Application of Wnt2 gene in buffalo ovarian granular cells

Abstract

The invention discloses application of a Wnt2 gene in buffalo ovarian granular cells, and belongs to the technical field of cell engineering and gene engineering. Wnt2 is taken as a research object, the Wnt2 overexpression vector or Wnt2 siRNA is transfected to the ovarian granulosa cells respectively by constructing the Wnt2 overexpression vector and synthesizing small interfering RNA (Wnt2 siRNA), then changes of signal channel gene mRNA and protein level related to ovarian granulosa cell apoptosis, proliferation and E2 and P4 secretion are detected, and finally phenotypic changes of the ovarian granulosa cells are detected. Results show that Wnt2 can inhibit apoptosis of ovarian granular cells and promote proliferation and E2 and P4 secretion. By exploring the application of the Wnt2 in the ovarian granular cells, the application has good application in researching an ovarian follicle atresia mechanism, initial estrus initiation and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering and gene engineering, and relates to the application of Wnt2 gene in buffalo ovarian granulosa cells. Background Art

[0002] Granulosa cells are an important component of follicles and the main site of estrogen synthesis. During follicular development, they gradually proliferate from a single flat layer to a multi-layer columnar layer, and produce cytokines, growth factors, and estrogens related to follicular development, promoting the maturation of follicles. It is currently believed that the recruitment, selection, and ovulation of follicles are essentially the proliferation and differentiation of granulosa cells, while the apoptosis of granulosa cells represents the atresia and degeneration of follicles.

[0003] The Wnt gene was first discovered in mouse breast cancer, so it was named integration 1 (Int1). Later, it was found that the gene was a homologous gene to the wingless gene (Wg) of fruit flies, so the two were combined and named Wnt genes. The Wnt gene is composed of a family of secreted glycoproteins rich in cysteine. It participates in a variety of signal transduction pathways, regulates cell proliferation, differentiation and apoptosis, and is an important regulatory factor for the growth and development of organisms. The Wnt2 gene is an important member of the Wnt gene family and is the second Wnt gene to be identified. The Wnt signaling pathway is considered to be an intraovarian regulatory factor, and the components of this pathway are regulated and expressed at specific stages of ovarian follicle development in adult animals. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the Wnt2 gene in buffalo ovarian granulosa cells in response to the above problems. The present invention takes Wnt2 as the research object, constructs a Wnt2 overexpression vector and a synthetic small interfering RNA (Wnt2siRNA), and transfects the Wnt2 overexpression vector or Wnt2siRNA into ovarian granulosa cells, respectively, and then detects changes in the mRNA and protein levels of signaling pathway genes related to ovarian granulosa cell apoptosis, proliferation, and E2 and P4 secretion, and finally detects phenotypic changes in ovarian granulosa cells. The results show that Wnt2 can inhibit apoptosis of ovarian granulosa cells and promote proliferation and E2 and P4 secretion. By exploring the application of Wnt2 in ovarian granulosa cells, the present invention has a good application in studying the mechanism of ovarian follicle atresia and the initiation of puberty.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] One object of the present invention is to provide the application of Wnt2 gene in buffalo ovarian granulosa cells, and the application of Wnt2 gene in ovarian granulosa cell proliferation, apoptosis, E2 secretion and / or progesterone secretion in vitro.

[0007] To further explain, the application is:

[0008] In vitro, increasing the Wnt2 gene can promote the proliferation of ovarian granulosa cells, and / or decreasing the Wnt2 gene can inhibit the proliferation of ovarian granulosa cells;

[0009] In vitro, increasing the Wnt2 gene can inhibit apoptosis of ovarian granulosa cells, and / or decreasing the Wnt2 gene can promote apoptosis of ovarian granulosa cells;

[0010] In vitro, increasing the Wnt2 gene can inhibit E2 secretion in ovarian granulosa cells, and / or decreasing the Wnt2 gene can promote E2 secretion in ovarian granulosa cells;

[0011] In vitro, increasing the Wnt2 gene can promote the secretion of progesterone by ovarian granulosa cells, and / or decreasing the Wnt2 gene can inhibit the secretion of progesterone by ovarian granulosa cells; the progesterone is progesterone.

[0012] The present invention also provides another purpose of using the Wnt2 gene in preparing a drug for promoting the secretion of progesterone by ovarian granulosa cells, specifically by increasing exogenous Wnt2 gene.

[0013] It is further explained that the increase of exogenous Wnt2 gene is achieved by the following method: connecting the Wnt2 gene to a vector to construct an overexpression vector containing the Wnt2 gene; and then transfecting the overexpression vector containing the Wnt2 gene into ovarian granulosa cells.

