CN114854756A - Application of miR-370 to regulation of GLI1 expression in porcine ovarian granulosa cells - Google Patents
Application of miR-370 to regulation of GLI1 expression in porcine ovarian granulosa cells Download PDFInfo
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Abstract
The invention discloses application of miR-370 regulation GLI1 expression in porcine ovarian granulosa cells, and belongs to the technical field of cell engineering and genetic engineering. The miR-370 is used as a research object, and the application of the miR-370in the porcine ovarian granulosa cells is researched by adopting a molecular and cell biological method, and the result shows that the miR-370 can down-regulate GLI1 expression, inhibit apoptosis of the ovarian granulosa cells, promote proliferation and E2 secretion. By researching the application of miR-370in ovarian granulosa cells, the method has good application value in researching ovarian follicular atresia, follicular development and other mechanisms. The technical scheme of the invention is thoroughly designed and has reliable results. In order to verify the influence of miR-370 on the proliferation, apoptosis and E2 secretion of the ovarian granulosa cells, the invention is verified from multiple levels and angles, verified at the levels of related signal pathway genes and mRNA, and finally verified on the phenotype of the ovarian granulosa cells.
Description
Technical Field
The invention belongs to the technical field of cell engineering and genetic engineering, and particularly relates to application of miR-370in regulation of GLI1 expression in porcine ovarian granulosa cells.
Background
microRNAs (miRNAs) are endogenous non-coding small RNA molecules with the length of about 22nt, and degrade or inhibit the translation of target mRNA in the cell growth and differentiation process by combining a seed sequence and a 3' -untranslated region (UTR) of the target mRNA to regulate gene expression. The expression of mirnas in the ovary varies with cell type, function and oestrus cycle. miRNAs are involved in primordial follicle formation, follicle recruitment and selection, follicular atresia, cumulus cell interactions and Granulosa Cell (GCs) function.
France sca et al found that the expression level of miR-370in GCs of PCOS patients was significantly down-regulated by expression analysis of miRNAs in GCs of PCOS patients, and correlated with the number of dominant follicles. MiR-370 can also mediate FSHR expression and thus be involved in folliculogenesis and oocyte maturation. GLI1 is one of the members of the GLI-Kruppel family, and is also a key member in the Hedgehog (HH) signaling pathway. Activation of the HH signal in mice can promote cell proliferation and follicle growth.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide the application of the miR-370in regulation of GLI1 expression in porcine ovarian granulosa cells.
It is still another object of the present invention to provide RNA small interference fragments (siRNAs) that inhibit the expression of GLI 1.
The purpose of the invention is realized by the following technical scheme:
the invention provides application of miR-370in regulation and control of GLI1 expression.
Under an in-vitro environment, increasing miR-370 to inhibit GLI1 expression, and/or decreasing miR-370 to promote GLI1 expression;
the invention provides an application of miR-370in porcine ovarian granulosa cells, and the application is specifically in the cycle process, proliferation, apoptosis and/or E2 secretion of the porcine ovarian granulosa cells; wherein the application environment is an in vitro environment;
under the in vitro environment, the increase of miR-370 can increase the proportion of swine ovarian granulosa cells in the S phase, so that the periodic process of the swine ovarian granulosa cells is promoted; and/or miR-370 is reduced, so that the proportion of swine ovarian granulosa cells in the S phase can be reduced, and the periodic process of the swine ovarian granulosa cells is delayed;
under the in-vitro environment, increasing miR-370 to promote the proliferation of the porcine ovarian granulosa cells, and/or reducing miR-370 to inhibit the proliferation of the porcine ovarian granulosa cells;
under the in-vitro environment, increasing miR-370 to inhibit the apoptosis of the porcine ovarian granulosa cells, and/or reducing miR-370 to promote the apoptosis of the porcine ovarian granulosa cells;
under the in vitro environment, increasing miR-370 promotes E2 secretion in porcine ovarian granulosa cells, and/or decreasing miR-370 inhibits E2 secretion in porcine ovarian granulosa cells.
The invention provides an application of GLI1 in porcine ovarian granulosa cells, in particular to an application in the cycle process, proliferation, apoptosis and/or E2 secretion of the porcine ovarian granulosa cells; wherein the application environment is an in vitro environment;
in an in vitro environment, reducing GLI1 promotes the cycle progression of porcine ovarian granulosa cells;
in an in vitro environment, reducing GLI1 promotes the proliferation of porcine ovarian granulosa cells;
in an in vitro environment, reducing GLI1 inhibits apoptosis of porcine ovarian granulosa cells;
in vitro, reduction of GLI1 promoted E2 secretion in porcine ovarian granulosa cells.
The invention provides application of miR-370 to regulation of GLI1 expression in porcine ovarian granulosa cells, and particularly relates to application in the cycle process, proliferation, apoptosis and/or E2 secretion of the porcine ovarian granulosa cells; wherein the application environment is an in vitro environment;
under an in-vitro environment, the expression of GLI1 is inhibited by increasing miR-370, and the cycle process of the porcine ovarian granulosa cells can be promoted; and/or by reducing miR-370 to promote expression of GLI1, the cycle process of the porcine ovarian granulosa cells can be delayed;
under an in-vitro environment, the expression of GLI1 is inhibited by increasing miR-370, and the proliferation of the porcine ovarian granulosa cells can be promoted; and/or by reducing miR-370 to promote expression of GLI1, proliferation of porcine ovarian granulosa cells can be inhibited;
under an in-vitro environment, by increasing miR-370 to inhibit GLI1 expression, the apoptosis of the swine ovarian granulosa cells can be inhibited; and/or by reducing miR-370 to promote expression of GLI1, the apoptosis of the swine ovary granular cells can be promoted;
under an in-vitro environment, the expression of GLI1 is inhibited by increasing miR-370, and the secretion of E2 in porcine ovarian granulosa cells can be promoted; and/or by reducing miR-370 to promote expression of GLI1, secretion of E2 in porcine ovarian granulosa cells can be inhibited.
