Application of miR-615 in ovarian granular cells

Abstract

The invention relates to the technical field of molecular biology, and particularly discloses application of miR-615 in ovarian granular cells. The miR-615 is applied to any one of the following items: preparing a product for promoting the activity and / or proliferation of ovarian granular cells; preparing a product for promoting generation of estradiol and / or progesterone; preparing a product for promoting the expression of the 3 beta-HSD and / or StAR gene; preparing a product for inhibiting ACSL4 gene expression; and preparing a product competitively combined with the lnc-GAM. According to the invention, porcine ovarian granular cells are used as a material, the expression level of lnc-GAM-miR-615 / ACSL4 regulatory axis is up-regulated or down-regulated in the porcine ovarian granular cells by using gene overexpression and interference technologies, and the expression of the lnc-GAM-miR-615 / ACSL4 regulatory axis is determined by detecting cell proliferation activity, hormone secretion level and apoptosis gene expression in combination with a dual-luciferase reporter gene test. And the regulation effect of lnc-GAM mediation on the apoptosis of the porcine ovarian granular cells is researched.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to the application of miR-615 in ovarian granulosa cells. Background Art

[0002] During the development of sow ovaries, a large number of atretic follicles cannot ovulate and fertilize, which seriously affects the reproductive capacity of sows. Therefore, exploring the molecular mechanism of follicular atresia is of great significance for rescuing follicular atresia, improving ovarian utilization and sow reproductive performance. At present, many studies have shown that sow follicular atresia involves the regulation of multiple genes, and many scholars have identified multiple genes and non-coding RNAs involved in regulating follicular atresia. In recent years, with the development of high-throughput sequencing technology, lncRNA and circRNA have been proven to be able to regulate the transcription and translation of target genes as ceRNA of miRNA, and ultimately regulate the process of follicular atresia.

[0003] It has been reported that the multi-tissue expression of miR-615 (including brain, ovary, testis, embryonic stem cells, embryo) continues to increase during mouse embryonic development and persists in newborn mice. In the ovaries of pregnant goats, the expression level of miR-615 was found to be significantly higher than that of non-pregnant goats. Some scholars have conducted research on cooperative pig testes and pointed out that miR-615 may be related to the regulation of precocious puberty in boars. In pig testicular interstitial cells, miR-615 induces an increase in cell number, but this phenotype has nothing to do with miR-615 inhibiting cell apoptosis and cell proliferation. Studies have also shown that lncRNA can act as a molecular sponge for specific miRNAs and exert regulatory effects by competitively binding to the expression of miRNAs. Summary of the invention

[0004] One of the purposes of the present invention is to provide an application of miR-615 in any one of the following (1)-(5):

[0005] (1) Preparing products that promote the activity and / or proliferation of ovarian granulosa cells;

[0006] (2) preparing products that promote the production of estradiol and / or progesterone;

[0007] (3) preparing products that promote 3β-HSD and / or StAR gene expression;

[0008] (4) preparing a product that inhibits ACSL4 gene expression;

[0009] (5) Prepare products that competitively bind to lnc-GAM.

[0010] Preferably, the target gene of miR-615 is ACSL4 gene.

[0011] Preferably, the primer nucleic acid sequence of miR-615 is AATGCACTCCGAGCCTGGGT.

[0012] The second object of the present invention is to provide a use of a miR-615 inhibitor in any one of the following (1)-(4):

[0013] (1) Preparing products that inhibit the activity and / or proliferation of ovarian granulosa cells;

[0014] (2) Preparation of products that inhibit the production of estradiol and / or progesterone;

[0015] (3) preparing products that inhibit 3β-HSD and / or StAR gene expression;

[0016] (4) Prepare products that promote the expression of ACSL4 gene.

[0017] Preferably, the nucleic acid sequence of the miR-615 inhibitor is AGAGGGAGACCCAGGCUCGGA.

