CN117866956A - LncRNA SFFD and application thereof in porcine ovarian granulosa cells - Google Patents
LncRNA SFFD and application thereof in porcine ovarian granulosa cells Download PDFInfo
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Abstract
The invention discloses lncRNA SFFD and application thereof in porcine ovarian granulosa cells, belonging to the technical fields of cell engineering and genetic engineering. The invention takes lncRNA SFFD as a research object, and constructs an lncRNA SFFD overexpression vector and synthesizes siRNA, and respectively transfects the lncRNA SFFD overexpression vector or the siRNA into the ovarian granulosa cells, then detects the changes of signal path gene mRNA and protein level related to apoptosis, proliferation and E2 secretion of the ovarian granulosa cells, and finally detects the phenotype changes of the ovarian granulosa cells. The results indicate that lncRNA SFFD can inhibit apoptosis and promote proliferation of ovarian granulosa cells and E2 secretion. The invention has good application value for researching the mechanisms such as ovarian follicle locking, sexual maturity delay and the like by exploring the influence of lncRNA SFFD on ovarian granulosa cells.
Description
Technical Field
The invention belongs to the technical fields of cell engineering and genetic engineering, and particularly relates to lncRNA SFFD and application thereof in porcine ovarian granulosa cells.
Background
Growth and function of Granulosa Cells (GCs) play a dominant role in follicle maturation. Proliferation and E2 secretion of GCs promote follicular growth, whereas excessive apoptosis of GCs results in massive follicular occlusion. It is well known that lncRNA competitively adsorbs mirnas by base pairing, thereby modulating GCs growth, function and follicular development. For example, in sheep, lncRNA FDNCR promotes GCs apoptosis via the miR-543-3p/DCN/TGF- β axis. Downregulation of lncRNA ZNF674-AS1 expression significantly inhibited GCs proliferation and follicular growth.
Disclosure of Invention
To overcome the disadvantages and shortcomings of the prior art, a primary object of the present invention is to provide an lncRNA SFFD.
Another object of the invention is to provide the use of lncRNA SFFD as described above in porcine ovarian granulosa cells.
It is a further object of the present invention to provide small interfering RNA fragments (siRNA) that inhibit the expression of lncRNA SFFD.
In the present invention, we found a novel lncRNA, which was named follicular development promoting factor (stimulatory factor of follicular development, SFFD) according to its function. LncRNA SFFD is located on pig mitochondrial chromosome, and the full length is 734bp.
The aim of the invention is achieved by the following technical scheme:
the invention provides a lncRNA SFFD, the nucleotide sequence of which is shown as SEQ ID NO: 1.
The biological material related to the lncRNA SFFD is any one or a combination of a plurality of the following biological materials;
1) A DNA molecule encoding the lncRNA SFFD;
2) An expression cassette comprising the DNA molecule of 1);
3) A recombinant vector comprising the DNA molecule described in 1), or a recombinant vector comprising the expression cassette described in 2);
4) A small interfering RNA fragment (siRNA) that inhibits SFFD expression of the lncRNA;
5) A recombinant cell comprising the DNA molecule of 1), or a recombinant cell comprising the expression cassette of 2), or a recombinant cell comprising the recombinant vector of 3), or a recombinant cell transfected with the small interfering RNA fragment of 4).
Further, the DNA molecule of 1) can be prepared by: extracting RNA of the porcine ovary granulosa cells, reversely transcribing the RNA into cDNA, and carrying out PCR amplification by taking the cDNA as a template to obtain DNA molecules.
Further, the primers used for PCR amplification are as follows:
lncRNASFFD Forward：
lncRNASFFD Reverse：
further, the recombinant vector in 3) can be prepared by the following method: inserting the DNA molecule between HindIII and KpnI cleavage sites of the pcDNA3.1 vector to obtain a recombinant vector;
the sequence of the DNA molecule is shown in SEQ ID NO: 2.
Further, the small interfering RNA fragments of 4) are as follows:
si-lnc SFFD-1：5′-GGAACAAUAGUAAGCACAAUC-3′；
si-lnc SFFD-2：5′-GUAGCCCAUUUCUUUCCAA-3′；
the lncRNA SFFD or the biological material related to the lncRNA SFFD is applied to the porcine ovarian granulosa cells.
In an in vitro environment, lncRNA SFFD can promote proliferation of porcine ovarian granulosa cells and/or inhibit apoptosis of porcine ovarian granulosa cells.
Further, the lncRNA SFFD or the biological material related to the lncRNA SFFD is applied to the preparation of medicines for regulating and controlling the proliferation and/or apoptosis of porcine ovarian granulosa cells.
The proliferation and/or apoptosis of the pig ovary granular cells are regulated by the following modes:
increasing lncRNA SFFD to promote proliferation of pig ovary granular cells and inhibit apoptosis of pig ovary granular cells; or reduce lncRNA SFFD to inhibit proliferation of pig ovary granular cells and promote apoptosis of pig ovary granular cells;
further, in particular any one or a combination of the following applications:
a) Increasing lncRNA SFFD, and promoting proliferation of porcine ovary granulosa cells;
b) Increasing lncRNA SFFD, and inhibiting apoptosis of porcine ovarian granulosa cells;
c) Reduce lncRNA SFFD, inhibit proliferation of porcine ovary granulosa cells;
d) Reduce lncRNA SFFD and promote apoptosis of porcine ovary granulosa cells.
The use of the lncRNA SFFD or the lncRNA SFFD-related biomaterial described above for modulating E2 (estradiol) production in porcine ovarian granulosa cells.
The regulation of E2 (estradiol) generation in the porcine ovary granular cells is realized by the following modes:
increasing lncRNA SFFD promotes E2 production; and/or reducing lncRNA SFFD inhibits E2 production.
The increase of the lncRNA SFFD is realized by increasing exogenous lncRNA SFFD;
the lncRNASFFD is realized by the way of inhibiting siRNA expressed by lncRNA SFFD from being transfected into porcine ovary granular cells.
The exogenous lncRNASFFD is increased by the following method: connecting lncRNA SFFD to pcDNA3.1 vector to construct super-expression vector containing lncRNASFFD; the overexpression vector containing lncRNA SFFD was then transfected into porcine ovarian granulosa cells.
The invention provides siRNA for inhibiting lncRNA SFFD expression, which has the following sequence:
si-lnc SFFD-1：5′-GGAACAAUAGUAAGCACAAUC-3′；
si-lnc SFFD-2：5′-GUAGCCCAUUUCUUUCCAA-3′。
the verification result of the invention is as follows:
1. 2 pairs of small fragments of interference lncRNA SFFD/control (si-lnc SFFD/siRNA-NC) were synthesized, screened and tested for interference efficiency. As a result, the gene interference small fragment is transfected into the ovary granular cells, and the si-lnc SFFD-2 small fragment with better interference effect is finally screened by qRT-PCR means for subsequent experiments.
si-lnc SFFD-2：5′-GUAGCCCAUUUCUUUCCAA-3′；
2. We transfected pcDNA3.1-lnc SFFD or si-lnc SFFD (si-lnc SFFD-2) into ovarian granulosa cells, and detected the effect of lncRNA SFFD on granulosa cell proliferation-related gene expression and proliferation by qRT-PCR, WB and Edu methods, respectively. qRT-PCR and WB results showed that pcDNA3.1-lnc SFFD promoted the expression levels of cell cycle related genes (PCNA, CDK1, CCNA1 and CCND 1). EdU staining showed that the cell proliferation rate of the pcDNA3.1-lnc SFFD group was significantly higher than that of the pcDNA3.1 group. Meanwhile, si-lnc SFFD inhibits the expression levels of PCNA, CDK1, and CCND 1. The cell proliferation rate of the si-lnc SFFD group was significantly lower than that of the siRNA-NC group. In conclusion, lncRNA SFFD can promote proliferation of porcine ovarian granulosa cells.
3. We transfected pcDNA3.1-lncSFFD or si-lncSFFD-2 into ovary granulosa cells, and detected the effect of lncRNA SFFD on granulosa cell apoptosis-related gene expression and apoptosis by qRT-PCR, WB and Annexin V-FITC method, respectively. qRT-PCR and WB results showed that pcDNA3.1-lnc SFFD inhibited the expression levels of the apoptosis-related genes (Caspase 3, caspase8 and Caspase 9). Flow cytometry analysis showed that the apoptosis rate (early apoptosis + late apoptosis) of the pcdna3.1-lnc SFFD group was significantly lower than that of the pcdna3.1 group. Meanwhile, si-lnc SFFD promotes the expression levels of Caspase3, caspase8 and Caspase 9. The apoptosis rate of the si-lnc SFFD group is significantly higher than that of the siRNA-NC group. In conclusion, lncRNASFFD can inhibit apoptosis of porcine ovarian granulosa cells.
4. We transfected pcDNA3.1-lnc SFFD or si-lnc SFFD (si-lnc SFFD-2) into ovarian granulosa cells, and tested the effects of lncRNA SFFD on granulosa cell E2 secretion-related gene expression and E2 secretion by qRT-PCR, WB and ELISA methods, respectively. qRT-PCR and WB results showed that pcDNA3.1-lnc SFFD promoted the expression level of genes related to E2 secretion by cells (CYP 11A1, HSD17B1 and HSD3B 1). ELISA results showed that the E2 concentration of pcDNA3.1-lnc SFFD group was significantly higher than that of pcDNA3.1 group. Meanwhile, si-lnc SFFD inhibits the expression levels of CYP11A1, HSD17B1, and HSD3B 1. The E2 concentration of the si-lnc SFFD group was significantly lower than that of the siRNA-NC group. In conclusion, lncRNA SFFD can promote secretion of porcine ovarian granulosa cells E2.
Compared with the prior art, the invention has the following advantages and effects:
(1) The lncRNASFFD may directly or indirectly participate in follicular locking, follicular maturation and sexual maturation, and the application of the lncRNASFFD in porcine ovarian granulosa cells is researched by taking the lncRNASFFD (see SEQ ID NO: 1) as a research object and adopting a molecular and cell biological method: lncRNA SFFD can inhibit apoptosis and promote proliferation and E2 secretion of ovarian granulosa cells. Has good application value for researching ovarian follicle locking, sexual maturity delay and the like.
(2) The technical scheme of the invention has detailed design and reliable result. To demonstrate the effect of lncRNA SFFD on ovarian granulosa cell proliferation, apoptosis and E2 secretion, the invention validated at multiple levels, angles, at related signaling pathway genes, mRNA and protein levels, and finally on the phenotype of ovarian granulosa cells.
Drawings
FIG. 1 is a graph showing the overexpression and interference efficiency of qRT-PCR detection of lncRNA SFFD; wherein A is an overexpression efficiency map of pcDNA3.1-lnc SFFD, and B is a si-lnc SFFD interference efficiency map.
FIG. 2 is a graph showing the effect of lncRNA SFFD on granulosa cell proliferation; wherein A is the effect of overexpression and interference of lncRNASFFD on mRNA expression level of genes related to proliferation of granulosa cells; b is the influence of overexpression and interference lncRNA SFFD on the protein expression level of genes related to proliferation of granulosa cells; c overexpression and interference of lncRNA SFFD on granulosa cell proliferation rate.
FIG. 3 is a graph showing the effect of lncRNA SFFD on granulosa apoptosis; wherein A is the effect of overexpression and interference of lncRNA SFFD on mRNA expression level of genes related to apoptosis of granulosa cells; b is the influence of overexpression and interference lncRNA SFFD on the protein expression level of genes related to apoptosis of granular cells; c is the effect of overexpression and interference of lncRNA SFFD on granulosa apoptosis rate.
FIG. 4 is a graph showing the effect of lncRNA SFFD on granulosa cell E2 secretion; wherein A is the effect of overexpression and interference of lncRNA SFFD on the mRNA expression level of genes related to E2 secretion of granulosa cells; b is the influence of overexpression and interference of lncRNA SFFD on the protein expression level of genes related to E2 secretion of granulosa cells; c is the effect of overexpression and interference of lncRNA SFFD on granulosa cell E2 secretion.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. The experimental procedure, in which specific conditions are not noted in the examples below, is generally followed by conventional conditions.
The results of 3 independent experiments in each example were analyzed using a statistical method in the present invention, and "mean ± standard deviation" was calculated, respectively, and a single factor analysis of variance was used for the analysis of significance of the differences (in the figure, "x" represents P <0.05, "x" represents P < 0.01).
EXAMPLE 1 construction of the overexpression vector of lncRNA SFFD
(1) Amplifying lncRNA SFFD by using cDNA of the extracted pig granular cells as a template through 5 'and 3' RACE; the amplified fragment was purified, recovered, ligated to pMD18T vector (purchased from Takara Co.), transformed, screened, and sequenced to identify the correct plasmid.
(2) The BioEdit software analysis found that the full-length sequence of lncRNASFFD had no HindIII and KpnI restriction sites, whereas pcDNA3.1 vector had HindIII and KpnI restriction sites. HindIII and KpnI cleavage site sequences are added to the upstream and downstream primers respectively. PCR amplification by taking lncRNASFFD recombinant pMD18T common plasmid as a template; the fragment was purified and recovered, digested with two enzymes, ligated with pcDNA3.1 vector, transformed, screened, sequenced and identified correctly, and then the endotoxin-free plasmid was extracted (the endotoxin-free plasmid miniprep kit was purchased from Magen, USA) and designated pcDNA3.1-lnc SFFD.
The lncRNA SFFD primer used in the invention:
lncRNASFFD Forward：
lncRNASFFD Reverse：
note that: the black bolded font is the protecting base, and the underlined is the cleavage site.
EXAMPLE 2 culture and transfection of ovarian granulosa cells
(1) Collecting fresh ovaries of healthy sows, placing the ovaries in PBS buffer solution (on ice) containing 1% of double antibodies, and rapidly transporting the ovaries back to a laboratory for treatment;
(2) Firstly, cleaning an ovary for 3-5 times by using PBS containing 2% of double antibodies outside a cell room, placing the ovary in a beaker, and placing the beaker in a transmission window;
(3) Alcohol wipes the cell room super clean bench, forceps clamp the ovary, the follicular fluid is sucked by a syringe, the ovarian follicular fluid is pumped into a centrifuge tube containing 5mL of DMEM culture fluid, and the follicular fluid is pumped to 9mL in each tube;
(4) Centrifuging at 1000rpm for 5min, discarding supernatant, adding 5mL PBS, blowing, mixing, and cleaning twice;
(5) Preparing a complete culture medium: 89% of DMEM, 10% of serum and 1% of diabody, and mixing the components upside down;
(6) Adding 5mL of complete culture medium to resuspend cell pellet;
(7) Adding 10mL of complete culture medium into a 75mL culture bottle, and then adding the heavy suspension;
(8) Microscopic observation is carried out and then the mixture is placed at 37 ℃ and 5% CO 2 Culturing in an incubator, observing the growth condition of granulosa cells after 24 hours, waiting for the granulosa cells to grow to about 90%, pouring out the culture medium, and washing 3 times by using preheated PBS containing 1% of double antibodies;
(9) Adding trypsin for digestion, placing in an incubator for about 3min, observing that most cells float under a microscope, immediately adding an equivalent stop solution for stopping digestion;
(10) DMEM wash 2 times, during which centrifugation at 1000rpm for 5min;
(11) Gently resuspending the cell pellet with complete medium, uniformly dividing into each well, supplementing volume with complete medium, gently shaking, and culturing in incubator;
(12) Observing the cell state for about 24 hours, and preparing transfection when the cell confluence reaches about 80%;
(13) Transfection procedure was performed as per Invitrogen corporation3000 kit instructions, 3 replicates per group were set;
(14) The transfected well plate was placed at 37℃with 5% CO 2 Culturing in an incubator, and after transfection, observing the cell state for 24-72 hours, and collecting the cells after good growth.
The double antibodies are penicillin and streptomycin.
Example 3qRT-PCR
The qRT-PCR detection of the gene in the invention adopts a Maxima SYBR Green qPCR Master Mix (2X) kit (Thermo Scientific company). The experiment adopts a Ct value comparison method to detect the content of the sample genes, and the specific calculation formula is as follows:
gene expression level=2- { < the experiment set target gene Ct value- > (the experiment set reference gene Ct value- > (the control set target gene Ct value- > (the control set reference gene Ct value)
GAPDH is used as an internal reference for detecting genes, and qRT-PCR primers used in the invention are as follows:
qRT-PCR-SFFD Forward：5′-GCTGAATTGGCAAGGGTTGG-3′；
Reverse：5′-AGCTAGGACCCAAACTGGGA-3′；
qRT-PCR-Caspase3 Forward：5′-ACATGGAAGCAAATCAATGGAC-3′；
Reverse：5′-TGCAGCATCCACATCTGTACC-3′；
qRT-PCR-Caspase8 Forward：5′-GAGCCTGGACTACATCCCAC-3′；
Reverse：5′-GTCCTTCAATTCCGACCTGG-3′；
qRT-PCR-Caspase9 Forward：5′-GCTGAACCGTGAGCTTTTCA-3′；
Reverse：5′-CCTGGCCTGTGTCCTCTAAG-3′；
qRT-PCR-BAX Forward：5′-ACTTCCTTCGAGATCGGCTG-3′；
Reverse：5′-AAAGACACAGTCCAAGGCGG-3′；
qRT-PCR-BCL2 Forward：5′-GATGCCTTTGTGGAGCTGTATG-3′；
Reverse：5′-CCCGTGGACTTCACTTATGG-3′；
qRT-PCR-PCNA Forward：5′-TCGTTGTGATTCCACCACCAT-3′；
Reverse：5′-TGTCTTCATTGCCAGCACATTT-3′；
qRT-PCR-CDK1 Forward：5′-AGGTCAAGTGGTAGCCATGAA-3′；
Reverse：5′-TCCATGAACTGACCAGGAGG-3′；
qRT-PCR-CCNA1 Forward：5′-GCGCCAAGGCTGGAATCTAT-3′；
Reverse：5′-CCTCAGTCTCCACAGGCTAC-3′；
qRT-PCR-CCNB1 Forward：5′-ACGGCTGTTAGCTAGTGGTG-3′；
Reverse：5′-GAGCAGTTCTTGGCCTCAGT-3′；
qRT-PCR-CCND1 Forward：5′-CTTCCATGCGGAAGATCGTG-3′；
Reverse：5′-TGGAGTTGTCGGTGTAGATGC-3′；
qRT-PCR-CYP19A1 Forward：5′-CTGAAGTTGTGCCTTTTGCCA-3′；
Reverse：5′-CTGAGGTAGGAAATTAGGGGC-3′；qRT-PCR-CYP11A1 Forward：5′-TCCCCTCTCCTGGTGACAAT-3′；
Reverse：5′-GCCACATCTTCAGGGTCGAT-3′；
qRT-PCR-STAR Forward：5′-CGACGTTTAAGCTGTGTGCT-3′；
Reverse：5′-ATCCATGACCCTGAGGTTGGA-3′；
qRT-PCR-HSD17B1 Forward：5′-GTCTGGCATCTGACCCATCTC-3′；
Reverse：5′-CGGGCATCCGCTATTGAATC-3′；
qRT-PCR-HSD3B1 Forward：5′-ATCTGCAGGAGATCCGGGTA-3′；
Reverse：5′-CCTTCATGACGGTCTCTCGC-3′；
qRT-PCR-GAPDH Forward：5′-TCACCAGGGCTGCTTTTAACT-3′；
reverse:5'-CTTGACTGTGCCGTGGAACT-3'; total RNA extraction of cells was carried out according to the Takara TRIzol protocol, and the procedure was as follows: (1) granulosa cells are added directly to TRIzol;
(2) Standing at room temperature for 10min to completely lyse cells, centrifuging for 5min at 12000g, removing precipitate, and sucking supernatant into a new RNase-free tube;
(3) Adding 0.2mL of chloroform (1 mL of TRIzol) into the mixture, shaking the mixture vigorously for 15 to 30 seconds, standing the mixture at room temperature for 5 minutes, and centrifuging the mixture at the temperature of 12000g for 15 minutes at the temperature of 4 ℃;
(4) Sucking the upper water phase and placing the upper water phase in a new RNase-free EP tube;
(5) 0.5mL of isopropanol (1 mL of TRIzol) was added, the mixture was gently mixed upside down, left at room temperature for 10min, and centrifuged at 12000g at 4℃for 10min;
(6) After discarding the supernatant, the mixture was left at room temperature, 1mL of 75% ethanol-DEPC (1 mL of TRIzol each) was added along the tube wall to wash RNA, and after centrifugation at 12000g at 4℃for 5min, the supernatant was discarded as much as possible;
(7) Vacuum drying for 5-10 min, taking care to avoid excessive drying of RNA precipitation;
(8) DEPC water was added to dissolve RNA precipitate.
PrimeScript using TaKaRa Corp TM The RT Master Mix (Perfect Real Time) cDNA reverse transcription kit reverse transcribes total RNA.
Example 4Western Blot
(1) Extraction and quantification of total protein from monolayer adherent cells (ovarian granulosa cells in example 2): the cell culture solution was poured off, and the cells were washed three times by adding an appropriate amount of pre-chilled PBS to wash the culture solution. 100-200 mu L of protein lysate and 10 mu L of 100mM PMSF are added to each well of the 6-well plate cells, and the cells are lysed for 30min. The cell lysate was collected and transferred to a 1.5mL centrifuge tube and centrifuged at 14000rpm at 4℃for 5min. Protein sample concentrations were determined using BCA method.
(2) SDS-PAGE electrophoresis: 20 mug total protein and 5 Xloading buffer were mixed in 5:1 and boiled for 5min. SDS-PAGE electrophoresis is carried out until bromophenol blue just goes out of the bottom of the gel;
(3) Transferring: pretreating PVDF film with methanol for 3-5 s, and soaking in transfer liquid for 30min. Taking out the gel, and placing the gel on filter paper to form a gel transfer printing accumulation layer sandwich structure. This operation must remove the bubbles completely. Constant pressure of 100V for 60-120 min;
(4) Immunoblotting: the hybridization membrane was removed, rinsed for 5min with TBST, and repeated three times. The 5% nonfat dry milk solution was blocked at room temperature for 90min and TBST rinsed for 5min and repeated three times. Membranes were incubated overnight at 4 ℃ with diluted primary antibody: CCND1 (26939-1-AP, proteontech, 1:1000), caspase3 (19677-1-AP, proteontech, 1:1000), HSD17B1 (25334-1-AP, proteontech, 1:2000) and Tubulin (11224-1-AP, proteontech, 1:5000). After 3 washes of the membranes with TBST, the membranes were incubated with goat anti-rabbit (ab 205718, abcam, 1:10000) or goat anti-mouse (ab 6789, abcam, 1:5000) secondary antibodies for 2h at room temperature. After ECL luminophor treatment, protein bands were visualized using Odyssey Fc image system, and finally analyzed using ImageJ software.
Example 5 granulosa apoptosis assay
The invention relates to an Annexin V-FITC technology for detecting apoptosis, which refers to an Annexin V-FITC Apoptosis Detection Kit operation instruction of BioVision company, and comprises the following specific operation steps:
(1) Placing the cell culture plate at room temperature, slightly rinsing cells in the culture plate with 2mL of PBS solution, and removing the PBS solution;
(2) Adding EDTA-free pancreatin to digest the cells, and gently resuspending the cells in the medium of step (1) to a density of about 1X 10 6 cells/mL;
(3) 0.5mL of the cell suspension was removed from the cell culture plate (about 5X 10) 5 Individual cells) were transferred to a clean centrifuge tube and 500 μl of 1×binding Buffer was added;
(4) Add 5. Mu.L Annexin V-FITC and room temperature 5. Mu. L propidium iodide;
(5) Reacting for 5min at room temperature in a dark place;
(6) The analysis was immediately examined with a FACSCalibur flow cytometer (three replicates per group).
EXAMPLE 6 granulosa cell proliferation assay
The invention uses EdU method to detect Cell proliferation, and refers to Cell-Light of Ruibo company TM The EdU Apollo 567In vitro Kit comprises the following specific operation steps:
(1) Preparation of 50. Mu.M EdU medium: cell culture medium was used to control the cell culture medium at 1: diluting the EdU solution in a ratio of 1000;
(2) The culture solution is discarded when the cell fusion degree is 50-80%, and 100 mu L of 50 mu M EdU culture medium is added for 2h of incubation;
(3) Fixing cells: discarding the culture solution, adding 100 mu L of cell fixing solution (PBS of 4% paraformaldehyde) into each hole, and incubating for 15-30 min at room temperature;
(4) 2mg/mL glycine was incubated for 10min, and washed 2 times with PBS;
(5) The supernatant was discarded, 100. Mu.L of permeabilizing agent (0.5% (v/v) Triton X-100 in PBS) was added and the cells were washed 1 time with PBS;
(6) EdU detection: add 100. Mu.L of 1XStaining the reaction solution, incubating for 30min at room temperature in dark place, washing for 1 time by PBS, precipitating cells, and discarding the supernatant;
(7) DNA staining: 100 mu L of DAPI reaction solution is added into each hole, and the mixture is incubated for 30min at room temperature in a dark place;
(8) 100. Mu.L of penetrant (0.5% (v/v) Triton X-100 PBS) was added to wash 3 times, and the DAPI reaction solution was eluted;
(9) Fluorescence microscopy (three replicates per group).
Example 7ELISA method for determining E2 content in pig ovarian granulosa cell supernatant samples
E2 concentration detection, referring to a pore E2 ELISA kit of Jiangsu Jingmei biological company, comprises the following specific operation steps: 50 mu L of standard substances with different concentrations are respectively added into the standard holes, and 50 mu L of samples to be detected are added into the sample holes. Then 100. Mu.L horseradish peroxidase (HRP) was added and incubated at 37℃for 60min. After washing, 50. Mu.L of each substrate A, B was added and incubated at 37℃for 15min. Finally, a stop solution was added and the OD was measured at 450 nm.
