Patients

Gastric cancer patients with cachexia who underwent surgery in Zhongshan Hospital of Fudan University from June 2019 to December 2020 were included in this study. Inclusive criteria for the study were: (1) patients diagnosed with gastric adenocarcinoma cancer (not gastric stromal tumor or lymphoma); (2) patients received surgical treatment without preoperative radiotherapy or chemotherapy; (3) patients with abdominal CT examinations and complete clinical data; (4) patients with weight loss > 5% in recent 6 months before surgery. This study was approved by the Ethics Committee of Zhongshan Hospital of Fudan University (B2019-193R). Written informed consents were obtained from all patients.

Clinical data collection

Height, weight, gender, and age were extracted from the preoperative medical records. Body mass index (BMI) was calculated as body weight (kg)/height2 (m2). Cachexia-related indicators were extracted from preoperative blood biochemical examinations. Inflammatory markers were measured with ELISA kit by expert from clinical laboratory. Areas of SAT and VAT from CT scans at the third lumbar vertebra were measured as described before [13].

Human tissue specimens

Adipose tissues were obtained from enrolled patients. At the beginning of the operation, about 500 mg of SAT near the median abdominal incision was obtained. About 500 mg of omental adipose tissue was taken as VAT within 30 min after gastric cancer specimens were isolated. The adipose tissue was immediately stored in liquid nitrogen at − 80 °C or transferred into tissue fixative for further analysis.

Mouse model of cancer cachexia

The mouse model of cancer cachexia refers to the previous methods [4]. In brief, Cachectic mice were induced by subcutaneous injection of colon-26 adenocarcinoma cells into the right flank of the mice. The littermate control mice received PBS injection only. Mice were euthanized at day 21 post-injection and were dissected to harvest inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT). Weight of iWAT and eWAT was recorded at day 0 (control mice) and day 21 (cachectic mice). The proportion of adipose tissues loss was calculated by weight change (weight at day 0 subtract weight at day 21) divided to initial weight. All animal studies were performed in accordance with the guidelines provided by Animal Care Committee of Fudan University.

RNA sequencing

The total RNA was extracted from 3 paired SAT and VAT from gastric cancer patients with cachexia. After quantification and qualification, a total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina, USA) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. Fragments per kilobase million (FPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene. The raw sequencing dataset that supported the results of this study was deposited in the NCBI GEO database. The data are accessible through GEO: GSE186466.

RNA sequencing data analysis

Differential expression analysis of two groups was performed using the DESeq2 R package. Differentially expressed transcripts between the two groups were identified when |log Fold Change| > 0 and the p value < 0.05. Gene Ontology (GO) enrichment analysis of DEGs was implemented by the ClusterProfiler R package, in which gene length bias was corrected. We also used ClusterProfiler R package to test the statistical enrichment of differential expression genes in KEGG pathways. GO terms and KEGG pathways with corrected p value < 0.05 were considered significantly enriched by differential expressed genes.

Conventional enrichment analysis based on hypergeometric distribution depends on significantly up-regulated or down-regulated genes, and it is easy to omit some genes with insignificant differential expression but important biological significance. Gene set enrichment analysis (GSEA) does not need to specify a clear differential gene threshold. All genes are sorted according to the degree of differential expression in the two groups of samples, and then statistical methods are used to test whether the preset gene set is enriched at the top or low section of the sorting table. GSEA mainly includes three steps: calculation of enrichment score; estimation of the significance level of enrichment score; multiple hypothesis tests.

The PPI network of DEGs was predicted using the Search Tool for the Retrieval of Interacting Genes (STRING) database. The interaction score threshold of 0.4 was set as the cut-off criterion. The PPI network was constructed using Cytoscape. Comprehensive experimentally validated miRNA-gene interaction data were collected from TargetBase. Transcription factor and gene target data derived from the ENCODE ChIP-seq data. Only peak intensity signal < 500 and the predicted regulatory potential score < 1 is used (using BETA Minus algorithm).