[0018] The third object of the present invention is to provide an application of lnc-GAM in any one of the following (1)-(5):

[0019] (1) preparing a product that competitively binds to miR-615;

[0020] (2) preparing products that inhibit the activity and / or proliferation of ovarian granulosa cells;

[0021] (3) Preparation of products that inhibit the production of estradiol and / or progesterone;

[0022] (4) preparing products that inhibit 3β-HSD and / or StAR gene expression;

[0023] (5) Prepare products that promote the expression of ACSL4 gene.

[0024] Preferably, the above-mentioned ovarian granulosa cells are porcine ovarian granulosa cells.

[0025] Preferably, the above-mentioned product is a medicine or a reagent.

[0026] The present invention found that follicle-stimulating hormone (FSH) promoted the expression of miR-615 and inhibited the expression of lnc-GAM and ACSL4, indicating that lnc-GAM, miR-615 and ACSL4 may be involved in the regulation of the development process of pig follicles.

[0027] The present invention uses porcine ovarian granulosa cells as materials, utilizes gene overexpression and interference technology, up-regulates or down-regulates the expression level of the lnc-GAM-miR-615 / ACSL4 regulatory axis in porcine ovarian granulosa cells, and systematically explores the regulatory effect of lnc-GAM-mediated ceRNA network on porcine ovarian granulosa cell apoptosis by detecting cell proliferation activity, hormone secretion level and expression of apoptosis genes, combined with dual luciferase reporter gene assay.

[0028] The present invention overexpressed and inhibited miR-615 in porcine ovarian granulosa cells, and found that miR-615 can significantly promote the proliferation and vitality of porcine ovarian granulosa cells, and the overexpression of miR-615 can promote the production of estradiol and progesterone, and also promote the expression of steroid hormone secretion-related genes 3β-HSD and StAR.

[0029] At the same time, the expression level of ACSL4 in pig ovarian granulosa cells was positively correlated with lnc-GAM. The inhibitors of miR-615 and lnc-GAM were co-transfected into pig ovarian granulosa cells, and it was found that lnc-GAM could act as a ceRNA to competitively bind to miR-615, thereby releasing the inhibitory effect of miR-615 on ACSL4, thereby inhibiting the proliferation and vitality of pig ovarian granulosa cells, reducing hormone secretion levels, and promoting apoptosis gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a comparison of the nuclear and cytoplasmic separation of porcine ovarian granulosa cells and the treatment with follicle-stimulating hormone. A is the subcellular localization of lnc-GAM, miR-615 and ACSL4; BD are the effects of adding FSH on the expression of lnc-GAM, miR-615 and ACSL4.

[0031] Figure 2 The figure is a comparison of the effects of miR-615 on the viability and proliferation of porcine ovarian granulosa cells. A is the efficiency test of miR-615 overexpression and inhibitors; B is the CCK8 test result; C is the EdU test result (the unit coordinate ruler represents 100nm).

[0032] Figure 3 This is a comparison chart of the effects of miR-615 on steroid hormone synthesis and related genes. AB are the effects of miR-615 on the secretion levels of E2 and P4, and CD are the effects of miR-615 on 3β-HSD and StAR genes.

[0033] Figure 4This is a diagram of the dual luciferase gene reporter test to verify that ACSL4 and miR-615 have a direct targeting relationship. A is the relative expression level of ACSL4 gene mRNA after overexpression / inhibition of miR-615; B is the wild-type / mutant site of ACSL4 and its dual luciferase detection with miR-615.

[0034] Figure 5 This is a comparison chart of miR-615 targeting ACSL4 to regulate the proliferation of pig ovarian granulosa cells.

[0035] Among them, AB respectively represent that transfection of miR-615inhibitor can release the inhibition of miR-615 on ACSL4 gene and protein expression levels; CD respectively represents the reversal of the inhibitory effect of miR-615inhibitor on porcine ovarian granulosa cell proliferation by interfering with ACSL4 expression (the unit coordinate ruler represents 100 nm).

