MiRNA signatures related to invasiveness and recurrence in patients with non-functioning pituitary adenomas

doi:10.21203/rs.3.rs-1656363/v1

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MiRNA signatures related to invasiveness and recurrence in patients with non-functioning pituitary adenomas

https://doi.org/10.21203/rs.3.rs-1656363/v1

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Purpose: The aim of the present work was to analyse and identify differentially expressed miRNAs in Bulgarian patients with non-functioning pituitary adenomas (NFPA). The relationship between deregulated miRNAs and tumor size, invasiveness, and recurrence was determined.

Methods: Twenty patients with NFPAs were selected and fresh pituitary tumor tissues were collected. RNA containing miRNAs were isolated using miRNAeasy mini kit and analysed with LNA miRNA Cancer Focus PCR Panel (Qiagen) by quantitative real-time PCR.

Results: Three miRNAs (miR-210-3p, miR-149-3p and miR-29b-3p) were deregulated in invasive compared to non-invasive NFPAs. Differential expression of 4-miRNA signature - miRNA-17, miR-19, miR-106a and miR-20 was informative as a recurrence biomarker. ROC analysis showed that the selected miRNAs could be used as potential biomarkers for the diagnosis, prognosis and therapy monitoring of NFPAs.

Conclusion: In this pilot study, we selected a unique miRNA expression profile correlating with NFPA invasiveness and recurrence. Moreover, some of the selected miRNAs are reported for the first time in patients with NFPAs and might elucidate the molecular mechanisms involved in pituitary pathogenesis. The selected miRNAs showed a promising biomarker potential and could be studied for future miRNAs-based anti-NFPAs therapy.

miRNA

non-functioning pituitary adenomas

invasive pituitary adenomas

aggressive pituitary tumors

Pituitary adenoma (PA) is the most frequent tumor in the sellar area. With a prevalence of 16.7% it is the third common intracranial tumor which can cause remarkable morbidity (1). Ongoing improvements in diagnostic technology have shown a rising trend in the incidence rates of this disease. Non-functioning pituitary adenomas (NFPAs) represent a heterogeneous group of tumors characterized by the lack of endocrine symptoms related to hypersecretion of adenohypophyseal hormones. They account for 15–30% of pituitary adenomas (2). Generally, PAs are benign lesions, however, a small subset of pituitary tumors does not respond to standard medical treatment and presents with rapid growth, invasiveness and multiple local recurrences (aggressive pituitary tumors) and in extremely rare occasion with metastases (pituitary carcinomas) (3). After complete or near-complete surgical resection, 12–58% of non-functional pituitary adenoma patients still experience regrowth, which cannot adequately be controlled by available therapeutics (4). Pituitary carcinomas are even rarer with an incidence of 0.1–0.2% of all pituitary tumors (5).

Because of the complicated pathogenesis of pituitary adenomas, it was believed that the risk factors include genetic events, hormonal stimulation, and growth factors in general. These three factors can promote tumor cell growth and proliferation (6). The progression from pituitary adenoma to carcinoma is accompanied by cumulative changes in molecular abnormalities (7), however mechanisms elucidating pituitary malignancy remain elusive. MicroRNA expression profiling by molecular techniques provides a powerful tool to elucidate involvement of these tiny molecules in pituitary tumor development and progression (7). MiRNAs are small single-stranded non-coding RNAs (containing about 18–25 nucleotides) that are involved in post-transcriptional regulation of gene expression (8). They function via base-pairing between “seed” sequence and its complementary sequences within messenger RNA (mRNA) molecules. As a result, these mRNA molecules are silenced, by one or more of the following processes: cleavage of the mRNA strand, destabilization of the mRNA through shortening of its poly (A) tail, and less efficient translation of the mRNA into proteins by ribosomes. Given their small size and relative stability, they show superior biomarker potential as compared to mRNA expression (9). MiRNAs have been implicated in many cellular processes, including cell proliferation, apoptosis, cell adhesion and metabolism, and have a role in many developmental processes, including stem cell and germline maintenance, development, and differentiation (10). An increasing number of studies have demonstrated that overexpressed miRNAs may function as oncogenes while downregulated miRNAs mainly are tumor suppressors. Thus, alterations in miRNAs expression can potentially be involved in the development of human neoplasia. Indeed, miRNAs are aberrantly expressed in various types of tumors, such as the liver, pancreas, esophageal, stomach, colon, hematopoietic, ovarian, breast, prostate, thyroid, testicular, and brain cancers (10–12).

