Patients and tissue samples
We performed a retrospective study involving 43 patients with COVID-19 as the study group, who were hospitalized in Renmin Hospital of Wuhan University from February 5 to Apri 5, 2020. All cases were laboratory-confirmed as SARS-CoV-2 positive using quantitative RT-PCR (qRT-PCR) on nasal and pharyngeal swab specimens. The diagnosis of COVID-19 and the severity was determined according to the New Coronavirus Pneumonia Prevention and Control Program (7th edition) published by the National Health Commission of China. Briefly, patients with accompanying respiratory failure (mechanical ventilation needed), shock or multiple organ dysfunctions were diagnosed as critical type. The control group came from the population who previously received pituitary function evaluation and were classified as normal in the same center. 45 age – and gender-matched healthy controls were randomly selected and the data of their pituitary hormones were collected. Patients with a history of pituitary disease and/or renal insufficiency were excluded from the study. All the patients who underwent a complete pituitary hormone assessment and their matched-controls did not have a pituitary MRI.
A total of 114 PitNETs from a single expert center were collected. The cohort comprised of 4 normal pituitary tissues obtained from autopsy. This consecutive series encompassed PitNET types, including 24 (20.9%) lactotrophs, 16 (13.9%) somatotrophs, 13 (11.3%) corticotrophs, 31 (27.0%) gonadotrophs, 23 (20.0%) null-cells, 4 (3.5%) plurihormonal PIT1-positive PitNETs, 3 (2.6%) mixed growth hormone/prolactin (GH-PRL), and 1 (0.9%) thyrotroph. The study was approved by the Ethical Review Board in Renmin Hospital of Wuhan University and Ruijin Hospital of Shanghai Jiao Tong University School of Medicine.
Pituitary hormone assessment
In the study group, prolactin (PRL), somatotropin (GH), adrenocorticotropic hormone (ACTH), cortisol (8AM), thyroid stimulating hormone (TSH), free triiodothyronine (FT3), free thyroxine (FT4), luteinizing hormone (LH) and follicle stimulating hormone (FSH) were detected by electrochemiluminescent immunoassays according to the instructions from the manufacturer. In the control group, the data of serum PRL, GH, ACTH, cortisol, TSH, FT3, FT4, LH and FSH levels were retrieved from the dataset already kept in the same medical center. All the cases in the two groups had not received corticosteroid therapy or medications that could suppress TSH, ACTH and PRL within 5 days before pituitary hormone assessment.
Public datasets acquisition and analysis
RNA-seq data of normal tissues and PitNETs were downloaded from the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo). 46 normal tissues, which included pituitary glands (5 tissues), distributed in different organs and 5 lactotroph adenomas were obtained for this study.
Quantitative real-time PCR (qPCR)
Total RNA of tumor tissues was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. 5μg RNA was reversely transcribed into cDNA using the cDNA Synthesis Kit (TaKaRa, Shiga, Japan). The mRNA expression level was quantified using qPCR with the SYBR Green real-time PCR Master Mix kit (TaKaRa Bio). PCR primers used were as follows: human ACE2 forward, 5’-CATTGGAGCAAGTGTTGGATCTT-3’; reverse: 5’-GAGCTAATGCATGCCATTCTCA-3’; human angiotensin II receptor type 1 (AGTR1) forward, 5’-GATGATTGTCCCAAAGCTGG-3’; reverse, 5’-TAGGTAATTGCCAAAGGGCC-3’; human proto-oncogene receptor (MAS) forward, 5’- TTG TTG AGG AAC CCA CGA AC-3’; reverse, 5’- CCA CTG GGG AGA TGC TCA TA-3’; human β-actin forward, 5’-GGATGCAGAAGGAGATCACTG-3’; reverse: 5’-CGATCCACACGGAGTACTTG-3’ [12-15]. The amplification reaction was carried out on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Warrington, UK). The mRNA expression of the target genes was normalized to that of the endogenous reference gene β-actin and expressed as the fold-difference (2–ΔΔCt). Low and high measurable levels were defined by ratio of specific/beta-actin transcripts<1 and ≥ 1, respectively .
Immunohistochemistry (IHC) and scoring
Tissue samples were fixed in 4% formalin, embedded in paraffin, and sliced into 4-mm-thick sections. IHC was conducted using the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA). Rabbit primary antibody for ACE2 (1:200, ab108252) was purchased from Abcam (Cambridge, MA, USA). Finally, the slides were developed with DAB and counterstained with hematoxylin.