Further, in the above-mentioned case,
the functional phenotype caused by miR-370 reduction and used for delaying the cell cycle process of the porcine ovarian granulosa cells can be recovered after the porcine ovarian granulosa cells are supplemented with GLI 1-siRNA;
the functional phenotype for inhibiting the proliferation of the porcine ovarian granulosa cells, caused by the reduction of miR-370, can be recovered after the porcine ovarian granulosa cells are supplemented with GLI 1-siRNA;
the functional phenotype for promoting the apoptosis of the porcine ovarian granulosa cells caused by the reduction of miR-370 can be recovered after the porcine ovarian granulosa cells are supplemented with GLI 1-siRNA;
the functional phenotype caused by miR-370 reduction for inhibiting E2 secretion in pig ovarian granulosa cells can be recovered after the pig ovarian granulosa cells are supplemented with GLI 1-siRNA.
The GLI1-siRNA is GLI1-siRNA-1 or GLI 1-siRNA-2;
GLI1-siRNA-1：5′-CCACAGGGCAGCAGCACUA-3′；
GLI1-siRNA-2：5′-GAGAAUGUCACCAUGGAUACC-3′；
the miR-370 is increased by the following method: the miR-370 simulant miR-370mimic is transfected into the porcine ovarian granule cells.
The miR-370 reduction is realized by the following method: the miR-370inhibitor is transfected into porcine ovarian granulosa cells.
The reduction of GLI1 is realized by a mode that siRNA for inhibiting GLI1 expression is transfected into pig ovarian granule cells.
The invention provides siRNA for inhibiting expression of GLI1, which has the following sequence:
GLI1-siRNA-1：5′-CCACAGGGCAGCAGCACUA-3′；
GLI1-siRNA-2：5′-GAGAAUGUCACCAUGGAUACC-3′；
wherein, the related sequence of miR-370 is as follows:
miR-370 mimic：5′-GCCUGCUGGGGUGGAACCUGGU-3′；
miR-370 inhibitor：5′-ACCAGGUUCCACCCCAGCAGGC-3′；
the verification results of the invention are as follows:
1. synthesizing a miR-370 mimic/miR-370 inhibitor, and detecting the overexpression or interference efficiency of the miR-370 mimic/miR-370 inhibitor. As can be seen from the result of FIG. 1A, the qRT-PCR result shows that miR-370 micic significantly promotes the expression of miR-370, and miR-370inhibitor significantly inhibits the expression of miR-370, so that the method can be used for subsequent experiments. GLI 13 'UTR-WT and GLI 13' UTR-MUT vectors and miR-370 mimic/mimic NC are co-transfected into ovarian granule cells respectively, and the results of dual-luciferase activity analysis show that the inhibition effect of miR-370 on the fluorescence activity can be recovered by mutating the binding site of GLI1 and miR-370. Meanwhile, as can be seen from the results shown in fig. 1C and D, miR-370 micic decreased the mRNA and protein levels of GLI1 in granulosa cells, while miR-370inhibitor increased the mRNA and protein levels of GLI1 in granulosa cells.
2. 3 pairs of small fragments/controls (GLI1-siRNA/siRNA-NC) that interfere with GLI1 were synthesized, screened and tested for their interference efficiency. As shown in the result of FIG. 2A, the gene interference small fragment is transfected into the ovary granular cell, and finally the GLI1-siRNA-1 small fragment with better interference effect is screened by qRT-PCR means for subsequent experiments.
GLI1-siRNA-1：5′-CCACAGGGCAGCAGCACTA-3′；
3. NC, miR-370mimic, miR-370inhibitor or miR-370inhibitor and GLI1-siRNA (GLI1-siRNA-1) are transfected into ovarian granular cells respectively, and the influence of miR-370 and GLI1-siRNA on the expression, the period and the proliferation of genes related to the proliferation of the granular cells is detected by utilizing qRT-PCR, Annexin V-FITC and Edu methods respectively. The qRT-PCR result shows that miR-370 micic remarkably promotes the expression level of cell cycle related genes (PCNA, CDK2, CCNB1 and CCND 1). Flow cytometry analysis results show that the S-phase cell proportion of the miR-370mimic group is obviously higher than that of the NC control group, and EdU staining shows that the cell proliferation rate of the miR-370mimic group is obviously higher than that of the NC control group. Meanwhile, miR-370inhibitor inhibits the expression levels of PCNA, CCNA1, CCNB1 and CCND 1. The S-phase cell proportion and the cell proliferation rate of the miR-370inhibitor group are obviously lower than those of an NC control group. Compared with the miR-370inhibitor group, the miR-370inhibitor + GLI1-siRNA promotes the expression levels of CCNB2 and CCND 1. The S-phase cell proportion and the cell proliferation rate of the miR-370inhibitor + GLI1-siRNA group are obviously higher than those of the miR-370inhibitor group. In conclusion, miR-370 promotes the proliferation of swine ovarian granulosa cells.
4. NC, miR-370mimic, miR-370inhibitor or miR-370inhibitor and GLI1-siRNA (GLI1-siRNA-1) are transfected into ovarian granular cells respectively, and qRT-PCR and Annexin V-FITC are utilized to detect the influence of miR-370 and GLI1-siRNA on granular cell apoptosis-related gene expression and apoptosis respectively. The qRT-PCR result shows that miR-370 micic remarkably inhibits the expression level of apoptosis-related gene (Caspase 3). Flow cytometry analysis results show that the apoptosis rate (early apoptosis + late apoptosis) of the miR-370mimic group is significantly lower than that of the NC control group. Meanwhile, miR-370inhibitor promotes the expression levels of Caspase3, Caspase8 and BAX. The cell apoptosis rate of the miR-370inhibitor group is obviously higher than that of the NC control group. Compared with the miR-370inhibitor group, the miR-370inhibitor + GLI1-siRNA significantly inhibits the expression levels of Caspase3 and Caspase 8. The apoptosis rate of the miR-370inhibitor + GLI1-siRNA group is obviously lower than that of the miR-370inhibitor group. In conclusion, miR-370 can inhibit apoptosis of swine ovarian granulosa cells.
