and DU145 cell line leads to a decreased in vitro cell proliferation and invasion, as well as a reduced in vivo cell growth in NUDE-mice tumor xenografts. Finally, nc886 expression associates with the prostate cancer cell cycle progression gene signature in TCGA-PRAD. CONCLUSIONS: Our data suggest a tumor suppressor role for nc886 in the prostate, whose expression is epigenetically silenced in cancer leading to an increase in cell proliferation and invasion. Nc886 might hold clinical value in prostate cancer due to its association with clinical parameters and with a clinically validated gene signature.

MiRNA-146b-5p upregulates migration and invasion of different Papillary Thyroid Carcinoma cells.

BACKGROUND: Tumor invasiveness is directly related to the ability of tumor cells to migrate and invade surrounding tissues, usually degrading extracellular matrix. Despite significant progress in the knowledge about migration and invasion, there is much more to elucidate about their regulatory mechanisms, especially in cancer cells. MicroRNAs (miRs) were recently described as important regulators of migration. Differential expression of miRs in cancer is frequently associated with progression, invasion and metastasis. In papillary thyroid carcinoma (PTC), miR-146b-5p is highly expressed and positively correlated to the degree of malignancy. METHODS: This study aimed to investigate the role of miR-146b-5p on the migratory and invasive behaviors of thyroid cells, using a non tumor rat thyroid follicular cell line (PCCl3) transfected with the miR-146b-5p genomic region, and two PTC cell lines (TPC-1 and BCPAP, bearing distinct oncogenic backgrounds), which express high levels of miR-146b-5p, after miR-146b inhibition by antagomiR and miR-146b overexpression by mimics-miR. Migration and invasion were studied by time-lapse and transwell assays (with and without Matrigel®). Gelatin degradation assays were also employed, as well as F-actin staining. RESULTS: Migration and invasion of PCCl3 were increased 2-3x after miR-146b-5p overexpression (10X) and large lamellipodia were evident in those cells. After miR-146b-5p inhibition, TPC-1 and BCPAP migration and invasion were significantly reduced, with cells showing several simultaneous processes and low polarity. Gelatin degradation was inhibited in TPC-1 cells after inhibition of miR-146b-5p, but was unaffected in BCPAP cells, which did not degrade gelatin. The inhibition of miR-146b-5p in PCCl3 also inhibited migration and invasion, and additional (exogenous) overexpression of this miR in TPC-1 and BCPAP cells increased migration and invasion, without effects on cell morphology or gelatin degradation. The overexpression of SMAD4 in BCPAP cells, a validated target of miR-146b-5p and key protein in the TGF-ß signaling pathway, inhibited migration similarly to the effects observed with the antagomiR 146b-5p. CONCLUSIONS: miR-146b-5p positively regulates migration and invasion of thyroid normal and tumor follicular cells (independently from their original mutation, either BRAF or RET/PTC), through a mechanism that involves the actin cytoskeleton but not an increased capacity of matrix degradation.

The Impact of a Non-Functional Thyroid Receptor Beta upon Triiodotironine-Induced Cardiac Hypertrophy in Mice.

BACKGROUND/AIMS: Thyroid hormone (TH) signalling is critical for heart function. The heart expresses thyroid hormone receptors (THRs); THRα1 and THRß1. We aimed to investigate the regulation mechanisms of the THRß isoform, its association with gene expression changes and implications for cardiac function. METHODS: The experiments were performed using adult male mice expressing TRßΔ337T, which contains the Δ337T mutation of the human THRB gene and impairs ligand binding. Cardiac function and RNA expression were studied after hypo-or hyperthyroidism inductions. T3-induced cardiac hypertrophy was not observed in TRßΔ337T mice, showing the fundamental role of THRß in cardiac hypertrophy. RESULTS: We identified a group of independently regulated THRß genes, which includes Adrb2, Myh7 and Hcn2 that were normally regulated by T3 in the TRßΔ337T group. However, Adrb1, Myh6 and Atp2a2 were regulated via THRß. The TRßΔ337T mice exhibited a contractile deficit, decreased ejection fraction and stroke volume, as assessed by echocardiography. In our model, miR-208a and miR-199a may contribute to THRß-mediated cardiac hypertrophy, as indicated by the absence of T3-regulated ventricular expression in TRßΔ337T mice. CONCLUSION: THRß has important role in the regulation of specific mRNA and miRNA in T3-induced cardiac hypertrophic growth and in the alteration of heart functions.

