Application of miR-145-3p as target spot in preparation of products for regulating and controlling proliferation of goat testicular interstitial cells

Abstract

The invention relates to the technical field of genetic engineering, in particular to application of miR-145-3p as a target spot in preparation of products for regulating and controlling proliferation of goat testicular interstitial cells. According to the invention, Inhibitor and Mimics sequences taking the miR-145-3p gene as a target spot are constructed, and it is proposed for the first time that overexpression of the miR-145-3p gene can significantly inhibit proliferation of goat testicular interstitial cells, and inhibition of the miR-145-3p gene can significantly promote proliferation of goat testicular interstitial cells. The invention has important significance for improving the development of the goat testis, and provides technical support for researching and developing related products for regulating and controlling the proliferation of the goat testis interstitial cells by taking the miR-145-3p gene as a target spot.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of genetic engineering, and particularly relates to an application of miR-145-3p as a target in preparation of a product for regulating proliferation of goat testis interstitial cells. BACKGROUND

[0002] Testis interstitial cells (LCs) are key cells in the process of testis development and function maintenance, and play an important role in androgen synthesis and regulation of spermatogenesis. In the process of testis development, the functional activity of LCs is crucial, and moderate proliferation and steady state maintenance of LCs are conducive to maintaining normal serum testosterone levels, and when LCs dysfunction occurs, it can lead to spermatogenesis failure, which is often manifested as azoospermia. At present, the role of testis interstitial cells in gluconeogenesis regulation and cell proliferation is still lacking systematic understanding, and the mechanism of causal relationship between metabolic programming and reproductive function imbalance still needs to be further elucidated.

[0003] Studies have shown that miR-145-3p plays an important role in regulating cell proliferation and inflammation. In prostate cancer cells, the decrease of miR-145-3p can promote the expression of proliferation genes MELK, NCAPG, BUB1 and CDK1, and then promote cell proliferation. In rats, miR-145-3p can regulate the balance of Th1 and Treg cells through the NFATc2 / NF-κB axis, and then reduce the inflammation of traumatic brain injury rats. In mice, overexpression of miR-145-5p in glial cells can inhibit the inflammatory response and maintain M2 macrophage polarization by targeting TLR4 / NF-κB signal, and then promote the repair of spinal cord injury tissue in mice. The above studies show that miR-145-3p affects the development of the body by reducing the occurrence of inflammation and regulating cell function. Although miR-145-3p has the above important biological functions as a target, its role in goat testis interstitial cells is still unknown.

[0004] Therefore, the application provides an application of miR-145-3p gene as a target in regulating proliferation of goat testis interstitial cells, aiming to overcome the shortcomings of the prior art and provide technical support for research and development of products related to miR-145-3p gene as a target for regulating proliferation of goat testis interstitial cells and gluconeogenesis. SUMMARY

[0005] The application aims to provide an application of miR-145-3p gene as a target in preparation of a product for regulating proliferation of goat testis interstitial cells.

[0006] Another objective of this invention is to provide the application of the miR-145-3p 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 gluconeogenesis of 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-145-3p gene as a target in the preparation of products that regulate the proliferation of goat testicular interstitial cells.

[0008] Preferably, in the application of the miR-145-3p gene as a target in the preparation of a product for regulating the proliferation of goat testicular interstitial cells, the product for regulating the proliferation of goat testicular interstitial cells targets the miR-145-3p gene and promotes the proliferation of goat testicular interstitial cells by inhibiting the expression of the miR-145-3p gene through a miR-145-3p inhibitor.

[0009] In a further preferred embodiment, in the application of the miR-145-3p gene as a target in the preparation of products regulating the proliferation of goat testicular interstitial cells, the miR-145-3p inhibitor is an inhibitor miR-145-3p, the sequence of which is shown in SEQ ID NO.1.