5. NC, miR-370mimic, miR-370inhibitor or miR-370inhibitor and GLI1-siRNA (GLI1-siRNA-1) are transfected into ovarian granular cells respectively, and the influence of miR-370 and GLI1-siRNA on E2 secretion related gene expression and E2 secretion of the granular cells is detected by utilizing qRT-PCR and ELISA methods respectively. The qRT-PCR result shows that miR-370mimic remarkably promotes the expression level of an E2 secretion-related gene (HSD17B 1). The ELISA result shows that the E2 concentration of the miR-370mimic group is higher than that of the NC control group. Meanwhile, the miR-370inhibitor can obviously inhibit the expression levels of CYP19A1 and CYP11A 1. The E2 concentration of the miR-370inhibitor group is significantly lower than that of the NC control group. Compared with the miR-370inhibitor group, the miR-370inhibitor + GLI1-siRNA significantly promotes the expression level of CYP19A 1. The E2 concentration of the miR-370inhibitor + GLI1-siRNA group is obviously higher than that of the miR-370inhibitor group. In conclusion, miR-370 can promote E2 secretion of swine ovarian granulosa cells.
Compared with the prior art, the invention has the following advantages and effects:
(1) the miR-370 can directly or indirectly participate in follicular atresia and follicular development, and the application of the miR-370in porcine ovarian granulosa cells is researched by taking the miR-370 as a research object and adopting a molecular and cell biological method: the miR-370 can inhibit apoptosis of ovarian granulosa cells and promote proliferation and E2 secretion. Has good application value for researching ovarian follicular atresia, follicular development and the like.
(2) According to the invention, a miR-370mimic (miR-370 micic) and an inhibitor (miR-370 inhibitor) are synthesized to construct a wild type (GLI 13 ' UTR-WT) of 3 ' UTR of GLI1 and a mutant type carrier (GLI 13 ' UTR-MUT), firstly, miR-370 micic and GLI 13 ' UTR-WT or GLI 13 ' UTR-MUT are co-transfected to ovarian granular cells, and the influence of miR-370 on GLI1 fluorescence activity and expression level is detected. Then respectively transfecting miR-370mimic, miR-370inhibitor and miR-370inhibitor + GLI1-siRNA to the ovarian granular cells, then detecting the change of the signal path gene mRNA level related to apoptosis and proliferation of the ovarian granular cells and E2 secretion, and finally detecting the phenotype change of the ovarian granular cells. The result shows that miR-370 can reduce GLI1 expression, inhibit apoptosis of ovarian granulosa cells and promote proliferation and E2 secretion. By researching the application of miR-370in ovarian granulosa cells, the method has good application value in researching ovarian follicular atresia, follicular development and other mechanisms.
(3) The technical scheme of the invention is thoroughly designed and has reliable results. In order to verify the influence of miR-370 on the proliferation, apoptosis and E2 secretion of the ovarian granulosa cells, the invention is verified from multiple levels and angles, verified at the levels of related signal pathway genes and mRNA, and finally verified on the phenotype of the ovarian granulosa cells.
Drawings
FIG. 1 is a graph of miR-370 regulating expression of GLI 1; wherein A is a miR-370 micic overexpression efficiency and miR-370inhibitor interference efficiency graph; b is the influence of miR-370 micic on the transcriptional activity of GLI 1; c is the influence of miR-370mimic and miR-370inhibitor on the mRNA level of GLI 1; d is the influence of miR-370 micic and miR-370inhibitor on the protein level of GLI 1.
FIG. 2 is a graph of the effect of miR-370 on granular cell cycle and proliferation; wherein A is an interference efficiency graph of GLI 1; b is the influence of overexpression and interference of miR-370 and interference of GLI1 on the mRNA expression level of the granulosa cell proliferation related gene; c is the influence of overexpression, miR-370 interference and GLI1 interference on the cell cycle process of the granule; d is the influence of overexpression and miR-370 interference and GLI1 interference on the proliferation rate of the granular cells.
FIG. 3 is a graph of the effect of miR-370 on granulosa cell apoptosis; wherein A is the influence of overexpression and interference of miR-370 and interference of GLI1 on the mRNA expression level of the granular cell apoptosis related gene; and B is the influence of overexpression and miR-370 interference and GLI1 interference on the apoptosis rate of the granular cells.
FIG. 4 is a graph of the effect of miR-370 on the secretion of granulosa cell E2; wherein A is the influence of overexpression and interference of miR-370 and interference of GLI1 on the mRNA expression level of the granulocyte E2 secretion-related gene; b is the influence of overexpression and miR-370 interference and GLI1 interference on the secretion of granulosa cells E2.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions.
In the present invention, the results of 3 independent experiments in each example were analyzed by a statistical method, and the "mean ± standard deviation" was calculated, and the analysis of significance of the difference was performed by a one-way variance analysis (in the figure, "+" indicates P <0.05, and "+" indicates P < 0.01).
Example 1 culture and transfection of ovarian granulosa cells
(1) Collecting fresh ovaries of healthy sows, placing the ovaries in PBS (ice) buffer solution containing 1% double antibody, and quickly transporting the ovaries back to a laboratory for treatment;
(2) cleaning ovaries for 3-5 times by using PBS (phosphate buffer solution) containing 2% double antibodies outside a cell room, placing the ovaries in a beaker, and placing the ovary in a transfer window;
(3) wiping a cell room superclean bench with alcohol, clamping an ovary by using forceps, sucking follicular fluid by using an injector, pumping the follicular fluid into a centrifuge tube containing 5mL of DMEM culture fluid, and extracting the follicular fluid to 9mL per tube;
(4) centrifuging at 1000rpm for 5min, discarding the supernatant, adding 5mL PBS, blowing, mixing, and cleaning twice;
(5) preparing a complete culture medium: 89% of DMEM, 10% of serum and 1% of double antibody are inverted from top to bottom and mixed evenly;
(6) 5mL of complete medium was added to resuspend the cell pellet;
(7) adding 10mL of complete culture medium into a 75mL culture bottle, and then adding the resuspension;
(8) observing with microscope, and standing at 37 deg.C and 5% CO 2 Culturing in an incubator, observing the growth condition of granular cells after 24h, pouring out the culture medium when the granular cells grow to about 90%, and washing for 3 times by using preheated PBS (phosphate buffer solution) containing 1% double antibody;
(9) adding trypsin for digestion, placing in an incubator for about 3min, observing under a microscope until most cells float, and immediately adding equivalent stop solution to stop digestion;
(10) washing with DMEM for 2 times, and centrifuging at 1000rpm for 5 min;
(11) lightly resuspending the cell sediment with complete culture medium, uniformly distributing the cell sediment into each hole, supplementing the volume with complete culture medium, lightly shaking up, and culturing in an incubator;
(12) observing the cell state after about 24 hours, and preparing transfection when the confluence degree of the cells reaches about 80%;
(13) transfection method Invitrogen
3000 kit instructions, each set 3 replicates;
(14) the transfected well plates were placed at 37 ℃ in 5% CO 2 Culturing in an incubator, observing the cell state 24-72 h after transfection, and collecting the cells after good growth.
