Application of miR-27b-3p in preparation of products for regulating cell proliferation and testosterone synthesis

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to application of miR-27b-3p in preparation of products for regulating cell proliferation and testosterone synthesis. The present application constructs Inhibitor and Mimics sequences with miR-27b-3p gene as a target, and for the first time proposes that overexpression of miR-27b-3p gene can significantly inhibit proliferation and testosterone synthesis of goat testicular interstitial cells, and inhibition of miR-27b-3p gene can significantly promote proliferation and testosterone synthesis of goat testicular interstitial cells. The present application has important significance for improving development of goat testis, and provides technical support for research and development of products for regulating proliferation and testosterone synthesis of goat testicular interstitial cells with miR-27b-3p gene as a target.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of miR-27b-3p in the preparation of products that regulate cell proliferation and testosterone synthesis. Background Technology

[0002] Interstitial cells of the testis are a crucial type of endocrine cell in the male reproductive system. Their primary function is to synthesize and secrete androgens, most notably testosterone. Testosterone plays a vital role in promoting spermatogenesis, maintaining secondary sexual characteristics, promoting reproductive organ development, and anabolism. In male animal reproduction, the normal function of interstitial cells is essential for male fertility and sexual behavior. Impaired function of interstitial cells can lead to decreased testosterone levels, impaired spermatogenesis, and reduced reproductive capacity. Currently, a systematic understanding of the role of interstitial cells in the regulation of testosterone synthesis and cell proliferation is lacking, and the causal relationship between their miRNAs and reproductive dysfunction requires further elucidation.

[0003] Studies have shown that miR-27b-3p plays a crucial role in regulating cell proliferation and hormone synthesis. Regarding cell proliferation regulation: in breast cancer, miR-27b-3p inhibits ccND1 and E2F2, thus blocking the G1 / S phase transition; in liver cancer, miR-27b-3p targets KRAS, inhibiting the MAPK pathway and thereby reducing cell proliferation. Regarding hormone synthesis regulation: in ovarian granulosa cells, miR-27b-3p inhibits CYP19B1, reducing estrogen synthesis; in adrenal cortex cells, it targets StAR, affecting progesterone and testosterone production. These studies indicate that miR-27b-3p influences organismal development by inhibiting cell proliferation and regulating cellular function. Despite these important biological functions as a target, the role of miR-27b-3p in goat testicular interstitial cells remains unclear.

[0004] Based on this, the present invention provides the application of miR-27b-3p gene as a target in regulating the proliferation of goat testicular interstitial cells and testosterone synthesis, aiming to overcome the shortcomings of existing technologies and provide technical support for the research and development of products related to regulating the proliferation of goat testicular interstitial cells and testosterone synthesis by targeting miR-27b-3p gene. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the miR-27b-3p gene as a target in the preparation of products that regulate the proliferation of goat testicular interstitial cells and testosterone synthesis.

[0006] Another objective of this invention is to provide the application of the miR-27b-3p gene as a target in the preparation of products that regulate the expression of marker genes ccNB1 and ccNE2 related to the proliferation of goat testicular interstitial cells and products that regulate the synthesis of testosterone in goat testicular interstitial cells.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The application of the miR-27b-3p gene as a target in the preparation of products that regulate the proliferation of goat testicular interstitial cells and testosterone synthesis.

[0008] Preferably, in the application of the miR-27b-3p gene as a target in the preparation of products that regulate the proliferation of goat testicular interstitial cells and testosterone synthesis, the products that regulate the proliferation of goat testicular interstitial cells and testosterone synthesis target the miR-27b-3p gene. By inhibiting the expression of the miR-27b-3p gene through miR-27b-3p inhibitors, the proliferation of goat testicular interstitial cells and testosterone synthesis are promoted.

[0009] In a further preferred embodiment, in the application of the miR-27b-3p gene as a target in the preparation of products that regulate the proliferation of goat testicular interstitial cells and testosterone synthesis, the miR-27b-3p inhibitor is an inhibitor miR-27b-3p, the sequence of which is shown in SEQ ID NO. 1.

