Method for recovering adipogenic gene expression and adipogenic capacity of bovine immortalized adipocytes based on siRNA

Abstract

The invention discloses a method for recovering adipogenic gene expression and adipogenic capacity of bovine immortalized adipocytes based on siRNA (small interfering Ribonucleic Acid). In order to solve the problems that bovine immortalized adipocytes established by overexpression of SV40 T-antigen genes are inhibited in adipogenesis related gene expression and abnormal in adipogenesis ability, the invention inhibits the expression of SV40 virus T-antigen in bovine adipocytes by designing, screening and identifying specific efficient interfering siRNA (small interfering ribonucleic acid) aiming at the SV40 virus T-antigen. Therefore, the inhibition effect of SV40 on adipogenic gene expression is relieved, the expression level and the adipogenic capacity of the adipogenic gene in the bovine immortalized adipocytes are recovered, and the method plays a key role in breaking through the bottleneck problem of developing species-specific adipodifferentiation mechanism research by utilizing the bovine immortalized adipocytes and getting rid of the current situation of developing research by depending on a mode animal cell line for a long time.

Description

Technical Field

[0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to a method for restoring adipogenic gene expression and adipogenic ability of immortalized bovine adipocytes based on siRNA. Background Art

[0002] Fat deposition ability is a key factor affecting the economic value of beef cattle. During the fattening process of beef cattle, excessive subcutaneous fat deposition will reduce the feeding reward of beef cattle and cause waste of resources; while more intramuscular fat deposition will improve the quality of beef and enhance economic benefits. In the market, the price of butter is one-tenth of the price of normal beef, while high-grade beef can be sold for about thirty times the price of normal beef. Therefore, it is a difficult problem that needs to be solved urgently to cultivate beef cattle breeds with low subcutaneous and high intramuscular fat deposition ability to maximize the economic value of beef cattle.

[0003] Correctly and fully understanding the mechanism of fat deposition is a prerequisite for improving the fat deposition capacity and location of beef cattle at the molecular level. Currently, there is a lack of immortalized cell lines that can be used to study the mechanism of fat deposition in cattle, and research can only be carried out by relying on cell lines of model animals such as mice. Due to differences between species, there are serious limitations in the current understanding of the mechanism of fat deposition in cattle. Establishing an immortalized bovine fat cell line is currently the key to correctly understanding the mechanism of fat deposition in cattle.

[0004] There are many methods to achieve cell immortalization. Common methods include telomerase activation, viral gene transfer, radioactive treatment and spontaneous immortalization. Among them, the use of lentivirus carrying simian virus 40 (SV40) to carry out primary cell immortalization research is one of the most commonly used methods, and it has been successful in the establishment of cell lines of multiple species. However, the overexpression of the SV40 gene will inhibit cell differentiation while ensuring the immortal proliferation of cells. In the process of immortalization of bovine adipocytes, we found that the overexpression of the SV40 gene would inhibit the expression of bovine adipogenesis-related genes, which made it difficult to conduct in-depth research on the mechanism of bovine adipogenesis differentiation.

[0005] In order to restore the expression of adipogenic genes and adipogenic ability of immortalized bovine adipocytes, the present invention restores the expression of key adipogenic genes of immortalized adipocytes by inhibiting the gene expression of SV40 after obtaining an immortalized bovine adipocyte cell line induced by SV40 with unlimited proliferation. Specifically, multiple siRNAs targeting the expression of SV40 T-antigen are designed, and the siRNA with the highest interference efficiency is identified to carry out interference effect research. In addition, transcriptome sequencing analysis is used to successfully find siRNAs that can rescue the expression of key adipogenic genes of bovine adipocytes inhibited by SV40. The siRNA plays a key role in inducing adipogenic differentiation of bovine adipocytes after immortalizing bovine adipocytes using SV40, and can quickly advance the research on the mechanism of bovine fat deposition ability and promote the breeding of beef cattle varieties with high economic value. Summary of the invention