[0014] The present invention also provides a third purpose of using the Wnt2 gene in buffalo ovarian granulosa cells. In vitro, adding the Wnt2 gene can improve the maturation rate, cleavage rate, blastocyst rate and blastocyst quality of buffalo ovarian granulosa cells.

[0015] It is further explained that the increase of Wnt2 in the application of the present invention is achieved by the following method: constructing an overexpression vector containing the Wnt2 gene, and transfecting the overexpression vector into buffalo ovarian granulosa cells; the reduction of Wnt2 is achieved by the following method: transfecting buffalo ovarian granulosa cells with siRNA that inhibits the expression of the Wnt2 gene.

[0016] The fourth object of the present invention is to provide an application of siRNA for inhibiting Wnt2 gene in buffalo ovarian granulosa cells, wherein the application environment is an in vitro environment; siRNA can inhibit apoptosis of ovarian granulosa cells, promote proliferation of ovarian granulosa cells, inhibit E2 secretion in ovarian granulosa cells, and promote progesterone secretion in ovarian granulosa cells;

[0017] The sequence of siRNA that inhibits Wnt2 gene is as follows:

[0018] siRNA-Wnt2-3:

[0019] si Wnt2-3-sense(5'-3'):CAGUUUGGAAUCUGUGCAA,

[0020] si Wnt2-3-antisense(5'-3'):UUGCACAGAUUCCAAACUG.

[0021] Due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0022] The results obtained by the inventors through research are: overexpression of Wnt2 in buffalo granulosa cells promotes the expression of genes such as Axin2, CTNNB1, Gsk3B, Caspase3, Bax, Bcl2, Cyp19a1, CCND2, Cart, DVL1, Wnt10, etc., and inhibits the expression of C-myc and SFRP1 genes. Overexpression of Wnt2 in buffalo granulosa cells inhibits p-GSK3β and promotes the expression of proteins such as β-catenin, p-AKT, and LEF1. Overexpression of Wnt2 in buffalo granulosa cells promotes granulosa cell proliferation, reduces the expression of E2, and promotes the expression of progesterone. The granulosa cells of oocytes in three groups, namely, the Wnt2 overexpression group, the Wnt2 interference group, and the control group, were matured. The Wnt2 overexpression group significantly improved the maturation rate, cleavage rate, blastocyst rate, and blastocyst quality (OCT4 expression was positively correlated with embryo quality), while the Wnt2 interference group had the opposite effect. It shows that overexpressing the Wnt2 gene can promote the proliferation of granulosa cells and the expression of E2, thereby inhibiting follicular atresia and increasing the synchronized estrus ovulation rate; on the other hand, making a single layer of granulosa cells that overexpress the Wnt2 gene can promote the maturation rate, cleavage rate, blastocyst rate and blastocyst quality, thereby improving the efficiency of embryo culture in vitro, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparison diagram of the overexpression and interference time screening of Wnt2 in buffalo granulosa cells of the present invention. The upper left figure is the overexpression time screening figure; the upper right figure is the interference time screening figure; the lower figure is the transfection red fluorescence expression figure.

[0024] Figure 2 This is a diagram showing the gene expression changes of overexpressed Wnt2 in buffalo granulosa cells of the present invention.

[0025] Figure 3 The diagram is a protein expression change diagram of overexpression and interference of Wnt2 in buffalo granulosa cells of the present invention. The left diagram is a WB diagram; the right diagram is a WB relative expression level.

[0026] Figure 4This is a comparison diagram of the promotion of cell proliferation by overexpression of Wnt2 in buffalo granulosa cells of the present invention.

[0027] Figure 5 The diagram is a comparison of the changes in the levels of Wnt2 hormone overexpressed in buffalo granulosa cells of the present invention. The left diagram is a comparison of the effects on the secretion of progesterone hormone; the right diagram is a comparison of the effects on the secretion of E2 hormone.

[0028] Figure 6 This is a comparison diagram of the effects of overexpression or interference of Wnt2 in buffalo granulosa cells on IVF embryo culture. The upper diagram is a comparison diagram of the number of mature, cleavage, and blastocysts; the lower diagram is an immunofluorescence diagram of the effects on Oct4 protein expression. DETAILED DESCRIPTION

[0029] The specific implementation of the invention is further described below with reference to the accompanying drawings.

[0030] 1. Construction of buffalo Wnt2 overexpression vector

[0031] Primerpremier 5.0 software was used to design primers to clone it and eGFP into pDC316. The primers are as follows:

[0032] Wnt2-pDC316-F(5'-3'): acgcagatcgaattcATGAACGCCTGTCTCGTTGG,

[0033] Wnt2-pDC316-R(5'-3'):tgctcaccatTGTCGGAGCAGCCCAGTCG,

[0034] EGFP-Wnt2-pDC316-F(5'-3'):tgctccgacaATGGTGAGCAAGGGCGAGG,

[0035] EGFP-Wnt2-pDC316-R (5'-3'): gacgagctcaagcttTTACTTGTACAGCTCGTCCATGCC. Extract buffalo ovarian RNA, reverse transcribe into cDNA, and perform PCR amplification; use pEGFP-N1 as a template to amplify eGFP, construct pDC316-Wnt2-eGFP vector, send for sequencing, and screen the vector with the correct sequence fragment for subsequent experiments.

