CN116656699A - Application of SESN2 and chi-miR-130b-3p in regulation of zearalenone-induced apoptosis in granulosa cells - Google Patents

Abstract

The invention discloses application of SESN2 and chi-miR-130b-3p in regulation of granular cell apoptosis induced by zearalenone. The research finds that SESN2 and chi-miR-130b-3p are involved in regulating oxidative stress injury and apoptosis caused by ZEA in goat GCs. And the over-expression of SESN2 or the inhibition of the expression of chi-miR-130b-3p can reduce the cell damage of ZEA on goat GCs. Therefore, SESN2 and chi-miR-130b-3p can be used as potential therapeutic targets for reducing the toxicity of ZEA, so that the damage effect of ZEA on germ cells is weakened in the actual production process, and the reproductive capacity of female animals is improved.

Description

SESN2 and chi-miR-130b-3p regulate zearalenone-induced granulation Applications in Apoptosis

technical field

The invention belongs to the technical field of bioengineering, and specifically relates to the application of SESN2 and chi-miR-130b-3p in regulating the apoptosis of granulosa cells induced by zearalenone.

Background technique

Mycotoxins are toxic secondary metabolites produced by fungi that can contaminate growing crops or stored food. Zearalenone (ZEA), as a widely distributed and potent mycotoxin, has brought a lot of trouble to the animal husbandry and food processing industries. In addition to estrogen-like effects (Poor, et al., 2015), ZEA also causes the accumulation of reactive oxygen species (ROS) in cells (Feng et al., 2022), leading to oxidative stress and apoptosis (Zhu et al., 2022).

Granulosa cells (GCs) are crucial to the maturation of oocytes and the development of follicles. In the early stage of follicular development, GCs will form gap junctions with oocytes (Hasegawa, et al., 2007) for information exchange and material exchange, providing a suitable microenvironment for oocytes (Zhou, et al., 2019 ). As follicles develop, GCs begin to differentiate into MGCs and CCs (Fan, et al., 2009). MGCs secrete steroid hormones and growth regulators that promote the growth and development of the entire follicle (Yamochi et al., 2021). CCs form COCs with oocytes, and nutrients such as cAMP and amino acids are transported from GCs to oocytes through gap junctions (Regassa, et al., 2011). Therefore, once GCs are damaged due to external stress conditions, the entire female reproductive system will be adversely affected.

Sestrins (SESNs) are an evolutionarily highly conserved family of stress-induced proteins (Kumar, et al., 2020), which consist of Sesterins 1-3 and are upregulated under stress conditions (Lee, et al., 2013). As a metabolic regulator induced by various stresses, SESN2 can help cells maintain cellular homeostasis by activating catabolic responses, stopping anabolic activities, and initiating cellular repair mechanisms (Pasha, et al., 2017). KEAP1/NRF2 is a canonical SESN2-regulated signaling pathway that reduces intracellular ROS levels (Tu et al., 2019). Normally, NRF2 binds to KEAP1 in the cytoplasm to form a complex, where NRF2 maintains a relatively low expression level (Yamamoto et al., 2018). However, under oxidative stress conditions, NRF2 translocates into the nucleus and binds to antioxidant response elements (AREs), thereby activating the transcription of a series of downstream oxidoreductases, including SOD, CAT, HO-1, and GPX. Play antioxidant function (Yang, et al., 2019). However, the specific response mechanism of SESN2 to the damage caused by ZEA exposure in GCs has not been thoroughly studied.

miRNAs are small non-coding RNA sequences ranging in size from 18 to 25 nucleotides that play key roles in post-transcriptional regulation of gene expression. With the rapid development of high-throughput sequencing technology, the functions of miRNAs are increasingly known. According to research reports, several miRNAs have been confirmed to regulate the expression of SESN2, including miR-182-5p (Zhang, et al., 2022), miR-138-5p (An, et al., 2021) and miR- 199a-5p (Yang, et al., 2022) et al. Since ZEA has been shown to cause ROS accumulation and upregulate the expression level of SESN2 in GCs (Qin, et al., 2015), this study wanted to understand whether miRNAs were also involved in this process.

Contents of the invention

The purpose of the present invention is to provide the application of SESN2 or/and chi-miR-130b-3p in regulating GCs apoptosis.

Another object of the present invention is that SESN2 or/and chi-miR-130b-3p regulates ROS accumulation and oxidative stress damage in GCs.