[0036] Figure 6 Figure 2 shows miR-615 targeting ACSL4 to regulate steroid hormone synthesis and related gene expression. AB respectively reverses the inhibitory effect of miR-615inhibitor on E2 and P4 secretion by interfering with ACSL4 expression; CD respectively reverses the inhibitory effect of miR-615inhibitor on 3β-HSD and StAR gene mRNA expression by interfering with ACSL4 expression.

[0037] Figure 7 The figure shows the effect of lnc-GAM on steroid hormone synthesis and related genes. A is the efficiency test of lnc-GAM siRNA; B is the expression level of lnc-GAM; C is the effect of lnc-GAM on the secretion levels of E2 and P4; D is the effect of lnc-GAM on hormone synthesis related genes.

[0038] Figure 8 The figure shows the effect of lnc-GAM on the viability and proliferation of porcine ovarian granulosa cells. A is the result of EdU detection (the unit coordinate ruler represents 100nm); B is the effect of lnc-GAM on the expression of apoptosis-related genes.

[0039] Fig. 9 This is a comparison chart of lnc-GAM regulating the proliferation of porcine ovarian granulosa cells by acting as the ceRNA of miR-615. A is the effect of lnc-GAM on the expression of ACSL4 gene; BC are the effects of inhibiting lnc-GAM and miR-615 on the expression levels of ACSL4 gene and protein; D is the result of CCK8 detection; E is the result of EdU detection (the unit coordinate ruler represents 100nm).

[0040] Fig.10Comparison of lnc-GAM regulating steroid hormone synthesis and related gene expression through miR-615. AB shows the effect of inhibiting lnc-GAM and miR-615 on E2 and P4 secretion; CD shows the effect of inhibiting lnc-GAM and miR-615 on 3β-HSD and StAR gene expression.

[0041] Figure 1-Figure 10 In the table, * represents P < 0.05; ** represents P < 0.01. DETAILED DESCRIPTION

[0042] The present invention will be further explained below in conjunction with specific embodiments.

[0043] Example 1

[0044] ExKineTM Nuclei Extraction Kit was used to lyse porcine ovarian granulosa cells, separate the nuclei and cytoplasm of porcine ovarian granulosa cells, and then the Trizol method was used to extract total RNA from the whole cell lysate, nuclei and cytoplasm, respectively.

[0045] The steps for nuclear-cytoplasmic separation are as follows:

[0046] (1) Cell collection: The adherent cells were digested with trypsin and then centrifuged at 500 × g for 5 min.

[0047] (2) Resuspend the pelleted cells in pre-chilled PBS and then transfer the cells to a 1.5 mL centrifuge tube.

[0048] (3) Centrifuge at 500 × g for 2-3 min and carefully remove PBS using a pipette to make the cell pellet as dry as possible.

[0049] (4) Add 3 mL of pre-cooled 1× Lysis Buffer to the cell pellet. Vortex the cells at 0.5 times the maximum speed for 10 seconds to completely suspend the pelleted cells.

[0050] (5) Incubate the lysed cells on ice for 5 min. During this process, 1-5 μL of the cell lysate should be examined under a microscope to ensure that the cells have been evenly lysed and that uniform and round nuclei are present.

[0051] (6) Cytoplasmic extraction: Centrifuge at 500 × g for 5 min at 4°C. The supernatant contains cytoplasmic components, so transfer the supernatant to a pre-cooled 1.5 mL centrifuge tube and store at -80°C for subsequent use.

[0052] (7) Nuclear extraction: Resuspend the cell pellet in 1 mL of pre-cooled 1× Lysis Buffer. Vortex the cells at 0.5 times the maximum speed for 10 seconds. Centrifuge at 500×g for 5 minutes at 4°C. Aspirate the supernatant and place the nuclear pellet on ice.