DLK1-DIO3 region as a source of tumor suppressor miRNAs in papillary thyroid carcinoma.

BACKGROUND: In previous studies, we demonstrated the downregulation of several miRNAs from the DLK1-DIO3 genomic region in papillary thyroid carcinoma (PTC). Due to the large number of miRNAs within this region, the individual contribution of these molecules to PTC development and progression remains unclear. OBJECTIVE: In this study, we aimed to clarify the contribution of DLK1-DIO3-derived miRNAs to PTC. METHODS: We used different computational approaches and in vitro resources to assess the biological processes and signaling pathways potentially modulated by these miRNAs. RESULTS: Our analysis suggests that, out of more than 100 mature miRNAs originated from the DLK1-DIO3 region, a set of 12 miRNAs accounts for most of the impact on PTC development and progression, cooperating to modulate distinct cancer-relevant biological processes, such as cell migration, extracellular matrix remodeling, and signal transduction. The restoration of the expression of one of these miRNAs (miR-485-5p) in a BRAFT199A-positive PTC cell line impaired proliferation and migration, suppressing the expression of GAB2 and RAC1, validated miR-485-5p targets. CONCLUSIONS: Overall, our results shed light on the role of the DLK1-DIO3 region, which harbors promising tumor suppressor miRNAs in thyroid cancer, and open prospects for the functional exploration of these miRNAs as therapeutic targets for PTC.

Profile of MicroRNAs Associated with Death Due to Disease Progression in Metastatic Papillary Thyroid Carcinoma Patients.

Papillary thyroid carcinoma (PTC) is the most common neoplasm of the endocrine system and has an excellent long-term prognosis, with low rates of distant metastatic disease. Although infrequent, there are cases of deaths directly related to PTC, especially in patients with metastatic disease, and the factors that could be associated with this unfavorable outcome remain a major challenge in clinical practice. Recently, research into genetic factors associated with PTC has gained ground, especially mutations in the TERT promoter and BRAF gene. However, the role of microRNAs remains poorly studied, especially in those patients who have an unfavorable outcome at follow-up. This paper aims to evaluate molecular markers related to the different pathological processes of PTC, as well as the histological characteristics of the neoplasm, and to compare this profile with prognosis and death from the disease using an analysis of patients treated for metastatic disease in a single tertiary cancer center. Evaluation of microRNA expression in paraffin-embedded tumor specimens was carried out by quantitative PCR using the TaqMan® Low Density Array (TLDA) system. Metastatic patients who died from progression of PTC had higher expressions of miR-101-3p, miR-17-5p, and miR-191-5p when compared to patients with stable metastatic disease. These findings are of great importance but should be considered as preliminary because of the small sample.

Gene Editing with CRISPR/Cas Methodology and Thyroid Cancer: Where Are We?

Important advances on the role of genetic alterations in thyroid cancer have been achieved in the last two decades. One key reason is linked to the development of technical approaches that allowed for the mimicking of genetic alterations in vitro and in vivo and, more recently, the gene editing methodology. The CRISPR/Cas methodology has emerged as a tangible tool for editing virtually any DNA sequence in the genome. To induce a double-strand break and programmable gene editing, Cas9 endonuclease is guided by a single-guide RNA (sgRNA) that is complementary to the target sequence in DNA. The gene editing per se occurs as the cells repair the broken DNA and may erroneously change the original DNA sequence. In this review, we explore the principles of the CRISPR/Cas system to facilitate an understanding of the mainstream technique and its applications in gene editing. Furthermore, we explored new applications of CRISPR/Cas for gene modulation without changing the DNA sequence and provided a Dry Lab experience for those who are interested in starting "CRISPRing" any given gene. In the last section, we will discuss the progress in the knowledge of thyroid cancer biology fostered by the CRISPR/Cas gene editing tools.

Let-7, Lin28 and Hmga2 Expression in Ciliary Epithelium and Retinal Progenitor Cells.