Another object of the present invention is to provide a method for alleviating the cell damage caused by ZEA to GCs.

The purpose of the present invention can be achieved through the following technical solutions:

The application of SESN2 or/and chi-miR-130b-3p in the following (1) or (2) or (3),

(1) regulate GCs apoptosis;

(2) regulate the accumulation of ROS and oxidative stress damage in GCs;

(3) Alleviate the cell damage caused by ZEA to GCs.

Preferably, overexpressing said SESN2 or/and interfering with said chi-miR-130b-3p weakens GCs apoptosis; inhibiting said SESN2 or/and overexpressing said chi-miR-130b-3p aggravates GCs apoptosis. Further preferably, the GCs apoptosis is GCs apoptosis induced by ZEA.

Preferably, overexpressing the SESN2 or/and interfering with the chi-miR-130b-3p can reduce the accumulation of ROS and oxidative stress damage in GCs; inhibit the SESN2 or/and overexpress the chi -miR-130b-3p aggravates ROS accumulation and oxidative stress damage in GCs. Further preferably, the accumulation of ROS and oxidative stress damage in GCs is the accumulation of ROS and oxidative stress damage in GCs induced by ZEA. Further preferably, said SESN2 or/and chi-miR-130b-3p affects the accumulation of ROS and oxidative stress damage in GCs induced by ZEA through the KEAP1/NRF2 signaling pathway.

Preferably, overexpressing the SESN2 or/and interfering with the chi-miR-130b-3p can reduce the cell damage caused by ZEA to GCs.

A method for regulating GCs apoptosis induced by ZEA, overexpressing said SESN2 or/and interfering with said chi-miR-130b-3p weakens GCs apoptosis induced by ZEA; inhibiting said SESN2 or/and Overexpression of the chi-miR-130b-3p aggravated the apoptosis of GCs induced by ZEA.

A method for regulating the accumulation of ROS and oxidative stress damage in GCs induced by ZEA, overexpressing the SESN2 or/and interfering with the chi-miR-130b-3p can alleviate the ROS in GCs induced by ZEA Accumulation and oxidative stress injury; Inhibition of the SESN2 or/and overexpression of the chi-miR-130b-3p aggravated the accumulation of ROS and oxidative stress injury in GCs induced by ZEA.

A method for alleviating the cell damage caused by ZEA to GCs, overexpressing SESN2 or/and interfering with chi-miR-130b-3p alleviates the cell damage caused by ZEA to GCs.

Beneficial effects of the present invention:

With the continuous expansion of the scale of intensive farming, conventional livestock species such as cattle and sheep are gradually transferred to large-scale sheds for centralized breeding. Therefore, the occurrence of ZEA poisoning incidents is becoming more and more frequent. ZEA not only disrupts the reproductive cycle of female animals, but also damages reproductive cells including granulosa cells and oocytes, greatly affecting the reproductive ability of female animals. This study found that SESN2 and chi-miR-130b-3p were involved in the regulation of ZEA-induced oxidative stress injury and apoptosis in goat GCs. And overexpressing SESN2 or inhibiting the expression of chi-miR-130b-3p can alleviate the cellular damage caused by ZEA on goat GCs. Therefore, SESN2 and chi-miR-130b-3p can be used as potential therapeutic targets to alleviate the toxicity of ZEA, weaken the damage effect of ZEA on germ cells in the actual production process, and improve the reproductive ability of female animals.

Description of drawings

Figure 1 is the screening of miRNAs with potential interaction with SESN2;

Among them: (A) The effect of chi-miR-17-5p, chi-miR-20b and chi-miR-130b-3p on the expression of SESN2 at the mRNA level. (B) Effects of chi-miR-17-5p, chi-miR-20b and chi-miR-130b-3p on the expression of SESN2 at the protein level.

Figure 2 shows the detection of the expression levels of SESN2 and chi-miR-130b-3p and the prediction of their interaction;

Among them: (A) FPKM values of NR4A1, CHAC1, TRIB3, PPP1R15A and POLR2E in negative control group and ZEA-treated group. (B) The expression levels of NR4A1, CHAC1, TRIB3, PPP1R15A and POLR2E mRNA in the negative control group and ZEA treatment group. (C) Localization of SESN2 in the differential gene volcano map.