Cell culture and differentiation

The mouse immortalized white preadipocytes were kindly provided by Professor Qiurong Ding from the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences. This cell line has been described previously and used in several studies to assess the effects of different factors on adipose differentiation and function [19]. The culture and differentiation methods of preadipocytes cell lines were as previously reported [18]. Mouse preadipocyte was cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. All cells were kept in an atmosphere of 5% CO2 and 95% oxygen at 37 °C. Differentiation of preadipocytes was initiated by an induction medium (0.5 mM isobutyl-1-methylxanthine (IBMX), 5 mM Dexamethasone, 1 μm Rosiglitazone, 5 ug/ml Insulin), and replaced with a maintenance medium (5ug/ml Insulin) after 2 days for further differentiation.

Plasmid and siRNA construction

The iroquois homeobox 1 (IRX1) expression plasmid pcDNA3.1þ/IRX1 and empty plasmid pcDNA3.1þ were designed and synthesized by GenePharma (Shanghai, China). Small interfering RNAs (siRNAs) targeting IRX1 were also designed by GenePharma. Cell transfection was conducted using Lipofectamine RNAiMAX Transfection Reagent kit (Invitrogen, USA) or Lipofectamine 2000 Transfection Reagent (Invitrogen, USA) according to the manufacturer’s instruction. Lipofectamine™ RNAiMAX transfection reagent was used to transfect 100 nM of siRNAs when cell density was approximately 70%.

Hematoxylin-eosin and immunohistochemical staining

All the samples were transferred to tissue fixative after being harvested. The protocols were previously described [18]. In brief, histological sections of adipose tissue were stained with hematoxylin–eosin to evaluate morphological changes and the adipocyte cross-sectional area (CSA). A total of 10 randomly selected fields for each section were captured and analyzed to evaluate adipocyte CSA with a computerized imaging software (ImageJ, USA). For immunohistochemistry, the positive cells in 10 randomly selected fields per section were counted and evaluated by two independent researchers. And the mean number of positive cells per field was calculated.

Oil Red O (ORO) staining

Mature adipocytes were fixed with 4% formaldehyde for 30 min, then washed twice with PBS. They were stained with 0.3% ORO solution and washed three times with distilled water. To assess lipid accumulation, the dye retained in the cells was dissolved in isopropanol and the absorbance of the resulting solution at 520 nm was examined.

RNA isolation and qRT-PCR

Total RNA was isolated from adipose tissues and adipocytes using TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s recommendations. cDNA was synthesized from 1 µg total RNA using FastKing RT Kit (Tiangen, China). Gene expression analysis was performed using Prime-Script RT master mix (Takara, Japan) in StepOnePlus Real-Time system (Applied Biosystems, USA). Expression levels of targeted genes were normalized to the expression of GAPDH. qRT-PCR was performed according to the manufacturer’s instructions and the relative fold change was calculated by the 2−ΔΔCt method. Primers were designed and synthesized by Sangon Biotech (Shanghai, China) and are listed in Additional file 2: Table S2. All experiments were repeated three times.

Western blot analysis

Preparation of total protein lysates and western blot analysis were performed as previously described [16]. Primary antibodies against IRX1 (Immunoway, YT2412), CEBPα (Cell signaling technology, #2295), Adiponectin (Cell signaling technology, #2789), and FABP4 (Cell signaling technology, #2120) were used. Tubulin expression was used as an endogenous control.

Statistical analyses

Statistical analyses were performed using GraphPad Prism software. Data calculated from independent experiments were presented as the mean ± standard deviation and a student’s t-test was performed to compare the differences between two groups. To analyze the correlation between IRX1 mRNA levels in SAT and the clinicopathological factors in gastric cancer patients with cachexia, we divided 61 patients into two groups according to the IRX1 expression in SAT compared to VAT. Comparisons between these two groups were made using the t-test for continuous data and χ2 test for categorical data. p < 0.05 was considered statistically significant.