As in other human tumors, there is an increasing interest in the study of miRNAs in pituitary adenomas and carcinomas (13). Several aberrant expression patterns of miRNAs in pituitary adenomas as compared to normal pituitary have been identified up to date which brought about a revolution in pituitary research. MiRNAs have been proposed as a promising biomarker which contributes to the primary diagnosis, prognosis, postoperative follow-up, and early prediction of postoperative recurrence for pituitary adenoma (14). Butz et al. have indicated a definite correlation between miRNAs expression levels and tumor size in NFPAs (15). Wu et al. suggested that miRNAs could be closely related to the invasive growth of aggressive PAs (16).

The aim of this study was to expound the pathogenesis of the NFPAs by investigating their miRNAs expression profile. We analyzed a broad spectrum of microRNAs in patients with NFPA thus selecting a deregulated microRNA profile reflecting disease aggressiveness, tumor size and recurrence.

Design

This was a prospective study in 20 patients with NFPAs − 13 men (65%) and 7 women (35%), with a mean age of 58.5 ± 16.7 years. Only patients with pituitary macroadenomas (tumor size > 10 mm) were included to the study, 5 of them (25%) were with giant adenomas (tumor size > 40 mm). Three patients (15%) had recurrent adenomas.

Patient’s History And Physical Examination

In 3 patients (15%) NFPAs were incidentally visualized (incidentalomas) on MRI whereas in 17 (85%) macroadenomas were diagnosed because of symptoms due to tumor compression on surrounding structures (headaches, decrease of visual acuity, visual field deficit, diplopia, signs of partial or panhypopitarism). Pituitary apoplexy manifested with a sudden onset of severe headache, nausea, vomiting, ophthalmoplegia, and pituitary disfunction was registered in 1 patient. MR imaging detected intratumoral haemorrhage in one more patient. The main presenting symptoms are listed in Table 1.

Table 1

Main symptoms of patients with NFPAs
Presenting symptoms	N (%)
Headache	17 (85%)
Visual disturbances	17 (85%)
Asteno-adynamia	6 (30%)
Loss of consciousness	2 (10%)
Weight loss	1 (5%)
Pituitary apoplexy	1 (5%)
Epileptic seizure	1 (5%)
Palpebral ptosis	1 (5%)
Vertigo	1 (5%)
Decreased libido	1 (5%)
Ischemic stroke	1 (5%)

Patients’ history was taken and full physical examination with a special focus on specific clinical manifestations of adenohypophyseal hypersecretion (galactorrhea, menstrual disturbances, decreased libido, typical features for acromegalia, “moon facies”, catabolic striae, etc.) was performed in all study participants. None of the patients manifested signs of anterior pituitary hypersecretion.

Hormonal Analysis

Diagnosis NFPA was confirmed by preoperative hormonal analysis which did not reveal adenohypohyseal hyperfunction. Circulatory levels of prolactin (PRL), growth hormone (GH), insulin-like growth factor-1 (IGF-1), luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (in men), oestradiol (in women), thyroid stimulating hormone (TSH), free thyroxine (fT4), adrenocorticotropic hormone (ACTH) and cortisol level were measured. Hypopituitarism (partial or panhypopituitarism) was found in 9 patients (45%).

Mri/assessment Of Invasiveness

Based on preoperative MRI suprasellar expansion was observed in 15 patients (75%), parasellar – in 8 patients (40%) and infrasellar in 8 patients (40%). Based on Modified Knosp Grading system for cavernous sinus-invading NFPAs twelve tumors (60%) were categorized as non-invasive and eight (40%) as invasive pituitary adenomas.

Sample Collection

The study was approved by the Ethics Committee for Scientific Research at the Medical University of Sofia (KENIMUS). All patients (n = 20) provided written informed consent before all study procedures. Fresh tumor tissues from PA were collected using transsphenoidal surgery and immediately placed in RNA latter solution for RNA protection (Qiagen, Germany). Samples were stored at -80°С until the further analysis.

