Cell cultureThe pancreatic β cell line NIT-1 cells (Ginio Biotechnology Co., Ltd., Guangzhou, China) were cultured in an incubator with saturated humidity, a temperature of 37 °C, and a CO2 concentration of 5%. The culture medium consisted of 1640 medium (Life Technologies/Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Life Technologies/Gibco, Grand Island, NY, USA). Additionally, 100 Unit/mL penicillin and 100 μg/mL streptomycin (New Cell & Molecular Biotech Co., Ltd, Suzhou, China) were added to the culture medium on a routine basis.Cell modelsCadmium-induced damage model15: CdSO4 was used as the test material in this study. It is the most abundant in the environment, is highly soluble in water and poses a serious pollution risk. NIT-1 cells were treated with varying concentrations (0 μmol/L, 1 μmol/L, 2 μmol/L, 4 μmol/L, 8 μmol/L, 16 μmol/L, 32 μmol/L and 64 μmol/L) of CdSO4 for 24 h. Cell viability was detected by CCK-8 assay. Three concentrations exhibiting cell viability above 70% were selected for the induction of oxidative damage. Finally, the CdSO4 concentrations were determined to be 0 μmol/L, 1 μmol/L, 2 μmol/L and 4 μmol/L.Antioxidant model: NIT-1 cells were treated with N-Acetyl-L-cysteine (NAC, Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) at concentrations of 0 mmol/L, 0.625 mmol/L, 1.25 mmol/L, 2.5 mmol/L, 5 mmol/L, 10 mmol/L, and 20 mmol/L for 24 h. The concentration of NAC with the highest cell viability was selected as the pretreatment concentration. The experiments were divided into four groups: a control group, a NAC group, a CdSO4 group, and a NAC + CdSO4 group. In the NAC group and NAC + CdSO4 group, 5 mmol/L NAC was added to the cells for 2 h pretreatment. Following the removal of the liquid, 4 μmol/L CdSO4 was added to the CdSO4 group and the NAC + CdSO4 group and placed in an incubator for 24 h. At this point, a series of measurements were taken.Differentially expressed gene screeningTotal RNA was prepared from the control group (0 μmol/L CdSO4) and the treatment group (4 μmol/L CdSO4) with qualified quality. The transcriptome expression profile was detected on the Clariom D mouse microarray from Affymetrix GeneChip by Shanghai Baygene Biotechnology Co., Ltd. The procedure was followed: total RNA preparation, hybridization, image scanning, and raw data acquisition. Subsequently, the raw data underwent RMA correction, with probe information being annotated in the process. Finally, the differentially expressed genes (DEGs) were analyzed by intergroup moderated T-test based on sample groups. The fold change (FC) of RNA expression in different groups was compared from the above DEGs, and the genes with upregulated or downregulated were identified for further analysis. Filter conditions: For up-regulation, the criteria were a FC of at least 2 and a false discovery rate (FDR) of less than 0.05. For down-regulation, the criteria were a FC of at least -2 and an FDR of less than 0.05. All gene expression data have been uploaded to the GEO database with the accession number GSE253072.RT-qPCRTotal RNA was extracted by using RNA simple total RNA kit (Tiangen Biotech Beijing Co., Ltd.) for mRNA detection. Total RNA with a length greater than 200 nt and less than 200 nt was extracted by using miRcute miRNA isolation kit (Tiangen Biotech Beijing Co., Ltd.). Less than 200 nt of total RNA was employed for miRNA detection and greater than 200 nt for LncRNA detection. Complementary DNA (cDNA) was synthesized following the FastKing gDNA dispelling RT superMix (Tiangen Biotech Beijing Co., Ltd.). cDNA was synthesized following the miRcute plus miRNA first-strand cDNA kit (Tiangen Biotech Beijing Co., Ltd.). RT-qPCR was performed on QuantStudio 3 (Thermo Fisher Scientific, Waltham, MA, USA). The mRNA and lncRNA expression levels were examined in accordance with the two-step method outlined in the instructions for the SuperReal premix plus (SYBR Green) kit (Tiangen Biotech Beijing Co., Ltd.). The miRNA levels were quantified using the miRcute plus miRNA qPCR Kit (Tiangen Biotech Beijing Co., Ltd.). The quality of the PCR process was evaluated based on the amplification and melting curves. And Gapdh was applied as internal control for mRNA and LncRNA, Rnu6 was applied as internal control for miRNA. The relative expression levels of the target genes were calculated by 2-△△Ct method. Primer sequences were listed in Table 1.