(D) The mRNA level expression of SESN2 in negative control group and ZEA treatment group. (E) The protein level expression of SESN2 in negative control group and ZEA treatment group. (F) Prediction of the relationship between SESN2 and miRNAs targeting. (G) FPKM values of chi-miR-130b-3p in negative control group and ZEA-treated group. (H) The expression level of chi-miR-130b-3p mRNA in negative control group and ZEA treatment group.

Figure 3 is the verification of the targeting relationship between SESN2 and chi-miR-130b-3p;

Wherein: (A) Construction of wild-type and mutant SESN2 overexpression plasmid sequences. (B) Chi-miR-130b-3p mimic efficiency validation. (C) Chi-miR-130b-3p interferer efficiency verification. (D) Dual-luciferase reporter results after co-transfection of chi-miR-130b-3p mimics with wild-type and mutant SESN2 overexpression plasmids. (E) The protein level expression of SESN2 in negative control group and chi-miR-130b-3p mimic treatment group. (F) The expression of SESN2 at the protein level in the negative control group and the chi-miR-130b-3p interferor treatment group.

Figure 4 is the verification of interference and overexpression efficiency of SESN2;

Among them: (A) The influence of three interference sequences SESN2 at different positions on mRNA level expression. (B)

The effect of SESN2 interference sequence on the expression level of SESN2 at the protein level. (C) The effect of SESN2 overexpression plasmid on the expression of SESN2 at the mRNA level. (D) The effect of SESN2 overexpression plasmid on the expression of SESN2 at the protein level.

Figure 5 shows the effect of SESN2 and chi-miR-130b-3p on the apoptosis of GCs induced by ZEA;

Among them: (A-D) The effect of combined overexpression/interference treatment of ZEA and SESN2/chi-miR-130b-3p on GCs cell viability and cell apoptosis flow cytometry results. (E-H) Effects of combined overexpression/interference treatment of ZEA and SESN2/chi-miR-130b-3p on the expression of apoptosis-related genes such as BAX, BCL-2, and PCNA at the protein level.

Figure 6 shows the effect of SESN2 and chi-miR-130b-3p on the oxidative stress of GCs induced by ZEA;

Among them: (A-D) Effects of combined overexpression/interference treatment of ZEA and SESN2/chi-miR-130b-3p on the level of ROS accumulation in GCs. (E-H) Effects of combined overexpression/interference treatment of ZEA and SESN2/chi-miR-130b-3p on the expression of apoptosis-related genes such as CAT and SOD2 at the protein level. (I-L) Effects of combined overexpression/interference treatment of ZEA and SESN2/chi-miR-130b-3p on oxidative stress indicators such as MDA, CAT, and SOD.

Figure 7 shows the influence of SESN2 and chi-miR-130b-3p on the KEAP1/NRF2 signaling pathway;

Among them: (A-D) Effects of combined overexpression/interference treatment of ZEA and SESN2/chi-miR-130b-3p on the expression of KEAP1/NRF2 signaling pathway-related proteins, including KEAP1, NRF2 and HO-1.

Figure 8 is a schematic diagram of the effect of SESN2 and chi-miR-130b-3p on the KEAP1/NRF2 signaling pathway.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

1. Materials and methods

1.1 Test material

All operations in this study were strictly implemented in the "Regulations on the Management of Experimental Animals" (SYXK2011-0036) of Nanjing Agricultural University. Goat ovary GCs used in this study were collected from the Haimen Goat Research Institute in Haimen City, Jiangsu Province. GCs samples were collected and stored in liquid nitrogen (-196°C) for subsequent experiments.

1.2 Test method

1.2.1 Culture of GCs

GCs were cultured in 6-well culture plates supplemented with DMEM/F12 glutaMAX (Gibco, ThermoFisher Scientific, Waltham, MA, USA), 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin ( P/S) complete medium. All cells were cultured at 37°C in a 5% _CO2 environment, and the complete medium was replaced every 24 hours.

1.2.2 Overexpression vector construction and gene interference sequence design

The coding domain sequence (CDS) of SESN2 (XM_018056884.1) was cloned into pEX overexpression vector (Tsingke, Nanjing, China). Negative control (si-NC) for all small interfering RNA (siRNA), siRNAs for SESN2 (si-SESN2(740), si-SESN2(1322), si-SESN2(1661)), negative for mock Control (mimics-NC), chi-miR-130b-3p mimic (mimics-chi-miR-130db-3p), inhibitor negative control (inhibitor-NC) and chi-miR-130b-3p inhibitor ( Inhibitor-chi-miR-130b-3p) were designed and synthesized by GenePharma (Shanghai, China). The related sequences of siRNA and chi-miR-130db-3p are shown in Table 1 below, and the SESN2 overexpression sequences are shown in Table 2 below.