[0010] Preferably, in the application of the miR-145-3p gene as a target in the preparation of a product for regulating the proliferation of goat testicular interstitial cells, the product for regulating the proliferation of goat testicular interstitial cells targets the miR-145-3p gene, promotes the expression of the miR-145-3p gene by overexpressing miR-145-3p mimics, and inhibits the proliferation of goat testicular interstitial cells.

[0011] In a further preferred embodiment, in the application of the miR-145-3p gene as a target in the preparation of products regulating the proliferation of goat testicular interstitial cells, the miR-145-3p overexpression mimic is Mimics-miR-145-3p, the sequence of which is shown in SEQ ID NO. 2.

[0012] The application of the miR-145-3p 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-145-3p gene as a target in the preparation of products that regulate gluconeogenesis in goat testicular interstitial cells.

[0014] The beneficial effects of this invention are: This invention constructs inhibitor and mimic sequences targeting miR-145-3p and proposes for the first time that inhibiting miR-145-3p gene expression can significantly promote gluconeogenesis and proliferation of goat testicular interstitial cells; conversely, overexpression of miR-145-3p gene can significantly inhibit gluconeogenesis and proliferation of goat testicular interstitial cells. This invention is of great significance for improving goat testicular development and provides technical support for the research and development of products related to miR-145-3p-targeted regulation of goat testicular interstitial cell proliferation. Attached Figure Description

[0015] Figure 1 The effect of miR-145-3p as a target on the expression of proliferation-related genes CCNB1 and CCNE1 in goat testicular interstitial cells (Figure: A shows the expression results of proliferation-related genes CCNB1 and CCNE2 in goat testicular interstitial cells after miR-145-3p overexpression by RT-qPCR; BC shows the expression results of PCK1, CCNB1, and CCNE2 proteins after miR-145-3p overexpression; D shows the expression results of proliferation-related genes CCNB1 and CCNE2 in goat testicular interstitial cells after inhibitor-miR-145-3p; EF shows the expression results of PCK1, CCNB1, and CCNE2 proteins after inhibitor-miR-145-3p). Figure 2 The effect of miR-145-3p as a target on the proliferation of goat testicular interstitial cells (Figure: A is the CCK8 result after miR-145-3p overexpression; B is the Edu result after miR-145-3p overexpression; C is the CCK8 result after inhibitor-miR-145-3p; D is the Edu result after inhibitor-miR-145-3p). Figure 3 The effect of miR-145-3p as a target on glucose and lactate levels in goat testicular interstitial cells (Figure: A shows glucose level after miR-145-3p overexpression; B shows lactate level after miR-145-3p overexpression; C shows glucose level after miR-145-3p inhibitor; D shows lactate level after miR-145-3p inhibitor). Figure 4The effect of miR-145-3p as a target on mitochondrial membrane potential of goat testicular interstitial cells (Figure: A is the fluorescence image of mitochondrial membrane potential detection after miR-145-3p overexpression; B is the quantitative analysis result of mitochondrial membrane potential after miR-145-3p overexpression; C is the fluorescence image of mitochondrial membrane potential detection after inhibitor-miR-145-3p; D is the quantitative analysis result of mitochondrial membrane potential after inhibitor-miR-145-3p). Detailed Implementation

[0016] The technical solution of the present invention will be described in detail below with reference to specific embodiments. These 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-145-3p, a miR-145-3p gene-specific inhibitor that targets the miR-145-3p gene to promote the proliferation of goat testicular interstitial cells, has the sequence: 5'-AAGAACAGUAUUUCCAGGAAU-3'.

[0018] Example 2 use The transfection reagent was used to transfect the miR-145-3p gene expression specific inhibitor provided in Example 1 (final concentration of 100 nM) into goat testicular interstitial cells according to the manufacturer's protocol, upregulating the levels and protein expression of PCK1, CCNB1 and CCNE2 genes, and promoting gluconeogenesis and proliferation of goat testicular interstitial cells.

[0019] Example 3 Mimics-miR-145-3p, a miR-145-3p gene expression specific promoter that targets the miR-145-3p gene to inhibit the proliferation of goat testicular interstitial cells, has the sequence: 5'-AUUCCUGGAAAUACUGUUCUU-3'.