[0006] In view of the problem that the expression of adipogenic-related genes is inhibited and the adipogenic ability is abnormal in immortalized bovine adipocytes established by overexpressing the SV40 T-antigen gene, the present invention provides a method for restoring the adipogenic gene expression and adipogenic ability of immortalized bovine adipocytes based on siRNA. By designing and screening and identifying specific siRNA for SV40 virus T-antigen, its expression in bovine adipocytes is inhibited, thereby relieving the inhibitory effect of SV40 on the expression of adipogenic genes, and restoring the adipogenic gene expression and adipogenic ability of immortalized bovine adipocytes.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The method for preparing bovine immortalized adipocytes with normal expression of adipogenic genes and adipogenic ability comprises infecting bovine primary preadipocytes with a lentivirus containing SV40 T-antigen gene, screening, passage and identification with purine toxin (preferably at a concentration of 2 μg / mL) to obtain bovine immortalized adipocytes, and then restoring adipogenic gene expression by siRNA, wherein the sequence of the siRNA is 5'-CCAUGAAUGAGUACAGUGU-3'. The adipogenic genes include LSS, TM7SF2, FADS2, HSD17B12, CPT1C, FADS1, FABP7, PLTP, PPARGC1A, SLC2A4 and LOC528769.

[0009] The invention discloses an siRNA for restoring the adipogenic gene expression and adipogenic ability of immortalized bovine adipocytes, wherein the siRNA targets the 1580bp-1598th region of the SV40 virus T-antigen gene, and the sequence thereof is 5'-CCAUGAAUGAGUACAGUGU-3'.

[0010] Compared with the prior art, the present invention has the following advantages or technical effects:

[0011] At present, a bovine immortalized fat cell line has not been established. The present invention utilizes SV40 to achieve immortalization of bovine primary fat cells. However, since SV40 overexpression reduces the expression level of adipogenic genes and inhibits cell adipogenic differentiation, the immortalized cells cannot be used for subsequent research on the mechanism of adipogenic differentiation. It is necessary to restore their adipogenic ability on the basis of immortalization. At present, there are no relevant reports on this bottleneck problem. The present invention is the first to design and screen siRNA with a high-efficiency interference effect on SV40, knock down the expression level of SV40, thereby restoring the adipogenic gene expression level and cell adipogenic ability of the bovine immortalized fat cells. The present invention has unique innovations in terms of siRNA sequence characteristics and interference regions for SV40. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 .Effects of three siRNAs on the expression level of SV40 T-antigen mRNA.

[0013] Figure 2 .Detection of cell adipogenesis after siRNA interference. SV40 indicates bovine immortalized adipocytes established by overexpression of SV40 T-antigen gene, and si-SV40 indicates bovine immortalized adipocytes interfered with by siRNA3.

[0014] Figure 3 .The effect of siRNA3 on the transcriptome of bovine immortalized adipocytes. A is the principal component analysis diagram, and B is the differential analysis volcano diagram. siT.antigen represents the siRNA3-treated group, and T.antigen represents the untreated group; down represents genes with significant differences and downregulation, insignificant represents genes with insignificant differences, and up represents genes with significant differences and upregulation.

[0015] Figure 4 .The expression of key genes in the adipogenesis process after siRNA interference. Control represents primary bovine adipocytes, SV40 represents immortalized bovine adipocytes established by overexpressing the SV40 T-antigen gene, and si-SV40 represents immortalized bovine adipocytes interfered with by siRNA3. DETAILED DESCRIPTION

[0016] Example 1 Preparation of immortalized bovine adipocytes

[0017] 1. Lentivirus packaging and infection

[0018] pLVX-IRES-Puro-SV40LT (purchased from Changsha Abiwei Biotechnology Co., Ltd., catalog number HG-VXY0602) was used as the lentiviral core plasmid, and pMD2.G and psPAX2 were selected as the three-plasmid expression system for packaging auxiliary plasmids for lentiviral packaging.

[0019] 1.1 Lentiviral packaging

[0020] 293T cell inoculation: Select 293T cells with normal morphology, no contamination, and in the logarithmic growth phase and inoculate them into a six-well cell culture plate (the number of cells is about 5×10 5 The cells were placed in a 37°C, 5% CO2 incubator for future use.

[0021] Preparation of plasmids and transfection reagents: Three plasmid DNA solutions were mixed in a mass ratio of 1:3:4 for pMD2.G:psPAX2:core plasmid.