Table 1 siRNA and chi-miR-130b-3p related sequences Table 1Sequences of siRNA and chi-miR-130b-3p mimics/inhibitor used in this study

Table 2 SESN2 overexpression sequence Table 2 Overexpression sequence of SESN2 used in this study

1.2.3 GCs treatment and transfection

GCs were seeded into 6-well plates for transfection. When the GCs density reached 60-70%, according to the protocol of the transfection reagent instructions, the SESN2 interference sequence/overexpression plasmid (built and synthesized by Nanjing Qingke Company, NJ0049585- 1) or mimics/inhibitors of chi-miR-130b-3p were added to 6-well plates. ZEA powder (Selleck, Shanghai, China) was dissolved in 200 mM dimethyl sulfoxide (DMSO) (Invitrogen, Shanghai, China) as a concentrated stock solution, and was diluted to the treatment concentration (200 μM) with DMEM/F12 for subsequent experiments. ZEA was added to the complete medium 12 hours before harvesting RNA and protein cell samples. Cells were collected 24 hours after transfection for RNA extraction and 48 hours after transfection for protein extraction.

1.2.4 CCK-8 Determination of Cell Viability

GCs were inoculated in 96-well culture plates, and the medium contained ZEA at specified concentrations (0, 50, 100 and 200 μmol/L) and diluted in dimethyl sulfoxide (di-methyl sulfoxide, DMSO). After treatment with ZEA for 8 hours, 10 μL of cell counting kit-8 (CCK-8) reagent was added to each well and incubated at 37°C, 5% CO ₂ for about 4 hours. Absorbance at 450 nm was detected by a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

1.2.5 Analysis of GCs apoptosis by flow cytometry

Apoptosis assay samples: Collect all cells in the 6-well plate, including those suspended in complete medium, and store them in DPBS. Samples were sent to Nanjing Liangwei Biotechnology Co., Ltd. for flow analysis within 2 hours.

1.2.6 Dual luciferase reporter carrier assay

293T cells were seeded in 24-well plates with Lipofectamine 3000 and 100 nM of mimics-NC or mimics-chi-miR-130b-3p and 0.5ng SESN2 3'UTR wild-type (WT) or mutant (MUT) overexpression Plasmid transfection, the detailed sequence is shown in Table 3 below. Cell samples were collected 48 hours after transfection, and the fluorescence intensity of the samples was measured using a dual-luciferase reporter gene assay system (Vazyme, Nanjing, China) according to the kit instructions.

Table 3SESN2-3'UTR vector sequence Table 3The sequences of SESN2-3'UTR vector

1.2.7 ROS level detection

After transfection and ZEA treatment, the ROS level of GCs was assessed using a ROS assay kit (S0033S, Beyotime, Nanjing, China) according to the instructions. The fluorescence intensity of ROS was analyzed using Image J software (Wayne Rasband, MD, USA).

1.2.8 ELISA detection of oxidative stress-related indicators

After 48 h of transfection and 12 h of ZEA treatment, cell supernatants were collected, and cellular oxidative stress-related indices including MDA, SOD, and CAT were measured using an ELISA assay (Amresco, Shanghai, China) according to the protocol of the instruction manual .

1.2.9 Extraction and reverse transcription of total RNA

(1) Extraction of total RNA

1) Collect GCs in a 6-well plate, place in a 2mL centrifuge tube, add 1mL Trizol reagent to homogenate, and place at room temperature for 5 minutes;

2) Add 0.2mL chloroform to 1), mix the centrifuge tube up and down for 10s, place it at room temperature for 6min, and centrifuge at 12000g for 15min;

3) Add an equal volume of isopropanol to the supernatant, mix up and down, let stand at room temperature for 10 minutes; centrifuge at 12000g for 10 minutes;

4) Aspirate the supernatant liquid, leaving a white precipitate, add 1mL of 75% ethanol, gently upside down to wash the centrifuge tube wall. Centrifuge at 12000g for 5min at 4°C;

5) Repeat step 4);

6) Discard the supernatant, let it stand for 10 minutes, and dry the residual alcohol in the EP tube. Add 10-50 μL (estimated according to the amount of RNA precipitation) RNase-free water to dissolve the RNA precipitation.