Purpose: Ciliary epithelium (CE) of adult mammalian eyes contains quiescent retinal progenitor/stem cells that generate neurospheres in vitro and differentiate into retinal neurons. This ability doesn't evolve efficiently probably because of regulatory mechanisms, such as microRNAs (miRNAs) that control pluripotent, progenitor, and differentiation genes. Here we investigate the presence of Let-7 miRNAs and its regulator and target, Lin28 and Hmga2, in CE cells from neurospheres, newborns, and adult tissues. Methods: Newborn and adult rats CE cells were dissected into pigmented and nonpigmented epithelium (PE and NPE). Newborn PE cells were cultured with growth factors to form neurospheres and we analyzed Let-7, Lin28a, and Hmga2 expression. During the neurospheres formation, we added chemically modified single-stranded oligonucleotides designed to bind and inhibit or mimic endogenous mature Let-7b and Let-7c. After seven days in culture, we analyzed neurospheres size, number and expression of Let-7, Lin28, and Hmga2. Results: Let-7 miRNAs were expressed at low rates in newborn CE cells with significant increase in adult tissues, with higher levels on NPE cells, that does not present the stem cells reprogramming ability. The Lin28a and Hmga2 protein and transcripts were more expressed in newborns than adults cells, opposed to Let-7. Neurospheres presented higher Lin28 and Hmga2 expression than newborn and adult, but similar Let-7 than newborns. Let-7b inhibitor upregulated Hmga2 expression, whereas Let-7c mimics upregulated Lin28 and downregulated Hmga2. Conclusions: This study shows the dynamic of Lin28-Let-7-Hmga regulatory axis in CE cells. These components may develop different roles during neurospheres formation and postnatal CE cells.

Thyroid Follicular Cell Loss of Differentiation Induced by MicroRNA miR-17-92 Cluster Is Attenuated by CRISPR/Cas9n Gene Silencing in Anaplastic Thyroid Cancer.

Background: Loss of the expression of thyroid differentiation markers such as sodium iodide symporter (NIS) and, consequently, radioiodine refractoriness is observed in aggressive papillary thyroid cancer and anaplastic thyroid cancer (ATC) that may harbor the BRAFV600E mutation. Activation of the BRAFV600E oncogene in thyroid follicular cells induces the expression of the miR-17-92 cluster that comprises seven mature microRNAs (miRNAs). miRNAs are a class of endogenous small RNAs (∼22 nt) that regulate gene expression post-transcriptionally. Indeed, miR-17-92 is overexpressed in ATC, and in silico prediction shows the potential targeting of thyroid transcription factors and tumor suppressor pathways. In this study, we aimed to investigate the role of the miR-17-92 cluster in thyroid cell differentiation and function. Methods:miR-17-92 silencing was performed using CRISPR/Cas9n-guided genomic editing of the miR-17-92 gene in the KTC2 ATC cell line, and miR-17-92 cluster or individual miRNAs were overexpressed in PCCl3 thyroid cells to evaluate the influence in thyroid cell differentiation and cell function. Results: In this study, we demonstrate that CRISPR/Cas9n gene editing of the miR-17-92 cluster results in promotion of thyroid follicular cell differentiation (NIS, thyroperoxidase, thyroglobulin, PAX8, and NKX2-1 expression) in the KTC2 ATC cell line and inhibits cell migration and proliferation by restoring transforming growth factor beta (TGF-ß) signaling pathway responsiveness. Moreover, induction of the miR-17-92 cluster in normal thyroid follicular cells strongly impairs thyroid differentiation and induces a pro-oncogenic effect by blocking TGF-ß signaling and increasing cell migration. Conclusions:miR-17-92 is a potent regulator of thyroid follicular cell differentiation, and CRISPR/Cas9n-mediated editing of the miR-17-92 gene efficiently blocks miR-17-92 expression in the KTC2 ATC cell line, resulting in improvement of thyroid differentiation. Thus, targeting miR-17-92 could provide a potential molecular approach to restoring thyroid cell differentiation and NIS expression in aggressive thyroid cancer.

Genetic Mutations and Variants in the Susceptibility of Familial Non-Medullary Thyroid Cancer.

Thyroid cancer is the most frequent endocrine malignancy with the majority of cases derived from thyroid follicular cells and caused by sporadic mutations. However, when at least two or more first degree relatives present thyroid cancer, it is classified as familial non-medullary thyroid cancer (FNMTC) that may comprise 3-9% of all thyroid cancer. In this context, 5% of FNMTC are related to hereditary syndromes such as Cowden and Werner Syndromes, displaying specific genetic predisposition factors. On the other hand, the other 95% of cases are classified as non-syndromic FNMTC. Over the last 20 years, several candidate genes emerged in different studies of families worldwide. Nevertheless, the identification of a prevalent polymorphism or germinative mutation has not progressed in FNMTC. In this work, an overview of genetic alteration related to syndromic and non-syndromic FNMTC is presented.

