Anna Kubiak

Application of solid phase extraction procedures for rare earth elements determination in environmental samples

Determination of rare earth elements in environmental samples requires often pre-concentration and separation step due to a low metal content and high concentration of the interfering matrix components. A solid phase extraction technique with different kind of solid sorbents offers a high enrichment factor, rapid phase separation and the possibility of its combination with various detection techniques used either in on-line or off-line mode. The recent developments in this area published over the last five years are presented and discussed in this paper.

Variants in the ATM-CHEK2-BRCA1 Axis Determine Genetic Predisposition and Clinical Presentation of Papillary Thyroid Carcinoma

The risk of developing papillary thyroid carcinoma (PTC), the most frequent form of thyroid malignancy, is elevated up to 8.6‐fold in first‐degree relatives of PTC patients. The familial risk could be explained by high‐penetrance mutations in yet unidentified genes, or polygenic action of low‐penetrance alleles. Since the DNA‐damaging exposure to ionizing radiation is a known risk factor for thyroid cancer, polymorphisms in DNA repair genes are likely to affect this risk. In a search for low‐penetrance susceptibility alleles we employed Sequenom technology to genotype deleterious polymorphisms in ATM, CHEK2, and BRCA1 in 1,781 PTC patients and 2,081 healthy controls. As a result of the study, we identified CHEK2 rs17879961 (OR = 2.2, P = 2.37e‐10) and BRCA1 rs16941 (odds ratio [OR] = 1.16, P = 0.005) as risk alleles for PTC. The ATM rs1801516 variant modifies the risk associated with the BRCA1 variant by 0.78 (P = 0.02). Both the ATM and BRCA1 variants modify the impact of male gender on clinical variables: T status (P = 0.007), N status (P = 0.05), and stage (P = 0.035). Our findings implicate an important role of variants in the ATM‐ CHEK2‐ BRCA1 axis in modification of the genetic predisposition to PTC and its clinical manifestations.

Family of microRNA-146 Regulates RARβ in Papillary Thyroid Carcinoma

Retinoic acid is a promising tool in adjuvant cancer therapies, including refractory thyroid cancer, and its biological role is mediated by the retinoic acid receptor beta (RARβ). However, expression of RARβ is lowered in papillary thyroid carcinoma (PTC), contributing to promotion of tumor growth and inefficiency of retinoic acid and radioactive iodine treatment. The causes of aberrant RARB expression are largely unknown. We hypothesized that the culpable mechanisms include the action of microRNAs from the miR-146 family, previously identified as significantly upregulated in PTC tumors. To test this hypothesis, we assessed the expression of RARB as well as miR-146a-5p and miR-146b-5p in 48 PTC tumor/normal tissue pairs by Taqman assay to reveal that the expression of RARB was 3.28-fold decreased, and miR-146b-5p was 28.9-fold increased in PTC tumors. Direct interaction between miRs and RARB was determined in the luciferase assay and further confirmed in cell lines, where overexpression of miR-146a-5p and miR-146b-5p caused a 31% and 33% decrease in endogenous RARB mRNA levels. Inhibition of miR-146a and miR-146b resulted in 62.5% and 45.4% increase of RARB, respectively, and a concomitant decrease in proliferation rates of thyroid cancer cell lines, analyzed in xCELLigence system.We showed that two microRNAs of the miR-146 family directly regulate RARB. Inhibition of miRs resulted in restoration of RARB expression and decreased rates of proliferation of thyroid cancer cells. By restoring RARB levels, microRNA inhibitors may become part of an adjuvant therapy in thyroid cancer patients.

Next-generation sequencing reveals microRNA markers of adrenocortical tumors malignancy

Background Adrenocortical carcinoma is a rare finding among common adrenocortical tumors, but it is highly aggressive and requires early detection and treatment. Still, the differential diagnosis between benign and malignant lesions is difficult even for experienced pathologists and there is a significant need for novel diagnostic methods. In this study we aimed to reveal a complete set of microRNAs expressed in the adrenal gland and to identify easily detectable, stable and objective biomarkers of adrenocortical malignancy. Methods We employed next-generation sequencing to analyze microRNA profiles in a unique set of 51 samples, assigned to either a learning dataset including 7 adrenocortical carcinomas (ACCs), 8 adrenocortical adenomas (AAs) and 8 control samples (NAs), or a validation dataset including 8 ACCs, 10 AAs and 10 NAs. The results were validated in real-time Q-PCR. Results We detected 411 miRNAs expressed in 1763 length isoforms in the examined samples. Fifteen miRNAs differentiate between malignant (ACC) and non-malignant (AA + NA) tissue in the test set of independent samples. Expression levels of 6 microRNAs, miR-503-5p, miR-483-3p, miR-450a-5p, miR-210, miR-483-5p, miR-421, predict sample status (malignancy/non-malignancy) with at least 95% accuracy in both datasets. The best single-gene malignancy marker, miR-483-3p, has been validated by real-time RT PCR. Conclusions As a result of the study we propose clinically valid and easily detectable biomarkers of adrenocortical malignancy that may significantly facilitate morphological examination. Since microRNAs can be detected in blood, the study brings tools for development of non-invasive diagnostics of adrenocortical carcinomas.