(2) RNA quality inspection

The concentration and purity of RNA are detected by UV spectrophotometer, and the 260/280 ratio must be between 1.8-2.0, and follow-up experiments can be carried out.

(3) Synthetic cDNA

RNA was reverse transcribed into cDNA using Novizym’s reverse transcription kit. See Table 4 and Table 5 for the reaction system. Where 1* indicates that the volume of RNA added is determined according to the concentration of RNA in the reaction system to ensure that the amount of total RNA remains consistent (1 μg).

Table 4 Removal of Genomic DNA Reaction System

Table 4 The reaction of removing genomic DNA

Program: 42°C, 2min; 4°C storage.

Table 5 Reverse transcription reaction system

Table 5 The reverse transcription reaction

Program: 37°C for 15 minutes, 85°C for 5s, constant temperature at 4°C.

1.2.10 Real-time fluorescence quantification

Novizym’s fluorescence quantification kit was used and operated according to the instruction manual. The reaction program was: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, a total of 40 cycles; final extension at 72°C for 10 min. After the reaction, GAPDH was used as an internal reference gene, and the expression of the target gene was analyzed by the 2 ^-ΔΔCt method. The detailed sequence information of the primers for fluorescent quantitative PCR is shown in Table 6 below.

Table 6 Primer sequence information for fluorescent quantitative PCR

Table 6 Sequences used for qRT-PCR

1.2.11 Collection and denaturation of protein samples

(1) protein lysate: protease inhibitor: phosphatase inhibitor (100:2:1, v:v:v) mixes and cracks the sample;

(2) After standing on ice for 10 minutes, centrifuge in a high-speed centrifuge at 12,000g for 10 minutes at 4°C, and collect the supernatant as the cell protein stock solution;

(3) Determine the protein concentration according to the instructions of the BCA kit;

(4) According to the extracted protein concentration, add an appropriate amount of protein stock solution, ultrapure water, 4× protein loading buffer and 10× reducing agent to make the protein concentration in the denaturation system consistent;

(5) Denature at 70°C for 10 minutes and store at -20°C.

1.2.12 Western blot

(1) Sample loading: unplug the comb from the precast gel, add 10 μL of denatured protein sample to each gel hole, and reserve a hole on each side to add protein markers;

(2) Electrophoresis: voltage 200V, 40min;

(3) Film transfer: semi-dry transfer at 25V constant voltage for 13min;

(4) Sealing: the PVDF membrane after membrane transfer was sealed with 5% (v/v) skimmed milk powder for 2h;

(5) Primary antibody incubation: Incubate the blocked PVDF membrane with primary antibody at 4°C overnight;

(6) Secondary antibody incubation: wash the PVDF membrane 3 times after the primary antibody incubation; then incubate the secondary antibody at room temperature for 1 hour; then wash 6 times;

(7) ECL development: Soak the PVDF membrane in the ECL luminescence solution for 30s, expose and take pictures in the gel imager;

(8) Densitometric analysis: Image J software was used to perform grayscale analysis of each band.

The antibody information of the involved primary antibody and secondary antibody is shown in Table 7.

Table 7 Antibody Information Sheet

1.3 Statistical methods

Data were analyzed using SPSS 24.0 software (SPSS Inc., Chicago, IL, USA). The t test was used for the data between two groups, and the significance of the difference between samples was compared by one-way ANOVA for the data of three or more groups. Each experiment was repeated at least three times. Data are expressed as "mean ± standard error (SEM)". p

<0.05(*) and p<0.01(**) indicate significant difference and extremely significant difference, respectively.

2. Results and Analysis

2.1 miRNA screening targeting SENS2

Using the sequencing results, this study selected three miRNAs that were most likely to interact with SENS2 for experimental verification. The three miRNAs were chi-miR-17-5p, chi-miR-20b and chi-miR-130b-3p (see Table 8 for the sequences). In this study, the mRNA and protein levels were detected, and the quantitative results showed that only chi-miR-130b-3p could significantly reduce the expression of SESN2 at the mRNA level (p<0.01) (Figure 1A). -miR-130b-3p can significantly reduce the expression of SESN2 at the protein level (p<0.01) (Figure 1B), so chi-miR-130b-3p was selected as the research object for subsequent dual fluorescent reporter carrier experiments and miRNA functional verification.