The double-resistant is penicillin and streptomycin.
Example 2 construction of 3' UTR wild type and mutant vectors of GLI1 and Dual fluorescence Activity analysis
(1) Primers were designed using Primer5, and the 3' UTR of GLI1 (Gene ID: 100520490) was amplified using extracted DNA from porcine granulosa cells as a template; the amplified fragment was purified, recovered, ligated with pMD18T vector (purchased from Takara), transformed, screened, sequenced and identified as correct, and then the ordinary plasmid was extracted. Analysis by BioEdit software found that these sequences have no SacI or SacI restriction enzyme cutting sites, while the pGL3 vector has SacI and SacI cutting sites. The sequences of SacI and SaI enzyme cutting sites are added to the upstream and downstream primers respectively. Then, taking each recombinant pMD18T common plasmid as a template, and carrying out PCR amplification by using each enzyme digestion primer; the fragment is purified and recovered, subjected to double digestion, connected with pGL3-basic vector, transformed, screened, sequenced and identified to be correct, and then extracted to obtain endotoxin-free plasmid (the endotoxin-free plasmid miniprep kit is purchased from American magenta), which is named as GLI 13' UTR-WT. The binding site of the 3 'UTR of GLI1 and miR-370 was amplified and mutated in the same way, and named GLI 13' UTR-MUT.
The primer for constructing the GLI 13' UTR wild type and mutant vector used by the invention comprises the following steps:
GLI1 3’UTR-WT Forward：
Reverse：
GLI1 3’UTR-MUT
Forward：5′-GTATTGGCCGCGCTGCCTGCTGGGGAGTGGTCACTGCTGC-3′；
Reverse：5′-GCAGCAGTGACCACTCCCCAGCAGGCAGCGCGGCCAATAC-3′；
note: the black bold font is the protecting base, the underlined is the restriction site.
(2) The Luciferase Reporter gene activity detection refers to a Dual-Luciferase Reporter Assay System kit of Promega company, and the specific operation steps are as follows: washing twice with PBS, adding 100 μ L Glo lysine Buffer into each well of cell, shaking slightly for 5min at room temperature, and collecting cell lysate; 30 μ L of cell lysate was added to a 96-well plate, and 75 μ L of Dual-
And (3) uniformly mixing the Luciferase Assay Reagent, and standing for 15-30 min at 20-25 ℃. Detecting a luminous value on a multifunctional microplate reader of Synergy 2 of BioTek company, wherein the luminous value corresponds to the expression level of the firefly luciferase; then 75. mu.L of Stop was added&
And (3) uniformly mixing the Reagent, and standing for 15-30 min at 20-25 ℃. Detecting a luminescence value corresponding to the level of renilla luciferase expression; the ratio of the expression amount of the firefly luciferase to the renilla luciferase is the relative activity of the firefly luciferase.
Example 3qRT-PCR
In the present invention, the qRT-PCR detection of the gene was performed using Maxima SYBR Green qPCR Master Mix (2X) kit (Thermo Scientific Co.). The content of the sample gene is detected by adopting a Ct value comparison method in the experiment, and the specific calculation formula is as follows:
relative gene expression level 2- { (Ct value of target gene of experimental group) -in experimental group (Ct value of reference gene of experimental group) -in control group (Ct value of target gene of control group) -in control group
GAPDH is used as an internal reference for detecting genes, and qRT-PCR primers used by the invention are as follows:
qRT-PCR-GLI1 Forward：5′-TTCAACTCGATGACCCCACC-3′；
Reverse：5′-CTGTGGGGACCAGACATGAG-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-CDK2 Forward：5′-AAAAGATCGGAGAGGGCACG-3′；
Reverse：5′-GCAGTACTGGGTACACCCTC-3′；
qRT-PCR-CCNA1 Forward：5′-GCGCCAAGGCTGGAATCTAT-3′；
Reverse：5′-CCTCAGTCTCCACAGGCTAC-3′；
qRT-PCR-CCNA2 Forward：5′-GTACTGAAGGCCGGGAACTC-3′；
Reverse：5′-AGCTGGCCTCTTTTGAGTCT-3′；
qRT-PCR-CCNB1 Forward：5′-ACGGCTGTTAGCTAGTGGTG-3′；
Reverse：5′-GAGCAGTTCTTGGCCTCAGT-3′；
qRT-PCR-CCNB2 Forward：5′-TGGAAATCGAGTTACAACCAGA-3′；
Reverse：5′-TGGAGCCAACATTTCCATCTGT-3′；
qRT-PCR-CCND1 Forward：5′-CTTCCATGCGGAAGATCGTG-3′；
Reverse：5′-TGGAGTTGTCGGTGTAGATGC-3′；
qRT-PCR-CCND2 Forward：5′-TTCCCCAGTGCTCCTACTTC-3′；
Reverse：5′-CACAACTTCTCAGCCGTCAG-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 performed according to the instructions of TRIzol of Takara, and the following steps were performed:
(1) adding the granular cells into TRIzol directly;
(2) standing at room temperature for 10min to fully lyse cells, centrifuging at 12000g for 5min, discarding the precipitate, and taking the supernatant in a new RNase-free tube;
(3) adding 0.2mL of chloroform (1 mL of TRIzol) and shaking vigorously for 15-30 s, standing at room temperature for 5min, and centrifuging at 4 ℃ and 12000g for 15 min;
(4) absorbing the upper aqueous phase and placing the upper aqueous phase in a new RNase-free EP tube;
(5) adding 0.5mL of isopropanol (per 1mL of TRIzol), gently inverting and mixing, standing at room temperature for 10min, and centrifuging at 4 ℃ at 12000g for 10 min;
(6) discarding the supernatant, placing at room temperature, adding 1mL 75% ethanol-DEPC (per 1mL TRIzol) along the tube wall to wash RNA, centrifuging at 4 ℃ at 12000g for 5min, and discarding the supernatant as much as possible;
(7) vacuum drying for 5-10 min, and taking care to avoid excessive drying of RNA precipitate;
(8) DEPC water was added to dissolve the RNA pellet.