[0022] Plasmid transfection: Use In vitro DNA transfection kit (Polyplus-transfection company) was used to introduce the lentiviral expression system into 293T cells. The specific steps were carried out according to the instructions:

[0023] (1) Transfection was performed when the confluence of 293T cells reached 70%;

[0024] (2) 1 h before transfection, fresh high-glucose DMEM medium containing 10% serum was replaced to stimulate morphological changes of 293T cells;

[0025] (3) Preparation of transfection complex in each well: Add 200 μL jetPRIME Buffer to each of three 1.5 mL sterile centrifuge tubes, add 2 μg of pre-incubated lentiviral expression system mixed plasmid to each tube, gently pipette to mix, add 5 μL jetPRIMEreagent transfection reagent, and gently pipette to mix to form a transfection complex;

[0026] (4) Incubate at room temperature for 15 min;

[0027] (5) Add plasmid DNA-liposome complex to the cell culture medium and shake to make it uniform;

[0028] (6) Collection of lentivirus fluid: The cell supernatants were collected at 24 h, 48 h, and 72 h, respectively, and filtered through a 0.45 μm cell filter to obtain SV40-overexpressing lentivirus.

[0029] 1.2 Lentiviral infection

[0030] (1) The primary bovine preadipocytes were revived to a 60 mm cell culture dish and transfected with SV40 virus at a density of 65% to 75%;

[0031] (2) Replace with 1 mL of fresh high-glucose DMEM medium containing 10% serum, add 1 mL of SV40 overexpression lentivirus, and add lentivirus infection enhancement reagent Polybrene with a final concentration of 8 μg / mL; mix well and place in a 5% CO2, 37°C cell culture incubator for culture;

[0032] 2. Screening of optimal concentration of puromycin

[0033] Untransfected bovine primary adipocytes were plated into 96-well plates and cultured overnight, and the old cell culture medium was removed the next day. Cell culture medium containing different concentrations of puromycin (1μg / mL, 1.5μg / mL, 2μg / mL, 2.5μg / mL, 3μg / mL, 3.5μg / mL, 4μg / mL, 4.5μg / mL, 5μg / mL, 5.5μg / mL) was set up and added to 96-well cell culture plates. Fresh cell culture medium containing puromycin was replaced every 48h, and the cell survival rate was observed daily. The minimum puromycin concentration was the lowest screening concentration that killed all cells within 1 to 4 days from the start of screening. Through comparative analysis, it was found that 2μg / mL was the optimal concentration for screening positive bovine immortalized adipocytes.

[0034] 3. Screening of positive bovine immortalized adipocytes

[0035] The bovine adipocytes transfected with SV40 virus were plated into a 60 mm cell culture dish, and fresh culture medium containing 10% serum high glucose DMEM containing 2 μg / mL puromycin was added, and the fresh culture medium containing puromycin was replaced every 48 hours; the survival rate of the cells was observed daily, and the screening was continued for 4 days. The cells that finally survived and did not deform were subcultured. At the same time, the primary bovine adipocytes that were not transfected with SV40 were used as the control. The cells that were subcultured for more than 20 generations and maintained normal morphology and had normal expression of key genes (PREF-1, a precursor adipocyte marker gene) were preliminarily defined as immortalized bovine adipocytes.

[0036] 4. Detection of key adipogenic gene expression and adipogenic ability of immortalized bovine adipocytes

[0037] The immortalized bovine adipocytes were digested with 0.25% pancreatin, spread in a culture dish at an appropriate density, cultured with DMEM high-glucose medium containing 10% fetal bovine serum to a cell density of about 70%, and the cells were collected and sent to a sequencing company for transcriptome sequencing. Non-immortalized bovine primary adipocytes were used as controls to detect overall changes in genes at the transcriptome level. At the same time, when the remaining cell density reached about 90%, complete medium containing induction reagent A (1.0 μmol / L rosiglitazone, 1.0 μmol / L dexamethasone, 10 mg / L insulin, 0.5 mmol / L IBMX) was used for 2 days of culture, and then replaced with maintenance induction reagent B (1.0 μmol / L rosiglitazone, 10 mg / L insulin) for maintenance induction. On the fourth day, Bodipy staining was used to evaluate the formation of lipid droplets. The results showed that the adipogenic ability of immortalized bovine adipocytes was abnormal and almost incapable of adipogenesis.

[0038] Example 2 Design of siRNA for restoring adipogenic gene expression

[0039] 1. Design siRNA

[0040] (1) Determination of target gene sequence

[0041] First, the target gene sequence of SV40-T antigen was determined through the public gene database. This step is the basis of siRNA design and ensures the accuracy and effectiveness of subsequent design.