Table 8 Three miRNAs sequences

2.2 ZEA changed the expression of SESN2 and chi-miR-130b-3p in GCs

To explore the exact mechanism of ZEA's cytotoxic effect on GCs and to find potential therapeutic targets to alleviate ZEA toxicity, whole transcriptome sequencing of GCs before and after ZEA treatment was performed in this study. To verify the accuracy of the sequencing results, five genes were randomly selected to detect their expression trends. The qRT-PCR results showed that the expression trends of the five selected genes were completely consistent with the sequencing results, proving that the sequencing results were reliable (Fig. 2A, B). According to the sequencing results, this study found that 459 mRNAs were upregulated and 3,620 mRNAs were downregulated after ZEA treatment. The expression level of SESN2 in the ZEA group was extremely significantly upregulated (p<0.01) (Fig. 2C-E), indicating that ZEA may interfere with the cellular homeostasis of GCs. Analysis of miRNA sequencing results also indicated that several miRNAs may be involved in the regulation of SESN2 expression (Fig. 2F). According to the predicted results of miRNA-mRNA interaction, this study found that chi-miR-130b-3p was most likely to bind SESN2. Therefore, this study detected the expression level of chi-miR-130b-3p, and found that in the ZEA group, the expression level of chi-miR-30b-3p was extremely significantly decreased (p<0.01) (Fig. 2G, H). In the ZEA group, the trend of decreased chi-miR-130b-3p expression and increased SESN2 expression was preliminarily consistent with the prediction that there might be an interaction between chi-miR-30b-3p and SESN2.

2.3chi-miR-130b-3p targets the 3'UTR region of SESN2 mRNA

To confirm that chi-miR-130b-3p can indeed regulate the expression of SENS2, a dual-luciferase reporter assay was performed in this study. Sequencing results showed that chi-miR-130b-3p could bind to the 3'UTR region of SESN2 mRNA. Based on this result, this study designed dual luciferase reporter vectors targeting chi-miRNA-130b-3p and SESN2 binding sequences, including wild-type (WT) and mutant (MUT) (Fig. 3A). First, this study confirmed the validity of the mock sequence and interference sequence designed for chi-miR-130b-3p through qRT-PCR results (Fig. 3B, C). Thereafter, this study co-transfected 293T cells with the mimetic sequence of chi-miR-130b-3p and the WT/MUT plasmid of SESN2. The results of the dual-luciferase reporter assay showed that the mimetic sequence of chi-miR-130b-3p could significantly reduce the fluorescence intensity of WT-SESN2 (p<0.05), while that of MUT-SESN2 was not changed (Fig. 3D). Furthermore, mimetic sequence of chi-miR-130b-3p could significantly reduce SESN2 protein expression in GCs (p<0.05) (Fig. 3E), while chi-miR-30b-3p interference sequence could significantly increase SESN2 protein expression in GCs (p<0.05). Taken together, these results suggest that chi-miR-130b-3p can bind to the 3'UTR of SESN2 mRNA and downregulate the expression of SESN2.

2.4 SESN2 interference sequence and overexpression plasmid efficiency verification

In order to ensure the accuracy of the subsequent functional verification research on SESN2, this study verified the interference and overexpression efficiency of SESN2. For the CDS sequence of SESN2, three interference sequences were designed in this study, and the sites were located at 740bp, 1332bp, and 1661bp of the SESN2 CDS sequence. The results of fluorescence quantification showed that both the interference sequences at 1332bp and 1661bp could significantly reduce the expression of SESN2 at the mRNA level (p<0.01) (Figure 4A), but the interference sequence at 1332bp had a better effect. On this basis, the protein level was verified in this study, and the WB results showed that the interference sequence located at 1332bp can also significantly reduce the expression of SENS2 at the protein level (p<0.01) (Figure 4B), so follow-up experiments in this study will be The interference sequence located at 1332bp of the SESN2 CDS sequence was defined as the si-SESN2 treatment group. The same study also verified the overexpression efficiency of SESN2, and the quantitative results showed that the overexpression plasmid could significantly increase the mRNA level (p<0.01) (Figure 4C); the protein level increased by about 2 times (p<0.01) ( Figure 4D). In summary, the interference sequence and overexpression plasmid constructed for SESN2 have significant effects and can be used for subsequent experimental verification.

