ABSTRACT The molecule mechanisms of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in human diseases have been broadly studied recently, therefore, our research aimed to assess the

effect of lncRNA taurine upregulated gene 1 (TUG1)/miR-187-3p/tescalcin (TESC) axis in pituitary adenoma (PA) by regulating the nuclear factor-kappa B (NF-κB) signaling pathway. We observed

that TUG1 was upregulated in PA tissues and was associated with invasion, knosp grade and tumor size. TUG1 particularly bound to miR-187-3p. TUG1 knockdown inhibited cell proliferation,

invasion, migration, and epithelial–mesenchymal transition, promoted apoptosis, and regulated the expression of NF-κB p65 and inhibitor of κB (IκB)-α in PA cells lines in vitro, and also

sponges miR-187-3p to affect PA development by elevating TESC and regulating the NF-κB signaling pathway. SIMILAR CONTENT BEING VIEWED BY OTHERS LNCRNA LINC00473 IS INVOLVED IN THE

PROGRESSION OF INVASIVE PITUITARY ADENOMA BY UPREGULATING KMT5A VIA CERNA-MEDIATED MIR-502-3P EVASION Article Open access 05 June 2021 LNCRNA LINC00667 AGGRAVATES THE PROGRESSION OF

HEPATOCELLULAR CARCINOMA BY REGULATING ANDROGEN RECEPTOR EXPRESSION AS A MIRNA-130A-3P SPONGE Article Open access 14 December 2021 SP1-MEDIATED UP-REGULATION OF LNCRNA TUG1 UNDERLINES AN

ONCOGENIC PROPERTY IN COLORECTAL CANCER Article Open access 04 May 2022 INTRODUCTION Arising from the adenohypophysial cells of the anterior pituitary1, pituitary adenoma (PA) constitutes at

least 15% of intracranial neoplasms. The anterior pituitary is comprised of some hormone-producing cell types that can result in a rise to tumors, causing the heterogeneous group of

neoplasms encompassed by the diagnosis of PA2. Microadenomas (diameter <10 mm) accounts for ~60% of PAs and macroadenomas (diameter >10 mm) accounts for ~40%, and these tumors can be

ulteriorly grouped into functional (hormone secreting) and non-functional3. Although there is a high prevalence of PA in general people, these tumors are always benign and perform

characteristics of differentiated pituitary cell function and premature proliferative arrest4. Previous studies have clarified that intrinsic and extrinsic factors can lead to the risks of

PA5,6,7. Currently, treatments of PA include observation, medical therapy, surgery, and radiotherapy, which are depended on the nature and classification of the tumors1. However, these

treatments are not available for many patients. Thus, the investigation to find a possible molecular target of PA might contribute to finding a novel treatment for this disease. Long

non-coding RNAs (lncRNAs) function as transcriptional activators and suppressors consistent with different chromatin modifiers8. The functions of many lncRNAs have been discussed in PA, such

as lncRNA IFNG-AS19 and lncRNA CCAT210. LncRNA taurine upregulated gene 1 (TUG1), which is situated at chromosome 22q12, is initially verified as a part of photoreceptors and retinal

development in murine retinal cells11. TUG1 has been revealed to regulate carcinogenesis in some malignancies, including malignant melanoma12 and glioma13. Although TUG1 exhibits differently

high expression in tumors, its effect on PA remains largely unknown. Considering the oncogenic role of TUG1 in human brain tumors14,15, we speculated that it may participate in the

progression of PA. LncRNAs are known to regulate the expression of microRNAs (miRNAs) through the decoy or sponge effect16. miRNAs are endogenous non-coding RNAs that play regulatory roles

by complementary base pairing to 3′-untranslated region (3′-UTR) of protein-coding mRNA17. MiR-187-3p has been identified in human tumors, including hemangioma18 and hepatocellular carcinoma

(HCC)19. Nevertheless, its role in PA remains to be further explored. Additionally, it has been reported that TUG1 sponges miR-29a in cholangiocarcinoma20 and sponges miR-498 in esophageal

squamous cell carcinoma21. We found through bioinformatic prediction that there were binding sites between TUG1 and miR-187-3p, and this binding relationship has been scarcely investigated.

Moreover, tescalcin (TESC) is a highly expressed gene in many cancer tissues and is thereby considered as an oncogene22. Although the impacts of TESC on malignancies such as renal cell

carcinoma (RCC)23 and colorectal cancer (CRC)24 have been previously studied, its effect on PA as well as the target relation between TESC and miR-187-3p remains unexplored. Furthermore,

nuclear factor-kappa B (NF-κB) is a nuclear transcription factor that is broadly expressed in the cytoplasm of higher eukaryotes25, and the NF-κB signaling pathway participates in human

pituitary neoplasm26 and murine pituitary corticotroph tumor27. This research aimed to clarify the role of lncRNA TUG1/miR-187-3p/TESC axis in PA by regulating the NF-κB signaling pathway,

and we hypothesized for the first time that TUG1 could competitively bind with miR-187-3p to regulate the development of PA by targeting TESC and modulating the NF-κB signaling pathway.