[0010] Preferably, in the application of the miR-27b-3p gene as a target in the preparation of products regulating the proliferation of goat testicular interstitial cells and testosterone synthesis, the products regulating the proliferation of goat testicular interstitial cells and testosterone synthesis target the miR-27b-3p gene, promote the expression of the miR-27b-3p gene by overexpressing miR-27b-3p mimics, and inhibit the proliferation of goat testicular interstitial cells and testosterone synthesis.

[0011] In a further preferred embodiment, in the application of the miR-27b-3p gene as a target in the preparation of products that regulate the proliferation of goat testicular interstitial cells and testosterone synthesis, the miR-27b-3p overexpression mimic is Mimics-miR-27b-3p, the sequence of which is shown in SEQ ID NO. 2.

[0012] The application of the miR-27b-3p gene as a target in the preparation of products that regulate the expression of marker genes ccNB1 and ccNE2 related to the proliferation of goat testicular interstitial cells.

[0013] The application of the miR-27b-3p gene as a target in the preparation of products containing 3β-HSD and StAR expression marker genes that regulate testosterone synthesis in goat testicular interstitial cells.

[0014] The beneficial effects of this invention are: This invention constructs inhibitor and mimic sequences targeting miR-27b-3p and proposes for the first time that inhibiting miR-27b-3p gene expression can significantly promote the proliferation of goat testicular interstitial cells and testosterone synthesis; while overexpression of the miR-27b-3p gene can significantly inhibit the proliferation of goat testicular interstitial cells and testosterone synthesis. This invention is of great significance for improving goat testicular development and provides technical support for the research and development of products related to the regulation of goat testicular interstitial cell proliferation using miR-27b-3p as a target. Attached Figure Description

[0015] Figure 1 The effect of miR-27b-3p as a target on the expression of proliferation-related genes ccNB1 and ccNE1, and testosterone synthesis-related genes 3β-HSD and StAR in goat testicular interstitial cells (Figure: A shows the expression results of proliferation-related genes ccNB1, ccNE2 and testosterone synthesis-related genes 3β-HSD and StAR in goat testicular interstitial cells after miR-27b-3p overexpression by RT-qPCR; B shows the expression results of StAR and ccNE2 proteins after miR-27b-3p overexpression; C shows the expression results of proliferation-related genes ccNB1, ccNE2 and testosterone synthesis-related genes 3β-HSD and StAR in goat testicular interstitial cells after inhibitor-miR-27b-3p; D shows the expression results of StAR and ccNE2 proteins after inhibitor-miR-27b-3p). Figure 2 The effect of miR-27b-3p as a target on the proliferation of goat testicular interstitial cells (Figure: A is the CCK-8 result after overexpression of miR-27b-3p; B is the CCK-8 result after inhibitor miR-27b-3p). Figure 3 The effect of miR-27b-3p as a target on testosterone levels in goat testicular interstitial cells (Figure: A shows the testosterone level detection results in testicular interstitial cells after overexpression of miR-27b-3p; B shows the testosterone level detection results in testicular interstitial cells after inhibitor-miR-27b-3p). Detailed Implementation

[0016] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0017] Example 1 Inhibitor-miR-27b-3p, a miR-27b-3p gene-specific inhibitor that targets the miR-27b-3p gene to promote the proliferation of goat testicular interstitial cells, has the sequence: 5'-GCAGAACTTAGCCACTGTGAA-3'.

[0018] Example 2 Use Lipofectamine TM According to the manufacturer's protocol, the 3000 transfection reagent was used to transfect the miR-27b-3p gene expression specific inhibitor (final concentration of 100 nM) provided in Example 1 into goat testicular interstitial cells. This upregulated the gene levels and protein expression of testosterone synthesis-related genes 3β-HSD and StAR, as well as testicular interstitial cell proliferation-related genes ccNB1 and ccNE2, thereby promoting the proliferation of goat testicular interstitial cells and testosterone synthesis.