[0053] (8) Gently vortex for 5 seconds to release the nuclear precipitate. Add 200 μL of pre-chilled Nuclei Storage Buffer and carefully pipette to resuspend the nuclei. Store at -80°C until needed.

[0054] The expression of lnc-GAM, miR-615 and ACSL4 was detected by RT-qPCR ( Figure 1 A), it was found that the three were expressed in the nucleus and cytoplasm, but lnc-GAM and miR-615 were mainly distributed in the cytoplasm, and ACSL4 was mainly distributed in the nucleus. The results showed that lnc-GAM and miR-615 may play a role in the cytoplasm, providing a basis for further research on the regulatory mechanism of lnc-GAM, miR-615 and ACSL4.

[0055] Previous studies have shown that follicle-stimulating hormone (FSH) can regulate the level of glutamine in follicular fluid to regulate ovulation. The adverse reaction of the ovaries to FSH can lead to infertility symptoms, specifically manifested as ovarian failure or ovarian defects, which seriously affect reproductive potential.

[0056] After culturing porcine ovarian granulosa cells in a 6-well plate for 24 or 48 hours, the culture medium was discarded, and the cells were washed three times with serum-free culture medium to remove dead cells that were not attached to the wall. The cells were cultured with serum-free DMEM / F12 culture medium. When the cell confluence reached 70-80%, 5 IU / mL and 10 IU / mL of FSH were added to the experimental group, and the same volume of PBS was added to the control group. The cells were cultured for 24 hours, and RNA was extracted and RT-qPCR was performed. The primer sequences for the RT-qPCR test are shown in Table 1.

[0057] Table 1 RT-qPCR test primer sequences

[0058]

[0059]

[0060] RT-qPCR results showed that lnc-GAM (such as Figure 1 B) and ACSL4 (as Figure 1 D) was significantly lower than that in the blank control group, while miR-615 (as shown in Figure 1The results showed that lnc-GAM, miR-615 and ACSL4 were regulated by the reproductive hormone FSH, which further indicated that the above factors may be involved in the reproductive process, laying the foundation for subsequent research.

[0061] Example 2

[0062] The miR-615 overexpression and inhibitor (miR-615mimics and miR-615inhibitor) were synthesized. The design and synthesis of the required miR-615mimics / inhibitor and the respective blank control mimics / inhibitor NC were completed by Beijing Qingke Biotechnology Co., Ltd. The specific sequence information is shown in Table 1.

[0063] Table 1. Mimics / inhibitor sequences of miR-615

[0064]

[0065] The dry powder centrifuge tube containing the above sequence was centrifuged at 2000 rpm for 2 min, RNase-free ddH2O was added to prepare a 20 μL solution and then stored at -20°C.

[0066] When the density of porcine ovarian granulosa cells in the culture dish reached about 40%, it was transfected into the porcine ovarian granulosa cells, and the cells were collected after 24 hours for subsequent experiments.

[0067] RT-qPCR detection showed that after transfection with miR-615mimics, the expression level of miR-615 in pig ovarian granulosa cells was significantly increased compared with the mimics NC group, and the expression level of miR-615 was significantly inhibited after transfection with miR-615inhibitor (e.g. Figure 2 A).

[0068] Through CCK-8 test (such as Figure 2 As shown in B): It was confirmed that miR-615 mimics could significantly promote cell viability, while the viability of porcine ovarian granulosa cells in the miR-615 inhibitor group was significantly inhibited.

[0069] EdU staining (eg Figure 2 C), it was found that the miR-615 mimics group significantly increased the proportion of positive proliferative cells. The above results indicate that miR-615 can promote the viability and proliferation of porcine ovarian granulosa cells.