PrimeScript from TaKaRa was used 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 adherent cells monolayer (ovarian granulosa cells in example 1): the cell culture was decanted and the cells were washed three times with an appropriate amount of pre-cooled PBS to wash out the culture. And adding 100-200 mu L of protein lysate and 10 mu L of 100mM PMSF into each well of 6-well plate cells, and lysing the cells for 30 min. The cell lysate was collected and transferred to a 1.5mL centrifuge tube and centrifuged at 14000rpm at 4 ℃ for 5 min. Protein sample concentrations were determined using the BCA method.
(2) SDS-PAGE electrophoresis: mu.g of total protein per group was mixed with 5 Xloading buffer at a ratio of 5:1 and boiled for 5 min. Performing SDS-PAGE electrophoresis until bromophenol blue just comes out of the bottom of the gel;
(3) film transfer: and (3) pretreating the PVDF membrane for 3-5 s by using methanol, and soaking the PVDF membrane in a transfer printing liquid for 30 min. Taking out the gel, and placing the gel on filter paper to form a sandwich structure of a gel transfer printing accumulation layer. 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 in TBST and repeated three times. 5% skimmed milk powder solution was blocked at room temperature for 90min, and rinsed with TBST for 5min, repeated three times. Membranes were incubated overnight at 4 ℃ with primary antibody diluted as follows: GLI1(66905-1-Ig, proteintech, 1:5000) and Tubulin (11224-1-AP, proteintech, 1: 5000). After washing the membrane 3 times with TBST, the membrane was incubated with goat anti-rabbit (ab205718, Abcam, 1:10000) or goat anti-mouse (ab6789, Abcam, 1:5000) secondary antibody for 2h at room temperature. And (3) after ECL luminous liquid treatment, visualizing the protein band by using an Odyssey Fc image system, and finally analyzing the protein band by using ImageJ software.
Example 5 granular cell apoptosis assay
The Annexin V-FITC technology for detecting the Apoptosis refers to the Annexin V-FITC Apoptosis Detection Kit operating instruction of BioVision, and comprises the following specific operating steps:
(1) placing the cell culture plate at room temperature, slightly rinsing cells in the culture plate by using 2mL of PBS solution, and removing the PBS solution;
(2) adding pancreatin without EDTA to digest the cells, and gently resuspending the cells in the medium of step (1) to a density of about 1X 10 6 cell/mL;
(3) 0.5mL of cell suspension was removed from the cell culture plate (approximately 5X 10) 5 Individual cells) were transferred into a clean centrifuge tube and 500 μ L of 1 × Binding Buffer was added;
(4) adding 5 μ L Annexin V-FITC and 5 μ L propidium iodide at room temperature;
(5) reacting at room temperature in dark for 5 min;
(6) analysis was immediately performed using a FACSCalibur flow cytometer (triplicates per group).
Example 6 granulosa cell proliferation assay
The present invention uses the EdU method to detect Cell proliferation, refer to Cell-Light of Ruibo corporation TM The EdU Apollo 567 In vitro Kit detection Kit comprises the following specific operation steps:
(1) preparation of 50 μ M EdU medium: culturing the cells in a cell culture medium at a temperature of 1: diluting the EdU solution at a ratio of 1000;
(2) discarding the culture solution when the cell fusion degree is 50-80%, and adding 100 mu L of 50 mu M EdU culture medium to incubate for 2 h;
(3) fixing the cells: discarding the culture solution, adding 100 μ L of cell fixing solution (4% paraformaldehyde PBS) into each well, and incubating at room temperature for 15-30 min;
(4)2mg/mL glycine incubation for 10min, PBS washing 2 times;
(5) discarding the supernatant, adding 100 μ L of penetrant (0.5% (v/v) TritonX-100 PBS) to permeabilize the cells, and washing with PBS for 1 time;
(6) EdU detection: adding 100 μ L of
Dyeing reaction liquid, incubating for 30min at room temperature in a dark place, washing for 1 time by PBS, precipitating cells, and removing supernatant;
(7) DNA staining: adding 100 mu L of DAPI reaction solution into each hole, and incubating for 30min at room temperature in a dark place;
(8) adding 100 μ L of penetrating agent (0.5% (v/v) TritonX-100 PBS) to wash for 3 times, eluting DAPI reaction solution;
(9) fluorescence microscopy (triplicates per group).
Example 7 determination of E2 content in porcine ovarian granulosa cell supernatant samples by ELISA
E2 concentration detection, referring to porcine E2 ELISA kit of Jiangsu Jingmei biology company, the specific operation steps are as follows: respectively adding 50 mu L of standard substances with different concentrations into the standard hole, and adding 50 mu L of samples to be detected into the sample hole. Then 100. mu.L of horseradish peroxidase (HRP) was added and incubated at 37 ℃ for 60 min. After washing, 50. mu.L of each substrate A, B was added and incubated at 37 ℃ for 15 min. Finally, a stop solution was added and the OD was measured at 450 nm.