[0042] (2) Selection of siRNA targets

[0043] Within the coding region (CDS) of SV40 T-antigen, we selected three potential siRNA targets located at positions 749-767, 1055-1073, and 1580-1598 of the gene sequence. These regions were selected based on their avoidance of 5' and 3' untranslated regions (UTRs) and sequences near the start codon to reduce the risk of nonspecific silencing.

[0044] (3) Calculation of GC content

[0045] The GC content of the siRNA sequence was ensured to be in the range of 30% to 52% to maintain the stability and silencing efficiency of the siRNA.

[0046] (4) Avoid unfavorable sequence characteristics

[0047] During the design process, care was taken to avoid including consecutive single bases and inverted repeats to prevent the siRNA from forming a hairpin-like structure, which may affect its silencing efficiency.

[0048] (5) Specificity evaluation

[0049] Use sequence alignment tools such as BLAST to ensure that the designed siRNA sequence has low homology with non-target gene sequences to avoid nonspecific silencing.

[0050] (6) Design of siRNA sequences

[0051] According to the above principles, the sense strand (Table 1) and antisense strand sequences of siRNA were designed with a length of 19 nucleotides to ensure the best silencing effect.

[0052] (7) Synthesis of siRNA

[0053] We submitted the designed siRNA sequence to a synthesis company for siRNA synthesis.

[0054] Table 1. Information of three siRNAs

[0055]

[0056] 2. Screening of siRNA for restoring adipogenic gene expression

[0057] (1) Under sterile conditions, immortalized bovine adipocytes were implanted in two 6-well plates and divided into four groups: si-NC (purchased from Ruibo Biotechnology, catalog number siN0000001-1-5), si-RNA1 (si-1), si-RNA2 (si-2) and si-RNA3 (si-3), with three replicates in each group. Immortalized bovine adipocytes were cultured in 2 ml of complete medium (DMEM containing 10% FBS) to a density of 60-70% (24 h).

[0058] (2) Preparation of siRNA and Lipofectamine 3000 complex: Take 20 μM siRNA storage solution placed at -20°C and centrifuge it to allow the siRNA to sink to the bottom. Take 5 μL of si-NC, si-RNA1, si-RNA2 and si-RNA3 storage solutions and Lipofectamine 3000, add them to 125 μL of Opti-MEM serum-free medium, and mix them gently. The diluted siRNA working solution (working concentration is 50 nM) and Lipofectamine 3000 were incubated for 3 minutes, then mixed in a 1:1 ratio and mixed to remove bubbles. After incubation at room temperature for 15 minutes, the complex of siRNA and Lipofectamine3000 was formed. During the incubation period, the bovine immortalized adipocytes were replaced with 1740 μL of complete medium.

[0059] (3) Add the above complexes to the immortalized bovine adipocytes according to the groups, add 260 μL of the complexes to each well, and ensure that the cells in each well are covered with the complexes. Gently shake the culture dish to evenly distribute the complexes.

[0060] (4) Place the culture dish back into the cell culture incubator and continue to culture the cells at 37°C and 5% CO2 to ensure siRNA transfection and gene silencing. Replace with complete culture medium (DMEM containing 10% FBS) after 6-12 hours to promote cell recovery and siRNA production.

[0061] (5) 36 hours after transfection, cells or cell lysates were collected and subjected to RT-qPCR experiments with primers as shown in Table 2 to evaluate siRNA transfection efficiency and gene silencing effect.

[0062] Table 2. Quantification primer sequences

[0063]

[0064]

[0065] Fluorescence quantitative PCR: The target gene (SV40T) was quantitatively detected according to the instructions of ChamQ Universal SYBR qPCR Master Mix, a product of Novozymes.

[0066] Table 3. Fluorescence quantitative PCR reaction system

[0067]

[0068] Table 4. Fluorescence quantitative PCR reaction program

[0069]

[0070] (6) Perform statistical analysis on the experimental data to determine the effect of siRNA transfection and compare it with the si-NC group to verify the specificity and efficiency of siRNA.

[0071] We systematically evaluated the interference effects of three small interfering RNAs (siRNAs) on the expression level of SV40 T-antigen mRNA. By fluorescence quantitative PCR, we found that the mRNA expression levels of the three siRNA treatment groups, si-1, si-2 and si-3, were significantly reduced to 0.6180, 0.7199 and 0.4510 ( Figure 1), with decreases of 38.2%, 28.0% and 54.9%, respectively. This phenomenon was further verified by statistical methods, in which the differences between the si-1, si-2 and si-3 groups and the si-NC group were analyzed by t-test, with P values ​​of 0.0004, 0.0066 and <0.0001, respectively, all below the significance level of 0.05, indicating that these differences were statistically significant (Table 5). In particular, the si-3 group showed the strongest interference effect, with a P value of less than 0.0001, which was significantly lower than the other two groups of siRNA, indicating that si-RNA3 has a higher potential in interfering with SV40 T-antigen expression (Table 5).