[0070] Example 3

[0071] To further understand the effect of miR-615 on steroid hormone production in porcine ovarian granulosa cells, miR-615 mimics, miR-615 inhibitor and respective controls were transfected into porcine ovarian granulosa cells. After 24 hours, porcine ovarian granulosa cells and culture dish supernatants were collected for subsequent detection. Elisa results showed that miR-615 mimics could significantly promote the expression of estradiol (such as Figure 3 A) and progesterone (as Figure 3 B) content; and miR-615inhibitor can significantly inhibit the levels of estradiol and progesterone.

[0072] The expression of steroid hormone secretion-related genes 3β-HSD and StAR was detected by RT-qPCR, and the results showed that miR-615 mimics could significantly promote 3β-HSD (such as Figure 3 C) and StAR (as Figure 3 D). After transfection with miR-615inhibitor, the expression of these two genes was significantly inhibited. This indicates that miR-615 can promote the production of steroid hormones in porcine ovarian granulosa cells and promote the expression of hormone-related 3β-HSD and StAR genes.

[0073] Example 4

[0074] miR-615 mimics and miR-615 inhibitor were transfected into porcine ovarian granulosa cells, and the effects of overexpression and inhibition of miR-615 on the expression level of ACSL4 were verified by RT-qPCR (e.g. Figure 4 (A) It was found that after overexpression of miR-615, the expression of ACSL4 was extremely significantly inhibited, while inhibiting the expression of miR-615 significantly promoted the expression level of ACSL4.

[0075] To elucidate whether miR-615 directly targets the 3'-UTR region of ACSL4, wild-type (ACSL4-wt) and mutant (ACSL4-mut) plasmids containing miR-615 binding sites were constructed and co-transfected with miR-615 mimics or mimics NC into 293T cells for dual luciferase activity assay (Fig. Figure 4 B).

[0076] The results showed that miR-615 mimics could significantly inhibit the fluorescence activity of ACSL4 wild-type vector, while the mutant vector had no significant change, indicating that ACSL4 is a direct target gene of miR-615.

[0077] Example 5

[0078] The design and synthesis of the ACSL4 interference fragment siRNA were completed by Beijing Tsingke Biotechnology Co., Ltd. (Tsingke, Beijing, China). The sequence information of siRNA and negative control (Negative Control, NC) is shown in Table 2.

[0079] Table 2 siRNA sequences for RNA interference assay

[0080]

[0081] miR-615inhibitor and ACSL4-siRNA were co-transfected into porcine ovarian granulosa cells. The results showed that the expression of ACSL4 in the porcine ovarian granulosa cells co-transfected with miR-615inhibitor and ACSL4-siRNA was significantly higher than that in the co-transfected miR-615inhibitor and NC-siRNA group, while the expression of ACSL4 in the porcine ovarian granulosa cells co-transfected with inhibitor NC and NC-siRNA was significantly lower than that in the co-transfected miR-615inhibitor and NC-siRNA group. This indicates that the increase in the expression level of ACSL4 gene and protein induced by miR-615inhibitor was downregulated by ACSL4-siRNA (such as Figure 5 A and Figure 5 B).

[0082] CCK-8 and EdU staining showed that knockdown of ACSL4 inhibited the reduction of cell viability and proliferation induced by miR-615inhibitor (e.g. Figure 5 C and Figure 5 The above results indicate that miR-615 regulates the viability and proliferation of porcine ovarian granulosa cells by targeting the ACSL4 gene.

[0083] Example 6

[0084] Hormone detection was performed on pig ovarian granulosa cells co-transfected with miR-615inhibitor and ACSL4-siRNA. The results showed that knockdown of ACSL4 inhibited the extremely significant downregulation of estradiol and progesterone induced by miR-615inhibitor (such as Figure 6 A and Figure 6 B).

[0085] The expression of hormone secretion-related genes 3β-HSD and StAR was detected by RT-qPCR, and it was found that the expression of related genes in pig ovarian granulosa cells co-transfected with miR-615inhibitor and ACSL4-siRNA showed a very significant up-regulation trend compared with the co-transfected NC-siRNA group (such as Figure 6 C and Figure 6 D). This indicates that miR-615 can target ACSL4 to inhibit steroid hormone production and hormone-related gene expression in porcine ovarian granulosa cells.