And (4) analyzing results:
1. synthesizing a miR-370 mimic/miR-370 inhibitor, and detecting the overexpression or interference efficiency of the miR-370 mimic/miR-370 inhibitor. As can be seen from the result of FIG. 1A, the qRT-PCR result shows that miR-370 micic significantly promotes the expression of miR-370, and miR-370inhibitor significantly inhibits the expression of miR-370, so that the method can be used for subsequent experiments. GLI 13 'UTR-WT and GLI 13' UTR-MUT vectors and miR-370 mimic/mimic NC are co-transfected into ovarian granule cells respectively, and the results of dual-luciferase activity analysis show that the inhibition effect of miR-370 on the fluorescence activity can be recovered by mutating the binding site of GLI1 and miR-370 (figure 1B). Meanwhile, as can be seen from the results shown in fig. 1C and D, miR-370 micic decreased the mRNA and protein levels of GLI1 in granulosa cells, while miR-370inhibitor increased the mRNA and protein levels of GLI1 in granulosa cells.
2. 3 pairs of small fragments/controls (GLI1-siRNA/siRNA-NC) that interfere with GLI1 were synthesized, screened and tested for their interference efficiency. As shown in the result of FIG. 2A, the gene interference small fragment is transfected into the ovary granular cell, and finally the GLI1-siRNA-1 small fragment with better interference effect is screened by qRT-PCR means for subsequent experiments.
GLI1-siRNA-1：5′-CCACAGGGCAGCAGCACUA-3′；
GLI1-siRNA-2：5′-GAGAAUGUCACCAUGGAUACC-3′；
GLI1-siRNA-3：5′-GGACGGCUGCAGUCAGGAAUU-3′；
The small interference fragments are synthesized by Ribo Biotech, Inc., Guangzhou; control siRNA-NC was from Ribo Biotech, Inc., Guangzhou.
3. NC, miR-370mimic, miR-370inhibitor or miR-370inhibitor and GLI1-siRNA (GLI1-siRNA-1) are transfected into ovarian granular cells respectively, and the influence of miR-370 and GLI1-siRNA on the expression, the period and the proliferation of genes related to the proliferation of the granular cells is detected by utilizing qRT-PCR, Annexin V-FITC and Edu methods respectively, and the result is shown in figure 2. The qRT-PCR result shows that miR-370 micic remarkably promotes the expression level of cell cycle related genes (PCNA, CDK2, CCNB1 and CCND 1). Flow cytometry analysis results show that the S-phase cell proportion of the miR-370mimic group is obviously higher than that of the NC control group, and EdU staining shows that the cell proliferation rate of the miR-370mimic group is obviously higher than that of the NC control group. Meanwhile, miR-370inhibitor inhibits the expression levels of PCNA, CCNA1, CCNB1 and CCND 1. The S-phase cell proportion and the cell proliferation rate of the miR-370inhibitor group are obviously lower than those of an NC control group. Compared with the miR-370inhibitor group, the miR-370inhibitor + GLI1-siRNA promotes the expression levels of CCNB2 and CCND 1. The S-phase cell proportion and the cell proliferation rate of the miR-370inhibitor + GLI1-siRNA group are obviously higher than those of the miR-370inhibitor group, namely the functional phenotype caused by the miR-370inhibitor, which is used for delaying the cell cycle process of the porcine ovarian granulosa cells and/or inhibiting the proliferation of the porcine ovarian granulosa cells, can be recovered after the porcine ovarian granulosa cells are supplemented with GLI 1-siRNA. In conclusion, miR-370 promotes the proliferation of swine ovarian granulosa cells.
4. NC, miR-370mimic, miR-370inhibitor or miR-370inhibitor and GLI1-siRNA (GLI1-siRNA-1) are transfected into ovarian granular cells respectively, and the influence of miR-370 and GLI1-siRNA on granular cell apoptosis-related gene expression and apoptosis is detected by qRT-PCR and Annexin V-FITC respectively, and the result is shown in figure 3. The qRT-PCR result shows that miR-370 micic remarkably inhibits the expression level of apoptosis-related gene (Caspase 3). Flow cytometry analysis results show that the apoptosis rate (early apoptosis + late apoptosis) of the miR-370mimic group is significantly lower than that of the NC control group. Meanwhile, miR-370inhibitor promotes the expression levels of Caspase3, Caspase8 and BAX. The cell apoptosis rate of the miR-370inhibitor group is obviously higher than that of the NC control group. Compared with the miR-370inhibitor group, the miR-370inhibitor + GLI1-siRNA significantly inhibits the expression levels of Caspase3 and Caspase 8. The cell apoptosis rate of the miR-370inhibitor + GLI1-siRNA group is obviously lower than that of the miR-370inhibitor group, namely the functional phenotype caused by the miR-370inhibitor for promoting the apoptosis of the porcine ovarian granule cells can be recovered after the GLI1-siRNA is supplemented in the porcine ovarian granule cells. In conclusion, miR-370 can inhibit apoptosis of swine ovarian granulosa cells.