MATERIALS AND METHODS ETHICS STATEMENT Written informed consents were acquired from all patients before this study. The protocol of this study was confirmed by the Ethic Committee of

Shandong Provincial Hospital Affiliated to Shandong First Medical University (ethical number: 201851124). The protocol of animal experiments was approved by the Institutional Animal Care and

Use Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University (ethical number: 201860913). STUDY SUBJECTS Fifty-five PA specimens were harvested from PA

patients (30 males and 25 females, aging 16–69 years, mean age of 51 years) that had accepted transsphenoidal surgery in Shandong Provincial Hospital Affiliated to Shandong First Medical

University. According to the types of hypophyseal hormones, the cases were divided into 16 prolactin (PRL) tumors, 5 adrenocorticotrophic hormone (ACTH) tumors, 11 growth hormone (GH)

tumors, and 23 non-functional tumors. Among the specimens, 10 cases <1 cm, 31 cases varied from 1 to 3 cm and 14 cases >3 cm in diameter. All the patients were diagnosed as PA by

clinicopathological examination. Based on the standard of knosp classification28, the PA specimens were grouped in to I (10 cases), II (22 cases), III (17 cases), and IV (6 cases) stages.

Specimens in III–VI stages were defined as the invasive group (24 cases) and those in 0–II stages were named as the non-invasive group (31 cases). The baseline data of these patients were

recorded and patients with other malignancies or had accepted tumor therapies (drug therapy, radiotherapy, gamma knife, etc.) were excluded. In addition, 11 normal human anterior pituitary

glands (the normal group) were collected from the donation process. CELL CULTURE PA cell lines (HP75 and GH3) were purchased from Beijing Zhongyuan Ltd. (Beijing, China). PA cell lines were

cultured in media supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Inc., CA, USA), streptomycin (100 μg/mL), and penicillin (100 units/mL). The above cells

were cultured at 37 °C in a humidified atmosphere of 5% CO2. CELL GROUPING AND TRANSFECTION The GH3 and HP75 cells were divided into seven groups: the Blank (no treatment), short hairpin RNA

(sh)-negative control (NC) (transfection with NC of shRNA plasmid), sh-TUG1 (transfection with TUG1 shRNA plasmid), mimic NC (transfection with miR mimic NC), miR-187-3p mimic (transfection

with miR-187-3p mimic), sh-TUG1 + inhibitor NC (transfection with TUG1 shRNA plasmid and miR inhibitor NC), and sh-TUG1 + miR-187-3p inhibitor (transfection with TUG1 shRNA plasmid and

miR-187-3p inhibitor) groups. Oligonucleotides and plasmids used for transfection were obtained from GenePharma Co., Ltd. (Shanghai, China). All cell transfection was performed using

Lipofectamine 3000 following the instructions of the manufacturer (Thermo Fisher Scientific). 3-(4,5-DIMETHYL-2-THIAZOLYL)-2,5-DIPHENYL-2-H-TETRAZOLIUM BROMIDE (MTT) ASSAY Cells were seeded

onto a 96-well plate at 1 × 104 cells/mL. Each well was appended with 180 μL MTT solution when the cells were incubated for 24 h, 48 h, and 72 h. After incubated continuously for 2 h with

the supernatant removed, each well was added with 150 μL dimethyl sulfoxide and shaken for 10 min. A microplate reader (Tecan M1000, Invitrogen) was used to measure the cell viability at 570

 nm. The experiment was independently repeated for three times. COLONY FORMATION ASSAY Cells were trypsinized and made into cell suspension, which was seeded and incubated for 2–3 weeks. The

incubation was stopped and the medium was discarded when the colonies could be observed by eyes. The cells were fixed, stained by Giemsa dye for 60 min and dried. The colonies were counted

under a microscope. The experiment was independently repeated for three times. FLOW CYTOMETRY Cell cycle distribution and apoptosis were assessed as previously described29, and a flow

cytometer (Biosciences, San Jose, CA) was used for the analysis. The experiment was independently repeated for three times. TRANSWELL ASSAY Cells (3 × 104) were seeded into an 8-μm pore

membrane with or without Matrigel (1 : 8, Becton, Dickinson and Company, NJ, USA), and the basolateral chamber was set with medium with 10% FBS. Incubated for 24 h, cells on the apical layer

were removed and those on the lower layer were fixed in Methanol for 20 min and stained with 0.1% crystal violet dye solution for 20 min. Five fields of view were randomly photographed and

the transmembrane cells were counted. The experiment was independently repeated for three times. REVERSE TRANSCRIPTION QUANTITATIVE POLYMERASE CHAIN REACTION Total RNA in tissues and cells

was extracted by Trizol method (Invitrogen) and RNA was reversely transcribed into cDNA. The PCR was conducted by SYBR Green method. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used

as the loading control of TUG1 and TESC, and U6 was used as the loading control of miR-187-3p, Bcl-2, Bax, N-Cadherin, Vimentin, and E-Cadherin. The primers (Table 1) were synthetized by

Genechem Co., Ltd. (Shanghai, China). The analysis was conducted by a PCR instrument (ABI 7500, ABI, CA, USA) and the data were analyzed by 2−ΔΔCt method. The experiment was independently

repeated for three times. WESTERN BLOT ANALYSIS Cells and tissues were lysed with 1 mL lysate plus 10 μL of PMSF (100 mM) for 30 min, and the lysate was centrifuged at 12,000 rpm for 5 min

at 4 °C. The supernatant was placed in a 0.5-mL centrifuge tube and placed at −20 °C. Protein concentration was determined using the bicinchoninic acid method (Solarbio Science &

Technology Co., Ltd., Beijing, China). The extracted proteins were conducted with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Boster) and transferred onto membranes, which

were blocked by 5% bovine serum albumin for 1 h and appended with primary antibodies TESC (1 : 1000, Santa Cruz Biotechnology Inc., CA, USA), NF-κB p65 (ab32536, 1: 2000), IκB-α (ab32518, 1

 : 1000), and β-actin (ab8226, 1 : 5000, all from Abcam Inc., MA, USA), then incubated at 4 °C overnight. Afterwards, the membranes were supplemented with corresponding secondary antibody

(Miaotong Biotechnology Co., Ltd., Shanghai, China) for 1-h incubation. The membranes were developed by enhanced chemiluminescent reagent and Bio-rad Gel Doc EZ imager (Bio-rad Laboratories,

CA, USA), and the gray values of the protein bands were analyzed by Image J software (National Institutes of Health, MD, USA). The experiment was independently repeated for three times.