[0086] Example 7

[0087] Three lnc-GAM-siRNAs were synthesized to interfere with the expression of lnc-GAM. The design and synthesis of the required lnc-GAM interference fragment siRNA were completed by Beijing Qingke Biotechnology Co., Ltd. (Tsingke, Beijing, China). The siRNA sequence information is shown in Table 3.

[0088] Table 3 siRNA sequences for RNA interference assay

[0089]

[0090] The interference efficiency was verified by RT-qPCR, and it was found that lnc-GAM-siRNA-1 could significantly inhibit the expression of lnc-GAM (such as Figure 7 A), and used it as an effective lnc-GAM-siRNA for subsequent experiments.

[0091] At the same time, the lnc-GAM overexpression vector lnc-GAM-OE of pig ovarian granulosa cells was constructed based on the pcDNA3.1 plasmid, and the overexpression efficiency of lnc-GAM-OE was further verified. It was found that the expression level of lnc-GAM in the pig ovarian granulosa cells transfected with lnc-GAM-OE was significantly increased compared with that in the control group (pcDNA3.1) (e.g. Figure 7 B), indicating the effectiveness of lnc-GAM overexpression and interference vector in pig ovarian granulosa cells. Elisa results showed that lnc-GAM overexpression can significantly inhibit the production of estradiol and progesterone (as shown in Figure 7 C), and inhibited the expression of hormone secretion-related genes 3β-HSD and StAR (as shown in Figure 7 D).

[0092] Example 8

[0093] Further EdU staining showed that overexpression of lnc-GAM significantly inhibited the proliferation of porcine ovarian granulosa cells (e.g. Figure 8 A). RT-qPCR detection of apoptosis-related genes (as shown in Figure 8 B), it was found that overexpression of lnc-GAM can promote the expression of pro-apoptotic genes such as Bax, BAD, and AIF, inhibit the expression of Bcl2 gene, and increase the Bax / Bcl2 ratio. The above results show that lnc-GAM can inhibit the proliferation of porcine ovarian granulosa cells and the production of steroid hormones, and promote the progression of cell apoptosis.

[0094] Example 9

[0095] RT-qPCR was used to verify the effects of lnc-GAM overexpression and interference on ACSL4. It was found that when lnc-GAM was overexpressed, the expression level of ACSL4 increased, and when lnc-GAM expression was interfered, the expression of ACSL4 was also downregulated (e.g. Fig. 9 A), preliminarily confirmed the expression relationship between lnc-GAM and ACSL4.

[0096] Subsequently, miR-615inhibitor and lnc-GAMsiRNA were co-transfected into porcine ovarian granulosa cells. RT-qPCR detection revealed that lnc-GAMsiRNA could significantly reduce the expression level of ACSL4 in porcine ovarian granulosa cells, but miR-615inhibitor could reverse this process (e.g. Fig. 9 B), and WB results showed that lnc-GAM siRNA could significantly reduce the protein expression level of ACSL4 in pig ovarian granulosa cells, but miR-615 inhibitor could reverse this effect (as shown in Fig. 9 C).

[0097] Through CCK-8 detection and EdU staining, it was found that inhibiting the expression of lnc-GAM could significantly increase cell viability and proliferation, and miR-615inhibitor could significantly reverse the effect of lnc-GAM siRNA (such as Fig. 9 D and Fig. 9 E).

[0098] Example 10

[0099] Hormone detection was performed on pig ovarian granulosa cells co-transfected with miR-615inhibitor and lnc-GAM-siRNA. The results showed that compared with the inhibitor NC and NC-siRNA groups, the introduction of lnc-GAM-siRNA led to a decrease in the levels of estradiol and progesterone in pig ovarian granulosa cells, while after miR-615inhibitor was transferred into pig ovarian granulosa cells, the hormone levels showed a significant reversal compared with the inhibitor NC and lnc-GAM-siRNA co-transfection group, and the hormone content increased (such as Fig.10 A and Fig.10 B).