5. NC, miR-370mimic, miR-370inhibitor or miR-370inhibitor and GLI1-siRNA (GLI1-siRNA-1) are transfected into ovarian granular cells respectively, and the influence of miR-370 and GLI1-siRNA on E2 secretion-related gene expression and E2 secretion of the granular cells is detected by qRT-PCR and ELISA methods respectively, and the result is shown in figure 4. The qRT-PCR result shows that miR-370mimic remarkably promotes the expression level of an E2 secretion-related gene (HSD17B 1). The ELISA result shows that the E2 concentration of the miR-370mimic group is higher than that of the NC control group. Meanwhile, the miR-370inhibitor can obviously inhibit the expression levels of CYP19A1 and CYP11A 1. The E2 concentration of the miR-370inhibitor group is significantly lower than that of the NC control group. Compared with the miR-370inhibitor group, the miR-370inhibitor + GLI1-siRNA significantly promotes the expression level of CYP19A 1. The E2 concentration of the miR-370inhibitor + GLI1-siRNA group is obviously higher than that of the miR-370inhibitor group, namely the functional phenotype caused by the miR-370inhibitor for inhibiting E2 secretion in the porcine ovarian granulosa cells can be recovered after GLI1-siRNA is supplemented in the porcine ovarian granulosa cells. In conclusion, miR-370 can promote E2 secretion of swine ovarian granulosa cells.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
Sequence listing
<110> southern China university of agriculture
Application of miR-370in regulation of GLI1 expression in porcine ovarian granulosa cells
<160> 51
<170> SIPOSequenceListing 1.0
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<211> 19
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<213> Artificial Sequence (Artificial Sequence)
<220>
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<223> miR-370 inhibitor
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<213> Artificial Sequence (Artificial Sequence)
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<223> GLI1 3’UTR-WT Forward
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cgagctccaa gcaccagaat cggaccc 27
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<213> Artificial Sequence (Artificial Sequence)
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<223> GLI1 3’UTR-WT Reverse
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> GLI1 3’UTR-MUT Forward
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> GLI1 3’UTR-MUT Reverse
<400> 9
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<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-GLI1 Forward
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<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-GLI1 Reverse
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-Caspase3 Forward
<400> 12
acatggaagc aaatcaatgg ac 22
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-Caspase3 Reverse
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tgcagcatcc acatctgtac c 21
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-Caspase8 Forward
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<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-Caspase8 Reverse
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-Caspase9 Forward
<400> 16
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-Caspase9 Reverse
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<213> Artificial Sequence (Artificial Sequence)
<220>
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-BAX Reverse
<400> 19
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-BCL2 Forward
<400> 20
gatgcctttg tggagctgta tg 22
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-BCL2 Reverse
<400> 21
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-PCNA Forward
<400> 22
tcgttgtgat tccaccacca t 21
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-PCNA Reverse
<400> 23
tgtcttcatt gccagcacat tt 22
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CDK1 Forward
<400> 24
aggtcaagtg gtagccatga a 21
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CDK1 Reverse
<400> 25
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CDK2 Forward
<400> 26
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CDK2 Reverse
<400> 27
gcagtactgg gtacaccctc 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCNA1 Forward
<400> 28
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCNA1 Reverse
<400> 29
cctcagtctc cacaggctac 20
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCNA2 Forward
<400> 30
gtactgaagg ccgggaactc 20
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCNA2 Reverse
<400> 31
agctggcctc ttttgagtct 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCNB1 Forward
<400> 32
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCNB1 Reverse
<400> 33
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCNB2 Forward
<400> 34
tggaaatcga gttacaacca ga 22
<210> 35
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCNB2 Reverse
<400> 35
tggagccaac atttccatct gt 22
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCND1 Forward
<400> 36
<210> 37
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCND1 Reverse
<400> 37
tggagttgtc ggtgtagatg c 21
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCND2 Forward
<400> 38
ttccccagtg ctcctacttc 20
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CCND2 Reverse
<400> 39
<210> 40
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CYP19A1 Forward
<400> 40
ctgaagttgt gccttttgcc a 21
<210> 41
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CYP19A1 Reverse
<400> 41
ctgaggtagg aaattagggg c 21
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CYP11A1 Forward
<400> 42
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CYP11A1 Reverse
<400> 43
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-STAR Forward
<400> 44
<210> 45
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-STAR Reverse
<400> 45
atccatgacc ctgaggttgg a 21
<210> 46
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-HSD17B1 Forward
<400> 46
gtctggcatc tgacccatct c 21
<210> 47
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-HSD17B1 Reverse
<400> 47
cgggcatccg ctattgaatc 20
<210> 48
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-HSD3B1 Forward
<400> 48
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-HSD3B1 Reverse
<400> 49
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-GAPDH Forward
<400> 50
tcaccagggc tgcttttaac t 21
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> qRT-PCR-GAPDH Reverse
<400> 51
Claims (10)
- The application of miR-370in the swine ovary granular cells is characterized in that: the application is particularly the application in the cycle process, proliferation, apoptosis and/or E2 secretion of the porcine ovarian granulosa cells; wherein the application environment is an in vitro environment.
- 2. Use according to claim 1, characterized in that:under the in vitro environment, the increase of miR-370 can increase the proportion of swine ovarian granulosa cells in the S phase, so that the periodic process of the swine ovarian granulosa cells is promoted; and/or miR-370 is reduced, so that the proportion of swine ovarian granulosa cells in the S phase can be reduced, and the periodic process of the swine ovarian granulosa cells is delayed;under the in-vitro environment, increasing miR-370 to promote the proliferation of the porcine ovarian granulosa cells, and/or reducing miR-370 to inhibit the proliferation of the porcine ovarian granulosa cells;under the in-vitro environment, increasing miR-370 to inhibit the apoptosis of the porcine ovarian granulosa cells, and/or reducing miR-370 to promote the apoptosis of the porcine ovarian granulosa cells;under the in vitro environment, increasing miR-370 promotes E2 secretion in porcine ovarian granulosa cells, and/or decreasing miR-370 inhibits E2 secretion in porcine ovarian granulosa cells.
- The application of miR-370in the regulation of GLI1 expression is characterized in that: under the in-vitro environment, increasing miR-370 inhibits GLI1 expression, and/or decreasing miR-370 promotes GLI1 expression.
- Use of GLI1 in porcine ovarian granulosa cells, characterized in that: the application is particularly the application in the cycle process, proliferation, apoptosis and/or E2 secretion of the porcine ovarian granulosa cells; wherein the application environment is an in vitro environment.
- 5. Use according to claim 4, characterized in that:in an in vitro environment, reducing GLI1 promotes the cycle progression of porcine ovarian granulosa cells;in an in vitro environment, reducing GLI1 promotes the proliferation of porcine ovarian granulosa cells;in an in vitro environment, reducing GLI1 inhibits apoptosis of porcine ovarian granulosa cells;in an in vitro environment, reduction of GLI1 promotes E2 secretion in porcine ovarian granulosa cells.
- The application of miR-370 to regulation of GLI1 expression in porcine ovarian granulosa cells is characterized in that: the application is particularly the application in the cycle process, proliferation, apoptosis and/or E2 secretion of the porcine ovarian granulosa cells; wherein the application environment is an in vitro environment.