DUAL LUCIFERASE REPORTER GENE ASSAY The bioinformatic website https://cm.jefferson.edu/rna22/Precomputed/ (or http://www.targetscan.org/vert_72/) was used to measure the binding sites of

TUG1 and miR-187-3p (or binding sites of miR-187-3p and TESC). Luciferase reporter gene vectors (pGL3-Control vector, Promega, WI, USA) containing wild-type (Wt) or mutant (Mut) TUG1 and the

3′-UTR of Wt or Mut TESC were co-transfected with miR-187-3p mimic and mimic NC. After 48 h, dual luciferase reporter assay was performed based on the manufacturer’s instructions (Promega).

The Renilla luciferase signal was normalized to the firefly luciferase signal for each individual analysis. The experiment was independently repeated for three times. RNA PULL-DOWN ASSAY

The miR-187-3p, miR-187-3p-Mut, and NC probes were synthesized and biotinylated by GenePharma. The RNA pull-down assay was conducted using the Magnetic RNA-Protein Pull-Down Kit (Thermo

Fisher) according to the manufacturer’s protocol. Cells were transfected with biotinylated miRNA and the M-280 streptavidin magnetic beads (Invitrogen) were used to incubate with cell

lysates. Then, TUG1 expression was assessed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The experiment was independently repeated for three times.

SUBCUTANEOUS TUMORIGENESIS IN NUDE MICE BALB/c nude mice (aging 6 weeks, weighing 18–20 g) were gained from the Laboratory Animal Center of Shandong University (Shandong, China). The mice

were classified into 2 large groups and 14 small groups: the blank (injection of GH3 and HP75 cells without any treatment), sh-NC (injection of GH3 and HP75 cells stably transfected with NC

of TUG1 shRNA plasmid), sh-TUG1 (injection of GH3 and HP75 cells stably transfected with TUG1 shRNA plasmid), mimic NC (injection of GH3 and HP75 cells stably transfected with miR mimic NC),

miR-187-3p mimic (injection of GH3 and HP75 cells stably transfected with miR-187-3p mimic), sh-TUG1 + inhibitor NC (simultaneous injection of GH3 and HP75 cells stably transfected with

TUG1 shRNA plasmid and miR inhibitor NC) and sh-TUG1 + miR-187-3p inhibitor (simultaneous injection of GH3 and HP75 cells stably transfected with TUG1 shRNA plasmid and miR-187-3p inhibitor)

groups. Cell suspension (100 μL) was injected into the nude mice at the armpit of right forelimb of nude mice 0.3–0.5 cm away from the back. After 24-day incubation, the mice were

euthanized by CO2 with the tumor tissues collected for subsequent experiment. HEMATOXYLIN–EOSIN STAINING The xenografts were washed with normal saline and fixed in 4% paraformaldehyde for

30–50 min, and then were washed, dehydrated, permeabilized, waxed, embedded, and sliced. After that, the tissue sections were put on the slide, dried in a 45 °C incubator, dewaxed, soaked in

gradient ethanol, and washed with distilled water for 5 min. Stained with hematoxylin for 5 min, the sections were washed with running water for 3 s, differentiated with 1% hydrochloric

acid ethanol for 3 s, and stained with 5% eosin solution for 3 min. Subsequently, the sections were dehydrated, permeabilized, sealed, and observed under a microscope. STATISTICAL ANALYSIS

The experimental data and image preprocessing were analyzed by SPSS 20 statistical software (IBM, USA) and GraphPad Prism7.0 software (La Jolla, USA). All data were shown as mean ± standard

deviation (SD). Student’s _t_ test was used to analyze differences between the two groups, and the differences between multiple groups were compared by one-way ANOVA with a post hoc Tukey

test. _P_ value < 0.05 was indicative of statistically significant difference. RESULTS EXPRESSION OF TUG1 IN PA AND ITS CORRELATION WITH TUMORIGENESIS TUG1 has been found to be

upregulated in multiple cancers and participate in the proliferation, migration, invasion, and drug-resistance of cancer cells30,31,32. Moreover, it has been reported that TUG1 was

up-expressed in glioblastoma15. We detected the expression of TUG1 in PA tissues and normal pituitary tissues using RT-qPCR to verify its abnormal expression in PA, and the results suggested

that versus the normal pituitary tissues, the expression of TUG1 was heightened in PA tissues (Fig. 1A). Based on this finding, we further analyzed the association between TUG1 and PA

tumorigenesis. The PA tissues were classified into the invasive group and the non-invasive group. It was found that TUG1 expression in the invasive group was higher than the non-invasive

group (Fig. 1B). We further determined the relations of TUG1 expression with knosp grade and tumor size, and the outcomes indicated that the higher knosp grade suggested a higher expression

of TUG1 in PA tissue (Fig. 1C) and the larger tumors had a higher expression of TUG1 (Fig. 1D), while TUG1 expression was not changed with age and gender (Fig. 1E, F). TUG1 BINDS TO