Elemental characterization of oral cavity squamous cell carcinoma and its relationship with smoking, prognosis and survival.

Oral cancer squamous cell carcinoma (OCSCC) mainly affects individuals aged between 50 and 70 years who consume tobacco and alcohol. Tobacco smoke contains hundreds of known toxic and carcinogenic molecules, and a few studies have sought to verify the relationship of such trace elements as risk or prognostic factors for head and neck cancer. We obtained 78 samples of tumor tissues from patients with OCSCC, and performed a qualitative elemental characterization using the micro X-Ray Fluorescence technique based on synchrotron radiation. We found the presence of magnesium, phosphorus, sulfur, chlorine, potassium, calcium, chromium, manganese, iron, zinc, cobalt, nickel, copper, arsenic and bromine in OCSCC samples. Magnesium, chlorine, chromium, manganese, nickel, arsenic and bromine are associated with smoking. We observed a significant association between relapse and chlorine and chromium. The presence of chlorine in the samples was an independent protective factor against relapse (OR = 0.105, CI = 0.01-0.63) and for best disease-free survival (HR = 0.194, CI = 0.04-0.87). Reporting for the first time in oral cancer, these results suggest a key relationship between smoking and the presence of certain elements. In addition, chlorine proved to be important in the context of patient prognosis and survival.

Interplay of TGFß signaling and microRNA in thyroid cell loss of differentiation and cancer progression.

Thyroid cancer has been rapidly increasing in prevalence among humans in last 2 decades and is the most prevalent endocrine malignancy. Overall, thyroid-cancer patients have good rates of long-term survival, but a small percentage present poor outcome. Thyroid cancer aggressiveness is essentially related with thyroid follicular cell loss of differentiation and metastasis. The discovery of oncogenes that drive thyroid cancer (such as RET, RAS, and BRAF), and are aligned in the MAPK/ERK pathway has led to a new perspective of thyroid oncogenesis. The uncovering of additional oncogene-modulated signaling pathways revealed an intricate and active signaling cross-talk. Among these, microRNAs, which are a class of small, noncoding RNAs, expanded this cross-talk by modulating several components of the oncogenic network - thus establishing a new layer of regulation. In this context, TGFß signaling plays an important role in cancer as a dual factor: it can exert an antimitogenic effect in normal thyroid follicular cells, and promote epithelial-to-mesenchymal transition, cell migration, and invasion in cancer cells. In this review, we explore how microRNAs influence the loss of thyroid differentiation and the increase in aggressiveness of thyroid cancers by regulating the dual function of TGFß. This review provides directions for future research to encourage the development of new strategies and molecular approaches that can improve the treatment of aggressive thyroid cancer.

The Highly Expressed FAM83F Protein in Papillary Thyroid Cancer Exerts a Pro-Oncogenic Role in Thyroid Follicular Cells.

Thyroid cancer is the most common endocrine cancer with predominant prevalence of papillary thyroid cancer (PTC) histotype. MAPK signaling genetic alterations are frequent in PTC, affecting more than 80% of cases. These alterations constitutively activate MAPK signaling cross-regulating different pro-oncogenic pathways. However, additional molecular alterations associated with thyroid cancer are not completely understood. In this extent, the new family of proteins named FAM83 (FAMily with sequence similarity 83) was recently identified as mediator of oncogenic signaling in different types of cancer. Here we report FAM83F as a novel highly expressed protein in PTC. We evaluated FAM83F levels in 106 PTC specimens, 34 goiter, and 41 adjacent non-tumoral human thyroid, and observed FAM83F cytoplasmic overexpression in 71% of PTC (76 of 106) while goiter tissues showed nuclear positivity and normal thyroid showed no staining by immunohistochemistry. Moreover, TSH-induced goiter and BRAF T1799A -induced PTC animal models also showed FAM83F activation. In vitro, we generated a stable thyroid cell line PCCL3 with FAM83F overexpression and observed that FAM83F deregulates thyroid follicular cell biology leading to loss of thyroid differentiation genes such as Sodium-Iodide Symporter (NIS), reactivation of stem cell markers such as LIN28B and SOX2, induction of cell migration and resistance to doxorubicin-induced apoptosis. Moreover, FAM83F activates MAPK signaling through interaction with BRAF and RAF while impairs TGFß antiproliferative signaling transduction. In this study, we showed FAM83F as a new pro-oncogenic protein overexpressed in thyroid cancer that modulates thyroid follicular cell biology and differentiation through cross-regulation of MAPK and TGFß signaling.