Analysis of results:
1. to study the effect of lncRNA SFFD on ovarian granulosa cell function, we transfected lncRNA SFFD overexpression vector (pcDNA3.1-lncSFFD) or small interfering RNA (si-lncSFFD) into ovarian granulosa cells to explore the effect of lncRNA SFFD on ovarian granulosa cell proliferation, apoptosis and E2 secretion; the construction method of the lncRNA SFFD super expression vector comprises the following steps: firstly, amplifying lncRNA SFFD by 5 'and 3' RACE, extracting common plasmid after sequencing verification is correct, and extracting endotoxin-free plasmid after double enzyme digestion, connection, transformation and single clone sequencing verification of common plasmid and pcDNA3.1 vector are correct. And (3) carrying out double enzyme digestion identification on the extracted endotoxin-free plasmid, and verifying whether the lncRNA SFFD super expression vector is successfully constructed. Finally, the successfully constructed pcDNA3.1-lnc SFFD is transfected into the ovary granular cells, and the expression level of lncRNASFFD is also increased along with the increase of the concentration of the pcDNA3.1-lnc SFFD vector as shown in FIG. 1A through qRT-PCR and WB method verification.
2. 2 pairs of small fragments/controls (si-lnc SFFD/siRNA-NC) interfering with lncRNASFFD were synthesized, screened and tested for interfering efficiency. As shown in FIG. 1B, the gene interference small fragment is transfected into the ovary granular cells, and the si-lnc SFFD-2 fragment with better interference effect is finally screened through qRT-PCR and WB means for subsequent experiments.
si-lnc SFFD-1：5′-GGAACAAUAGUAAGCACAAUC-3′；
si-lnc SFFD-2：5′-GUAGCCCAUUUCUUUCCAA-3′；
The interference small fragment is synthesized by Sharpo biotechnology limited company in Guangzhou city; control siRNA-NC was from Sharp Biotech Inc. in Guangzhou.
3. We transfected pcDNA3.1-lncSFFD or si-lncSFFD (si-lncSFFD-2) into ovarian granulosa cells, and detected the effect of lncRNASFFD on granulosa cell proliferation-related gene expression and proliferation by qRT-PCR, WB and Edu methods, respectively. qRT-PCR and WB results are shown in FIG. 2A, B, pcDNA3.1-lnc SFFD promotes the expression levels of cell cycle related genes (PCNA, CDK1, CCNA1 and CCND 1). EdU staining as shown in FIG. 2C showed that the cell proliferation rate of the pcDNA3.1-lnc SFFD group was significantly higher than that of the pcDNA3.1 group. Meanwhile, as shown in fig. 2A, B, si-lnc SFFD inhibits the expression levels of PCNA, CDK1, and CCND 1. As shown in fig. 2C, the cell proliferation rate of si-lnc SFFD group was significantly lower than that of siRNA-NC group. In conclusion, lncRNASFFD can promote proliferation of porcine ovarian granulosa cells.
4. We transfected pcDNA3.1-lncSFFD or si-lncSFFD-2 into ovary granulosa cells, and detected the effect of lncRNASFFD on granulosa cell apoptosis-related gene expression and apoptosis by qRT-PCR, WB and Annexin V-FITC methods, respectively. qRT-PCR and WB results are shown in FIG. 3A, B, where pcDNA3.1-lnc SFFD inhibited the expression levels of the pro-apoptotic associated genes (Caspase 3, caspase8 and Caspase 9). The flow cytometer analysis results show that the apoptosis rate (early apoptosis + late apoptosis) of the pcdna3.1-lnc SFFD group was significantly lower than that of the pcdna3.1 group as shown in fig. 3C. Meanwhile, as shown in FIG. 3A, B, si-lnc SFFD promoted the expression levels of Caspase3, caspase8 and Caspase 9. As shown in fig. 3C, the apoptosis rate of si-lnc SFFD group was significantly higher than that of siRNA-NC group. In conclusion, lncRNA SFFD can inhibit apoptosis of porcine ovarian granulosa cells.