Mirna Extraction

MiRNA extraction

Total RNA including miRNA fraction were purified using miRNeasy mini kit (Qiagen, Germany) according to the manufacturer’s instructions. Extraction quality and cDNA synthesis were monitored using RNA spike-in kit (Qiagen, Germany). During the first step 1 µl of spike-in control mix (UniSp2, UniSp4 and UniSp5) with descending concentrations was added into QIAzol lysis buffer. RNA was eluted in 50 µl RNase-free water and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). RNAs with А260/А280 > 1,8 were included in the subsequent analysis.

Cdna Synthesis

MiRCURY LNA RT kit (Qiagen, Germany) was used for cDNA synthesis using poly(A) polymerase and the reverse transcriptase. During the RT reaction UniSP6 and cel-miR-39-3p spike-ins were used as a cDNA synthesis quality control. Synthesized cDNAs were stored at temperatures between − 30 and − 15°C until the further analysis.

Qpcr

We used miRCURY LNA miRNA Cancer Focus PCR Panel (Qiagen, Germany) for miRNA expression profiling. We analysed expression profiles of 84 cancer-specific and even low expressed miRNAs without preamplification step, by LNA (locked nucleic acids) approach. The qPCR protocol was conducted using a LightCycler 480 (Roche). For miRNA amplification control and inter-plate calibrator control was used a set of UniSp3.

Data analysis

The GeneGlobe Data Analysis tool (Qiagen, Germany) was used for selection of most stable reference miRNAs, miRNA normalization. Fold Change (FC) in miRNA expression was calculated using 2^-ΔΔCр method. The GeneGlobe Data Analysis platform uses “FR - Fold Regulation” instead of fold change and Fold Regulation = FC if > 1. If FC < 1, Fold Regulation = − 1/FC.

Statistical analysis

Two-tailed, un-paired t-test was used for miRNA expression analysis. The correlation analysis was used for analysis between the clinicopathological characteristics and deregulated miRNAs. ROC (Receiver Operating Characteristic) curves were used to assess the differentially expressed miRNAs and AUC (areas under the curve) were calculated. Data analyses were performed using SPSS v 26 (IBM, USA). Data are shown as mean ± SD. P < 0.05 was considered statistically significant.

The aim of our scientific work was to investigate the aberrant expression of miRNAs that correlate with recurrent and invasive nature of NFPAs. We assessed 84 cancer–specific miRNAs in fresh tumor tissues from Bulgarian patients. Additionally, we wanted to analyze and select a specific miRNA profile that could be used as a prognostic recurrence and size-related biomarker.

Invasion-related Mirnas

Expression analysis showed deregulated miRNAs when comparing invasive NFPA (n = 8) and non-invasive NFPA (n = 12) (Table 2). The most significantly down-regulated were miR-210-3p (p < 0.001), miR-149 (p < 0.05) and miR-29b (p < 0.001), miR-103а (p < 0.001), and miR-107 (p < 0.001) in invasive NFPA with fold regulation (FR) = -6.12; FR = -8.4; FR = -4.03, FR = -2.71, FR = -2.71, respectively. We found miR-100 slightly up-regulated, FR = 3.43 (Fig. 1).

Table 2

Deregulated miRNA in invasive NFPA compared to non-invasive NFPA
miRNA name	FR	p-value
miR-149-3p	-8.4	0.01
miR-210-3p	-6.12	(p < 0.001)
miR-29b-3p	-4.03	(p < 0.001)
miR-103a-3p	-2.71	(p < 0.001)
miR-107	-2.71	(p < 0.001)
miR-100	3.43	0.04
FR - fold regulation

Additionally, we observed aberrant expression of some short-noncoding RNAs, such as U6, SNORD49A, SNORD38B that were included in the panel as potential reference controls.

Recurrence-related Mirnas

Furthermore, we divided invasive NFPA into two groups. Recurrent tumors were compared to newly diagnosed tumors (Table 3). Significantly up-regulated were miR-20а, miR-19a, miR-16, miR-17, while miR-106a was slightly up-regulated (Fig. 2).

Table 3

Deregulated miRs in recurrent NFPAs compared to NFPAs without recurrence
miR name	FR	p-value
miR-20а	2.35	0.005
miR-16	2.04	0.009
miR-19a	2.07	0.007
miR-17	2.12	0.012
miR-106a	1.88	0.02
FR - fold regulation

Size-related Mirnas

Additionally, we compared miRNA expression in giant NFPAs (n = 5) and other macroadenomas (n = 15). We found that miR-186 (FR = -24.08, p = 0.001), miR-17 (FR = -24.88, p = 0.006) and miR-210 (FR = -22.77, p = 0.012) were the most significantly down-regulated, while miR-9 (FR = 51.94) (p = 0.0015) was the most highly expressed and up-regulated. MiR-9 is a brain specific miRNA that showed significantly increased expression in our patients with giant NFPAs (Table 4).