Functional analysis of a novel, thyroglobulin-embedded microRNA gene deregulated in papillary thyroid carcinoma.

MicroRNAs, non-coding regulators of gene expression, are known culprits of thyroid cancer. Using next-generation sequencing, we identified a novel microRNA gene, encoded within an important thyroid regulator – thyroglobulin, and analyzed its functionality in the thyroid gland. In vitro and in silico analyses proved that the novel miR-TG is processed from the precursor, and co-expressed with thyroglobulin. Both genes are specific for thyroid tissue and downregulated in papillary thyroid carcinoma by 44% (p = 0.04) and 48% (p = 0.001), respectively. Putative target genes for miR-TG were identified using in silico tools, which pinpointed MAP4K4, an oncogene upregulated in thyroid cancer. Analysis of transcriptome by RNA-seq revealed that overexpression of miR-TG in PTC-derived cell line led to downregulation of several genes, including MAP4K4 (fold change 0,82; p = 0.036). The finding was confirmed by SQ-PCR (fold change 071; p = 0.004). Direct interaction between miR-TG and MAP4K4 was confirmed in the luciferase assay (p = 0.0006). Functional studies showed increase proliferation in K1 cell line transfected with miR-TG. We propose that in normal thyroid miR-TG plays a fine-tuning effect on the maintenance of MAPK pathway, inhibiting the expression of miR’s target MAP4K4. This regulation is disturbed in cancer due to downregulation of the novel, thyroglobulin-embedded microRNA, characterized in this study.

MicroRNA Analysis Using the Quantitative Real-Time PCR Reaction

The analysis of microRNA expression patterns provides new insights into numerous cellular processes and their aberrances in diseases. Despite its potential pitfalls, the quantitative real-time polymerase chain reaction (qPCR) is the most commonly used tool for microRNA profiling. The method requires extraction and quality analysis of RNA, which is further reverse transcribed using specific primers and used as a template in a qPCR reaction. All these elements have been addressed in this chapter.

MicroRNA Analysis Using Next-Generation Sequencing

Next-generation sequencing (NGS) enables the analysis of both microRNA expression and sequence, allowing for elucidation of a comprehensive landscape of miRNAs in a given tissue and sample type. NGS analysis requires high-quality RNA extraction and preparation of microRNA libraries. In this chapter, we describe the methods used for RNA extraction from tissue specimens, serum, cytological slides, and formalin-fixed paraffin-embedded samples. Although the described library preparation and sequencing approaches are based on Illumina NextSeq 500 sequencing technology, the presented principles shall be compatible with other commercially available sequencing platforms.

The rs2910164 Genetic Variant of miR-146a-3p Is Associated with Increased Overall Mortality in Patients with Follicular Variant Papillary Thyroid Carcinoma

Aberrant expression of the sodium-iodide symporter (NIS) and the resistance to post-operative radioactive iodide treatment is a crucial cause of higher mortality of some thyroid cancer patients. In this study, we analyzed the impact of miR-146a on the expression and function of NIS and on the overall survival of thyroid cancer patients. The study included 2441 patients (2163 women; 278 men); including 359 cases with follicular variant of papillary thyroid carcinoma (fvPTC). miR:NIS interactions were analyzed in cell lines using in vivo binding and inhibition assays and radioactive iodine uptake assays. Tumor/blood DNA was used for rs2910164 genotyping. Overall survival was assessed retrospectively. In the results, we showed that miR-146a-3p directly binds to and inhibits NIS. Inhibition of miR-146a-3p restores the expression and function of NIS, increasing radioactive iodine uptake. Rs2910164 functional variant within miR-146a-3p is associated with increased overall mortality among fvPTC female patients. The deaths per 1000 person-years were 29.7 in CC carriers vs. 5.08 in GG/GC-carriers (HR = 6.21, p = 0.006). Higher mortality of CC vs. GG/GC carriers was also observed in patients with lower clinical stage (HR = 22.72, p < 0.001), smaller tumor size (pT1/pT2) (HR = 25.05, p < 0.001), lack of extrathyroidal invasion (HR = 9.03, p = 0.02), lack of nodular invasion (HR = 7.84, p = 0.002), lack of metastases (HR = 6.5, p = 0.005) and older (age at diagnosis >50 years) (HR = 7.8, p = 0.002). MiR-146a-3p underwent somatic mutations in 16.1% of analyzed specimens, mainly towards the deleterious C allele. In this report we propose a novel molecular marker of the clinical outcome of fvPTC patients. Rs2910164 increases the overall mortality with inhibition of NIS and disruption of radioiodine uptake as a possible mechanism.