MIR-187-3P, AND TESC IS TARGETED BY MIR-187-3P LncRNAs are known to function as competing endogenous RNA (ceRNAs) during tumourigenesis. The ceRNAs can interact with functional miRNAs to

regulate gene expression. It was predicted at the online analysis website RNA22 (https://cm.jefferson.edu/rna22/Precomputed/) that there existed particular binding region between the

sequences of TUG1 and miR-187-3p (Fig. 2A). Then, we detected the expression of miR-187-3p in PA tissues. The results showed that miR-187-3p was downregulated in PA tissues versus normal

pituitary tissues (Fig. 2B). Subsequently, TUG1 luciferase reporter plasmids with Wt and predicted mutant sites for miR-187-3p were constructed. We found that miR-187-3p mimic repressed the

luciferase activity of the Wt plasmid whereas did not affect the luciferase activity of the mutant plasmid (Fig. 2C). Moreover, the results of RNA pull-down assay illustrated that TUG1 was

pulled down by the target oligos (Fig. 2D). These data implied that TUG1 is a sponge for miR-187-3p. RNA22 (https://cm.jefferson.edu/rna22/Precomputed/0) was also used to identify the

potential targets of miR-187-3p. It was found that TESC may be a target of miR-187-3p (Fig. 2E). The expression of TESC in tissues was determined and we found that TESC was upregulated in PA

tissue versus normal pituitary tissues (Fig. 2F). The results of dual luciferase reporter gene assay suggested that (Fig. 2G) the co-transfection of TESC-Wt and miR-187-3p mimic decreased

the luciferase activity of the cells, while the luciferase activity was not broadly changed after the PA cells co-transfected with TESC-Mut and miR-187-3p mimic, showing a targeting

relationship between miR-187-3p and TESC. A study has revealed that TESC promoted NF-κB signaling pathway, thus facilitating tumorigenesis33. We detected the expression NF-κB p65 and IκB-α

in PA tissues, and it came out that the expression levels of NF-κB p65 were increased, while that of IκB-α were suppressed in PA tissues versus normal pituitary tissues (Fig. 2F). INHIBITED

TUG1 OR ELEVATED MIR-187-3P RESTRAINS PROLIFERATION AND PROMOTES CELL CYCLE PROGRESSION OF PA CELLS The viability, colony formation ability, and cell cycle distribution of PA cells were

determined respectively using MTT assay, colony formation assay, and flow cytometry to explore the roles of TUG1 or miR-187-3p on PA cells. MTT assay was used to evaluate the viability of

GH3 and HP75 cells, and the results (Fig. 3A, B) revealed that silenced TUG1 or elevated miR-187-3p repressed the cell viability, while the effect of silenced TUG1 on cell viability was

reversed by miR-187-3p inhibition. The colony formation ability of GH3 and HP75 cells was determined using colony formation assay and we found that (Fig. 3C–F) TUG1 downregulation or

miR-187-3p upregulation restrained the colony formation ability of cells, and the impact of TUG1 downregulation was abolished by miR-187-3p reduction. Flow cytometry was employed to assess

the cell cycle distribution of GH3 and HP75 cells, and the outcomes (Fig. 3G–J) reflected that TUG1 reduction or miR-187-3p elevation arrested cells at G0/G1 phase, while the effect of TUG1

reduction on cell cycle distribution was abrogated by miR-187-3p suppression. The above findings suggest that TUG1 promotes proliferation in PA cell lines by sponging miR-187-3p. INHIBITED

TUG1 OR ELEVATED MIR-187-3P ACCELERATES APOPTOSIS OF PA CELLS To investigate the effect of TUG1 or miR-187-3p on PA cells, we detected the apoptosis of PA cells using flow cytometry and

assessed expression of apoptotic factors in PA cells using RT-qPCR. The apoptosis rate of GH3 and HP75 cells was obtained from flow cytometry (Fig. 4A–D) and it came out that inhibited TUG1

or upregulated miR-187-3p promoted the apoptosis of GH3 and HP75 cells, and reduced miR-187-3p reversed the promotive role of inhibited TUG1 in cell apoptosis. The mRNA expression of

apoptotic proteins (Bax and Bcl-2) in GH3 and HP75 cells was detected by RT-qPCR. It was found that TUG1 silencing or miR-187-3p elevation increased Bax mRNA expression while decreased Bcl-2

mRNA expression in GH3 and HP75 cells; the alterations on Bax and Bcl-2 expression induced by TUG1 silencing could be reversed by miR-187-3p inhibition (Fig. 4E, F). These data indicated

that TUG1 inhibited apoptosis in PA cell lines by sponging miR-187-3p. INHIBITED TUG1 OR ELEVATED MIR-187-3P DECELERATES MIGRATION AND INVASION OF PA CELLS We detected the effect of TUG1 or

miR-187-3p on the migration and invasion abilities of GH3 and HP75 cells to evaluate whether TUG1 or miR-187-3p contributes to the metastasis of PA. Transwell assay was used to determine the

migration and invasion abilities of GH3 and HP75 cells (Fig. 5A–H), and we observed that the migration and invasion abilities of cells were restricted by TUG1 silencing or miR-187-3p

amplification, while the role of TUG1 silencing was reversed by miR-187-3p downregulation. The expression of EMT-related proteins (N-Cadherin, E-Cadherin, and Vimentin) was determined using