Table 4

Deregulated miRs in giant NFPAs compared to other macroadenomas
miRNA name	FR	p-value
Up-regulated
miR-9	51.94	0.0015
Down-regulated
miR-17	-24.88	0.006
miR-186	-24.08	0.001
miR-210-3p	-22.77	0.012
FR - fold regulation

The correaltion anaylsis between clinocopathological characteristics and miRNA expession showed a possitive correlation between miR-17, miR-210, miR-186, miR-29b, miR-20, miR-149, miR-106a (r > 0.7, p > 0.01) and invasiveness of NFPA.

Roc Curve Analysis

ROC (Receiver Оperating Characteristic) curves and AUC (Area Under the Curve) were used to calculate the biomarker power of the selected miRNAs and their diagnostic potential.

ROC analysis of deregulated miRNA showed that miR-210-3p (AUC = 0.903, p = 0.003, 95% Confidence Interval (CI) = 0.721-1), miR-149 (AUC = 1, p < 0.001, 95% CI = 1), and miR-29b (AUC = 0.898, p = 0.004, 95% CI = 1) could be used as diagnostic biomarkers to differentiate between invasive NFPA and non-invasive NFPA (Fig. 3).

ROC analysis of miRNA potential to differentiate recurrent NFPA showed that the combination of miR-17, miR-20 and miR-106a is a suitable biomarker (AUC = 1, p = 0.025, 95% CI = 1) (Fig. 4).

Target Analysis

The cell-signaling pathways regulated by some of the most disturbed miRNAs were analyzed by GeneGlobe tool. Some of the target genes of our disturbed miRNAs were important tumor-suppressors and cell-signaling regulators (Table 5).

Table 5

Selected putative miRNA targets of our selected deregulated miRNAs
miRNA	Some of the putative mRNA targets	Function of the targets
miR-186	PIK3R3, BCL2L11, DICER1, MAPK1, VEGFA, CDK5R1, WNT5A, PIK3- AKT	angiogenesis, cell proliferation and invasion
miR-20а	TP53INP1, RB1, CASP2, CASP7	tumor-suppressors, apoptosis
miR-17	TP53INP1, RB1, CASP7, VEGFA	tumor-suppressors, angiogenesis, apoptosis
miR-16	DICER1, TSC1, BCL2, FGFR1, NF1, TSC1	oncogenes, tumour-suppressors, gene expression
miR-106а	RB1, CASP2, CASP7	tumor-suppressors, apoptosis
miR-210-3p	ARMC1 BDNF B4GALT5 EFNA3, ISCU MID1IP1, NEUROD2, NDUFA4, SCN9A, FGFR	neural development and erythropoiesis, apoptosis, migration and cell division
miR-149-3p	MAPK, TGF	cell growth, proliferation
miR-29b-3p	TET3, COL1A1, DNMTs,	DNA methylation, cell connection
miR-9	PTCH1, HOXA11, MAP3K1,PIK3C2A, RAB11FIP4 SCN2B, RABs, EFNA1, IGF2BP2, KLF13	apoptosis, DNA reparation, development, cell growth, intracellular vesicular trafficking

In the current work we aimed to analyze the NFPAs that are the second most common PAs after prolactinomas in adults. Furthermore, up to 10% of PAs show aggressive nature with invasive growth, treatment resistance and recurrence after surgical resection, radiotherapy and chemotherapy (3, 17). The group of the NFPAs is of significant clinical importance as by now, no circulating hormones or other biomarkers for disease monitoring are available. Selection of informative molecular profile in patients with NFPAs regarding aggressiveness of the disease is an actual scientific problem. As miRNAs are tissue specific their expression profiles are disturbed during malignization and invasion. We assumed that a restricted miRNA number could be used as a potential biomarker for diagnosis, prognosis and monitoring of patients with NFPAs.

MiRNA signature associated with invasiveness

Our group analysed 84 onco-specific miRNAs. ROC analysis showed that deregulated miRNAs with potential clinical application for differentiation and stratification of invasive NFPAs are miR-210-3p, miR-149-3p, and miR-29b-3p. Interestingly, we observed deregulation in some short non-coding RNAs such as U6 snRNA, SNORD49A, and SNORD38B, that have been frequently used as reference controls. Our analyses show that they are not suitable for data normalisation and reference controls in tumor tissues from NFPAs, as they are significantly deregulated. We performed normalization with endogenous stably expressed miRNAs.

Decreased expression of miR-210-3p, observed in our cohort of invasive NFPAs, also correlates with EMT (epithelial–mesenchymal transition), cell migration and metastases and is related to disturbance of RAS-MAP and PIK3-pathways (18). It has been reported in ovarian and bladder cancer (19, 20). Restoration of miR-210-3p expression inhibits cell migration, probably by regulating processes as apoptosis, migration and cell division of its putative target FGFR (19, 20). Target analyses using GeneGlobe showed that one of the miR-210-3p target is the EFNA3, regulating neural development and erythropoiesis. Other authors have observed increased expression of miR-210-3p in patients with lung cancer, GBM, pancreas cancer and in metastases from breast and prostate cancer (21, 22). The expression level and clinical significance of miR-210-3p patients with NFPAs need to be further explored.

MiR-149-3p is significantly under expressed in NFPAs compared to functioning PAs and is suggested to be one of 17 down-regulated miRNAs which expression may be useful in predicting aggressive behavior of PAs (23). Restoration of miR-149-5p together with miR-99a-3p levels could inhibit invasiveness in PAs, tumor growth, angiogenesis and ETM (24). The down-regulated miR-149-3p in invasive NFPAs has also been associated with suppression of the malignant potential in spinal chordoma (25) and with development of gastric and esophageal tumors (26, 27), probably related to the regulated pathways as MAPK (Mitogen-activated protein kinase) pathway, apoptosis, TGF beta (Transforming growth factor beta) pathway.

MiR-29b- 3p was suppressed in NFPAs probably due to its function as a tumor – suppressor (28). MiR-29 family has been deregulated in osteosarcoma (29), breast cancer (30) and prolactinomas (31). Additionally, miR-29b-3p has been reported to increase the radioresistance in cell cultures and prolactinomas, regulating IGF-1, DNMT1, DNMT3B, Bcl-2, and PIK3R1/AKT2 pathways (31). These data supported the role of miR-29a-3p in the progression and invasiveness of NFPAs by regulating oncogene expression. Furthermore, miR-29b-3p could be a novel therapeutic target related to radiotherapy sensitivity.

Taken together, our selected miR-210-3p, miR-149-3p, and miR-29a-3p could be used as putative biomarkers for NFPAs invasiveness.

MiRNA signature associated with recurrence

To further investigate whether a deregulated miRNA profile could predict a recurrence, we analyzed patients with and without relapsed NFPAs. We observed up-regulation in miR-20а, miR-16, miR-19a, miR-17, and slightly in miR-106а. Interestingly, according to the ROC analysis, we found that our data are in concordance with Wei et al. who have reported up-regulation of miR-20a, miR-106b and miR-17 in non-functioning pituitary carcinomas with many metastases compared to primary neoplasms, as well as in atypical PAs than typical PAs (7). The observed up-regulation of miR-20a, miR-106b, miR-19 and miR-17 is presumably related to inhibition of their targets - PTEN and TIMP-2 (7). MiR-17 increased expression correlates with cell proliferation and invasiveness in PAs and is suggested as a proliferative biomarker (32). To the best of our knowledge, the present study is the first to provide evidence that miR-19a-3p was up-regulated in recurrent NFPAs. MiR-19 is up-regulated by estrogen (33) and has been observed up-regulated in bone metastasis from ER + breast cancer (34), colorectal cancer (35), lung cancer (36), renal cell carcinoma (37), gastric cancer (38) and other tumors. The results of the present and previous studies indicate that miR-19 can regulate the proliferation and progression of NFPAs, targeting PTEN (37, 39) and PI3K/AKT pathway (38). Probably, up-regulated miR-19a suppresses PTEN and activates PI3K/AKT pathway, thus promoting epithelial to mesenchymal transition and NFPA recurrence.

In our study, miR-16 was up-regulated in relapsed patients but ROC analysis showed weak diagnostic capability. Other data reported that up-regulation of miR-16 inhibits proliferation and activates apoptosis by regulating ERK/MAPK MEK1 and other pathways in PA (40).

