Upregulation of MAPKAPK5‐AS1, PXN‐AS1 and URB1‐AS1 lncRNAs in non‐functioning pituitary adenoma

Abstract Long non‐coding RNAs (lncRNAs) have been shown to be dysregulated in a variety of malignant and non‐malignant lesions including non‐functioning pituitary adenomas (NFPAs). In the current experimental study, we have selected six lncRNAs, namely MAPKAPK5‐AS1, NUTM2B‐AS1, ST7‐AS1, LIFR‐AS1, PXN‐AS1 and URB1‐AS1 to assess their expression in a cohort of Iranian patients with NFPA. MAPKAPK5‐AS1, PXN‐AS1 and URB1‐AS1 were shown to be over‐expressed in NFPA tissues compared with control samples (Expression ratios (95% CI) = 10 (3.94–25.36), 11.22 (4.3–28.8) and 9.33 (4.12–21.12); p values < 0.0001, respectively). The depicted ROC curves showed the AUC values of 0.73, 0.80 and 0.73 for MAPKAPK5‐AS1, PXN‐AS1 and URB1‐AS1, respectively. Relative expression level of PXN‐AS1 was associated with tumour subtype (p value = 0.49). Besides, relative expression levels of MAPKAPK5‐AS1 and LIFR‐AS1 were associated with gender of patients (p values = 0.043 and 0.01, respectively). Cumulatively, the current study indicates the possible role of MAPKAPK5‐AS1, PXN‐AS1 and URB1‐AS1 lncRNAs in the pathogenesis of NFPAs.


| INTRODUC TI ON
Pituitary adenomas (PA) include a wide variety of anterior pituitary masses. Based on the statistics, clinically important pituitary neoplasms have an incidence of 80-100 per 100,000 persons. 1 In a broad classification which is based on the levels of hormone release, pituitary neoplasms are classified as functional and nonfunctioning pituitary adenomas (NFPAs). The latter type includes about one third of all PAs. 2 Most of these tumours are considered as histologically benign tumours; however, the associated comorbidity and mortality of these tumours make them clinically important. 3 The process of development of NFPA is dependent on numerous molecular events and biomolecules, among them being long non-coding RNAs (lncRNAs). 4,5 These transcripts control expression of their targets at almost all supposed stages.
Notably, malfunction of these transcripts has been shown to speed up the carcinogenesis process in several tumours, including PA. [6][7][8][9] Recently, we have used an in silico method to find differentially expressed long non-coding RNAs (lncRNAs) in PAs versus normal samples and identify their relation with important signalling pathways. 10 In the current experimental study, we have

| Statistical analysis
Microarray data were processed using the R statistical programming language as described previously. 10 Batch effects were removed by applying the ComBat function from the R Package Surrogate Variable Analysis (SVA). Subsequently, quantile normalisation method was used to normalize data expression matrix. Quantile normalisation was performed using the preprocessCore R package. The Limma package in R language was used to identify differentially expressed lncRNAs between NFPA and normal samples.   Table 2 shows the information about the studied genes.
There was a significant positive association between age of NFPA patients and invasiveness of NFPA (χ 2 = 4.24, p value = 0.039).
Relative expression level of PXN-AS1 was associated with tumour subtype (p value = 0.49). Besides, relative expression levels of MAPKAPK5-AS1 and LIFR-AS1 were associated with gender of patients (p values = 0.043 and 0.01, respectively). Table 7 summarizes these results.

| DISCUSS ION
NFPAs are a group of histologically benign tumours that are associated with morbidity and mortality. 3

TA B L E 4
The results of expression study of six lncRNA genes in nonfunctional pituitary adenoma (NFPA) tissues compared with the adjacent normal tissues.

F I G U R E 2
The receiver operating characteristic (ROC) curve of PXN-AS1, URB1-AS1 and MAPKAPK5-AS1 lncRNA genes for discrimination of NFPA tumours from adjacent normal tissues. AUC indicates area under the ROC curve.   11 PXN-AS1-L has a role in progression of nasopharyngeal carcinoma through upregulation of SAPCD2. 12

ACK N O WLE D G E M ENTS
The authors would like to thank the clinical Research Development Unit (CRDU) of Loghman Hakim Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran for their support, cooperation and assistance throughout the period of study.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The analysed datasets generated during the study are available from the corresponding author upon reasonable request.