CISD2 protects against Erastin induced hepatocellular carcinoma ferroptosis by upregulating FSP1

Cell culture and ferroptosis inducing drugs treatment

The LO2 normal liver cell lines and HepG2, Bel-7404, and SMMC-7721 liver cancer cell lines were donated by the specimen bank (Guangxi, China) and cultured in complete medium containing 10% fetal bovine serum (GEMINI, America, 900-108) in a 37 °C, 5% constant CO₂ incubator (SANYO, Japan, 10050098). If the cells were treated with drug, the subsequent experiments were performed after 24 h of treatment with Erastin (TargetMOI, America, T1765, drug dissolved with DMSO, final concentration of DMSO below 0.1%).

Cell viability test

Cells were plated in a 96-well plate (4,000 cells/well) and incubated with the CCK-8 kit (MCE, America, HY-K0301). The absorbance value of the cells at 450 nm was then measured using a multi-function microplate reader (BERTHOLD, Germany, TriStar LB941), and the cell viability was calculated according to the formula in the instruction.

Detection of Fe content

The cells to be tested were collected and processed with the Fe assay kit (BaiOLaiBo, China, GL1920). The absorbance value of cells at 562 nm was then measured using a multi-function microplate reader, and the Fe content was calculated according to the formula in the instruction.

Detection of GSH content

The cells to be tested were collected and processed with a GSH content detection kit (Solarbio, China, BC1175). The absorbance value of the cells at 412 nm was then measured using a multi-function microplate reader, and the GSH content was calculated according to the formula in the instruction.

Detection of ROS content

Cells were plated in 6-well plates (2.5 × 10⁵ cells/well) and the probe was loaded with the ROS assay kit (Solarbio, China, CA1410). Cells were then visualized with an inverted multifunctional microimager (OLYMPUS, Japan, IX73 + DP80) at an excitation wavelength of 488 nm and emission wavelength of 525 nm (green fluorescence).

Western blot

The cells to be tested were collected, lysed into protein with RIPA lysate (RIPA, EpiZyme, China, PC101) and PMSF protease inhibitor (PMSF, Solarbio, China, P0100) and measured by protein concentration with BCA Protein Concentration Measurement kit (Beyotime, China, Cat #P0010). Samples, SDS-PAGE protein loading buffer (Beyotime, China, P0015) and appropriate PBS were mixed and boiled. Proteins were separated by gel electrophoresis using an SDS-PAGE solution (Beyotime, China, P0014B). After electrophoresis, proteins were transferred to the PVDF membranes (Merck Millipore, Germany, Cat# ISEQ00010) using Western membrane transfer fluid (Beyotime, China, P0021B) (Merck Millipore, Germany, Cat# ISEQ00010). Then, the PVDF membranes were blocked with the Western blocking fluid (Beyotime, China, P0252). Next, The PVDF membranes were incubated with CISD2 (1:1000 dilution), FSP1 (1:1,500 dilution), and GAPDH (Proteintech, China, 10494-1-AP, 1:30000 dilution) for incubation, And the PVDF membranes were further incubated with HRP-conjugated Affinipire Goat Anti-Rabbit IgG antibody (Proteintech, China, SA00001-2, 1:10000 dilution). Finally, the PVDF membranes were visualized using ECL ultrasensitive chemiluminescence detection kit (EpiZyme, China, SQ 201) and automatic chemiluminescence image analyzer (Tanon, China, Tanon-5200 multi). Grayscale analysis was performed with the Image J software.

RT-qPCR

Total RNA was extracted from the cells with a total RNA extraction kit (TIANGEN, China, DP419). Then, the purity and concentration of the total RNA were determined by a trace UV spectrophotometer (TIANGEN, China, TGEM Plus), and the total RNA was reverse transcribed into cDNA using the cDNA first strand synthesis kit (Beyotime, China, D7168L) and the gradient PCR instrument (eppendorf, Finland, mastercycler gradient). Finally, SYBR Green qPCR Mix (MonDNA, China, 130526), primers (GENEray, China), and fluorescent quantitative PCR instrument (Roche, Switzerland, LightCycle) were used to make the cDNA go through the process of pre-denaturation, denaturation, annealing and extension, and finally obtain the melting curve. Relative mRNA expression was calculated by the 2^−ΔΔCt method.