Osteoglycin post-transcriptional regulation by miR-155 induces cellular architecture changes in H9c2 cardiomyoblasts.

Several studies have demonstrated dysregulated cardiac microRNAs (miRNAs) following cardiac stress and development of cardiac hypertrophy and failure. miRNAs are also differentially expressed in the inflammation that occurs in heart failure and, among these inflammatory-related miRNAs, the miR-155 has been implicated in the regulation of cardiac hypertrophy. Despite these data showing the role of miRNA-155 in cardiomyocyte hypertrophy under a hypertrophic stimulus, it is also important to understand the endogenous regulation of this miRNA without a hypertrophic stimulus to fully appreciate its function in this cell type. The first aim of the present study was to determine whether, without a hypertrophic stimulus, miR-155 overexpression induces H9c2 cardiac cells hypertrophy in vitro. The second objective was to determine whether osteoglycin (Ogn), a key regulator of heart mass in rats, mice, and humans, is post-transcriptionally regulated by miR-155 with a potential role in inducing H9c2 cells hypertrophy. Here, we show that, without a hypertrophic stimulus, miR-155 significantly repressed Ogn protein levels, but induce neither alteration in morphological phenotype nor in the expression of the molecular markers that fully characterize pathological hypertrophy of H9c2 cells. However, most importantly, Ogn silencing in H9c2 cells mimicked the effects of miR-155 overexpression in inducing cellular architecture changes that were characterized by a transition of the cell shape from fusiform to rounded. This is a new role of the post-transcriptional regulation of Ogn by miR-155 in the maintenance of the cardiac cell morphology in physiological and pathological conditions.

MicroRNAs in thyroid development, function and tumorigenesis.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that modulate the vast majority of cellular processes. During development, the correct timing and expression of miRNAs in the tissue differentiation is essential for organogenesis and functionality. In thyroid gland, DICER and miRNAs are necessary for accurately establishing thyroid follicles and hormone synthesis. Moreover, DICER1 mutations and miRNA deregulation observed in human goiter influence thyroid tumorigenesis. The thyroid malignant transformation by MAPK oncogenes is accompanied by global miRNA changes, with a marked reduction of "tumor-suppressor" miRNAs and activation of oncogenic miRNAs. Loss of thyroid cell differentiation/function, and consequently iodine trapping impairment, is an important clinical characteristic of radioiodine-refractory thyroid cancer. However, few studies have addressed the direct role of miRNAs in thyroid gland physiology. Here, we focus on what we have learned in the thyroid follicular cell differentiation and function as revealed by cell and animal models and miRNA modulation in thyroid tumorigenesis.

Down-regulation of 14q32-encoded miRNAs and tumor suppressor role for miR-654-3p in papillary thyroid cancer.

Papillary thyroid carcinoma (PTC) is the most prevalent malignant neoplasia of the thyroid gland. A fraction of PTC cases show loss of differentiation and aggressive behavior, with radioiodine therapy resistance and metastasis. Although microRNAs (miRNAs) emerged as promising molecular markers for PTC, their role in the loss of differentiation observed during PTC progression remains to be fully understood. We performed the large-scale analysis of miRNA expression during PTC progression in BRAFT1799A-transgenic animals (Tg-Braf) and thyroid cancer cell lines and identified the marked downregulation of several miRNAs from the region 14q32. Data from The Cancer Genome Atlas (TCGA) confirmed the global downregulation of miRNAs from the 14q32 region in human PTC. The regulatory network potentially suppressed by these miRNAs suggests that key cancer-related biological processes such as cell proliferation, adhesion, migration and angiogenesis. Among the downregulated miRNAs, we observed that miR-654-3p levels decrease with long-term PTC progression in Tg-Braf mice and inversely correlate with EMT. The in vitro restoration of miR-654-3p decreased cell proliferation and migration and induced reprogramming of metastasis-related genes, suggesting a tumor suppressor role for this miRNA. In conclusion, we show global downregulation of 14q32-encoded miRNAs in an in vivo model of PTC progression. The potential circuitry in which these miRNAs are involved suggests that these miRNAs could play a key role in the pathophysiology of PTC and therefore be relevant for the development of new therapeutic strategies.

Osteoglycin inhibition by microRNA miR-155 impairs myogenesis.

Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3´-untranslated region (3´ UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80-90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3'UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3'UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells.