Application of PLPP3 gene in regulating autophagy in human ovarian granulosa cells

Abstract

The present invention discloses the application of the PLPP3 gene in regulating autophagy in human ovarian granulosa cells, belonging to the field of cell engineering and genetic engineering technology. The present invention discovered for the first time that overexpression of the PLPP3 gene can significantly increase the levels of autophagy and apoptosis in granulosa cells; while interfering with the expression of the PLPP3 gene significantly reduces the levels of autophagy, apoptosis and cell activity in granulosa cells. It was also found that the PLPP3 protein level was low in the ovarian tissue of middle-aged people; while the PLPP3 protein level was high in the ovarian tissue of the elderly. The PLPP3 protein level was positively correlated with the degree of human ovarian aging and can be used as a marker to diagnose / evaluate the level of human ovarian aging. The present invention has great application value in studying the mechanism of the influence of autophagy regulation on ovarian follicle development, reproductive capacity and ovarian-related diseases, and has important clinical value in protecting ovarian function and treating ovarian-related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical fields of cell engineering and genetic engineering, and particularly relates to the application of the PLPP3 gene in regulating autophagy in human ovarian granulosa cells. Background Art

[0002] Existing studies have shown that granulosa cell apoptosis, also known as programmed cell death, is generally considered to be the main cause of follicular atresia, which often leads to decreased ovarian follicle quality and premature ovarian failure in women. Autophagy is a lysosomal degradation mechanism that is essential for cell survival, differentiation, development, and homeostasis. Moderate cell autophagy can maintain the number of primordial follicles and the survival of germ cells. However, excessive cell autophagy will lead to cell death, also known as autophagic cell death (or type II cell death). Studies have shown that autophagy can promote cell apoptosis and participate in the regulation of primordial follicle development and follicular atresia. Autophagy has also been found to be involved in the regulation of polycystic ovary syndrome in women.

[0003] Phospholipid phosphatase 3 (PLPP3) belongs to the phosphatidic acid phosphatase family and is a transmembrane enzyme containing six transmembrane domains. As a glycoprotein localized to the cytoplasmic membrane, PLPP3 hydrolyzes lysophosphatidic acid and short-chain phosphatidic acid, converting phosphatidic acid into diacylglycerol, playing a role in the de novo synthesis of glycerolipids and receptor-activated signaling pathways. Furthermore, PLPP3 has been reported to hydrolyze ceramide phosphate and phosphatol. Through the production of these products, PLPP3 is involved in cell adhesion, cell-cell interactions, angiogenesis, inflammatory responses in related diseases, and cell invasion. Despite these important biological functions, the role of PLPP3 in the development of ovarian granulosa cells and follicles remains unclear. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide an application of the PLPP3 gene in regulating autophagy in human ovarian granulosa cells.

[0005] The present invention constructs the overexpression plasmid pcDNA3.1-PLPP3 (OE-PLPP3) of the PLPP3 gene and synthesizes its interference sequence si-PLPP3 (synthesized by Dongze Company). The autophagy level of granulosa cells was detected using autophagy double-labeled adenovirus (mRFP-GFP-LC3); the effect of PLPP3 gene on the mRNA and protein levels of autophagy signaling pathway marker genes was detected by qRT-PCR and Western blot (WB) experiments; the interaction between PLPP3 gene and autophagy signaling pathway marker genes at the mRNA and protein levels was detected by RNA immunoprecipitation (RIP) and co-immunoprecipitation (Co-IP) methods; the expression localization and interaction of PLPP3 gene and autophagy signaling pathway marker genes were detected by RNA and protein nucleocytoplasmic fractionation experiments; by constructing truncated fragments of GFP-PLPP3 fusion protein, namely Full (containing the full length of PLPP3 protein, 1aa-311aa), TransR-acidPPc (1aa-275aa) and TransR (1aa-125aa), the specific sites of interaction between autophagy marker proteins and PLPP3 protein were detected by Co-IP experiment; the localization and interaction of PLPP3 protein and autophagy marker proteins were detected at the tissue level by tissue immunofluorescence experiment; The V / PI experimental method was used to explore the effect of PLPP3 on the apoptosis of human ovarian granulosa cells; the EdU and CCK8 methods were used to detect the effect of PLPP3 on the proliferation and cell activity of human ovarian granulosa cells.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] Application of the PLPP3 gene in regulating autophagy in human ovarian granulosa cells, wherein the application is any one or more of the following applications:

[0008] I. Application of overexpression of exogenous PLPP3 gene to promote autophagy in ovarian granulosa cells in vitro;

[0009] II. Application of inhibiting PLPP3 gene expression to inhibit autophagy of ovarian granulosa cells in vitro.

[0010] Furthermore, overexpression of the exogenous PLPP3 gene significantly increased the number of autophagosomes and autolysosomes in ovarian granulosa cells, significantly decreased the mRNA and protein levels of the autophagy marker genes p62 and mTOR, and significantly increased the mRNA and protein levels of the autophagy marker genes LC3B and ATG7; inhibition of PLPP3 gene expression significantly decreased the number of autophagosomes and autolysosomes in ovarian granulosa cells, significantly increased the mRNA and protein levels of the autophagy marker genes p62 and mTOR, and significantly decreased the mRNA and protein levels of the autophagy marker genes LC3B and ATG7.

[0011] Furthermore, PLPP3 protein regulates autophagy in nidogenous cells by interacting with p62 protein.

[0012] The use of the PLPP3 gene in regulating the transcription and / or translation activity of the p62 gene in human ovarian granulosa cells, wherein the use is any one or more of the following:

[0013] III. Application of overexpression of exogenous PLPP3 gene to inhibit transcription and / or translation activity of p62 gene in vitro;

[0014] IV. Application of inhibiting PLPP3 gene expression to increase the transcription and / or translation activity of p62 gene in vitro.

[0015] Application of the PLPP3 gene in regulating the activity of human ovarian granulosa cells, wherein the application is any one or more of the following applications:

[0016] V. Application of overexpression of exogenous PLPP3 gene to inhibit ovarian granulosa cell activity in vitro;

[0017] VI. Application of inhibiting PLPP3 gene expression to enhance the activity of ovarian granulosa cells in vitro.

[0018] Furthermore, the overexpression of the exogenous PLPP3 gene is achieved through gene overexpression technology.

[0019] Furthermore, the gene overexpression plasmid used in the gene overexpression technology is prepared by the following method:

[0020] (1) cDNA was extracted from ovarian granulosa cells and PCR amplified using the cDNA as a template to obtain the target fragment;

[0021] (2) The target fragment was ligated to the pcDNA3.1 vector digested with restriction endonucleases XbaI and kpnl to obtain a recombinant plasmid.

[0022] Furthermore, the primers used for PCR amplification in step (1) are as follows:

[0023] Forward: 5′-GG GGTACC CCATGCTGATGGTCCTCCTTGTATC-3′;

[0024] Reverse:5′-GC TCTAGAG CCTACACCATGTTGTGGTGATTGTT-3′.

[0025] Furthermore, the inhibition of PLPP3 gene expression is achieved through RNA interference technology.

[0026] Furthermore, the small interfering fragment sequence used in the RNA interference technology is as follows:

[0027] si-PLPP3: 5′-CTGATGGGTCCTCCTTGTAT-3′.

[0028] Use of an agent for inhibiting PLPP3 gene expression in preparing an agent for inhibiting autophagy of human ovarian granulosa cells, an agent for promoting transcription and / or translation activity of p62 gene in ovarian granulosa cells, and / or an agent for promoting activity of human ovarian granulosa cells.

[0029] Furthermore, the agent for inhibiting PLPP3 gene expression is a small interfering fragment, the sequence of which is as follows:

[0030] si-PLPP3: 5′-CTGATGGGTCCTCCTTGTAT-3′.

[0031] Application of PLPP3 protein quantitative reagent in the preparation of human ovarian aging diagnosis / evaluation kit.

[0032] Furthermore, the PLPP3 protein level is positively correlated with the degree of human ovarian aging.

[0033] Furthermore, the diagnostic / evaluation kit comprises at least one of a PLPP3 protein-specific antibody and a PLPP3 protein immunoblotting reagent.

[0034] The present invention uses protein-protein interactions as a starting point and adopts cell biology methods to study the effect of the PLPP3 gene on the autophagy level of granulosa cells. Specifically, the present invention uses the changes in the number of autophagosomes and autolysosomes in granulosa cells and the expression regulation of autophagy signaling pathway marker genes by PLPP3 as research objects to analyze the effect of PLPP3 on the autophagy level of granulosa cells. It was verified that overexpression of PLPP3 increased the number of autophagosomes and autolysosomes in granulosa cells, reduced the mRNA and protein levels of the autophagy marker genes p62 and mTOR, and increased the mRNA and protein levels of LC3B and ATG7. Through RIP and Co-IP experiments, it was found that PLPP3 protein only interacted with both p62 mRNA and p62 protein; RNA and protein nuclear-cytoplasmic separation experiments showed that PLPP3 and p62 genes were localized and interacted in both the cytoplasm and the nucleus, and PLPP3 negatively regulated the expression of the p62 gene. Furthermore, through the Co-IP experiment of GFP-PLPP3 fusion protein, it was determined that the p62 protein interacts with the transmembrane domain (TransR) of the PLPP3 protein. At the same time, in human ovarian tissue, the interaction between the PLPP3 protein and p62 and LC3B proteins was verified again by Co-IP and immunofluorescence experiments. Finally, at the human granulosa cell level, it was found that overexpression of PLPP3 promoted granulosa cell apoptosis, inhibited cell proliferation, and reduced cell viability. The present invention explores how PLPP3 regulates the level of autophagy in granulosa cells by interacting with the autophagy marker gene p62, thereby increasing the level of granulosa cell apoptosis. This invention has great application value for studying the molecular mechanism of the involvement of the PLPP3 gene in the regulation of follicular development and ovarian aging.

[0035] The verification results of the present invention are as follows:

[0036] In vitro, overexpression of the PLPP3 gene significantly increased the number of autophagosomes and autolysosomes; while interfering with the expression of the PLPP3 gene significantly reduced the number of autophagosomes and autolysosomes.

[0037] In vitro, overexpression of PLPP3 can significantly reduce the mRNA and protein levels of autophagy marker genes p62 and mTOR, significantly increase the mRNA and protein levels of autophagy marker genes LC3B and ATG7, and significantly increase the mRNA levels of autophagy marker genes ATG5, BECN1 and ATG2B; while interfering with the expression of PLPP3 can significantly increase the mRNA and protein levels of autophagy marker genes p62 and mTOR, significantly reduce the mRNA and protein levels of autophagy marker genes LC3B and ATG7, and significantly reduce the mRNA levels of autophagy marker genes ULK1 and ATG2B.

[0038] In vitro, anti-PLPP3 antibodies can significantly enrich p62 mRNA and p62 protein, while anti-p62 antibodies can significantly enrich PLPP3 protein.

[0039] In vitro, PLPP3 can interact with p62 mRNA and p62 protein in the nucleus and cytoplasm, and can negatively regulate the expression of the p62 gene.

[0040] In vitro, p62 protein can bind to the transmembrane region (TransR) of PLPP3.

[0041] In vitro, anti-PLPP3 antibodies significantly enriched p62 protein in human ovarian tissue. PLPP3, p62, and LC3B proteins interact in human ovarian tissue, with PLPP3 negatively correlated with p62 protein levels but positively correlated with LC3B protein levels. In ovarian tissue from middle-aged individuals (approximately 40 years of age), PLPP3 and LC3B protein levels were low, but p62 protein expression was high. In ovarian tissue from elderly individuals (approximately 60 years of age), PLPP3 and LC3B protein levels were high, but p62 protein levels were low.

[0042] In vitro, overexpression of PLPP3 can significantly increase the apoptosis rate of granulosa cells, while interfering with the expression of the PLPP3 gene can significantly reduce the apoptosis rate of granulosa cells.

[0043] In vitro, overexpression of PLPP3 can significantly increase the mRNA levels of apoptosis signaling pathway marker genes CASP3, CASP8, CASP9, BID, p53, PLCY1, BAX and BIM in granulosa cells, and reduce the mRNA levels of MCL1 and CREB1; while interfering with the expression of PLPP3 can significantly reduce the mRNA levels of apoptosis signaling pathway marker genes CASP3, CASP8, CASP9, BID, p53, PLCY1, BAX and BIM, and increase the mRNA levels of MCL1 and CREB1; overexpression of PLPP3 can significantly increase the expression of pro-apoptotic marker proteins BIM, BAX and CASP9, and reduce the expression of anti-apoptotic marker protein MCL1, while si-PLPP3 can significantly reduce the expression of pro-apoptotic marker proteins BIM, BAX and CASP9, and increase the expression of anti-apoptotic marker protein MCL1.

[0044] In vitro, overexpression of the PLPP3 gene can significantly reduce the proliferation rate of human ovarian granulosa cells; while interfering with the expression of the PLPP3 gene can significantly increase the proliferation rate of granulosa cells.

[0045] In vitro, overexpression of PLPP3 can significantly reduce the viability of human ovarian granulosa cells, while interfering with the expression of the PLPP3 gene can significantly increase the viability of granulosa cells.

[0046] In vitro, overexpression of PLPP3 can significantly reduce the mRNA levels of proliferation signaling pathway marker genes CDK4, SP1, PCNA, IKBA and p65 in granulosa cells, and significantly reduce the protein levels of CDK4, PCNA and p65 genes; while interfering with the expression of the PLPP3 gene significantly increased the mRNA levels of proliferation signaling pathway marker genes CDK4, SP1, PCNA, IKBA and p65, and significantly increased the protein levels of CDK4, PCNA and p65 genes.

[0047] The present invention has the following advantages and effects compared to the prior art:

[0048] The technical solution of the present invention is well-designed and the results are reliable. To confirm the effect of PLPP3 on the function of human ovarian granulosa cells by regulating cellular autophagy levels, the present invention conducted multi-level and multi-faceted verification. First, the autophagic cell phenotype and the regulation of mRNA and protein levels of autophagy signaling pathway marker genes were verified. Then, RIP, Co-IP, and the construction of a GFP-PLPP3 fusion protein were used to verify the interaction between the proteins and detect specific regions of protein interaction. Finally, Co-IP and immunofluorescence experiments were used at the tissue level to further verify the localization and interaction of the proteins in the tissue. It was demonstrated that overexpression of PLPP3 increased the level of granulosa cell apoptosis and reduced the proliferation rate and viability of granulosa cells.

[0049] This study, published in the journal Nature Communications, demonstrates that overexpressing the PLPP3 gene significantly increases autophagy and apoptosis in granulosa cells, while disrupting PLPP3 expression significantly reduces these levels. Furthermore, the study found that PLPP3 protein levels are low in the ovarian tissue of middle-aged individuals (approximately 40 years old), while elevated in the ovarian tissue of elderly individuals (approximately 60 years old). This positive correlation between PLPP3 protein levels and the degree of ovarian aging in humans could provide a reference for biomarker diagnosis and assessment of ovarian aging.

[0050] The present invention has great application value in studying the influence mechanism of autophagy regulation on ovarian follicle development, reproductive capacity and ovarian-related diseases, and has important reference value in protecting ovarian function and treating ovarian-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1Figure 2 is a study result on the effect of PLPP3 on the autophagy level of granulosa cells; among them, a and b are the fluorescence images (a) and the statistical results of the changes in the number of autophagosomes and autolysosomes in granulosa cells caused by PLPP3; c is the effect of OE-PLPP3 and si-PLPP3 on the mRNA levels of autophagy signaling pathway marker genes mTOR, p62, LC3B, ATG5, ATG7, BECN1, ATG2B and ULK1; d is the effect of OE-PLPP3 and si-PLPP3 on the protein levels of autophagy signaling pathway marker genes mTOR, p62, LC3B and ATG7.

[0052] Figure 2 The following are the results of the study on the interaction between PLPP3 protein and p62 gene at the mRNA and protein levels; among them, a is the mRNA enrichment of autophagy signaling pathway marker genes on anti-PLPP3 antibody in RIP experiment; b is the protein enrichment of autophagy signaling pathway marker genes on anti-PLPP3 antibody in Co-IP experiment; c is the protein enrichment of PLPP3 protein on anti-p62 antibody in Co-IP experiment; d and e are the localization of PLPP3 and p62 genes in the cytoplasm and nucleus in RNA and protein nuclear-cytoplasmic separation experiments, and the effect of PLPP3 on p62 mRNA level (d) and p62 protein level (e).

[0053] Figure 3 This is a diagram showing the research results of the specific sites of interaction between p62 protein and PLPP3 protein; among them, a is a schematic diagram of the protein domain of PLPP3 and the truncation treatment of GFP-PLPP3 fusion protein; b is the interaction between p62 protein and PLPP3 protein specific sites in the Co-IP experiment.

[0054] Figure 4 Figure 2 is a study on the interaction between PLPP3, p62, and LC3B proteins in human ovarian tissue. Figure a shows the protein enrichment of the p62 gene in human ovarian granulosa cells in response to anti-PLPP3 antibodies. Figures b and c show the co-localization and interaction of PLPP3, p62, and LC3B proteins in the ovarian tissue of middle-aged and elderly individuals in tissue immunofluorescence experiments.

[0055] Figure 5Figure 1 is a study result showing the effect of PLPP3 on the apoptosis level of granulosa cells; wherein, a and b are the effects of OE-PLPP3 (a) and si-PLPP3 (b) on the apoptosis rate of granulosa cells; c is the effect of OE-PLPP3 and si-PLPP3 on the mRNA levels of apoptosis signaling pathway marker genes CASP3, CASP8, CASP9, BID, p53, PLCY1, BAX, BIM, MCL1 and CREB1; d is the effect of OE-PLPP3 and si-PLPP3 on the protein levels of apoptosis signaling pathway marker genes BIM, BAX and CASP9;

[0056] Figure 6 Figure 5 is a study result of the effect of PLPP3 on the proliferation rate and cell viability of granulosa cells; among them, a and b are the fluorescence image (a) and cell proliferation rate statistical graph (b) of the effect of OE-PLPP3 and si-PLPP3 on the proliferation rate of granulosa cells in the EdU experiment; c and d are the effects of OE-PLPP3 (c) and si-PLPP3 (d) on the viability of granulosa cells; e is the effect of OE-PLPP3 and si-PLPP3 on the mRNA levels of proliferation signaling pathway marker genes CDK4, SP1, PCNA, IKBA and p65; f is the effect of OE-PLPP3 and si-PLPP3 on the protein levels of proliferation signaling pathway marker genes CDK4, PCNA and p65. DETAILED DESCRIPTION

[0057] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0058] The experimental methods in the following examples without specific conditions are generally based on conventional conditions.

[0059] In the following examples, statistical methods were applied to analyze the results of three independent experiments in each example, and the "mean ± standard deviation" was calculated. The significance of the differences was analyzed using one-way analysis of variance ("*" indicates P < 0.05, and "**" indicates P < 0.01 in the figure).

[0060] Example 1: Cultivation of human ovarian cancer granulosa cell line COV434

[0061] The present invention uses COV434 cell line (ATCC) and complete culture medium (containing 10% calf serum and 1% double antibody), and cultured in a 37°C, 5% CO2 incubator. Transfection or drug treatment is performed when the cell confluence reaches 50%-70%, and subsequent experiments can be performed after 24 hours.

[0062] Example 2: Construction of PLPP3 overexpression vector and interference fragment

[0063] (1) Primers were designed using the NCBI website, and the CDS region of PLPP3 (Gene ID: 8613) was amplified using cDNA extracted from human granulosa cells as a template. The amplified fragment was purified, recovered, ligated to the pMD18T vector (purchased from Takara), transformed, screened, and sequenced to extract the common plasmid.

[0064] (2) Xba I and Kpn I restriction site sequences were added to the upstream and downstream primers of PLPP3, respectively. PCR amplification was performed using the recombinant pMD18T common plasmid of the PLPP3 CDS region as a template. The fragment was purified, recovered, double-enzyme digested, ligated to the pcDNA3.1 vector, transformed, screened, and sequenced to extract the endotoxin-free plasmid (Magen, USA) and named pcDNA3.1-PLPP3 (OE-PLPP3).

[0065] The PLPP3 gene CDS region primers used in the present invention are:

[0066] Forward: 5′-GG GGTACC CCATGCTGATGGTCCTCCTTGTATC-3′;

[0067] Reverse:5′-GC TCTAGAG CCTACACCATGTTGTGGTGATTGTT-3′.

[0068] (3) Designed and commissioned Dongze Company to synthesize the small interfering fragment sequence of the PLPP3 gene:

[0069] si-PLPP3: 5′-CTGATGGGTCCTCCTTGTAT-3′.

[0070] (4) Observe the cell status. When the cell confluence reaches about 50%-70%, 1000 ng / mL of PLPP3 overexpression vector (OE-PLPP3) and overexpression control (OE-NC) are transfected into the cells, and 100 nmol / L of PLPP3 interference fragment (si-PLPP3) and interference control (si-NC) are transfected into the cells, respectively. Other subsequent experiments are carried out after 24 h.

[0071] Example 3: Detection of autophagy levels

[0072] Hanheng Bio was commissioned to synthesize autophagy double-labeled adenovirus (HBAD-mRFP-GFP-LC3). When the cell confluence was 50%, the cells were washed twice with PBS, 1 / 2 new complete medium was added, and the cells were infected with 30MOI of autophagy double-labeled adenovirus. After 4 hours, the original medium was aspirated and new medium was added. At the same time, the cells were transfected with the overexpression vector and interference fragment of PLPP3, incubated in the incubator for 24 hours, and washed twice with PBS. A confocal fluorescence microscope was used to take pictures and count the number of cell autophagosomes and autolysosomes. mRFP-labeled autophagosomes are red, and GFP-labeled autophagosomes are green. After the fusion of autophagosomes and lysosomes, autolysosomes are formed, and GFP is quenched. The accumulation of mRFP dots represents autolysosomes, and the co-localization of mRFP and GEP dots indicates autophagy.

[0073] Example 4: qRT-PCR

[0074] The qRT-PCR detection of the genes in the present invention was performed using the YEASEN SYBR Green qPCR Master Mix (2X) kit. The experiment uses the comparative Ct value method to detect the content of sample genes. The specific calculation formula is as follows:

[0075] Relative gene expression level = 2-{〈﹙Ct value of target gene in experimental group﹚-﹙Ct value of internal reference gene in experimental group﹚〉-〈﹙Ct value of target gene in control group﹚-﹙Ct value of internal reference gene in control group﹚〉}

[0076] GAPDH was used as an internal reference gene for detection. The qRT-PCR primers used in the present invention are:

[0077] qPLPP3 Forward: 5′-GGAGGAGCAGGAGGAGCCG-3′;

[0078] Reverse: 5′-AGTCCAAGGGGAAGAGAGC-3′;

[0079] qP62 Forward: 5′-TGTGTAGCGTCTGCGAGGGAAA-3′;

[0080] Reverse: 5′-AGTGTCCGTGTTTCACCTTCCG-3′;

[0081] qLC3B Forward: 5′-GAGAAGCAGCTTCCTGTTCTGG-3′;

[0082] Reverse: 5′-GTGTCCGTTCACCAACAGGAAG-3′;

[0083] qATG5 Forward: 5′-GCAGATGGACAGTTGCACACAC-3′;

[0084] Reverse: 5′-GAGGTGTTTCCAACATTGGCTCA-3′;

[0085] qATG7 Forward: 5′-CGTTGCCCACAGCATCATCTTC-3′;

[0086] Reverse: 5′-CACTGAGGTTCACCATCCTTGG-3′;

[0087] qBECN1 Forward: 5′-CTGGACACTCAGCTCAACGTCA-3′;

[0088] Reverse: 5′-CTCTAGTGCCAGCTCCTTTAGC-3′;

[0089] qULK1 Forward: 5′-GCAAGGACTCTTCCTGTGACAC-3′;

[0090] Reverse: 5′-CCACTGCACATCAGGCTGTCTG-3′;

[0091] qmTOR Forward: 5′-AGCATCGGATGCTTAGGAGTGG-3′;

[0092] Reverse: 5′-CAGCCAGTCATCTTTGGAGACC-3′;

[0093] qATG2B Forward: 5′-CTTCAGATGGAGTTGGAGGAGAC-3′;

[0094] Reverse: 5′-AGTGGCTCCTTTCAGTCCTACG-3′;

[0095] qGAPDH Forward: 5′-TCACCAGGGCTGCTTTTAACT-3′;

[0096] Reverse: 5′-CTTGACTGTGCCGTGGAACT-3′;

[0097] The total RNA of cells was extracted according to the instructions of the RNA fast 200-total RNA rapid extraction kit of Shanghai Feijie Company. The specific steps are as follows:

[0098] (1) Add 500 μl of RA2 solution to the granulosa cells digested with trypsin and mix thoroughly by inversion for 1 minute.

[0099] (2) Aspirate or pour the sample lysate into the inner tube and centrifuge for 1 minute.

[0100] (3) Discard the liquid in the outer cannula, add 500 μl of washing solution to the inner cannula, centrifuge for 1 minute, and repeat this washing process once more.

[0101] (4) Remove the inner cannula, discard the liquid in the outer cannula, put it back into the inner cannula without adding washing solution, and centrifuge for 1 minute.

[0102] (5) Move the inner cannula into a new eppendorf tube, add 25-50 μl of elution buffer (or DEPC-treated water with a pH greater than 7.0) to the center of the membrane, let it stand at room temperature for 1 min, and centrifuge for 1 min to obtain total RNA.

[0103] Using PrimeScript from TaKaRa TM Total RNA was reverse transcribed using the RT Master Mix (Perfect Real Time) cDNA Reverse Transcription Kit.

[0104] Example 5: Western Blot

[0105] (1) Total cell protein extraction

[0106] ① Prepare protein lysis buffer: Add 1% proteasome inhibitor to RIPA protein lysis buffer (White Shark Company) and mix by inverting.

[0107] ② Add 120 μl of protein lysis buffer to each well (six-well plate), place on ice for 10-15 minutes, take out and shake gently in the middle to promote cell detachment.

[0108] ③ Collect the lysed blood cells, centrifuge at 12,000g, 4℃ for 10 min, carefully aspirate the supernatant, and remove the lower precipitate.

[0109] (2)SDS-PAGE:

[0110] ① Protein quantification (BCA protein quantification kit from Biyuntian): A multifunctional microplate reader was used to measure A 562 The OD value of each wavelength was used to draw a standard curve, and the concentration of each protein sample was calculated by the regression equation and the average OD value of each protein standard;

[0111] ②Sample preparation: Mix 20 μg of total protein with 5× loading buffer at a ratio of 5:1 and boil for 10 minutes;

[0112] ③ Preparation of gel sample: Add 20 μg protein to each well and run the gel at 120-150 V for about 40 minutes (stop electrophoresis when bromofen blue runs to about 1-2 cm from the bottom of the gel; or adjust the running voltage and time according to the specific situation);

[0113] ④ Cut the gel strip containing the target protein according to the molecular weight standard of the protein marker and select the transfer current of 300mA for 20min;

[0114] ⑤ After transfer, rinse the membrane gently with TBST for 5 minutes and block with 5% skim milk powder at room temperature for 2 hours;

[0115] ⑥ Gently rinse the membrane twice with TBST, 5 min each time, dilute the primary antibody PLPP3 (purchased from Beijing Biosun Company) with TBST according to the antibody instructions, and incubate at 4°C overnight;

[0116] ⑦ Gently rinse the membrane with TBST three times, 10 minutes each time, and incubate with the secondary antibody Goat anti-rabbit IgG (purchased from SAB) at a dilution of 1:10,000 at room temperature for 2 hours;

[0117] ⑧ Gently rinse the membrane with TBST three times, 10 min each time;

[0118] ⑨ The ultra-sensitive ECL chemiluminescence kit (Biyuntian, China) was used for color development, and the protein bands were observed and photographed using the Tianneng gel forming system (Tianneng, China). The protein bands in the film were analyzed using Image J software.

[0119] Example 6: RIP experiment

[0120] RIP assays were performed using the EZ-Magna RIP kit (Millipore, USA) according to the manufacturer's instructions. Pellets were collected from 6-well plates using trypsin and lysed with the lysis buffer provided in the kit. The cells were then divided into three groups: IP, IgG, and input. The IP group was immunoprecipitated overnight at 4°C using an anti-PLPP3 antibody, with homologous IgG used as a control. After washing and elution, the enriched RNA samples were analyzed by RT-qPCR. Specific primers for qRT-PCR are detailed in Example 4.

[0121] Example 7: Co-IP experiment

[0122] Co-IP experiments were performed using the EZ-Magna Co-IP Kit (Millipore, USA). Collected cells were immunoprecipitated overnight at 4°C with antibodies against PLPP3 (Boaosen, China), p62 (Affinity, USA), LC3B (Affinity, USA), ATG7 (Affinity, USA), ATG5 (Affinity, USA), and BECN1 (Affinity, USA). For negative validation, IgG was used as a control. Enriched proteins were detected by Western blotting.

[0123] Example 8: RNA nuclear-cytoplasmic separation experiment

[0124] Cytoplasmic and nuclear RNA extraction kits (Norgen Biotek, Canada) were used to extract and purify cytoplasmic and nuclear RNA. The specific steps are as follows:

[0125] ① Transfer the digested cells to an RNase-free tube and centrifuge for 10 minutes to collect the cell pellet. Add Lysis Buffer J to the pellet and vortex to fully lyse the cells.

[0126] ② Transfer the lysate to a new RNase-free microcentrifuge tube and centrifuge at maximum speed for 10 minutes. Transfer the supernatant containing the cytoplasmic RNA to another RNase-free tube. The precipitate is the nuclear RNA fraction.

[0127] ③ Extract and purify RNA. Add buffer to the supernatant containing cytoplasmic RNA and the pellet containing nuclear RNA, respectively. Vortex and mix for 10 seconds. Then add 96%-100% ethanol and vortex and mix for 10 seconds. Centrifuge for 1 minute, wash three times with Wash Solution A, and elute the purified RNA with Elution Buffer E.

[0128] ④ After the purified RNA is reverse transcribed into cDNA, the expression level of the target gene is detected by qRT-PCR.

[0129] Example 9: Protein nuclear-cytoplasmic separation experiment

[0130] NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, USA) were used to extract and purify cytoplasmic and nuclear proteins. The specific steps are as follows:

[0131] ① Add ice-cold Cytoplasmic Extraction Reagent I (CER I) to the collected cell pellet, vortex for 15 seconds to completely suspend the cell pellet, and incubate on ice for 10 minutes.

[0132] ② Add ice-cold Cytoplasmic Extraction Reagent (IICER II), vortex for 5 seconds, and incubate on ice for 1 minute. Vortex again and centrifuge to collect the supernatant, which is the cytoplasmic protein. Transfer it to a new centrifuge tube and store until further use. The cell pellet at the bottom is the nucleus.

[0133] ③ Add ice-cold nuclear extraction reagent NER to the nuclear pellet, vortex for 15 seconds every 10 minutes, vortex 4 times in total, and continue for 40 minutes.

[0134] ④ Centrifuge for 10 minutes, transfer the supernatant, i.e. nuclear protein, to a clean pre-cooled tube and store for later use.

[0135] ⑤ Add SDS to the extracted cytoplasm and cytoplasmic proteins for denaturation and use in subsequent WB experiments to detect the expression level of the target protein.

[0136] Example 10: Tissue Immunofluorescence

[0137] The specific steps are as follows:

[0138] ① Paraffin sections of human ovaries (obtained from the Gynecological Reproductive Medicine Center of the First Affiliated Hospital of Sun Yat-sen University, approved by the Medical Ethics Committee) were placed in dewaxing solution and anhydrous ethanol for 10-15 minutes respectively.

[0139] ② Place the washed ovarian slices in a retrieval box filled with citric acid antigen retrieval solution and perform antigen retrieval in a microwave oven.

[0140] ③ After cooling naturally, wash the slides three times with PBS on a decolorizing shaker for 5 minutes each. Use a histochemical pen to draw a circle around the tissue to prevent liquid loss.

[0141] ④ After sealing the slides with 3%-5% BSA for 30 minutes, incubate with the primary antibody at 4°C overnight and the secondary antibody at 37°C for 3 hours.

[0142] ⑤ Add DAPI dye solution and incubate at room temperature for 5 minutes in the dark to locate the target protein. Then seal the slices with a resin sealant and observe and collect slice images under a fluorescence microscope.

[0143] Example 11: Granulocyte apoptosis detection

[0144] The present invention uses Annexin V-FITC technology to detect cell apoptosis. Referring to the instructions of the Annexin V-FITC Apoptosis Detection Kit of BioVision, the specific operation steps are as follows:

[0145] (1) Cells were seeded in 6-well plates and cultured until the confluence reached 50%–70%. PLPP3 overexpression plasmids and interference fragments were transfected using the lip3000 reagent (Thermo Fisher Scientific, USA). The cells were cultured for 24 h and then washed with PBS.

[0146] (2) Digest the cells with EDTA-free trypsin and wash the cells with 2 mL of PBS solution;

[0147] (3) Take 0.5 mL of cell suspension (about 5×10 5 cells), add 500 μL 1× Binding Buffer;

[0148] (4) Add 5 μL Annexin V-FITC and 5 μL Propidium Iodide at room temperature and incubate at room temperature in the dark for 5 min;

[0149] (5) Immediately analyze the cells using flow cytometry (3 replicates per group).

[0150] Example 12: Cell proliferation detection

[0151] The present invention uses EdU kit (Ruibo, China) to detect granulosa cell proliferation, referring to the Cell-Light TM EdU Apollo 567 In vitro Kit instructions. The specific steps are as follows:

[0152] (1) Cells were seeded in 48-well plates and cultured until the confluence reached 50% to 70%. PLPP3 overexpression plasmids and interference fragments were transfected using the lip3000 reagent (Thermo Fisher Scientific, USA) and cultured for 24 h.

[0153] (2) Prepare 50 μM EdU culture medium by diluting the EdU solution 1000:1 with cell culture medium. Add 200 μL of 50 μM EdU culture medium to each well and incubate for 2 h. Wash the cells with PBS for 3–5 min each time.

[0154] (3) Cell fixation: Add 200 μL of cell fixative (80% acetone diluted with PBS) to each well and incubate at room temperature for 15–30 min. Wash the cells with PBS for 3–5 min each time.

[0155] (4) Cell permeabilization: 200 μL of permeabilization agent (0.5% Triton X-100 in PBS) was added to each well to permeabilize the cells. The cells were incubated for 10 min and then washed with PBS.

[0156] (5) EdU detection: Add 200 μL of The staining reaction solution (prepared in a dark place) was incubated in the cell culture in the dark for 30 min, and the cells were washed with PBS;

[0157] (6) Cell permeabilization again: add 200 μL of permeabilization agent (0.5% TritonX-100 in PBS) to each well to permeabilize the cells, incubate for 10 min, and then wash the cells with PBS;

[0158] (7) DNA staining: Add 200 μL of DAPI reaction solution to each well and incubate at room temperature for 30 min in the dark;

[0159] (8) Fluorescence microscopy (three replicates per group).

[0160] Example 13: Granulocyte Activity Detection

[0161] The present invention uses CCK-8 detection kit (Biyuntian, China) to detect cell activity. The specific operation steps are as follows:

[0162] (1) Cells were seeded in 96-well plates. When the cell confluence reached 50%-70%, PLPP3 overexpression plasmids and interference fragments were transfected using lip3000 reagent (Thermo Fisher Scientific, USA) and cultured for 24 h.

[0163] (2) Add 10 μL of CCK-8 solution (culture medium: CCK8 solution = 10:1) to each well, place in a cell culture incubator and incubate for 1 h, and measure the OD value using an A450 microplate reader.

[0164] result:

[0165] 1. PLPP3 significantly increased the level of autophagy in granulosa cells

[0166] The autophagy double-labeled adenovirus (mRFP-GFP-LC3) (Hanheng Biotechnology, China) was used to detect the effect of PLPP3 on the changes in the number of autophagosomes and autolysosomes in granulosa cells; qRT-PCR and Western blotting experiments were used to detect the effect of PLPP3 on the mRNA and protein levels of marker genes of the autophagy signaling pathway.

[0167] The results of autophagy double-labeled adenovirus assay showed that OE-PLPP3 significantly increased the number of autophagosomes and autolysosomes in granulosa cells ( Figure 1 a, b), while si-PLPP3 significantly reduced the number of autophagosomes and autolysosomes in granulosa cells ( Figure 1 a, b) in the above.

[0168] qRT-PCR results showed that OE-PLPP3 significantly reduced the mRNA levels of autophagy signaling pathway marker genes mTOR and p62, and significantly increased the mRNA levels of LC3B, ATG5, ATG7, BECN1 and ATG2B ( Figure 1 c), but had no significant effect on the ULK1 gene; while si-PLPP3 significantly increased the mRNA levels of mTOR and p62, key marker genes of the autophagy signaling pathway, and significantly reduced the mRNA levels of LC3B, ATG7, ATG2B and ULK1, marker genes of the autophagy signaling pathway, but had no significant effect on ATG5 and BECN1 genes ( Figure 1 c) in the above example.

[0169] WB results showed that OE-PLPP3 significantly reduced the protein levels of autophagy marker genes mTOR and p62 ( Figure 1 d), significantly increased the protein levels of LC3B and ATG7; while si-PLPP3 significantly increased the protein levels of autophagy marker genes mTOR and p62, and significantly decreased the protein levels of LC3B and ATG7 ( Figure 1 d) in the above.

[0170] 2. PLPP3 interacts with p62 gene at the mRNA and protein levels

[0171] RIP, Co-IP and nucleocytoplasmic fractionation experiments were used to detect the interaction between PLPP3 and autophagy signaling pathway marker genes at the mRNA and protein levels.

[0172] The results of RIP and Co-IP showed that compared with other genes in the autophagy signaling pathway, anti-PLPP3 antibody significantly enriched a large amount of p62 mRNA ( Figure 2 a) and p62 protein ( Figure 2 b), and anti-p62 antibody significantly enriched a large amount of PLPP3 protein ( Figure 2 c) in the

[0173] The results of RNA and protein nuclear-cytoplasmic separation experiments showed that PLPP3 interacted with p62 mRNA and p62 protein in both the cytoplasm and nucleus of granulosa cells, and OE-PLPP3 inhibited the transcription of the p62 gene ( Figure 2 d) and translation ( Figure 2 e) in the.

[0174] 3. p62 protein binds to the transmembrane region (TransR) of PLPP3 protein

[0175] GFP-PLPP3 fusion protein was used to treat Figure 3a) and perform Co-IP experiments to detect the specific binding regions of p62 and PLPP3 proteins.

[0176] Co-IP experiments showed that p62 protein interacted with the transmembrane region (TransR) (1-125aa) of PLPP3 ( Figure 3 b) in the above example.

[0177] 4. PLPP3 interacts with p62 protein in human ovaries

[0178] Co-IP and tissue immunofluorescence techniques were used to detect the localization and interaction of PLPP3 protein and p62 protein in the ovaries of middle-aged and elderly people.

[0179] Co-IP experiments showed that anti-PLPP3 antibody significantly enriched p62 protein in human ovarian granulosa cells ( Figure 4 a); Tissue immunofluorescence experiments showed that PLPP3 protein and p62 protein co-localized in human ovarian tissue, and in the ovarian tissue of middle-aged people (about 40 years old), the levels of PLPP3 and LC3B proteins were low, but the level of p62 protein was high ( Figure 4 b); whereas in ovarian tissue of elderly people (about 60 years old), PLPP3 and LC3B protein levels were high, but p62 protein level was low ( Figure 4 c) in the above example.

[0180] 5. PLPP3 gene significantly increased the apoptosis level of ovarian granulosa cells

[0181] Annexin V / PI was used to detect the effect of PLPP3 on the apoptosis rate of granulosa cells; qRT-PCR and Western Blot experiments were used to detect the effect of PLPP3 on the mRNA and protein levels of marker genes of the cell apoptosis signaling pathway.

[0182] Annexin V / PI experimental results showed that OE-PLPP3 significantly increased the apoptosis rate of granulosa cells ( Figure 5 a), while si-PLPP3 significantly reduced the apoptosis rate of granulosa cells ( Figure 5 b) in the above example.

[0183] qRT-PCR results showed that OE-PLPP3 significantly increased the mRNA levels of apoptosis signaling pathway marker genes CASP3, CASP8, CASP9, BID, p53, PLCY1, BAX, and BIM, and significantly decreased the mRNA levels of MCL1 and CREB1 ( Figure 5c), while si-PLPP3 significantly reduced the mRNA levels of apoptosis signaling pathway marker genes CASP3, CASP8, CASP9, BID, p53, PLCY1 and BIM, and significantly increased the mRNA level of MCL1, but had no significant effect on BAX and CREB1 ( Figure 5 c) in the

[0184] WB results showed that OE-PLPP3 significantly increased the protein levels of pro-apoptotic marker proteins BIM, BAX, and CASP9, and decreased the protein level of anti-apoptotic marker protein MCL1 ( Figure 5 d), while si-PLPP3 significantly reduced the protein levels of pro-apoptotic marker proteins BIM, BAX, and CASP9, and increased the protein level of the key anti-apoptotic protein MCL1 ( Figure 5 d) in the above.

[0185] 6. PLPP3 gene significantly reduced the proliferation rate and cell viability of granulosa cells

[0186] The effects of PLPP3 on the proliferation rate and cell viability of granulosa cells were detected using EdU (5-Ethynyl-2'-deoxyuridine) kit (Ribo, China) and CCK8 kit (Biyuntian, China), respectively. The effects of PLPP3 on the mRNA and protein levels of marker genes of cell proliferation signaling pathways were detected by qRT-PCR and Western Blot experiments.

[0187] The results of EdU experiments showed that OE-PLPP3 significantly reduced the proliferation rate of granulosa cells ( Figure 6 a, b), while si-PLPP3 significantly increased the proliferation rate of granulosa cells ( Figure 6 a, b) in the above.

[0188] The results of CCK8 experiments showed that OE-PLPP3 significantly reduced the viability of granulosa cells ( Figure 6 c), while si-PLPP3 significantly increased the viability of granulosa cells ( Figure 6 d) in the above.

[0189] qRT-PCR results showed that OE-PLPP3 significantly reduced the mRNA levels of cell proliferation signaling pathway marker genes CDK4, SP1, PCNA, IKBA, and p65; while si-PLPP3 significantly increased the mRNA levels of cell proliferation signaling pathway marker genes SP1, PCNA, IKBA, and p65, but had no significant effect on CDK4 ( Figure 5 e) in;

[0190] WB results showed that OE-PLPP3 significantly reduced the protein levels of CDK4 and PCNA, marker genes of the cell proliferation signaling pathway, but had no significant effect on the protein level of p65; while si-PLPP3 significantly increased the protein levels of PCNA, CDK4, and p65, marker genes of the cell proliferation signaling pathway ( Figure 5 f) in the above example.

[0191] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. PLPP3 The use of a gene expression specific inhibitor is characterized by: The application is PLPP3 Application of gene expression-specific inhibitors to inhibit autophagy in cultured ovarian granulosa cells in vitro.

2. The use according to claim 1, characterized in that: The PLPP3 The gene expression specific inhibitor is a small interfering fragment with the following sequence: si-PLPP3: 5′-CUGAUGGUCCUCCUUGUAU-3′.

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