The primer sequence used for RT-qPCR is as follows. GAPDH Forward primer sequence: 5′-GGACCTGACCTGCCGTCTAG-3′, GAPDH Reversed primer sequence: 5′-GTAGCCCAGGATGCCCTTGA-3′. CISD2 Forward primer sequence: 5′-GTGGCCCGTATCGTGAAGG-3′, CISD2 Reversed primer sequence: 5′-CTAGCGAACCCGGTAATGCTT-3′. FSP1 Forward primer sequence: 5′-TCTTGGTTTGGTGCTTCTG-3′, FSP1 Reversed primer sequence: 5′-CCCTCTTTGCCCGAGTA-3′.

Immunohistochemical staining

The cancer tissue and adjacent tissue wax blocks from 25 patients were collected and the tissue wax blocks were cut into thin pieces and attached to adhesive glass slides. After the glass slides with tissue were dewaxed, hydrated and repaired by antigen, were incubated with CISD2 antibody (CST, America, 60758S, 1:200 dilution) and FSP1 antibody (Proteintech, China, 2003130425h, 1:350 dilution) at room temperature. Next, the glass slides with tissues were dripped with immune chromogenic reagent (MaxVision, China, 200825S407m) and DAB staining solution (MaxVision, China, 2009270805M) for coloration, and then the processes of contrast staining, differentiation, blue-return, dehydration, and sealing piece were performed. Finally, immunohistochemistry scoring was performed using an upright fluorescence microscope (OLYMPUS, Japanese, BX 53).

The immunohistochemical staining results were scored as follows. FSP1 protein in cells was localized in the cytoplasm and cell membrane, and CISD2 protein in the cytoplasm was scored by pathologists according to the positive degree and the proportion of positive cells at high magnification (400×). According to the positive score, negative score is 0 point, pale yellow is one point, brown is two point, brown is three point and dark brown is four point. According to the proportion of positive cells, <5% is 0 point, 5–25% is one point, 26% is two point, 51–75% is three point, and >75% is four point. The total score of multiplying the degree of positive score and the proportion of positive cell score was taken as the final result.

Plasmid amplification

Empty vector plasmid, CISD2 overexpression vector plasmid and FSP1 overexpression vector plasmid were amplified using DH-5α competent cells (Huayueyang, China, 2110 # 09D211009D). Then, plasmid extraction was performed with an endotoxin-free plasmid bulk lift kit (TIANGEN, China, # DP117). Next, DNA concentration was determined using a trace UV spectrophotometer and purity was determined using an agarose horizontal electrophoresis tank (LIUYI, CHYA, DYCP-31DN) and an automated chemiluminescence image analyzer.

Cell transfection

The cells were plated into 6-well plates. When the cell density reached about 90%, the empty vector, CISD2 overexpression vector plasmid and FSP1 were transfected into the cells with lipofectamine 3,000 transfection reagent (invitrogen, America, 2328417), respectively.

Statistical method

The statistical analysis of data results was carried out by using the software of grahpad prism 8.0.2, and the line chart and histogram were drawn. Use the data results of repeated experiments for 3 times to verify whether the difference is statistically significant. The data in the figure is the mean ± standard deviation (x ± s). */**/*** in the figure represents p value<0.05/0.01/0.001, and it is considered that the difference is/is significant/has extremely significant statistical significance. T-test was used to analyze the differences between the two samples. The one-way ANOVA is used to analyze the differences between groups of multiple samples, and the two-way ANOVA is used to analyze the differences between groups of multiple samples with multiple factors.