[0020] Example 4 use The transfection reagent, in accordance with the manufacturer's protocol, transfected the miR-145-3p gene expression specific promoter Mimics-miR-145-3p (final concentration of 100 nM) provided in Example 3 into goat testicular interstitial cells, downregulating the levels and protein expression of PCK1, CCNB1 and CCNE2 genes, and inhibiting gluconeogenesis and proliferation of goat testicular interstitial cells.

[0021] To further verify the reliability of the present invention, the inventors conducted a series of experiments, as follows: 1. Materials 1.1 Animals After collecting goat testicles at the slaughterhouse, they were placed in a thermos containing 1 mL of sterile saline solution with penicillin-streptomycin (10000 IU / mL) and transported back to the laboratory within 1 hour. The collected testicles were rinsed three times with preheated saline solution to remove impurities and blood. Fat deposits, epididymis, and seminal vesicles were removed from the surface of the testicles with scissors. The trimmed testicles were then rinsed again with saline solution. The treated testicles were placed in a beaker containing 1 mL of penicillin-streptomycin (10000 IU / mL) saline solution and placed on a 37°C constant temperature platform for later use. Subsequently, the tunica albuginea tissue was peeled off and cut into 1... The cells were broken into small fragments. Next, they were digested with trypsin for 8 minutes. Then, complete culture medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, and 89% DMEM / F-12 was added to terminate the digestion process. The digested and separated cell solution was filtered through 250-mesh and 400-mesh sieves to collect the cells. Finally, the collected cells were cultured in a cell culture incubator until 80% adherence was achieved, and the culture medium was changed regularly for subsequent experiments.

[0022] 1.2 Drugs and Reagents The miR-145-3p overexpression sequence (Mimics-miR-145-3p) and the miR-145-3p inhibitor sequence (Inhibitor-miR-145-3p) were both synthesized by Shanghai Jiying Biotechnology Co., Ltd.; the lactate level detection kit was purchased from Nanjing Jiancheng Bioengineering Institute; the mitochondrial membrane potential detection kit and the glucose level detection kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; DMEM / F-12 was purchased from Thermofisher, Inc., USA; transfection reagents... The RNA extraction kit was purchased from Invitrogen, USA; the RNA extraction kit was purchased from Takara, Japan; and the reverse transcription kit was purchased from Aibimeng Technology Co., Ltd., China.

[0023] 2. Test methods This study constructed inhibitor and mimic sequences targeting miR-145-3p, with the PCK1 gene being the target gene of miR-145-3p. Transfection reagents were used to transfect Mimics-miR-145-3p (final concentration 100 nM) and Inhibitor-miR-145-3p (final concentration 100 nM) into goat testicular interstitial cells according to the manufacturer's protocol. The effects of miR-145-3p as a target on the mRNA and protein levels of proliferation pathway marker genes were detected using qRT-PCR and Western blot (WB) assays. The effect of miR-145-3p as a target on goat testicular interstitial cell proliferation was detected using the CCK8 assay. The effect of miR-145-3p as a target on mitochondrial membrane potential in goat testicular interstitial cells was detected using the JC-1 assay. Cellular lactate and glucose levels were detected using ELISA to observe the effect of miR-145-3p as a target on gluconeogenesis in goat testicular interstitial cells. Cell proliferation, mitochondrial membrane potential, lactate, and glucose levels were measured.

[0024] Sequence information: (1) Inhibitor-miR-145-3p (SEQ ID NO.1) sequence: 5'-AAGAACAGUAUUUCCAGGAAU-3'.

[0025] (2) Mimics-miR-145-3p (SEQ ID NO.2) sequence: 5'-AUUCCUGGAAAUACUGUUCUU-3'.

