-Studied population
Twenty-six fresh frozen adrenal tissues were collected prospectively from October 2019 to December 2020 from patients undergoing laparoscopic adrenalectomy either because of tumour size or suspicious imaging characteristics or autonomous hormonal secretion. All surgical procedures were performed by the same surgical team of the 3rd Department of Surgery in the General hospital “G. Gennimatas” in Athens. For every sample of the pathological adrenal tissue, the surgeon collected also a sample of the adjacent adrenal tissue, which was considered macroscopically normal, except for adrenocortical carcinomas (ACC) tissues. The adjacent adrenal tissue was used as “control” only when it was histologically confirmed by the pathologist as normal adrenal tissue.
The histopathological analysis of the surgical specimens was performed by two independent pathologists in the Department of Pathology of “G. Gennimatas” and “Evangelismos” General Hospitals in Athens. All adrenocortical neoplasms were classified according to the universal diagnostic criteria endorsed by the WHO classifications including the modified Weiss criteria [15]. All primary malignant adrenal tumours reviewed as part of this study demonstrated three or more of the histopathologic Weiss criteria needed for the diagnosis of ACC. The diagnosis of the autonomous hormonal secretion from the adrenal neoplasms was based on the history, clinical examination and preoperative endocrine tests performed in the same laboratory. Cortisol (basal and after 1-mg overnight dexamethasone test (ODST)) and 24-h urinary free cortisol (UFC) levels were measured by electrochemiluminescent bridging immunoassay (ECLIA) (Cobas 8000 e801, Hitachi, intra-assay CV < 3.9% and inter-assay CV < 3.8% for all hormones), adrenocorticotropic hormone (ACTH) was measured by chemiluminescent assay (Liaison, DiaSorin, intra-assay CV 4.3-7.5% and inter-assay 10-14.5%).
All patients gave written informed consent for the use of samples and clinical data. The protocol of this study was approved by the institutional Research Ethics Board of the National and Kapodistrian University of Athens (Laiko hospital, code 6545/22-4-2019) according to the guidelines of the Declaration of Helsinki.
-Materials
A quantity of 100–200 mg of adrenal tissue (adrenal tumour and normal adrenal tissue) was cut and stored immediately after surgery at −80 °C as previously described [16].
Cell culture:
The SW-13 ACC cell lines (kindly provided by Professor C. Stratakis, NIH, USA) were grown and maintained in high glucose DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum and 100 µg/ml penicillin/ streptomycin in a standard humidified incubator at 37°C in a 95% air and 5% CO2 atmosphere. Cell monolayers were sub-cultured into a 12-well plate for RNA extraction (4x105),for apoptosis analysis using Annexin PI (2 x 105 cells/well) and for wound healing assay (3 x 105 cells/well) and into a 96-multi-well plate (103 cells/well) for MTS assay. Cells were maintained in complete medium for 16 h pre- and for 48h post- transfection.
siRNA
Cells were seeded into 12-multi-well plates (1 × 105 cells/well) and after 16hous of incubation they were transfected either with control siRNA (siRNA scrambled) or with CHCHD2 siRNA (Thermo Fisher Scientific) using lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) following the manufacturer’s instructions and optiMEM (complete medium). After 48 hours, CHCHD2 levels were detected by RT-PCR and Western Blot analysis.
MTS assay:
The effect of CHCHD2 silencing on SW-13 cell viability was determined using MTS assay as previously described [17].
Would healing assay:
Cells were incubated in 12-well plates until about 70% confluent and then they were transfected with CHCHD2 siRNA. After 8 hours of transfection, a 10-μL pipette tip was used to create a scratch/wound with clear edges across the well. Cells were photographed with Olympus CKX53 microscope at 0 hours, 24 and 48 hours. All experiments were performed in triplicates.