5. We transfected pcDNA3.1-lnc SFFD or si-lnc SFFD (si-lnc SFFD-2) into ovarian granulosa cells, and tested the effects of lncRNASFFD on granulosa cell E2 secretion-related gene expression and E2 secretion by qRT-PCR, WB and ELISA methods, respectively. qRT-PCR and WB results are shown in FIG. 4A, B, pcDNA3.1-lnc SFFD promotes the expression levels of genes related to E2 secretion by cells (CYP 11A1, HSD17B1 and HSD3B 1). ELISA results as shown in FIG. 4C showed that the E2 concentration of pcDNA3.1-lnc SFFD group was significantly higher than that of pcDNA3.1 group. Meanwhile, as shown in fig. 4A, B, si-lnc SFFD inhibits the expression levels of CYP11A1, HSD17B1, and HSD3B 1. As shown in fig. 4C, the E2 concentration of si-lnc SFFD group was significantly lower than that of siRNA-NC group. In conclusion, lncrnas sffd can promote secretion of porcine ovarian granulosa cells E2.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. An lncRNA SFFD, characterized in that: the nucleotide sequence is shown as SEQ ID NO: 1.
2. The lncRNA SFFD-related biomaterial of claim 1, characterized in that: is any one of the following biological materials;
1) A DNA molecule encoding the lncRNA SFFD;
2) An expression cassette comprising the DNA molecule of 1);
3) A recombinant vector comprising the DNA molecule of 1);
4) siRNA that inhibits SFFD expression of the lncRNA;
the siRNA is shown as si-lnc SFFD-2 or si-lnc SFFD-1:
si-lnc SFFD-1：5′-GGAACAAUAGUAAGCACAAUC-3′；
si-lnc SFFD-2：5′-GUAGCCCAUUUCUUUCCAA-3′；
5) A recombinant cell comprising the DNA molecule of 1), or transfected with the siRNA of 4); the recombinant cells are porcine ovary granulosa cells.
3. The biomaterial according to claim 2, characterized in that:
3) Wherein the recombinant vector is a recombinant vector containing the expression cassette described in 2);
5) Wherein the recombinant cell containing the DNA molecule of 1) is a recombinant cell containing the expression cassette of 2) or a recombinant cell containing the recombinant vector of 3).
4. A biomaterial according to claim 2 or 3, characterized in that:
1) The DNA molecule is prepared by the following steps: extracting RNA of the porcine ovary granulosa cells, carrying out reverse transcription on the RNA to obtain cDNA, and carrying out PCR amplification by taking the cDNA as a template to obtain DNA molecules;
the primers used for PCR amplification are as follows:
lncRNA SFFD Forward：5′-CCAAGCTTACTACATGCTTGAGGAGGGTG-3′；
lncRNA SFFD Reverse：5′-GGGGTACCCGCGGTCATACGATTAACCCA-3′。
5. a biomaterial according to claim 2 or 3, characterized in that:
3) The recombinant vector is prepared by the following steps: the DNA molecule is inserted between HindIII and KpnI cleavage sites of the pcDNA3.1 vector to obtain a recombinant vector.
6. Use of lncRNA SFFD of claim 1 or the biomaterial of any one of claims 2-5 for modulating proliferation and/or apoptosis of porcine ovarian granulosa cells, characterized in that: the environment of the application is an in vitro environment.
7. Use of lncRNA SFFD of claim 1 or the biomaterial of any one of claims 2-5 in the manufacture of a medicament for modulating proliferation and/or apoptosis of porcine ovarian granulosa cells.
8. Use according to claim 6 or 7, characterized in that: for any of the following applications a), b), c), d), or a combination of a) and b), or a combination of c) and d):
a) Increasing lncRNA SFFD, and promoting proliferation of porcine ovary granulosa cells;
b) Increasing lncRNA SFFD, and inhibiting apoptosis of porcine ovarian granulosa cells;
c) Reduce lncRNA SFFD, inhibit proliferation of porcine ovary granulosa cells;
d) Reduce lncRNA SFFD and promote apoptosis of porcine ovary granulosa cells.
9. Use of lncRNA SFFD of claim 1 or the biomaterial of any one of claims 2-5 in the manufacture of a medicament for modulating E2 production in porcine ovarian granulosa cells.
10. The use according to claim 9, characterized in that:
the E2 generation in the pig ovary granular cells is regulated and controlled by the following modes:
increasing lncRNA SFFD promotes E2 production; or reduce lncRNA SFFD from inhibiting E2 production.
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