RT-qPCR and the results showed that reduced TUG1 or elevated miR-187-3p promoted the mRNA expression of E-Cadherin and suppressed the mRNA expression of N-Cadherin and Vimentin in GH3 and

HP75 cells, while these effects of reduced TUG1 were abolished by downregulated miR-187-3p (Fig. 5I, J). It could be concluded that TUG1 promotes invasion and migration in PA cell lines by

sponging miR-187-3p. TUG1 OR MIR-187-3P AFFECTS THE NF-ΚB SIGNALING PATHWAY Expression levels of TUG1, miR-187-3p, TESC, NF-κB/p65, and IκB-α in PA cells were assessed and the results (Fig.

6A–D) suggested that TUG1 knockdown downregulated TUG1, TESC, and NF-κBp65, while upregulated miR-187-3p and IκB-α; miR-187-3p elevation inhibited expression of TESC and NF-κBp65 and

promoted expression of miR-187-3p and IκB-α; the alterations on expression levels of miR-187-3p, TESC, NF-κB/p65, and IκB-α that induced by TUG1 knockdown were reversed by inhibited

miR-187-3p. INHIBITED TUG1 OR ELEVATED MIR-187-3P RESTRAINS TUMOR GROWTH OF PA IN VIVO We established a PA xenograft model to measure the role of TUG1 or miR-187-3p in vivo. The xenografts

in nude mice were shown in Fig. 7A–F. It was observed that mice accepted the injection of cell suspension treated with TUG1 silencing or miR-187-3p elevation had repressed tumor weight and

volume, and miR-187-3p inhibition abrogated the impact of TUG1 repression. Hematoxylin–eosin (HE) staining was employed to observe the pathological changes in the xenografts from nude mice,

and the results implied that a loose arrangement and a large necrotic region existed in xenografts from nude mice that accepted the injection of cell suspension treated with TUG1 silencing

or miR-187-3p elevation, while the effect of TUG1 knockdown was abolished by miR-187-3p reduction (Fig. 7G, H). DISCUSSION PA is a benign epithelial tumor that derived from the

adenohypophysial cells of the pituitary gland3. The tumor development is regulated by gene networks, and lncRNAs may interact with miRNAs, mRNAs, or other molecules. The ceRNA hypothesis

indicates that lncRNAs can act as ceRNAs to interact with miRNAs by miRNA response elements, thereby regulating the mRNA expression34. The present study aims to investigate the impact of the

lncRNA TUG1/miR-187-3p/TESC axis on the progression of PA, and we have found that lncRNA TUG1 was able to sponge miR-187-3p to modulate the development of PA by targeting TESC and

regulating the NF-κB signaling pathway (Fig. 8). We firstly measured the expression levels of TUG1, miR-187-3p, TESC, and NF-κB pathway-related proteins, and the results suggested that TUG1

and TESC were highly expressed and the NF-κB signaling pathway was activated, while miR-187-3p was downregulated in PA tissues and cell lines. Similarly, Long et al.12 have revealed that

TUG1 is overexpressed in melanoma specimens and cell lines, and it has been demonstrated that TUG1 is upregulated in glioma tissues13,14. Moreover, Liu et al. have discovered that miR-187-3p

expression is inhibited in infantile hemangioma tissues18, and a recent document has pointed out that miR-187-3p is downregulated in HCC tissues and cell lines19. As for the abnormal

expression of TESC, Luo et al.23 have found that contrasted to the normal tissues, TESC expression is elevated in RCC tissues, and it has been unraveled that the level of TESC is heightened

in the tissues and serum from CRC patients24. Furthermore, Lu et al.26 have figured out that the expression of NF-κB is amplified in HP75 cells. These data indicated the abnormal expression

of miR-187-3p, TUG1, and TESC, as well as the activated NF-κB signaling pathway in various tumors, providing a theoretical basis for our study. We have also clarified the regulatory relation

between TUG1 and miR-187-3p, and the target relation between miR-187-3p and TESC in PA, which have not been clearly identified before. Another vital finding in our study reflected that the

expression of TUG1 was negatively related to the prognosis of PA patients. Consistently, Wei et al.35 have proposed that higher expression of TUG1 indicates a worse prognosis of patients

with cervical cancer, and it has also been testified that TUG1 predicts a poor prognosis of prostate cancer patients36. These documents suit well with our finding that TUG1 acted as a

predictive role in tumor diagnosis and could be used as a diagnostic biomarker. In addition, evidence in our research revealed that the inhibited TUG1 and elevated miR-187-3p were able to

restrain the growth of PA cells. In line with this result, Hui et al.37 have verified that TUG1 inhibition could block the cell cycle and suppress the proliferation of pancreatic cancer

cells, and it has been demonstrated that TUG1 facilitates the proliferation and stemness of ovarian cancer cells38. As for the role of miR-187-3p, Chen et al.39 have figured out that the

overexpressed miR-187 has the ability to repress proliferation of gastric cancer cells and arrest the gastric cancer cells at the G0/G1 phase. Another publication has indicated that

miR-187-3p reduces the cell viability in infantile hemangioma18. Additionally, we have found that the downregulation of TUG1 and upregulation of miR-187-3p could restrict the migration and