Slices were scanned by Pannoramic 1000 slide digitalization system (3DHISTECH, Budapest, Hungary). Images were captured at ×20 magnification using CaseViewer 2.3(3DHISTECH, Budapest, Hungary). The field was selected with a good contrast of DAB chromogen and hematoxylin which is considered region of interest. Before capturing the images, the color density and white balance were standardized for all images. All the acquired images were saved as JPEG format. After IHC images acquisition, ImageJ 1.48 version (NIH, Bethesda, Maryland) analysis was performed and ACE2 immunostaining of pituitary glands and tumor tissues was scored as negative (0+), low positive (1+), positive (2+) and high positive (3+), as described in the previous study .
Cell culture and reagents
Pituitary cell lines MMQ (CRL-10609™), GH3 (CCL-82.1™), and AtT-20/D16v-F2 (AtT-20, CRL-1795™) were purchased from the American Type Culture Collection (Manassas, USA). The MMQ and GH3 cell lines were cultured in Dulbecco’s modified Eagle medium and F12 medium (Gibco, Grand Island, NY, USA), supplemented with 15% horse serum (Gibco) and 2.5% fetal bovine serum (FBS; Gibco). The AtT-20 cell line was cultured in RPMI1640 (Sangon Biotech, Shanghai, China) supplemented with 10% FBS (Gibco).
Primary human tumor cells were obtained from patients who underwent endoscopy surgery for PitNETs between March 2020 and August 2020 at the Department of Neurosurgery, Ruijin Hospital of Jiaotong University, Shanghai, China. As described in the previous study , the tumor cells were cultured in DMEM with 10% FBS and 100U/mL penicillin/streptomycin (Gibco) . Cells were seeded onto 10cm dishes at a density of 106 cells per well and were cultured for 48 hours before treatment.
All cell lines and primary tumor cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C. Diminazene aceturate (DIZE) was purchased from MedChemExpress (Shanghai, China).
Establishment of stably transfected cells
The recombinant plasmids (pCDH-puro-AGTR1) were constructed, and sequenced by Tongyong Biotechnologies (Anhui, China). AtT-20 cell lines stably expressing DEPTOR or empty vectors, were constructed using the lentiviral technique. After 48 h transduction with lentiviral supernatant, cells were selected with 2 μg/mL puromycin for 1–2 weeks for stable transfectants.
Total proteins were extracted using the Total Protein Extraction Kit (Millipore Co., Billerica, MA, USA). The cultured tumor cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China). An equivalent of 30 mg protein/sample was resolved by SDS-PAGE (Sangon Biotech, Shanghai, China) and transferred to polyvinylidene difluoride membranes (PVDF; Millipore). The membranes were blocked with 5% nonfat milk in Tris-buffered saline with 1% Tween-20 (TBST) buffer for 1 h and probed overnight with primary antibodies at 4 °C. The antibodies used were as follows: ACE2 (ab108252, Abcam), and POMC (ab210605, Abcam), Tubulin (ab7291, Abcam) and AGTR1 (25343-1-AP, Proteintech). The immunoreactive bands were detected using ECL detection reagent (Millipore) according to the manufacturer’s instructions. Kidney paracancer tissues from two renal cell carcinoma patients were obtained for use as a positive control. Protein expression was quantified using ImageJ software (Wayne Rasband, National Institute of Mental Health, Bethesda, MD, USA).
Cell proliferation assay
Cell proliferation was measured using a WST-8 Cell Counting Kit-8 (CCK8) (Bimake, Houston, TX, USA) according to the manufacturer’s instructions. MMQ, GH3, and AtT-20 and primary tumor cells were plated in 96-well plates at a density of 1×104 cells/well, respectively. All cells were treated with different concentrations of DIZE and assessed after 24h, 48h and 72h.
Enzyme-linked immunosorbent assay (ELISA)
The levels of the hormone in the cell culture supernatants were measured using ELISA kits (Signalway Antibody LLC, Maryland, MD, USA) at the indicated time points, with the appropriate treatment according to the manufacturer’s recommendations.
All data were analyzed using GraphPad Prism, version 5 (GraphPad Software, La Jolla, CA, USA). The differences among categorical variables were analyzed using independent-sample Student’s t-test or one-way analysis of variance (ANOVA). The immunoreactive scores of ACE2 were analyzed using the non-parametric Kruskal–Wallis H test. P < 0.05 indicated statistical significance.