[0019] Example 3 Mimics-miR-27b-3p, a miR-27b-3p gene expression specific promoter that targets the miR-27b-3p gene to inhibit the proliferation of goat testicular interstitial cells, has the sequence: 5'-TTCACAGTGGCTAAGTTCTGC-3'.

[0020] Example 4 Use Lipofectamine TM According to the manufacturer's protocol, the miR-27b-3p gene expression specific promoter Mimics-miR-27b-3p (final concentration of 100 nM) provided in Example 3 was transfected into goat testicular interstitial cells. This resulted in the inhibition of gene levels and protein expression of testosterone synthesis-related genes 3β-HSD and StAR, and testicular interstitial cell proliferation-related genes ccNB1 and ccNE2, as well as the inhibition of goat testicular interstitial cell proliferation and testosterone synthesis.

[0021] To further verify the reliability of the present invention, the inventors conducted a series of experiments, as follows: 1. Materials 1.1 Animals Fresh goat testicular tissue was collected from the slaughterhouse and immediately placed in sterile saline containing 10,000 IU / mL penicillin-streptomycin, then transported back to the laboratory at a constant temperature within 1 hour. The testes were rinsed three times with preheated saline to remove surface impurities and blood. Under aseptic conditions, the epididymis, seminal vesicles, and surface adipose tissue were trimmed and removed, followed by a second rinse. The processed testes were then placed in saline containing penicillin-streptomycin (37°C) for later use. The tunica albuginea was removed, and the testicular parenchyma was cut into pieces approximately 1 mm thick. 3Cell fragments were added and digested with trypsin for 8 min, followed by the addition of DMEM / F-12 complete medium containing 10% fetal bovine serum (FBS) to terminate the digestion. The cell suspension was filtered sequentially through 250-mesh and 400-mesh cell sieves. Cells in the filtrate were collected, seeded in culture dishes, and incubated at 37°C in a 5% CO2 incubator until the cell confluence reached 80% for subsequent experiments.

[0022] 1.2 Main Reagents The miR-27b-3p overexpression sequence (Mimics-miR-27b-3p) and the inhibitor sequence miR-27b-3p (Inhibitor-miR-27b-3p) were synthesized by Shanghai Jiying Biotechnology Co., Ltd.

[0023] Reagent kits: CCK-8 assay kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.); testosterone assay kit (purchased from Nanjing Jiancheng Bioengineering Institute); RNA extraction kit (RNAiso Plus) (purchased from Takara Corporation, Japan); reverse transcription kit and qPCR premix (purchased from Aibimeng Technology Co., Ltd., China).

[0024] General reagents: DMEM / F-12 culture medium, purchased from Thermo Fisher Scientific, USA; Lipofectamine TM 3000 transfection reagent was purchased from Invitrogen, USA.

[0025] 2. Test methods This study investigated the effects of miR-27b-3p and its target gene PPARG on the proliferation and testosterone synthesis of goat testicular interstitial cells by transfecting Mimics and Inhibitor. The proliferation of testicular interstitial cells was detected using the CCK-8 assay, testosterone secretion was detected using ELISA, and the expression of proliferation-related markers (ccNB1, ccNE2) and testosterone synthesis-related markers (3β-HSD, StAR) was detected by qRT-PCR and Western Blot.

[0026] Sequence information: (1) Inhibitor-miR-27b-3p sequence (SEQ ID NO.1) 5'-GCAGAACTTAGCCACTGTGAA-3'.

[0027] (2) Mimics-miR-27b-3p sequence (SEQ ID NO.2) 5'-TTCACAGTGGCTAAGTTCTGC-3'.