invasion of PA cells. Consistently, Guo et al. have unraveled that the inhibition of TUG1 could repress the migration and invasion of bladder cancer cells40, and TUG1 has been revealed to

promote the proliferation, migration, and invasion of HCC cells41. A former literature has unearthed that miR-187-3p mimic could inhibit the migration and invasion of non-small-cell lung

cancer (NSCLC) cells42. Moreover, the promotive effects of reduced TUG1 and elevated miR-187-3p on apoptosis of PA cells have been illuminated in our research as well. A similar outcome has

also been drawn out that the repression of TUG1 has the capacity to accelerate apoptosis of RCC cells43 and esophageal cancer cells44, and Sun et al.42 have clarified that miR-187-3p

elevation promotes the apoptosis of NSCLC cells. Except for that, we have also illustrated that the knockdown of TUG1 and elevation of miR-187-3p were able to suppress tumor growth of PA in

vivo. Consistent with this result, it has been recently identified that the inhibited TUG1 could restrain the tumor growth of melanoma in vivo12, and Cui et al.45 have demonstrated that

miR-187 could inhibit tumor growth in osteosarcoma. The above data implied the oncogenic role of TUG1 as well as the anti-tumor role of miR-187-3p during the progression of tumors, helping

us to identifying our findings. Altogether, we have testified that the inhibited TUG1 and elevated miR-187-3p were able to decelerate the growth of both PA cells and tumors by reducing TESC,

thereby repressing the progression of PA, which may be helpful for investigation on novel treatment for PA. Nevertheless, the role of TESC as well as its relation to the NF-κB signaling

pathway in PA was not fully explored here. We would explain the detailed molecular mechanisms in our future works. REFERENCES * Platta, C. S., Mackay, C. & Welsh, J. S. Pituitary

adenoma: a radiotherapeutic perspective. _Am. J. Clin. Oncol._ 33, 408–419 (2010). Article  PubMed  Google Scholar  * Hauser, B. M., Lau, A., Gupta, S., Bi, W. L. & Dunn, I. F. The

epigenomics of pituitary adenoma. _Front. Endocrinol._ 10, 290 (2019). Article  Google Scholar  * Naguib, M. M., Mendoza, P. R., Jariyakosol, S. & Grossniklaus, H. E. Atypical pituitary

adenoma with orbital invasion: case report and review of the literature. _Surv. Ophthalmol._ 62, 867–874 (2017). Article  PubMed  PubMed Central  Google Scholar  * Melmed, S. Pathogenesis of

pituitary tumors. _Nat. Rev. Endocrinol._ 7, 257–266 (2011). Article  CAS  PubMed  Google Scholar  * Dworakowska, D. & Grossman, A. B. The pathophysiology of pituitary adenomas. _Best.

Pract. Res. Clin. Endocrinol. Metab._ 23, 525–541 (2009). Article  CAS  PubMed  Google Scholar  * Levy, A. Molecular and trophic mechanisms of pituitary tumourigenesis. _Horm. Res.

Paediatr._ 76, 2–6 (2011). Article  CAS  PubMed  Google Scholar  * Yu, R. & Melmed, S. Pathogenesis of pituitary tumors. _Prog. Brain Res._ 182, 207–227 (2010). Article  CAS  PubMed 

Google Scholar  * Deng, W. et al. Long noncoding MIAT acting as a ceRNA to sponge microRNA-204-5p to participate in cerebral microvascular endothelial cell injury after cerebral ischemia

through regulating HMGB1. _J. Cell. Physiol._ https://doi.org/10.1002/jcp.29334 (2019). * Lu, G., Duan, J. & Zhou, D. Long-noncoding RNA IFNG-AS1 exerts oncogenic properties by

interacting with epithelial splicing regulatory protein 2 (ESRP2) in pituitary adenomas. _Pathol. Res. Pract._ 214, 2054–2061 (2018). Article  CAS  PubMed  Google Scholar  * Fu, D., Zhang,

Y. & Cui, H. Long noncoding RNA CCAT2 is activated by E2F1 and exerts oncogenic properties by interacting with PTTG1 in pituitary adenomas. _Am. J. Cancer Res._ 8, 245–255 (2018). CAS 

PubMed  PubMed Central  Google Scholar  * Duan, L. J. et al. Long noncoding RNA TUG1 alleviates extracellular matrix accumulation via mediating microRNA-377 targeting of PPARgamma in

diabetic nephropathy. _Biochem. Biophys. Res. Commun._ 484, 598–604 (2017). Article  CAS  PubMed  Google Scholar  * Long, J., Menggen, Q., Wuren, Q., Shi, Q. & Pi, X. Long noncoding RNA

Taurine-Upregulated Gene1 (TUG1) promotes tumor growth and metastasis through TUG1/Mir-129-5p/Astrocyte-Elevated Gene-1 (AEG-1) axis in malignant melanoma. _Med. Sci. Monit. Int. Med. J.

Exp. Clin. Res._ 24, 1547–1559 (2018). Google Scholar  * Li, J., An, G., Zhang, M. & Ma, Q. Long non-coding RNA TUG1 acts as a miR-26a sponge in human glioma cells. _Biochem. Biophys.