2.5 Effect of SESN2/chi-miR-130b-3p on GCs apoptosis induced by ZEA

Based on a large number of existing research reports that SESN2 has a positive regulatory effect on cell resistance to external stimuli (Li et al., 2021, Park et al., 2022, Wu et al., 2021), this study attempts to investigate whether SESN2 Cytotoxic damage induced by ZEA in GCs could also be alleviated. In addition, given that chi-miR-130b-3p acts as an upstream regulator of SESN2, this study speculated that the expression of chi-miR-130b-3p may also be involved in the ZEA-induced damage process of GCs. CCK-8 results showed that ZEA significantly decreased the cell viability of GC (p<0.01) (Fig. 5A-D). Both overexpression of SESN2 and interference of chi-miR-130b-3p had a tendency to restore the viability of damaged cells (Fig. 5A,B). Notably, overexpression of SESN2 had a very significant effect (p<0.01) on this process (Fig. 5A). In contrast, inhibition of SESN2 or overexpression of chi-miR-130b-3p exacerbated ZEA's impairment of cell viability in GCs, although this effect was not significant (Fig. 5C,D). In order to further explore the effect of SESN2 and chi-miR-130b-3p on the apoptosis of GCs induced by ZEA, flow cytometry analysis was performed to detect the level of apoptosis in this study. The results showed that overexpression (p<0.05) of SESN2 (Fig. 5E) or interference (p<0.05) of chi-miR-130b-3p could attenuate ZEA-induced apoptosis of GCs. However, inhibition of SESN2 (p<0.01) (Fig. 5G) and overexpression of chi-miR-130b-3p (p<0.01) (Fig. 5H) aggravated the apoptosis of GCs. To validate these phenotypic data, this study examined the expression levels of several genes related to cell viability and apoptosis, including BAX, BCL-2, and PCNA. Consistent with the results of CCK-8 and flow cytometry, on the basis of ZEA treatment, the expression of BAX followed the overexpression of SESN2 (p<0.05) (Fig. 5I) or the interference of chi-miR-130b-3p (p< 0.05) down-regulation (Fig. 5J), while the interference of SESN2 or the overexpression of chi-miR-130b-3p will further increase the expression of BAX. The trend of BCL-2 and PCNA is just opposite to that of BAX. Taken together, these results suggest that SESN2 is able to restore ZEA-induced damage to GC to a certain extent, whereas chi-miR-130b-3p exerts the opposite effect.

2.6 Effect of SESN2/chi-miR-130b-3p on ZEA-induced oxidative stress injury in GCs

The antioxidant capacity of SESN2 has been known since its initial discovery (Wang et al., 2021). At the same time, studies have shown that ZEA can cause oxidative stress damage by causing the accumulation of ROS in GCs (Zheng et al., 2019). Based on the results of these two studies, this study wanted to explore whether SESN2 and chi-miR-130b-3p would affect the accumulation of ROS and the process of oxidative stress injury in ZEA-induced GCs. First, this study measured the ROS levels in GCs of different treatment groups. This study observed that ZEA exposure resulted in a significant increase in ROS levels in GCs (p<0.01) (Fig. 6A-D), while overexpression of SESN2 (p<0.05) (Fig. p<0.01) (Fig. 6B) alleviated the accumulation of ROS in GCs, while SESN2 interference (p<0.01) (Fig. 6C) or overexpression of chi-miR-130b-3p (p<0.01) (Fig. 6D) Exacerbated the accumulation of ROS in GCs. Then, this study detected the expression levels of SOD2 and CAT by WB, and the results showed that ZEA inhibited the expression of CAT and SOD2 (Fig. 6E-H). However, this damage could be reversed by SESN2 overexpression (p<0.05) (Fig. 6E) or chi-miR-130b-3p interference (p<0.05) (Fig. 6F), while SESN2 (p<0.01) Inhibition (Fig. 6G) and overexpression (p<0.01) of chi-miR-30b-3p aggravated ZEA's inhibition of the expression of the above antioxidant enzymes (Fig. 6H). In addition, this study evaluated the oxidative stress level and antioxidant enzyme activity of ZEA-treated GCs using ELISA kits for MDA, SOD, and CAT, respectively. Consistent with the expected results of this study, the activities of SOD and CAT were consistent with the results of WB (Figure 6I-L), and the increased concentration of MDA by ZEA could be through the overexpression of SESN2 (p<0.05) (Figure 6I) or chi-miR- 130b-3p interference (p<0.01) to alleviate (Fig. 6J), but SESN2 inhibition (p<0.01) (Fig. 6K) and chi-miR-130b-3p overexpression (p<0.01) enhanced GCs The accumulation level of MDA (Fig. 6L). The above evidence indicated that SESN2 and chi-miR-130b-3p were involved in the process of ROS accumulation and oxidative stress injury induced by ZEA in GCs.

