RT-qPCR assessment of mRNA expression (ERBB4, NRG4 and major lipogenesis genes) in human liver with histologic liver images and biochemical analysis of blood serum

Description

ERBB4 and NRG4 have been shown to ameliorate steatosis and prevent the development of non-alcoholic steatohepatitis in mouse models, but little to nothing is known about their role in non-alcoholic fatty liver disease (NAFLD) in humans. This data is the first expression study of ERBB4, NRG4, ACACA, FASN, FTO, SREBF1 and TFEB mRNAs in the liver biospecimens of individuals with obesity, type 2 diabetes and biopsy-proven NAFLD. Against a background of increased BMI, ERBB4 and NRG4 mRNA levels decreased, while ACACA mRNA levels increased. No deregulation of the analyzed transcripts was detected in NAFLD (link to the article). The data also contains information on detailed blood biochemical parameters, the anthropometric information (including diagnosis) and liver histology images that can be used for various meta-analyses and the development of prognostic biomarkers. Verification of the diagnosis of patients was performed at the Regional Clinical Hospital in Kaliningrad, Russia. Patients underwent standard dietary adjustments before elective abdominal surgery, which was performed under general anesthesia. All obese patients discontinued medications affecting carbohydrate and lipid metabolism parameters 36 hours before surgery. A fasting blood sample was obtained the morning before surgery. During surgery, liver samples of up to 0.5 mL were collected for RNA extraction (immediately placed in 600 μL RNAlater solution (Ambion, USA)) and up to 1 mL for histologic analysis (immediately fixed in 5 mL neutral buffered formalin). The study was conducted in accordance with the World Medical Association Declaration of Helsinki (2000) and the Protocol to the Convention on Human Rights and Biomedicine (1999). The study was approved by the local ethics committee of the Immanuel Kant Baltic Federal University: IKBFU Ethics Committee Conclusion №40 of 26.06.2023. All of the participants provided signed informed consent. All interventions were performed by highly qualified medical professionals and posed no risk to the patients' health. Inclusion criteria: individuals over 21 years who have received a diagnosis of their health condition from an official healthcare provider and have been referred for planned abdominal surgery, and who declared their willingness to participate in the study and donate a small tissue sample and their blood by giving informed written consent. Exclusion criteria: presence of infectious liver disease, concomitant somatic and infectious diseases in the acute inflammatory stage, presence of a known HIV infection, presence of malignant or benign neoplasms, age under 21 years, refusal to undergo medical and laboratory examinations during the study, or refusal to sign the informed consent form.

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The analysis of biochemical parameters in blood serum was performed on an automated biochemical analyzer Furuno CA-180 (Furuno Electric Company, Japan) using DiaSys test systems (DiaSys Diagnostic Systems, Holzheim, Germany). Histological analysis of liver biopsies was performed to confirm NAFLD. Liver wedge biopsies (volume of up to 1 mL) were obtained intraoperatively by incisional biopsy from the left lobe of the liver and immediately fixed in neutral-buffered formalin. Paraffin sections of the liver, stained with hematoxylin and eosin, were examined by traditional histologic examination using a Leica DM3000 microscope (Leica Microsystems, Wetzlar, Germany). The biopsies examined contained at least four portal tracts and were informative. The histologic parameters (steatosis, ballooning, lobular inflammation, portal inflammation, fibrosis) were scored according to an adjusted “Steatosis – Activity – Fibrosis” (SAF) scoring system (the exact analytical parameters used by the histologist in this study are given below): • steatosis – 0 points: < 5% of the histological image area consists of lipid droplets; 1 point: 5-33%; 2 points: > 33-66%; 3 points: > 66%. • hepatocellular ballooning – 0 points: normal cell size; 1 point: pale cytoplasm, normal sized cells; 2 points: pale cytoplasm, enlarged cells; • lobular inflammation – 0 points: none; 1 point: < 2 foci (more than 2 cells, clusters); 2 points: 2-4 foci; 3 points: > 4 foci; • portal inflammation – 0 points: none or minimal; 1 point: mild; 2 points: more than mild; • fibrosis – 0 points: none; 1 point: perisinusoidal; 2 points: in 2 areas; 3 points: without complete partitions; 4 points: with partitions. The other part of liver samples were preserved in 600 μL RNAlater solution (Ambion, USA) and stored at –80°C. Total RNA was isolated from a portion of a liver sample of approximately 100 µL using the RNeasy Plus Mini Kit (Qiagen, Germany). The isolated total RNA was eluted in 45 µl of RNase-free water. RNA concentration was measured immediately after isolation using an Implen NanoPhotometer N (Implen, Germany). Samples were stored at –80°C until subsequent reverse transcription-polymerase chain reaction (RT-PCR). RT-qPCR for genes was performed as follows: universal reverse transcription was carried out using the MMLV RT kit (Evrogen, Russia) with the addition of the RNase inhibitor RiboCare (Evrogen, Russia) according to the manufacturer's protocol. For qPCR, HS SYBR PCR mix (Evrogen, Russia) was used. Amplification and reading of qPCR results were performed on a CFX96 thermal cycler (Bio-Rad, USA). After amplification, the melting curves were analyzed to verify the specificity of the reactions. An internal control, the reference gene RPLP0, was used to normalize gene expression data. Threshold cycles of transcripts were converted to relative expression values using the 2-ΔСt method.

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