Expression of cAMP and oxidative phosphorylation‐related lncRNAs in non‐functioning pituitary adenomas

Abstract Non‐functioning pituitary adenomas (NFPAs) are benign lesions in the pituitary gland with important morbidities. In this study, based on a bioinformatics analysis to identify the genes and pathways that display significant differences between tumour tissues of NFPA patients and normal pituitary tissues, we selected lncRNAs related to cAMP and oxidative phosphorylation pathways, namely DNAH17‐AS1, LINC00706 and SLC25A5‐AS1. Then, we aimed to investigate by means of RT‐qPCR, the expression of these lncRNAs along with two other lncRNAs, namely CADM3‐AS1 and MIR7‐3HG in NFPA samples compared to that in healthy tissues adjacent to the tumours. Transcripts of DNAH17‐AS1, LINC00706 and MIR7‐3HG were lower in NFPA samples compared with controls (Expression ratios (95% CI) = 0.43 (0.23–0.78), 0.58 (0.35–0.96) and 0.58 (0.35–0.96); p‐values = 0.009, 0.025 and 0.036, respectively). AUC values of ROC curves of DNAH17‐AS1, LINC00706 and MIR7‐3HG were 0.62, 0.61 and 0.62, respectively. Expression of CADM3‐AS1 was associated with the gender of patients in a way that it was lower in female patients (p‐value = 0.04). The level of SLC25A5‐AS1 was lower in subjects with disease duration lower than 1 year (p‐value = 0.048). We showed dysregulation of three lncRNAs in NFPA tissues and potentiates these lncRNAs as important regulators of pathogenic events in these tumours.

indicated for tumour syndrome, but there is still debate about other aspects of NFPA, including hormonal evaluation, follow-up (especially post-operative follow-up) and management of post-operative recurrence. 3ile in the case of hormone-secreting tumours, disease monitoring is possible by measuring pituitary hormones, the only option for clinical follow-up in non-functioning tumours is MRI.
Hence, identifying non-invasive biomarkers for non-functioning tumours is clinically useful for early diagnosis and follow-up. 4The evaluation of a perfect tumour biomarker should be easy, consistent, cost-effective and performable with a method that has high sensitivity and specificity.Such a biomarker should also be specific to a certain disease, be capable to distinguish between different physiological states being reliable among different ethnicities and genders. 5n-coding RNAs have many of these features, so they are often indicated as promising biomarkers.Non-coding RNAs are involved in many cellular processes and are divided into two groups: small non-coding RNAs (less than 200 base pairs in length) and long non-coding RNAs or lncRNA (with a length of more than 200 bp). 6In the pathobiology of cancer, thousands of intracellular factors are altered, including signalling pathways, energy metabolism, gene transcription regulators, etc. [7][8][9][10] Signalling pathways are often stimulated by signalling molecules found on cell membranes or within the cytoplasm, such as growth factors, hormones, or ions.For signal transduction, a variety of intracellular chemicals known as secondary messengers are used, an important one of which is cyclic adenosine monophosphate (cAMP). 11Apart from this, the observation that glycolysis is augmented in cancer cells compared to normal cells leads to the hypothesis that oxidative phosphorylation is generally reduced in cancer, which is true for many malignancies, but in some cancers including leukaemia and lymphoma, where mitochondrial metabolism is not disturbed, this assumption is challenged. 12 performed a bioinformatics analysis based on a previously conducted microarray experiment to identify the genes and pathways that display significant differences between tumour tissues of NFPA patients and normal pituitary tissusses of healthy individuals. 13We aimed to investigate by means of RT-qPCR, the expression of DNAH17-AS1, LINC00706, SLC25A5-AS1, CADM3-AS1 and MIR7-3HG in NFPA samples compared to that in healthy tissues adjacent to the tumours.
Based on the information above, our study will yield more comprehensive knowledge about the role of the aforementioned pathways and molecules in NFPA, which will have the potential to be used for better management of the disease.

Name Type Sequence
Primer length

PCR product length
TA B L E 1 Information about primers and amplicons.

| Statistical analysis
Statistical analyses and graphics were performed using SPSS v.22.0 (SPSS Inc.) and GraphPad Prism version 9.0 (GraphPad Software), respectively.Expression levels of DNAH17-AS1, LINC00706, SLC25A5-AS1, CADM3-AS1 and MIR7-3HG were compared between NFPA samples and adjacent non-cancerous tissues using the Efficiency adjusted method.The normal/gaussian distribution of the values was judged by the Shapiro-wilk test.
Wilcoxon matched-pairs signed rank test or paired t-test was used to recognize differentially expressed genes between two sets of samples.
The correlation between expressions of mentioned genes was evaluated using the Spearman correlation coefficient.Expression levels of genes were compared between different subgroups using the Mann-Whitney and Kruskal-Wallis one-way anova test.The chisquare test was used to find out the association between clinical parameters and gene expression levels.

| General information.
Clinical data of patients are summarized in Table S1.Table 2 displays location and other characteristics of studied genes.