[0026] 2.1 Cell passage and transfection Select cells in the late logarithmic growth phase with good morphology for passage. Passage can be performed when the cell density reaches 80%-90%. Before passage, discard the old culture medium, wash three times with PBS, and then add an appropriate amount of preheated 0.25% trypsin to the culture dish to cover the cells. Digest at room temperature for 2 min, then immediately add an appropriate amount of DMEM / F-12 medium (containing 10% FBS) to stop the digestion. Transfer the cells to a 4 mL sterile centrifuge tube, mix well, and centrifuge at 300×g for 5 min. After centrifugation, discard the supernatant, then resuspend the cells in 2 mL of DMEM / F-12 medium (containing 10% FBS). One cell line was seeded in a 10 cm culture dish and transferred to a CO2 incubator (5%). Incubate at 37℃ and 100% humidity for 48 hours.

[0027] Goat testicular interstitial cells were seeded at appropriate densities in 10 cm cell culture dishes. When the cell density reached 85%-90%, they were passaged. Cells / well and Goat testicular interstitial cells were seeded at a density of [number] cells / well in 12-well and 6-well cell culture plates, respectively, and then placed in a CO2 incubator (5%). Cells can be transfected when they have been cultured at 37°C and 100% humidity until the cell density is approximately 60%. Follow the transfection instructions. Mimics-miR-145-3p (final concentration 100 nM) and Inhibitor-miR-145-3p (final concentration 100 nM) were transfected into goat testicular interstitial cells. Cells were harvested 24 h after transfection.

[0028] 2.2 Cell RNA Extraction and Reverse Transcription (1) Extraction of cellular RNA RNA extraction was performed according to the instructions of Takara's RNAiso Plus reagent. The specific steps were as follows: (1) Cell collection: After 24 h of transfection, the supernatant was aspirated, washed 3 times with PBS, and 800 μL of RNAiso Plus reagent was added to each sample. The cells were then transferred to a 1.5 mL EP tube using a scraper and lysed at room temperature for 5 min. (2) Total RNA extraction: 200 μL of chloroform was added and the mixture was inverted and mixed. The mixture was allowed to stand for 30 s and then centrifuged at 12000×g for 10 min at 4℃. (3) RNA precipitation: The supernatant was carefully aspirated with a pipette and transferred to a 1.5 mL centrifuge tube without RNase. An equal volume of anhydrous ethanol was added, and the mixture was inverted and mixed. The mixture was then transferred to a nucleic acid adsorption column and centrifuged according to the instructions. The column was washed twice with 70% ethanol and then left to air dry. (4) RNA dissolution: The centrifuge column was placed in a 1.5 mL centrifuge tube without RNase. An appropriate amount of RNase-free water was added to dissolve the RNA. The mixture was allowed to stand for 1 min and then centrifuged at 10000×g for 1 min at 4℃. After 1 minute, discard the centrifuge column and use a UV spectrophotometer to detect the RNA quality. Qualified RNA will be used directly for reverse transcription or frozen and stored in a -80°C freezer for later use.

[0029] (2) Cellular mRNA reverse transcription Cellular mRNA reverse transcription was performed according to the instructions of the All-In-One 5X RT MasterMix kit from Abimon Technologies Co., Ltd. After preparing the reaction solution, the incubation times were 37°C for 10 min, 60°C for 5 min, and 95°C for 5 s.

[0030] 2.3 Analytical Methods (1) Detection of cell proliferation level The effects of Mimics-miR-145-3p and Inhibitor-miR-145-3p on the proliferation of goat testicular interstitial cells were detected using the CCK8 cell proliferation assay kit. The specific detection method is as follows: Goat testicular interstitial cells were... Cells were seeded at a density of 100 μL of culture medium per well into 96-well cell culture plates. After 12 h of cell adhesion, the cells were transfected. 24 h later, 10 μL of CCK8 was added to each well. The plates were incubated in the dark at 37°C for 2 h. The optical density (OD450) at 450 nm was measured using a microplate reader and statistical analysis was performed.