Erastin induced ferroptosis in hepatoma cells, and HepG2 hepatoma cell line was sensitive to Erastin

The chemical structural formula of Erastin is shown (Figure 1A). CCK-8 kit was used to measure the cell viability of HepG2, Bel-7404 and SMMC-7721 hepatoma cells treated with Erastin at different concentrations (0, 2.5, 5, 10 and 20 µM). Compared with the untreated control group, Erastin reduced the cell viability of the three hepatoma cells in a dose-dependent manner (Figure 1B). In addition, HepG2 cells with median lethal dose (LD50) of 10 µM were selected as the hepatoma cell line sensitive to Erastin. In the following experiments, Erastin with a concentration of 10 µM was selected to treat HepG2 cells. Fe accumulation, GSH consumption and lipid ROS production are three key signaling events of ferroptosis. Compared with the control group, the total Fe content increased significantly after Erastin treatment (Figure 1C), while the GSH content decreased significantly (Figure 1D). In addition, ROS levels increased significantly under the influence of Erastin (Figure 1E, 100×). Taken together, these data suggest that Erastin inhibits the cell viability of hepatoma cells and promotes ferroptosis in HepG2 cells.

Figure 1:

Erastin induced ferroptosis in hepatoma cells, and hepG2 hepatoma cell line was sensitive to Erastin. Chemical structure formula of Erastin from the drug instruction (A). The cell viability of HepG2, Bel-7404 and SMMC-7721 cells treated with Erastin (0, 2.5, 5, 10 and 20 µM) was detected using CCK-8 kit. (B) Erastin treated hepatoma cells sensitive to it, and the relative contents of (C) total Fe and (D) GSH and (E) ROS were detected using Fe detection kit, glutathione content detection kit and ROS detection kit respectively. *p < 0.05, **p< 0.005, ***p < 0.001.

The protein and mRNA expression of CISD2 and FSP1 changed significantly during the ferroptosis of HepG2 cells

Subsequently, the protein and mRNA expression of CISD2 and FSP1 after Erastin treatment were detected by Western blot and RT-qPCR. Compared with the control group, Erastin significantly up-regulated the protein and mRNA expression levels of CISD2 (Figure 2A and B), while down regulated the protein and mRNA expression levels of FSP1 (Figure 2C and D). These data showed that the protein and mRNA expression of CISD2 and FSP1 changed significantly during ferroptosis in HepG2 cells.

Figure 2:

The protein and mRNA expression of CISD2 and FSP1 changed significantly during the ferroptosis of HepG2 cells. The protein and mRNA expression levels of CISD2 in HepG2 cells treated with Erastin were detected by (A) Western blot and (B) RT-qPCR. And the protein and mRNA expression levels of FSP1 in HepG2 cells treated with Erastin were detected by (C) Western blot and (D) RT-qPCR. *p < 0.05, **p< 0.005, ***p < 0.001.

Overexpression of CISD2 reversed the effect of Erastin on ferroptosis of HepG2 cells and Partially Restored the Regulatory Effect of FSP1 in ferroptosis of HepG2 cells

Compared with the adjacent tissues, the protein expression level of CISD2 in HCC tissues was significantly increased (Figure 3A, 400×). In order to further study the specific role of CISD2 in HCC cells, we transfected CISD2 overexpression plasmid to increase the expression level of CISD2. After transfection, the protein expression level of CISD2 was significantly up-regulated, and the protein expression level of FSP1 was also increased (Figure 3B). Then, the cell viability was detected by CCK-8 kit. Compared with the OE-NC + 10 µM Erastin group, the up-regulation of CISD2 significantly increased the cell viability of HepG2 cells (Figure 3C). In addition, we also detected the contents of total Fe and GSH in cells to analyze the role of overexpression of CISD2 in ferroptosis of HCC. The effect of Erastin on total Fe and GSH content in HepG2 cells was reversed by overexpression of CISD2 (Figure 3D and E). In addition, compared with HepG2 cells stimulated only by Erastin, the production of ROS in HepG2 cells treated with Erastin after up-regulation of CISD2 was significantly inhibited (Figure 3F, 100×). Taken together, these results suggest that overexpression of CISD2 can reverse the effects of Erastin on cell viability of HepG2 cells and ferroptosis in HCC.

Figure 3:

Overexpression of CISD2 reversed the effect of Erastin on ferroptosis of HepG2 cells. The protein expression level of CISD2 in HCC tissues and paired adjacent tissues was detected by IHC (A). After transfection of CISD2 overexpression plasmid, the protein expression levels of CISD2 and FSP1 in HepG2 cells were detected by Western blot to verify the transfection efficiency (B). The cell viability of HepG2 cells overexpressing CISD2 and treated with Erastin was detected using CCK-8 kit (C). The relative contents of (D) total Fe and (E) GSH, and (F) ROS levels of HepG2 cells overexpressing CISD2 and treated with Erastin were detected using the Fe detection kit, GSH content detection kit, and ROS detection kit, respectively. *p < 0.05, **p< 0.005, ***p < 0.001.