LINC00706
Since we detected a significant difference in the expression of DNAH17-AS1, LINC00706 and MIR7-3HG between tumoral and non-tumoral tissues, we depicted ROC curves for these lncRNAs to show their diagnostic value (Figure 2).
Expression levels of LINC00706 were significantly correlated with all other lncRNAs except for MIR7-3HG in both NFPA tissues and adjacent tissues (Table 5).Moreover, expression levels of CADM3-AS1 were correlated with those of DNAH17-AS1 and SLC25A5-AS1 in both sets of tissues.
Finally, we assessed association between expression levels of DNAH17-AS1, LINC00706, SLC25A5-AS1, CADM3-AS1 and MIR7-3HG and clinicopathological data (Table 6).Expression of CADM3-AS1 was associated with gender of patients in a way that it was lower in female patients (p-value = 0.04).Expression of SLC25A5-AS1 was lower in subjects with disease duration lower than 1 year (p-value = 0.048).

| DISCUSS ION
Expression assays can lead to the identification of important regulators of cell proliferation, apoptosis and other features in each tissue since dysregulated genes are potentially involved in these processes.In the current study, we selected lncRNAs related to cAMP and oxidative phosphorylation pathways to assess their expression in NFPA and adjacent tissues.Expression levels of DNAH17-AS1, LINC00706 and MIR7-3HG were lower in NFPA samples compared with controls.DNAH17-AS1 has been shown to promote tumorigenesis and metastatic ability of non-small cell lung cancer (NSCLC) cells through regulation of miR-877-5p/ CCNA2 pathway. 14Another miRNA that regulates expression of CCNA2, namely miR-130b has been shown to be dysregulated in pituitary adenomas. 15In the pancreatic cancer cells, DNAH17-AS1 upregulation designates aggressive behaviour of tumour.When it is silenced in these cells, cell apoptosis is induced and viability, invasion and migration are decreased. 16It acts as a miR-432-5p sponge. 16Forthcoming studies are needed to find correlation between DNAH17-AS1, other miRNAs and CCNA2 in this type of tumour.
MIR7-3HG is regarded as a MYC-dependent regulator of cell proliferation that suppresses autophagy through a regulatory loop that involves AMBRA1. 17AMBRA1 has been shown to be among genes associated with recurrence in lactotroph lesions based on a CGH array study. 18However, relation of this gene with NFPA has not been evaluated yet.AUC values of ROC curves of DNAH17-AS1, LINC00706 and MIR7-3HG were 0.62, 0.61 and 0.62, respectively.Hence, none of these lncRNAs can be considered as appropriate markers for differentiation of NFPA from adjacent tissues.Expression of CADM3-AS1 was associated with gender of patients in a way that it was lower in female patients.CADM3-AS1 is an lncRNA which is associated with regulation of protein translation, targeting and localization.Expression of this lncRNA in patients with lung adenocarcinoma has been associated with overall survival. 19pression of SLC25A5-AS1 was lower in subjects with disease duration lower than 1 year.Downregulation of SLC25A5-AS1 in gastric cancer cells can facilitate cell growth and inhibit apoptosis through miR-19a-3p/PTEN/PI3K/AKT axis. 20This pathway has a fundamental role in the pathogenesis of tumours of endocrine system. 21However, the importance of SLC25A5-AS1 in the regulation of this pathway in NFPA needs to be elucidated.Finally, the observed correlations between a number of lncRNAs in this study suggest the presence of a functional network between these lncRNAs.
In brief, we reported dysregulation of three lncRNAs in NFPA.
The current study potentiates these lncRNAs as important regulators of pathogenic events in these tumours.We propose conduction of functional assays to elaborate the mechanism of dysregulation of these lncRNAs and the functional consequences of these events.| 4201

2. 1 |
Patients Expression of DNAH17-AS1, LINC00706, SLC25A5-AS1, CADM3-AS1 and MIR7-3HG lncRNAs was assessed in 46 pairs of NFPA specimens and paired non-cancerous specimens.Tissue samples were obtained during tumour excision from patients admitted to Loghman and Nikan hospitals during 2021-2022.Patients took no chemo/radiotherapy prior to lesion excision.The study protocol was approved by the ethical committee of Shahid Beheshti University of Medical Sciences.Informed consent forms were obtained from all participants.

F I G U R E 1
Levels of lncRNAs expression in non-functional pituitary adenoma (NFPA) tissues compared with adjacent non-tumoral tissues as described by -delta Ct values.Delta Ct Data were plotted as box-and-whisker plots showing median [line], mean [cross], interquartile range [box] and minimum and maximum values.Data were analysed using the Wilcoxon rank-sum test or paired t-test, and p < 0.05 was considered statistically significant.Asterisks designate significant differences between groups (*p-value < 0.05, **p-value < 0.01 ns; nonsignificant).

F I G U R E 2 4 TAHERI
ROC curves of DNAH17-AS1, MIR7-3HG and LINC00706 lncRNAs for discrimination of NFPA tumours from adjacent normal tissues.AUC designates an area under the ROC curve.ROC curve analysis of lncRNAs transcript levels for discrimination of NFPA tumours genes from adjacent non-tumoral tissues.et al.positive association between disease duration and CSF leak (χ 2 = 9.4, p-value = 0.043).Furthermore, a significant positive association was detected between Knosp classification and invasiveness of NFPA (χ 2 = 7.28, p-value = 0.026).Lastly, a significant positive association was reported between tumour size and Knosp classification (χ 2 = 14.4,p-value = 0.006) and invasiveness of NFPA (χ 2 = 8.17, p-value = 0.017).

Table 3
General features of studied genes.
The results of expression study of five lncRNAs in nonfunctional pituitary adenoma (NFPA) tissues compared with the neighbouring normal tissues.
TA B L E 3 Comparison of expression levels of lncRNAs in NFPA patients with different clinical features.Mann-Whitney and Kruskal-Wallis one-way anova tests were used for comparing gene expression levels.