Table 1 Primer sequences used for RT-qPCR.Western blotNIT-1 cells were inoculated in 6-well plates and allowed to adhere to the wall. The cells were then treated with CdSO4 and/or NAC for 24 h. At the conclusion of this period, 180 μL of a ready-made cell lysate was added. Subsequently, the reaction was conducted at 4 °C for 10 min after thorough agitation, and the supernatant was harvested by high-speed centrifugation at low temperature (4 °C, 13,000 rpm, 10 min) to obtain the protein samples. The protein concentration was determined by the BCA method (Beyotime Biotech. Inc., Shanghai, China).A total of 50 μg of protein samples were successively loaded onto SDS-PAGE gels at concentrations of 5% and 12% for separation purposes. Following this, the samples were transferred to polyvinylidene difluoride (PVDF) membranes. Following a two-hour incubation period at room temperature with 5% skimmed milk, the primary antibody to COL3A1 (Beijing Solarbio Science & Technology Co., Ltd.) was applied. On the subsequent day, the membranes were incubated with a secondary antibody conjugated to horseradish peroxidase (Beijing Zhongsui Jinqiao Biotechnology Co., 1:6000) for one hour at room temperature. Subsequently, the immunoreactive bands were visualised with an enhanced chemiluminescence reagent (Millipore, Temecula, CA) and imaged with a JS-M6 chemiluminescence imaging system (Peiqing Technology Co., Ltd., Shanghai, China). The intensity of the protein bands was quantified using the Image J software (National Institutes of Health, USA). GAPDH was employed as an internal control, and the fold change in protein expression was calculated based on the protein levels observed in the control group.CCK-8 assayThe inoculation of 96-well plates should be conducted in accordance with the density of 1 × 104 cells per well. CdSO4 or NAC should be added overnight following 24 h of wall attachment. The culture medium should be configured with 10% CCK-8, and the old medium discarded. The fresh medium should be added rapidly and the cells incubated for 3 h. Absorbance at 450 nm was measured by Multifunctional Fluorescent Plate Reader (Flash Biotechnology Co., Ltd., Shanghai, China), and the cell viability = A450 mean of the treated group / A450 mean of the control group × 100%.Oxidative Stress DetectionDetection of ROSAccording to the manual of the reactive oxygen species assay kit (Beyotime Biotech. Inc., Shanghai, China), cells were incubated (37 ℃, 20 min) after diluting the DCFH-DA probe (final concentration of 10 μmol/L). Cells were observed and photographed with a fluorescence microscope (excitation wavelength 488 nm, NIB610-FL, Ningbo Yongxin Optics Co., Ltd., Ningbo, China). The fluorescence intensity was quantified by Image Pro Plus, and the average fluorescence intensity of ROS = the sum of fluorescence intensity/ region area.Detection of MDAThe MDA content was measured by lipid peroxidation MDA assay kit (Beyotime Biotech. Inc., Shanghai, China). Firstly, a standard curve was prepared for the standards. Then, the reaction system was formulated in a 1.5 mL centrifuge tube following the guideline, mixing and heating at 100 ℃ for 15 min. Finally, the samples were cooled to room temperature and centrifuged (1000 g, 10 min), and 200 μL of the upper solution was used to measure the absorbance at 532 nm. A standard curve regression was established with the concentration of the standards as an independent variable (x), and the absorbance as a dependent variable (y). Absorbance of the samples were substituted into the regression equation and the MDA concentration of the samples were calculated.Detection of SODThe reaction system was prepared in a 96-well plate as per the instructions of the total superoxide dismutase assay kit with WST-8 (Beyotime Biotech. Inc., Shanghai, China). Then, the absorbance at 450 nm was measured after incubation at 37 ℃ for 30 min. The absorbance was used to calculate the percentage inhibition, which was (A blank control 1 – A sample)/(A blank control 1 – A blank control 2) × 100%. The SOD enzyme activity units in the sample to be tested = SOD enzyme activity units in the assay system = percentage inhibition/(1 – percentage inhibition).Detection of GSH-PxNIT-1 cells were inoculated into a 6-well plate, and protein was extracted after treatment. GSH-Px was detected by following the protocol of the cellular glutathione peroxidase assay kit with NADPH (Beyotime Biotech. Inc., Shanghai, China). The reaction system was prepared referring to the instructions and incubated at room temperature for 15 min. Lastly, each well was added with 10 μL of peroxide reagent solution and the absorbance at 340 nm was measured immediately by a Multifunctional Fluorescent Plate Reader, at which time the 0 min value was recorded. Six values were recorded at 2 min intervals. Glutathione peroxidase activity in the assay system = [(sample△A340 – blank△A340)/min]/(0.00622 × 0.276).Statistical analysisAll statistical analysis of data from the experiments was performed in SPSS 26.0. One-way ANOVA was performed for comparisons between multiple groups, and the LSD method was used for comparisons between groups. Kruskal–Wallis rank sum test was used when the variance was not equal, and Bonferroni method was used for two-way comparisons between groups. The significant level was set to 0.05.
Screening of differentially expressed RNAs and identifying a ceRNA axis during cadmium-induced oxidative damage in pancreatic β cells