Res. Commun._ 477, 743–748 (2016). Article  CAS  PubMed  Google Scholar  * Katsushima, K. et al. Targeting the Notch-regulated non-coding RNA TUG1 for glioma treatment. _Nat. Commun._ 7,

13616 (2016). Article  PubMed  PubMed Central  Google Scholar  * Cai, H. et al. Long non-coding RNA taurine upregulated 1 enhances tumor-induced angiogenesis through inhibiting microRNA-299

in human glioblastoma. _Oncogene_ 36, 318–331 (2017). Article  CAS  PubMed  Google Scholar  * Jiang, T. et al. The lncRNA MALAT1/miR-30/Spastin axis regulates hippocampal neurite outgrowth.

_Front. Cell. Neurosci._ 14, 555747 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cheng S., Zhu C. H., Zhang A. H. & Huang S. M. MiR-29b expression is altered in

crescent formation of HSPN and accelerates Ang II-induced mesangial cell activation. _World J. Pediatr._ 16, 201–212 (2019). * Liu, C., Zhao, Z., Ji, Z., Jiang, Y. & Zheng, J. MiR-187-3p

enhances propranolol sensitivity of hemangioma stem Cells. _Cell Struct. Funct._ 44, 41–50 (2019). Article  CAS  PubMed  Google Scholar  * Dou, C. et al. miR-187-3p inhibits the metastasis

and epithelial-mesenchymal transition of hepatocellular carcinoma by targeting S100A4. _Cancer Lett._ 381, 380–390 (2016). Article  CAS  PubMed  Google Scholar  * Liang, Y. et al. Exosomal

microRNA-144 from bone marrow-derived mesenchymal stem cells inhibits the progression of non-small cell lung cancer by targeting CCNE1 and CCNE2. _Stem Cell Res. Ther._ 11, 87 (2020).

Article  CAS  PubMed  PubMed Central  Google Scholar  * Jin, G., Yang, Y., Tuo, G., Wang, W. & Zhu, Z. LncRNA TUG1 promotes tumor growth and metastasis of esophageal squamous cell

carcinoma by regulating XBP1 via competitively binding to miR-498. _Neoplasma_ 67, 751–761 (2020). Article  CAS  PubMed  Google Scholar  * Kim, T. W. et al. Differential expression of

tescalcin by modification of promoter methylation controls cell survival in gastric cancer cells. _Oncol. Rep._ 41, 3464–3474 (2019). PubMed  Google Scholar  * Luo, A. J. et al. Suppression

of Tescalcin inhibits growth and metastasis in renal cell carcinoma via downregulating NHE1 and NF-kB signaling. _Exp. Mol. Pathol._ 107, 110–117 (2019). Article  CAS  PubMed  Google Scholar

  * Kang, J. et al. Tescalcin expression contributes to invasive and metastatic activity in colorectal cancer. _Tumour Biol._ 37, 13843–13853 (2016). Article  CAS  PubMed  Google Scholar  *

Liang, H., Yang, X., Liu, C., Sun, Z. & Wang, X. Effect of NF-kB signaling pathway on the expression of MIF, TNF-alpha, IL-6 in the regulation of intervertebral disc degeneration. _J.

Musculoskelet. Neuronal Interact._ 18, 551–556 (2018). CAS  PubMed  PubMed Central  Google Scholar  * Lu, B. et al. MicroRNA16/VEGFR2/p38/NFkappaB signaling pathway regulates cell growth of

human pituitary neoplasms. _Oncol. Rep._ 39, 1235–1244 (2018). CAS  PubMed  Google Scholar  * Li, R. et al. Triptolide suppresses growth and hormone secretion in murine pituitary

corticotroph tumor cells via NF-kappaB signaling pathway. _Biomed. Pharmacother._ 95, 771–779 (2017). Article  CAS  PubMed  Google Scholar  * Knosp, E., Steiner, E., Kitz, K. & Matula,

C. Pituitary adenomas with invasion of the cavernous sinus space: a magnetic resonance imaging classification compared with surgical findings. _Neurosurgery_ 33, 610–617 (1993). CAS  PubMed

  Google Scholar  * Kim, S. K. & Huang, L. Nanoparticle delivery of a peptide targeting EGFR signaling. _J. Control. Release_ 157, 279–286 (2012). Article  CAS  PubMed  Google Scholar  *

Liu, S., Liu, L. H., Hu, W. W. & Wang, M. Long noncoding RNA TUG1 regulates the development of oral squamous cell carcinoma through sponging miR-524-5p to mediate DLX1 expression as a

competitive endogenous RNA. _J. Cell. Physiol._ 234, 20206–20216 (2019). Article  CAS  PubMed  Google Scholar  * Yu, X. et al. Long non-coding RNA Taurine upregulated gene 1 promotes

osteosarcoma cell metastasis by mediating HIF-1alpha via miR-143-5p. _Cell Death Dis._ 10, 280 (2019). Article  PubMed  PubMed Central  CAS  Google Scholar  * Yu, G., Zhou, H., Yao, W.,

Meng, L. & Lang, B. lncRNA TUG1 promotes cisplatin resistance by regulating CCND2 via epigenetically silencing miR-194-5p in bladder cancer. _Mol. Ther. Nucleic Acids_ 16, 257–271

(2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kang, Y. H. et al. The EF-hand calcium-binding protein tescalcin is a potential oncotarget in colorectal cancer. _Oncotarget_

5, 2149–2160 (2014). Article  PubMed  Google Scholar  * Lyu, L. et al. Integrative analysis of the lncRNA-associated ceRNA network reveals lncRNAs as potential prognostic biomarkers in human

muscle-invasive bladder cancer. _Cancer Manag. Res._ 11, 6061–6077 (2019). Article  PubMed  PubMed Central  Google Scholar  * Wei, X. et al. Low expression of TUG1 promotes cisplatin

sensitivity in cervical cancer by activating the MAPK pathway. _J. BUON_ 24, 1020–1026 (2019). PubMed  Google Scholar  * Xu, T., Liu, C. L., Li, T., Zhang, Y. H. & Zhao, Y. H. LncRNA

TUG1 aggravates the progression of prostate cancer and predicts the poor prognosis. _Eur. Rev. Med. Pharmacol. Sci._ 23, 4698–4705 (2019). CAS  PubMed  Google Scholar  * Hui, B. et al.