[0100] The expression of hormone secretion-related genes 3β-HSD and StAR was detected by RT-qPCR, and it was found that the expression of related genes in pig ovarian granulosa cells co-transfected with miR-615inhibitor and lnc-GAM-siRNA showed a very significant down-regulation trend compared with the co-transfected NC-siRNA group (such as Fig.10 C and Fig.10 D). This indicates that lnc-GAM competitively binds to miR-615 and upregulates the expression of ACSL4, thereby inhibiting the production of steroid hormones and the expression of hormone-related genes in porcine ovarian granulosa cells.

[0101] In summary, the addition of FSH to the porcine ovarian granulosa cell culture system can affect the expression of lnc-GAM, miR-615 and ACSL4, which is specifically manifested in that FSH promotes the expression of miR-615 and inhibits the expression of lnc-GAM and ACSL4, indicating that lnc-GAM, miR-615 and ACSL4 may be involved in the regulation of the development of pig follicles. By overexpressing and inhibiting miR-615 in porcine ovarian granulosa cells, it was found that miR-615 can significantly promote the proliferation and vitality of porcine ovarian granulosa cells, and the overexpression of miR-615 can promote the production of estradiol and progesterone, and also promote the expression of steroid hormone secretion-related genes 3β-HSD and StAR. At the same time, the expression level of ACSL4 in porcine ovarian granulosa cells is positively correlated with lnc-GAM. Therefore, the inhibitors of miR-615 and lnc-GAM were co-transferred into porcine ovarian granulosa cells, and it was found that lnc-GAM could competitively bind to miR-615 as a ceRNA, thereby releasing the inhibitory effect of miR-615 on ACSL4, thereby inhibiting the proliferation and vitality of porcine ovarian granulosa cells, reducing hormone secretion levels, and promoting the expression of apoptosis genes.

[0102] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Application of miR-615 in any of the following (1)-(5): (1) Preparing products that promote the activity and / or proliferation of ovarian granulosa cells; (2) preparing products that promote the production of estradiol and / or progesterone; (3) preparing products that promote 3β-HSD and / or StAR gene expression; (4) preparing a product that inhibits ACSL4 gene expression; (5) Prepare products that competitively bind to lnc-GAM.

2. The application according to claim 1, characterized in that: The target gene of miR-615 is ACSL4 gene.

3. The application according to claim 1, characterized in that: The primer nucleic acid sequence of miR-615 is AATGCACTCCGAGCCTGGGT.

4. Use of miR-615 inhibitor in any of the following (1)-(4): (1) Preparing products that inhibit the activity and / or proliferation of ovarian granulosa cells; (2) Preparation of products that inhibit the production of estradiol and / or progesterone; (3) preparing products that inhibit 3β-HSD and / or StAR gene expression; (4) Prepare products that promote the expression of ACSL4 gene.

5. The use according to claim 4, characterized in that: The nucleic acid sequence of the miR-615 inhibitor is AGAGGGAGACCCAGGCUCGGA.

6. Application of lnc-GAM in any of the following (1)-(5): (1) preparing a product that competitively binds to miR-615; (2) preparing products that inhibit the activity and / or proliferation of ovarian granulosa cells; (3) Preparation of products that inhibit the production of estradiol and / or progesterone; (4) preparing products that inhibit 3β-HSD and / or StAR gene expression; (5) Prepare products that promote the expression of ACSL4 gene.

7. The use according to any one of claims 1 to 6, characterized in that: The ovarian granulosa cells are porcine ovarian granulosa cells.

8. The use according to any one of claims 1 to 6, characterized in that: The product is a drug or reagent.

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