2.7 Effect of SESN2/chi-miR-130b-3p on KEP1/NRF2 signaling pathway

SESN2 is well known for its antioxidant capacity, and previous studies have confirmed that SESN2 is involved in regulating the KEAP1/NRF2 signaling pathway to exert its antioxidant function (Fan, et al., 2020). Given the findings of this study on the effects of SESN2 and chi-miR-130b-3p on ROS accumulation and oxidative stress damage in GCs, this study sought to explore whether SESN2 and chi-miR-130b-3p regulate the KEAP1/NRF2 signaling pathway To influence ZEA-induced ROS accumulation and oxidative stress damage in GCs. To this end, this study used WB analysis to evaluate the expression levels of KEAP1, NRF2 and heme oxygenase 1 (haeme oxygenase 1, HO-1) protein. On the basis of ZEA treatment, the results are shown in Figure 7, the expression of KEAP1 decreased with the overexpression of SESN2 (Figure 7A) or the interference of chi-miR-130b-3p (p<0.05) (Figure 7B), but The expression of NRF2 and HO-1 increased with the inhibition of SESN2 (p<0.05) (Fig. 7C) or the overexpression of chi-miR-130b-3p (Fig. 7D), while the expression of NRF2 and HO-1 showed an opposite trend to that of KEAP1 (Fig. -D). The above results can confirm that SESN2 and chi-miR-130b-3p can affect the ROS accumulation and oxidative stress damage caused by ZEA in GCs through the KEAP1/NRF2 signaling pathway, and the specific regulatory effects are shown in Figure 8.

Claims (10)

1. The use of SESN2 or/and chi-miR-130b-3p in (1) or (2) or (3) below,

   (1) Modulating GCs apoptosis;

   (2) Regulating ROS accumulation and oxidative stress damage in GCs;

   (3) And the cell damage of ZEA to GCs is reduced.
2. 2. The use according to claim 1, wherein overexpression of SESN2 or/and interference with chi-miR-130b-3p reduces GCs apoptosis; inhibiting SESN2 or/and over-expressing chi-miR-130b-3p aggravates GCs apoptosis.
3. 3. The use according to claim 1 or 2, wherein the GCs apoptosis is a ZEA-induced GCs apoptosis.
4. 4. The use according to claim 1, wherein overexpression of SESN2 or/and interference with chi-miR-130b-3p reduces ROS accumulation and oxidative stress damage in GCs; inhibiting accumulation of ROS and oxidative stress injury in GCs by SESN2 or/and over-expressing chi-miR-130b-3 p.
5. 5. The use according to claim 1 or 4, wherein the accumulated and oxidative stress injury of ROS in GCs is an accumulated and oxidative stress injury of ROS in GCs induced by ZEA.
6. 6. The use according to claim 1 or 4, wherein SESN2 or/and chi-miR-130b-3p affects ROS accumulation and oxidative stress damage in GCs induced by ZEA via KEAP1/NRF2 signaling pathway.
7. 7. The use according to claim 1, wherein overexpression of SESN2 or/and interference with chi-miR-130b-3p reduces cell damage caused by ZEA to GCs.
8. 8. A method of modulating the apoptosis of GCs induced by ZEA, wherein overexpression of said SESN2 or/and interference with said chi-miR-130b-3p attenuate the apoptosis of GCs induced by ZEA; inhibiting said SESN2 or/and over-expressing said chi-miR-130b-3p exacerbates ZEA-induced apoptosis of GCs.
9. 9. A method of modulating ROS accumulation and oxidative stress damage in a ZEA-induced GCs, wherein overexpressing said SESN2 or/and interfering with said chi-miR-130b-3p is capable of alleviating ROS accumulation and oxidative stress damage in a ZEA-induced GCs; inhibiting said SESN2 or/and over-expressing said chi-miR-130b-3p exacerbates ROS accumulation and oxidative stress damage in ZEA-induced GCs.
10. 10. A method for reducing cell damage caused by ZEA to GCs, characterized in that overexpression of SESN2 or/and interference of chi-miR-130b-3p reduces cell damage caused by ZEA to GCs.