[0072] Table 5. Interference efficiency of siRNA on SV40 T-antigen mRNA expression level

[0073]

[0074] 3. Effect of siRNA3 on the adipogenic capacity of immortalized bovine adipocytes

[0075] When the density of immortalized bovine adipocytes interfered with by siRNA3 reached about 90%, they were cultured for 2 days in complete medium containing induction reagent A (1.0 μmol / L rosiglitazone, 1.0 μmol / L dexamethasone, 10 mg / L insulin, 0.5 mmol / L IBMX), and then replaced with maintenance induction reagent B (1.0 μmol / L rosiglitazone, 10 mg / L insulin) for maintenance induction. On the fourth day, Bodipy staining was performed to detect the induced adipogenesis. The results showed that fat droplets appeared in immortalized bovine adipocytes after SV40 interference on the fourth day, and part of the adipogenic ability was restored ( Figure 2 ).

[0076] 4. Effects of siRNA3 on the transcriptome of bovine immortalized adipocytes

[0077] (1) Transcriptome clustering analysis

[0078] TRIzol reagent was used to extract total RNA from cells in the siRNA3-treated and control groups, and RNA quality and integrity were tested. The extracted RNA samples were then sent to a sequencing service provider for transcriptome sequencing and principal component analysis (PCA) of the transcriptome. The principal component analysis diagram further revealed the differential distribution of samples in the control group and the siRNA3-treated group (labeled as the siT-antigen group) in the principal component analysis ( Figure 3 A), The analysis results showed that the two groups of samples showed a significant separation trend on the principal component axis, indicating that siRNA3 treatment had a significant impact on the transcriptome structure of bovine immortalized adipocytes.

[0079] (2) Volcano plot shows the variation of gene expression

[0080] Under the criteria of P < 0.05 and |log2FoldChange|>1, a total of 554 differentially expressed genes were identified, of which 395 genes were down-regulated and 159 genes were up-regulated in the siRNA3 treatment group ( Figure 3 B).

[0081] (3) Functions of key genes in adipogenesis and their expression after siRNA interference

[0082] Analysis of KEGG pathways significantly associated with fat deposition revealed that key genes in the process of adipogenesis, especially key genes in early adipogenesis, such as LSS, TM7SF2, FADS2, HSD17B12, CPT1C, FADS1, FABP7, PLTP, PPARGC1A, SLC2A4, and LOC528769, were significantly overexpressed after siRNA interference (Table 6, Figure 4 ). By using siRNA3 specific to SV40 virus T antigen, its expression in bovine adipocytes is inhibited, thereby relieving the inhibitory effect of SV40 on the expression of adipogenic genes in bovine immortalized adipocytes and restoring the expression level of adipogenic genes in bovine immortalized adipocytes.

[0083] Table 6. Significantly enriched fat deposition-related KEGG pathways and their significantly differentially expressed genes

[0084]

Claims

1. An siRNA for restoring adipogenic gene expression and adipogenic ability of immortalized bovine adipocytes, characterized in that: The sequence of the siRNA is 5'-CCAUGAAUGAGUACAGUGU-3', and the specific targeting region of the sequence is the 1580-1598 bp position of the SV40 T-antigen gene sequence.

2. A method for preparing immortalized bovine adipocytes with normal adipogenic gene expression and adipogenic ability, characterized in that: The bovine primary preadipocytes were infected with a lentivirus containing the SV40 T-antigen gene, and the bovine immortalized adipocytes were obtained through purine toxin screening, passage and identification, and then the adipogenic gene expression was restored by siRNA, wherein the sequence of the siRNA was 5'-CCAUGAAUGAGUACAGUGU-3'.

3. The method according to claim 2, characterized in that The concentration of the purine toxin is 2 μg / mL.

4. The method according to claim 2, characterized in that: The adipogenic genes include LSS, TM7SF2, FADS2, HSD17B12, CPT1C, FADS1, FABP7, PLTP, PPARGC1A, SLC2A4, LOC528769 .

Images