Association of disturbed expression of miR- 20а, miR-19a, miR-17 and miR-106a with recurrence could be of unique clinical importance. Patients with increased biomarker risk of relapse should be monitored more frequently and the therapy could be adjusted.

Size-related miRNAs

We observed significant increase in miR-9 expression in patients with giant NFPAs and down-regulation of miR-186, miR-17 and miR-210-3p. The last two miRNAs were also associated with recurrence and invasiveness in NFPAs discussed above. MiR-9 role in NFPAs is not clear but it is known that it plays an important role in brain development, synaptic plasticity, memory and is highly expressed in the adult brain (41, 42). MiR-9-5p has been down-regulated after cocaine treatment (43) and increased in pituitary due to fetal alcohol exposure leading to suppressed dopamine D2 receptor and increased prolactin (44). Furthermore, in breast cancer, lower miR-9 expression was associated with smaller tumors, improved overall survival and earlier stage (45).

The down-regulated miR-186 that we found was also size-related, acts as a tumor-suppressor regulating apoptosis, invasion and therapy resistance in PAs and other cancers. Suppression of miR-186 leads to increase levels of its target - SKP2 (F-box protein S-phase kinase-interacting protein-2) increasing the proliferation in human and mice pituitary tumor cells (46). Additionally, target analysis of miR-186 showed its regulation of PIK3-АКТ pathway, responsible for cell proliferation and invasion. In out cohort, ROC analysis showed weak diagnostic capability of the selected miRNAs to differentiate between giant adenomas and macroadenomas, probably due to the small number of patients.

Given that miRNAs expression seems to be tissue specific and have been found aberrantly expressed in various types of tumors (10–12, 34–38, 47) we suggested that a restricted miRNA signature could be used as a biomarker in NFPAs. It is known that miRNAs are deregulated in PAs compared to normal pituitary tissues (12) but few studies have been conducted so far to identify miRNAs associated with aggressiveness of NFPAs (7). The aim of our study was to select a unique and specific miRNAs profile associated with invasiveness and recurrence prediction that could facilitate diagnosis, prognosis, and therapy monitoring of NFPAs. Our results suggest that the selected miRNA signature might be a promising target in future therapy for patients with invasive NFPAs. However, their possible off-target effects should be precisely considered particularly before translation into clinical practice.

In conclusion, we selected miRNAs signature with diagnostic and prognostic biomarker potential. This is the first study analyzing deregulated miRNA profile in Bulgarian patients with NFPAs. The selected deregulated miRNA signature of miR-210-30, miR-149-3p, miR-29b-3p showed diagnostic and prognostic biomarker potential and could differentiate invasive and non-invasive NFPAs. Additionally, our miRNA signature of miR-20а, miR-19, miR-106а, miR-17 was informative for patient recurrence status highlighting its promising clinical application. Furthermore, we showed that short non-coding RNAs such as U6 snRNA, SNORD49A, SNORD38B, are deregulated in NFPAs and are not suitable for reference controls in NFPA tissues. The selected miRNAs signatures could be further analyzed as future targets for development of personalized therapy against invasive and recurrent NFPAs.

Funding

Medical University Sofia, grant number D-117/24.06.2020.

Competing interests

All authors declare that they have no competing interests or other interests that might be perceived to influence the results and/or discussion reported in this paper.

Research involving human rights

All procedures performed in this study were in accordance with the ethical standards of the institutional ethics committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All study participants signed written informed consent prior to all study procedures.

Author contributions

All authors contributed to the manuscript and approved the final version.

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No competing interests reported.

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MiRNA signatures related to invasiveness and recurrence in patients with non-functioning pituitary adenomas

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Abstract

Figures

Introduction

Materials And Methods

Design

Patient’s History And Physical Examination

Hormonal Analysis

Mri/assessment Of Invasiveness

Sample Collection

Mirna Extraction

Cdna Synthesis

Qpcr

Data analysis

Statistical analysis

Results

Invasion-related Mirnas

Recurrence-related Mirnas

Size-related Mirnas

Roc Curve Analysis

Target Analysis

Discussion

MiRNA signature associated with invasiveness

MiRNA signature associated with recurrence

Size-related miRNAs

Conclusion

Declarations

References

Additional Declarations

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