Then, compared with HepG2 cells stimulated only by Erastin, the protein expression level of CISD2 was significantly up-regulated in HepG2 cells treated with Erastin after up-regulation of CISD2, and the protein expression level of FSP1 was also significantly up-regulated (Figure 4A–C). Subsequently, the string database was used to predict the potential proteins interacting with CISD2 (Figure 4D). CISD3, CIAPIN1, MRFAP1, SFXN4, WFS1, SOD1, ITPR1, BECN1, COX4I1, GIMAP5 and so on were the potential proteins of CISD2. FSP1 was not found to be an important protein of CISD2, which was the first time that we found the relationship between CISD2 and FSP1. Based on the above findings, we conclude that overexpression of CISD2 can partially restore the regulatory effect of Erastin on FSP1 in HepG2 cells.

Figure 4:

Overexpression of CISD2 partially restored the regulatory effect of FSP1 in ferroptosis of HepG2 cells. Western blot was used to detect the protein expression levels of CISD2 and FSP1 in HepG2 cells overexpressing CISD2 and then treated with Erastin (A–C). STRING database (https://string-db.org/) showed (D) the protein-protein interaction network of CISD2. *p < 0.05, **p< 0.005, ***p < 0.001.

Overexpression of FSP1 reversed the effect of Erastin on ferroptosis of HepG2 cells

Compared with adjacent tissues, the level of FSP1 in HCC tissues was significantly reduced (Figure 5A, 400×). In addition, we also found that compared with normal liver cell line LO2, the mRNA expression level of FSP1 in HepG2, Bel-7404 and SMMC-7721 liver cancer cell lines was also significantly reduced (Figure 5B). In order to further study the specific role of FSP1 in HCC cells, we transfected FSP1 overexpression plasmid to increase the expression level of FSP1. After transfection, the protein expression level of FSP1 was significantly up-regulated (Figure 5C). Then, the cell viability was detected by CCK-8 kit. Compared with the OE-NC + 10 µM Erastin group, the up regulation of FSP1 significantly increased the cell viability of HepG2 cells (Figure 5D). In addition, we also detected the total Fe content in cells to analyze the role of FSP1 overexpression in ferroptosis of HCC. The effect of Erastin on total Fe content in HepG2 cells was reversed by overexpression of FSP1 (Figure 5E). In addition, compared with HepG2 cells stimulated only by Erastin, ROS production in HepG2 cells treated with Erastin after FSP1 up regulation was significantly inhibited (Figure 5F, 100×). Then, compared with HepG2 cells stimulated only by Erastin, the protein expression level of FSP1 in HepG2 cells treated with Erastin increased significantly after up-regulation of FSP1 (Figure 5G). Taken together, these results suggest that overexpression of FSP1 can reverse the effects of Erastin on cell viability of HepG2 cells and ferroptosis in HCC.

Figure 5:

Overexpression of FSP1 reversed the effect of Erastin on ferroptosis of HepG2 cells. The expression level of FSP1 protein in HCC tissues and matched adjacent tissues was detected by IHC (A). The mRNA expression level of FSP1 in normal liver cell line LO2, HepG2, Bel-7404 and SMMC-7721 was detected by RT-qPCR (B). After transfection of FSP1 overexpression plasmid, the protein expression level of FSP1 in HepG2 cells was detected by Western blot to verify the transfection efficiency (C). The cell viability of HepG2 cells overexpressing FSP1 and treated with Erastin was detected using CCK-8 kit (D). The relative content of (E) total Fe and (F) ROS in HepG2 cells treated with FSP1 and Erastin were detected using the Fe detection kit and ROS detection kit respectively. Western blot was used to detect the protein expression level of FSP1 in HepG2 cells overexpressing FSP1 and then treated with Erastin (G). *p < 0.05, **p< 0.005, ***p < 0.001.