[0028] 2.1 Cell Culture and Transfection Select healthy cells in the logarithmic growth phase for passage. Discard the old culture medium, wash three times with PBS, and digest with 0.25% trypsin at room temperature for about 2 min. Once the cells have shrunk and become rounded, immediately add DMEM / F-12 medium containing 10% FBS to stop the digestion. Collect the cell suspension, centrifuge at 300×g for 5 min, resuspend, and seed. Seed the cells in 6-well plates (2×10⁶ cells / well). 6 (cells / well) or 12-well plate (5×10) 5 (cells / well). When the cell confluence reaches approximately 60%, proceed according to Lipofectamine. TM According to the 3000 instructions, transfect Mimics-miR-27b-3p or Inhibitor-miR-27b-3p (both at a final concentration of 100 nM) into cells. Collect cells for further analysis 24 h after transfection.

[0029] 2.2 RNA extraction and cDNA synthesis (1) Total RNA extraction Strictly follow the Takara RNAiso Plus kit instructions. 24 h after transfection, wash cells with PBS, add 800 μL of RNAiso Plus to each well, and lyse at room temperature for 5 min. Collect the lysis buffer, add 200 μL of chloroform, mix vigorously by inverting, incubate for 30 s, and centrifuge at 12,000 × g for 10 min at 4 °C. Aspirate the supernatant, add an equal volume of anhydrous ethanol, and transfer to an adsorption column. After centrifugation and washing according to the instructions, dissolve the RNA in RNase-free water. Detect RNA concentration and purity using a UV spectrophotometer. Store qualified samples at -80 °C.

[0030] (2) Reverse transcription cDNA synthesis was performed using the All-In-One 5X RT MasterMix kit. Reaction program: 37℃, 10 min; 60℃, 5 min; 95℃, 5 s.

[0031] 2.3 Analytical Methods (1) Detection of cell proliferation level (CCK-8 assay) Goat testicular interstitial cells were used at a concentration of 3.0 × 10⁻⁶. 3 Seeds were planted at a density of 10 cells / well in 96-well plates and transfected after 12 h of adhesion. 24 h after transfection, 10 μL of CCK-8 was added to each well in the dark and incubated at 37°C for 2 h. The absorbance (OD value) at 450 nm was measured using a microplate reader and statistical analysis was performed.

[0032] (2) Detection of gene levels for cell proliferation and testosterone synthesis Twenty-four hours after transfection, goat testicular interstitial cells were used to detect the mRNA and protein levels of proliferation-related genes ccNB1 and ccNE2, and testosterone synthesis-related genes 3β-HSD and StAR, using qRT-PCR and Western blot methods. The qRT-PCR and Western blot methods are as follows: qRT-PCR uses cDNA as a template and follows the BlasTaq protocol developed by Albion Technology Co., Ltd. TM The 2X qPCR MasterMix kit was used according to the instructions. After preparing the reaction solution, the reaction program was as follows: 95℃, 30 s; 95℃, 10 s; 60℃, 30 s; 40 cycles; 95℃, 15 s; 60℃, 60 s; 95℃, 15 s, with β-actin as an internal control. Real-time fluorescence quantitative results were obtained using... The primer sequences were calculated using the method described in Table 1.

[0033] .

[0034] (3) Testosterone level detection in testicular interstitial cells Testicular interstitial cells were seeded into 6-well cell culture plates, and the cell supernatant was collected 24 h after transfection. Subsequently, testosterone (T) levels in mouse serum were detected using an ELISA kit. Follow the instructions provided in the kit for testing. The specific steps are as follows: 1) Equilibrate the ELISA kit at room temperature for 30 min to prepare the standard; 2) Set up blank wells, standard wells (for creating a standard curve), and sample wells according to experimental needs. Add 50 μL of standard and 50 μL of cell supernatant to the standard and sample wells, respectively; 3) Add 50 μL of biotin-HRP to each well containing the standard and sample, gently vortex to mix, seal with sealing film, and incubate at 37°C for 30 min. After incubation, discard the liquid in the wells and add diluted washing buffer to wash for 30 s / wash, for a total of 5 washes; 4) After washing, pat the plate dry. Add 50 μL of biotin-HRP to each well containing the standard and sample wells, gently vortex to mix, seal with sealing film, and incubate at 37°C for 30 min. After incubation, discard the liquid in the wells and add diluted washing buffer to wash for 30 s / wash, for a total of 5 washes; 5) After washing, pat the plate dry. Add 50 μL of colorimetric solution A to each well, then add 50 μL of colorimetric solution B. Gently shake to mix, then seal with sealing film and incubate at 37℃ for 10 min. 6) After incubation, add stop solution to each well to stop the reaction, and use a microplate reader to detect the absorbance value at 450 nm (OD450). The detection should be completed within 10 min. 7) Plot a standard curve based on the concentration and OD value of the standard, and calculate the testosterone concentration in the cell supernatant based on the standard curve.