Flow cytometric analysis:
Annexin PI: Apoptotic cell death was identified in SW-13 cells after CHCHD2 silencing by double supravital staining with recombinant FITC (fluorescein isothiocyanate) conjugated Annexin V and propidium iodide (PI), using the Annexin V-FITC Apoptosis Detection kit (4830-01-K) according to the manufacturer’s instructions.
Total RNA isolation and qPCR
Total RNA was isolated from 50 mg of adrenal lesion and normal tissue using NucleoSpin® RNA Plus kit (Macherey-Nagel, Düren, Germany). The purity and concentration of the extracted RNA was determined using the NanoDrop spectrophotometer 2000 (Thermo Scientific). One microgram of RNA was reverse transcribed into cDNA using a LunaScript® RT SuperMix Kit (New England Biolabs, Ipswich, MA, USA). The mRNA expression of Βax, Bcl-2, CHCHD2 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) as previously described [14]. Briefly, mRNA levels of the tioned genes were evaluated using Luna® Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) on the CFX96 Touch real-time PCR detection system (Bio-RAD, Hercules, CA, USA). All reactions were carried out in triplicates. Each qPCR run included a negative control. The pooled cDNA sample was used as an internal control to correct the inter-assay variation for samples run on different plates. The relative fold change was calculated using the 2(-Delta Delta C(T))(ΔΔCTs) after normalizing to the value of internal-control. The expression of each gene for every adrenal sample was normalized against Actin expression [18]. The sequences of primers used in this study are listed below:
Primer
|
Sequence
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Actin
|
|
Forward
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5’-AGAGCTACGAGCTGCCTGAC-3’
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Reverse
|
5’-AGCACTGTGTTGGCGTACAG-3’
|
Bax
|
|
Forward
|
5’- CCGCCGTGGACACAGAC-3’
|
Reverse
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5’- CAGAAAACATGTCAGCTGCCA-3’
|
Bcl-2
|
|
Forward
|
5’-GCTGAAGATTGATGGGATCG-3’
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Reverse
|
5’-TACAGCATGATCCTCTGTCAAG-3’
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CHCHD2
|
|
Forward
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5’-GTGCCGACTTGCAAACGGAT-3’
|
Reverse
|
5’-GGCCACACAAACATTTGCCC-3’
|
Protein extraction and western blot analysis
Whole protein was extracted from 50 mg adrenal tissues using 2X cell lysis buffer (CST. 9803) according to the manufacturer’s instruction, the protein concentration was determined by Bradford assay (Applichem). Thirty micrograms of extracted protein from each sample were resolved on the 12% or 15% SDS-PAGE gel by electrophoresis and immediately transferred to PVDF membrane. After blocking with 1X-TBST containing 5% dry milk for 1h, membranes were incubated overnight at 4 ◦C with anti-b-actin (MAB1501 Millipore, 1:5000), anti-Bax (#5023 Cell Signaling, 1:800), anti-Bcl-2 (#15071 Cell Signaling, 1:800), anti-CHCHD2 (HPA027407 Sigma-Aldrich, 1:1000). Subsequently, membranes were washed 3 times with 1X-TBST and incubated with HRP conjugated anti-mouse IgG (31430, Thermo Scientific) or anti-rabbit IgG (12–348, Millipore) secondary antibodies for 1 h at room temperature. The protein bands were visualized using the Clarity Western ECL Substrate (Bio-Rad) and quantified using ImageJ software. β-actin served as a loading control and an aliquot of pooled standard sample was loaded in each gel to correct the inter-assay variation for samples run in different gels.
Statistical analysis
All the data are reported median (IQR). Non-parametric parameters were analyzed with Mann–Whitney test for the comparison between ACCs patients and patients with ACAs or controls while Wilcoxon test was performed for the comparison of non-parametric paired values in the same group. Student t-test was applied to evaluate the difference of parametric parameters between the groups. For correlation analysis we used Spearmen rank correlation coefficient test. All tests were 2-sided with statistical significance set at 0.05 and all computation were made using PRISM 7.