[0031] Cell Edu proliferation assay: Goat testicular interstitial cells were used... Cells were seeded at a density of 100 μL / well in 96-well cell culture plates. After 12 h of cell adhesion, the cells were transfected. After 24 h, the cells were cultured according to the instructions of Beyotime Biotechnology Co., Ltd. The EdU-488 cell proliferation assay kit instructions are as follows: Add the prepared EdU solution to cell culture medium and incubate for 2 hours. Remove the culture medium and fix the cells with cell fixation solution for 30 minutes, followed by permeabilization with permeabilizer for 10 minutes, and then wash with PBS. Next, perform Apo110 staining, followed by Hoechst staining for nuclear DNA after PBS washing. Observe the cell staining under an inverted fluorescence microscope; the remaining steps are the same as above. Add 10 μL of CCK8 to each well, incubate in the dark at 37°C for 2 hours, and then measure the optical density (OD450) at 450 nm using a microplate reader and perform statistical analysis.

[0032] (2) Detection of cell proliferation gene levels Goat testicular interstitial cells were transfected for 24 h, and the mRNA and protein levels of proliferation-related genes CCNB1 and CCNE2 were detected using qRT-PCR and Western blot methods. The qRT-PCR and Western blot methods are as follows: qRT-PCR uses cDNA as a template, following the instructions of Aibimeng Technology Co., Ltd. The 2X qPCR MasterMix kit was used according to the instructions. After preparing the reaction solution, the reaction program was 95℃ for 30 s; 95℃ for 10 s; 60℃ for 30 s; 40 cycles; 95℃ for 15 s; 60℃ for 60 s; 95℃ for 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] ; (3) Detection of mitochondrial membrane potential in testicular interstitial cells Testicular interstitial cells were seeded into 6-well cell culture plates. After 24 h of transfection, the cells were washed three times with PBS. Then, JC-1 staining solution (10 μM) was added, and the cells were incubated at 37°C in the dark for 30 min. After incubation, the cells were washed three times with PBS solution for 5 min each time to remove the JC-1 dye. The samples were placed under a fluorescence microscope, and the fluorescent dye was excited at a specific wavelength. Images were taken using the same parameters. The fluorescence images of the testicular interstitial cells were processed using Image-J software, and the average fluorescence intensity was calculated. Independent samples t-tests were performed on the mean fluorescence intensity values ​​of the two groups, and the mean fluorescence intensity of the control group was reset to 1 to plot the statistical graph.

[0034] (4) Detection of lactate levels in testicular interstitial cells Testicular interstitial cells were seeded in 6-well cell culture plates. After 24 h of transfection, the cells were washed three times with PBS. Then, 0.25% EDTA-free trypsin was added to digest the cells for 2 min at room temperature. Immediately afterward, an appropriate amount of DMEM / F-12 medium (containing 10% FBS) was added to terminate the digestion. The cells were then transferred to 4 mL sterile centrifuge tubes and centrifuged at 300×g for 5 min. The supernatant was discarded after centrifugation, and lactate was measured according to the lactate detection instructions. Specifically, 0.5 mL of extraction buffer was added to the collected cells, and the cells were sonicated and centrifuged at 10000×g for 10 min. The reaction solution was prepared according to the corresponding instructions. After mixing the sample, the reaction was incubated at 37℃ in the dark for 30 min, and the OD value was read at 450 nm. Independent samples t-tests were performed on the OD values ​​of the two groups. The mean lactate level of the control group was reset to 1 for statistical plotting.

[0035] (5) Detection of glucose levels in testicular interstitial cells Testicular interstitial cells were seeded into 6-well cell culture plates. After 24 h of transfection, the cells were washed three times with PBS. Then, following the instructions of the glucose assay kit, 100 μL of cell lysis buffer was added to each well. The cells and lysis buffer were thoroughly mixed by pipetting, and then transferred to 1.5 mL centrifuge tubes. The mixture was centrifuged at 12000×g for 5 min, and the supernatant was collected into a new centrifuge tube. Next, 20 μL of the sample was added to 170 μL of glucose assay reagent, vortexed, and centrifuged at 5000×g for a few seconds to allow the liquid to settle to the bottom of the tube. The PCR tubes were heated at 95°C for 8 min and then cooled to 4°C. After cooling to 4°C, the PCR tubes were removed. 180 µL of the liquid was aspirated from each tube into a new 96-well plate, and the OD value was read at 450 nm. An independent samples t-test was performed on the OD values ​​of the two groups, with the mean glucose level of the control group reset to 1 for statistical plotting.