[0035] 3. Results All data in this study are expressed as mean ± standard deviation, with at least three independent replicates. Differences between two groups were analyzed using t-tests and ANOVA. All analyses were performed in GraphPad Prism 8. (The text in the figures is incomplete and likely refers to a separate section.) "Represents P" 0.05, "Represents P" 0.01.

[0036] 3.1 Effects of miR-27b-3p as a target on the expression of goat testicular interstitial cell proliferation genes ccNB1 and ccNE2, and testosterone synthesis-related genes 3β-HSD and StAR. The results are as follows Figure 1 As shown in the results, Mimics-miR-27b-3p significantly inhibited the mRNA levels of 3β-HSD, StAR, ccNB1, and ccNE2 in LCs. Figure 1A), the Western blot results were consistent with the RT-qPCR results ( Figure 1 B). The Inhibitor-miR-27b-3p group significantly promoted the mRNA levels of 3β-HSD and StAR, ccNB1 and ccNE2 in LCs (B). Figure 1 C), the Western blot results were consistent with the RT-qPCR results. Figure 1 D).

[0037] 3.2 Effects of miR-27b-3p as a target on the proliferation of goat testicular interstitial cells The results are as follows Figure 2 As shown in the results, Mimics-miR-145-3p significantly inhibited the viability of LCs cells. Figure 2 A). The Inhibitor-miR-145-3p group significantly promoted LC cell viability ( Figure 2 B).

[0038] 3.3 Effects of miR-27b-3p as a target on testosterone levels in goat testicular interstitial cells The results are as follows Figure 3 As shown in the results, Mimics-miR-27b-3p significantly inhibited testosterone levels in LCs ( Figure 3 A). Inhibitor-miR-27b-3p significantly increased testosterone levels in LCs (A). Figure 3 B). The above results indicate that inhibiting miR-27b-3p can promote testosterone synthesis in LCs.

[0039] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of miR-27b-3p gene as a target in the preparation of products that regulate the proliferation of goat testicular interstitial cells and testosterone synthesis.

2. The application according to claim 1, characterized in that, The product regulating goat testicular interstitial cell proliferation and testosterone synthesis targets the miR-27b-3p gene. By inhibiting the expression of the miR-27b-3p gene through miR-27b-3p inhibitors, it promotes the proliferation of goat testicular interstitial cells and testosterone synthesis.

3. The application according to claim 2, characterized in that, The miR-27b-3p inhibitor is Inhibitor-miR-27b-3p, and its sequence is shown in SEQ ID NO.

1.

4. The application according to claim 1, characterized in that, The product regulating goat testicular interstitial cell proliferation and testosterone synthesis targets the miR-27b-3p gene. It promotes miR-27b-3p gene expression by overexpressing miR-27b-3p mimics, thereby inhibiting goat testicular interstitial cell proliferation and testosterone synthesis.

5. The application according to claim 4, characterized in that, The miR-27b-3p overexpression mimic is Mimics-miR-27b-3p, and its sequence is shown in SEQ ID NO.

2.

6. Application of miR-27b-3p gene as a target in the preparation of products that regulate the expression of marker genes ccNB1 and ccNE2 related to the proliferation of goat testicular interstitial cells.

7. Application of miR-27b-3p gene as a target in the preparation of products containing 3β-HSD and StAR expression marker genes related to the regulation of testosterone synthesis in goat testicular interstitial cells.

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