Overexpressed long noncoding RNA TUG1 affects the cell cycle, proliferation, and apoptosis of pancreatic cancer partly through suppressing RND3 and MT2A. _OncoTargets Ther._ 12, 1043–1057

(2019). Article  CAS  Google Scholar  * Zhan, F. L., Chen, C. F. & Yao, M. Z. LncRNA TUG1 facilitates proliferation, invasion and stemness of ovarian cancer cell via miR-186-5p/ZEB1

axis. _Cell Biochem. Funct._ 38, 1069–1078 (2020). Article  CAS  PubMed  Google Scholar  * Chen, W. et al. Effects of downregulated expression of microRNA-187 in gastric cancer. _Exp. Ther.

Med._ 16, 1061–1070 (2018). PubMed  PubMed Central  Google Scholar  * Guo, P. et al. Upregulation of long noncoding RNA TUG1 promotes bladder cancer cell proliferation, migration, and

invasion by inhibiting miR-29c. _Oncol. Res._ 26, 1083–1091 (2018). Article  PubMed  PubMed Central  Google Scholar  * Zhao, W., Jiang, X. & Yang, S. lncRNA TUG1 promotes cell

proliferation, migration, and invasion in hepatocellular carcinoma via regulating miR-29c-3p/COL1A1 axis. _Cancer Manag. Res._ 12, 6837–6847 (2020). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Sun, C. et al. MicroRNA-187-3p mitigates non-small cell lung cancer (NSCLC) development through down-regulation of BCL6. _Biochem. Biophys. Res. Commun._ 471, 82–88 (2016).

Article  CAS  PubMed  Google Scholar  * Zhang, M. et al. Downregulation of the long noncoding RNA TUG1 inhibits the proliferation, migration, invasion and promotes apoptosis of renal cell

carcinoma. _J. Mol. Histol._ 47, 421–428 (2016). Article  CAS  PubMed  Google Scholar  * Zong, M., Feng, W., Wan, L., Yu, X. & Yu, W. LncRNA TUG1 promotes esophageal cancer development

through regulating PLK1 expression by sponging miR-1294. _Biotechnol. Lett._ 42, 2537–2549 (2020). PubMed PMID: 33009634. Article  CAS  PubMed  Google Scholar  * Cui, C. & Shi, X.

miR-187 inhibits tumor growth and invasion by directly targeting MAPK12 in osteosarcoma. _Exp. Ther. Med._ 14, 1045–1050 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references ACKNOWLEDGEMENTS We acknowledge and appreciate our colleagues for their valuable suggestions and technical assistance for this study. FUNDING: None AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * Department of Neurosurgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 250021, Jinan, Shandong, China Rui Zhang, Fan Yang, 

Haitao Fan, Haocong Wang, Qinghao Wang & Tao Song * Department of Neurosurgery, The People’s Hospital of Qingzhou, 262500, Qingzhou, Shandong, China Jianxin Yang Authors * Rui Zhang View

author publications You can also search for this author inPubMed Google Scholar * Fan Yang View author publications You can also search for this author inPubMed Google Scholar * Haitao Fan

View author publications You can also search for this author inPubMed Google Scholar * Haocong Wang View author publications You can also search for this author inPubMed Google Scholar *

Qinghao Wang View author publications You can also search for this author inPubMed Google Scholar * Jianxin Yang View author publications You can also search for this author inPubMed Google

Scholar * Tao Song View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS T.S. contributed to study design; F.Y. contributed to manuscript

editing; R.Z., H.F., and H.W. contributed to experimental studies; Q.W. and J.Y. contributed to data analysis. CORRESPONDING AUTHOR Correspondence to Tao Song. ETHICS DECLARATIONS CONFLICT

OF INTEREST The authors declare no competing interests. ETHICAL APPROVAL The protocol of this study was confirmed by the Ethic Committee of Shandong Provincial Hospital Affiliated to

Shandong First Medical University. Animal experiments were strictly in accordance with the Guide to the Management and Use of Laboratory Animals issued by the National Institutes of Health.

The protocol of animal experiments was approved by the Institutional Animal Care and Use Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University. INFORMED

CONSENT Written informed consents were acquired from all patients before this study. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional

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http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Zhang, R., Yang, F., Fan, H. _et al._ Long non-coding RNA TUG1/microRNA-187-3p/TESC

axis modulates progression of pituitary adenoma via regulating the NF-κB signaling pathway. _Cell Death Dis_ 12, 524 (2021). https://doi.org/10.1038/s41419-021-03812-7 Download citation *

Received: 27 August 2020 * Revised: 11 January 2021 * Accepted: 13 January 2021 * Published: 21 May 2021 * DOI: https://doi.org/10.1038/s41419-021-03812-7 SHARE THIS ARTICLE Anyone you share

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