- 7. Use according to claim 6, characterized in that:under an in-vitro environment, the expression of GLI1 is inhibited by increasing miR-370, and the cycle process of the porcine ovarian granulosa cells can be promoted; and/or by reducing miR-370 to promote expression of GLI1, the cycle process of the porcine ovarian granulosa cells can be delayed;under an in-vitro environment, the expression of GLI1 is inhibited by increasing miR-370, and the proliferation of the porcine ovarian granulosa cells can be promoted; and/or by reducing miR-370 to promote expression of GLI1, proliferation of porcine ovarian granulosa cells can be inhibited;under an in-vitro environment, by increasing miR-370 to inhibit GLI1 expression, the apoptosis of the swine ovarian granulosa cells can be inhibited; and/or by reducing miR-370 to promote expression of GLI1, the apoptosis of the porcine ovarian granulosa cells can be promoted;under an in-vitro environment, the expression of GLI1 is inhibited by increasing miR-370, and the secretion of E2 in porcine ovarian granulosa cells can be promoted; and/or by reducing miR-370 to promote expression of GLI1, secretion of E2 in porcine ovarian granulosa cells can be inhibited.
- 8. Use according to claim 7, characterized in that:the functional phenotype caused by miR-370 reduction and used for delaying the cell cycle process of the porcine ovarian granulosa cells can be recovered after the porcine ovarian granulosa cells are supplemented with GLI 1-siRNA;the functional phenotype for inhibiting the proliferation of the porcine ovarian granulosa cells, caused by the reduction of miR-370, can be recovered after the porcine ovarian granulosa cells are supplemented with GLI 1-siRNA;the functional phenotype for promoting the apoptosis of the porcine ovarian granulosa cells caused by the reduction of miR-370 can be recovered after the porcine ovarian granulosa cells are supplemented with GLI 1-siRNA;the functional phenotype caused by miR-370 reduction and inhibiting E2 secretion in the porcine ovarian granulosa cells can be recovered after the porcine ovarian granulosa cells are supplemented with GLI 1-siRNA;the GLI1-siRNA is GLI1-siRNA-1 or GLI 1-siRNA-2;GLI1-siRNA-1：5′-CCACAGGGCAGCAGCACUA-3′；GLI1-siRNA-2：5′-GAGAAUGUCACCAUGGAUACC-3′。
- 9. use according to claim 2, 3, 6 or 7, characterized in that:the miR-370 is increased by the following method: transfecting a miR-370mimic into a pig ovarian granule cell;the miR-370 reduction is realized by the following method: the miR-370inhibitor is transfected into porcine ovarian granulosa cells.
- 10. An siRNA that inhibits expression of GLI1, characterized by:the siRNA is GLI1-siRNA-1 or GLI1-siRNA-2, and the sequence is as follows:GLI1-siRNA-1：5′-CCACAGGGCAGCAGCACUA-3′；GLI1-siRNA-2：5′-GAGAAUGUCACCAUGGAUACC-3′。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115927321A (en) * | 2022-08-30 | 2023-04-07 | 华南农业大学 | Application of LncRNA IFA (LncRNA-induced fluorescence amplification) to adsorption of miR-26a in swine ovarian granulosa cells |
| CN117165696A (en) * | 2023-11-03 | 2023-12-05 | 四川省畜牧科学研究院 | Application of lncRNA related reagent in regulation and control of chicken follicular development |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100035966A1 (en) * | 2006-06-14 | 2010-02-11 | Rosetta Inpharmatics Llc | Methods and compositions for regulating cell cycle progression |
| US20100249213A1 (en) * | 2007-09-06 | 2010-09-30 | The Ohio State University Research Foundation | MicroRNA Signatures in Human Ovarian Cancer |
| US20150297575A1 (en) * | 2012-11-29 | 2015-10-22 | Strasspharma Llc | Methods of modulating follicle stimulating hormone activity |
| CN105567686A (en) * | 2016-01-19 | 2016-05-11 | 华南农业大学 | Application of miR-126-3p to porcine ovarian granular cells |
| CN110129428A (en) * | 2019-05-09 | 2019-08-16 | 安徽农业大学 | The application of miR-26a adjusting apoptosis of germ cells |
| CN110511903A (en) * | 2019-08-06 | 2019-11-29 | 安徽农业大学 | A kind of Study on Molecular Mechanism method and its application of miR-21-5p targeting Smad7 regulation pig ovary granular cell |
-
2022
- 2022-05-31 CN CN202210605257.4A patent/CN114854756B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100035966A1 (en) * | 2006-06-14 | 2010-02-11 | Rosetta Inpharmatics Llc | Methods and compositions for regulating cell cycle progression |
| US20100249213A1 (en) * | 2007-09-06 | 2010-09-30 | The Ohio State University Research Foundation | MicroRNA Signatures in Human Ovarian Cancer |
| US20150297575A1 (en) * | 2012-11-29 | 2015-10-22 | Strasspharma Llc | Methods of modulating follicle stimulating hormone activity |
| CN105567686A (en) * | 2016-01-19 | 2016-05-11 | 华南农业大学 | Application of miR-126-3p to porcine ovarian granular cells |
| CN110129428A (en) * | 2019-05-09 | 2019-08-16 | 安徽农业大学 | The application of miR-26a adjusting apoptosis of germ cells |
| CN110511903A (en) * | 2019-08-06 | 2019-11-29 | 安徽农业大学 | A kind of Study on Molecular Mechanism method and its application of miR-21-5p targeting Smad7 regulation pig ovary granular cell |
Non-Patent Citations (6)
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115927321A (en) * | 2022-08-30 | 2023-04-07 | 华南农业大学 | Application of LncRNA IFA (LncRNA-induced fluorescence amplification) to adsorption of miR-26a in swine ovarian granulosa cells |
| CN115927321B (en) * | 2022-08-30 | 2024-07-12 | 华南农业大学 | Application of LNCRNA IFA to adsorption of miR-26a in porcine ovarian granule cells |
| CN117165696A (en) * | 2023-11-03 | 2023-12-05 | 四川省畜牧科学研究院 | Application of lncRNA related reagent in regulation and control of chicken follicular development |
| CN117165696B (en) * | 2023-11-03 | 2024-02-06 | 四川省畜牧科学研究院 | Application of lncRNA related reagent in regulation and control of chicken follicular development |
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