[0036] 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.) "This represents P < 0.05", "This means P < 0.01.

[0037] 3.1 Effects of miR-145-3p as a target on the expression of proliferation genes CCNB1 and CCNE2 in goat testicular interstitial cells The results are as follows Figure 1 As shown in the results, Mimics-miR-145-3p significantly inhibited the mRNA levels of PCK1, CCNB1, and CCNE2 in LCs. Figure 1 A), the Western blot results were consistent with the RT-qPCR results ( Figure 1 B, 1C). The Inhibitor-miR-145-3p group significantly promoted the mRNA levels of gluconeogenesis genes PCK1, CCNB1, and CCNE2 in LCs (B, C). Figure 1 D), the Western blot results were consistent with the RT-qPCR results. Figure 1 E, 1F).

[0038] 3.2 Effects of miR-145-3p 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 2A), Edu assays revealed that Mimics-miR-145-3p significantly inhibited LC proliferation (A). Figure 2 B). The Inhibitor-miR-145-3p group significantly promoted LC cell viability (B). Figure 2 C), Edu assays revealed that Inhibitor-miR-145-3p significantly promoted LC proliferation (C). Figure 2 D).

[0039] 3.3 Effects of miR-145-3p as a target on glucose and lactate levels in goat testicular interstitial cells The results are as follows Figure 3 As shown. Gluconeogenesis is often accompanied by an increase in glucose, while the substrate lactate level is measured. This study found that Mimics-miR-145-3p significantly inhibited glucose levels in LCs ( Figure 3 A), lactate levels were significantly increased ( Figure 3 B). Inhibitor-miR-145-3p significantly increased glucose levels in LCs (B). Figure 3 C), lactate levels were significantly reduced ( Figure 3 D). The above results indicate that inhibiting miR-145-3p can promote gluconeogenesis in LCs.

[0040] 3.4 Effects of miR-145-3p as a target on mitochondrial membrane potential levels in goat testicular interstitial cells The results are as follows Figure 4 As shown. Gluconeogenesis is often accompanied by an increase in energy levels. This study found that Inhibitor-miR-145-3p significantly increased the mitochondrial membrane potential level in LCs (as shown). Figure 4 A, 4B). However, after Mimics-miR-145-3p, the mitochondrial membrane potential level in LCs was significantly reduced (…). Figure 4 C, 4D).

[0041] 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-145-3p gene as a target in the preparation of products that regulate the proliferation of goat testicular interstitial cells.

2. The application according to claim 1, characterized in that, The product regulating the proliferation of goat testicular interstitial cells targets the miR-145-3p gene. By inhibiting the expression of the miR-145-3p gene through miR-145-3p inhibitors, the proliferation of goat testicular interstitial cells is promoted.

3. The application according to claim 2, characterized in that, The miR-145-3p inhibitor is Inhibitor-miR-145-3p, and its sequence is shown in SEQ ID NO.

1.

4. The application according to claim 1, characterized in that, The product regulating the proliferation of goat testicular interstitial cells targets the miR-145-3p gene. It promotes the expression of the miR-145-3p gene by overexpressing miR-145-3p mimics, thereby inhibiting the proliferation of goat testicular interstitial cells.

5. The application according to claim 4, characterized in that, The overexpression miR-145-3p mimic is Mimics-miR-145-3p, and its sequence is shown in SEQ ID NO.

2.

6. Application of miR-145-3p 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-145-3p gene as a target in the preparation of products that regulate gluconeogenesis in goat testicular interstitial cells.

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