[0036] 2. Design of synthetic interference fragments

[0037] ThermoFisher siRNA design website (Invitrogen Block-iT RNAi Designer (thermofisher.com)) was used to design three buffalo siRNA fragments, namely

[0038] si Wnt2-1-sense(5'-3'):GCGGAGGAACGAGUUGAAA,

[0039] si Wnt2-1-antisense(5'-3'):UUUCAACUCGUUCCUCCGC;

[0040] si Wnt2-2-sense(5'-3'):GAAUCUUCACAACAACCGA,

[0041] si Wnt2-2-antisense(5'-3'):UCGGUUGUUGUGAAGAUUC;

[0042] si Wnt2-3-sense(5'-3'):CAGUUUGGAAUCUGUGCAA,

[0043] si Wnt2-3-antisense(5'-3'):UUGCACAGAUUCCAAACUG;

[0044] Control group siNC-sense (5′-3′): UUCUCCGAACGUGUCACGU;

[0045] siNC-antisense(5'-3'):ACGUGACACGUUCGGAGAA.

[0046] Synthesized by Zixi Biotechnology Co., Ltd.

[0047] 3. Isolation and Culture of Buffalo Oocytes and Granulosa Cells

[0048] Buffalo ovaries were obtained from a slaughterhouse and rinsed three times with 2× double-antibody saline. Follicles with a diameter of 2-4 mm were selected, and follicular fluid was extracted with a syringe to collect oocytes for subsequent experiments. Granulosa cells were collected by centrifugation of the remaining follicular fluid and cultured in a serum-free culture system until they adhered to the wall.

[0049] 4. Screening of interference fragments and interference time

[0050] Buffalo granulosa cells were collected at 1×10 41000 cells / well were inoculated in a 24-well plate. Transfection was performed according to the instructions of the siRNA transfection reagent LipofectamineRNAiMAX. Cells were collected 24 hours after transfection, and the best interference fragment was screened by detecting the effect of the interference fragment on the expression of the buffalo Wnt2 gene. The best interference fragment and the control fragment were transfected into buffalo granulosa cells respectively, and the cells were collected 24, 48 and 72 hours after transfection to screen the best interference time.

[0051] After screening, the best interference fragment was si-RNA-Wnt2-3. Figure 1 (Top left figure) It can be seen that the best overexpression transfection of buffalo granulosa cells at 24, 48 and 72 hours was significantly different from the control fragment, and the best time was 24 hours. Figure 1 (Upper right figure) It can be seen that the best interference fragment transfected buffalo granulosa cells at 24, 48 and 72 hours were significantly different from the control fragment, and the best time was 24 hours. Figure 1 (Figure below) It can be seen that red fluorescence is expressed at 24, 48 and 72 hours, and the fluorescence expression increases with time.

[0052] 5. Oocyte Maturation in vitro

[0053] Select COCs wrapped with two or more layers of cumulus cells, and place 10 to 20 COCs in 40 μL in vitro maturation medium (TCM199 + 10% FBS + 5 μg / mL FSH + 10 μg / mL LH + 0.2 mmol / L sodium pyruvate + 1 μg / mL LH) covered with paraffin oil. 2 +50μmol / L cysteine ​​+25ng / mL EGF) droplets were inoculated into granulosa cell monolayers, and the monolayers were divided into Wnt2 overexpression group, Wnt2 interference group and control group, at 38.5℃ and 5% CO 2 The oocytes were cultured under saturated humidity for 22-24 hours. The oocyte maturation was marked by the discharge of the first polar body.

[0054] Maturation rate (%) = number of oocytes with the first polar body released / total number of oocytes incubated × 100%

[0055] The cell maturation rate data are shown in the table below.

[0056]

[0057] Different lowercase letters on the shoulders indicate significant differences (P<0.05); different uppercase letters on the shoulders indicate extremely significant differences (P<0.01); the same letters on the shoulders indicate no significant differences (P>0.05). The same below.

[0058] 6. IVF embryo production

[0059] Sperm used for IVF was purchased from frozen semen of Mora or Nili-Lafite buffalo produced by the Guangxi Zhuang Autonomous Region Animal and Poultry Breeding Station. Sperm was thawed, climbed and centrifuged, and then inoculated into oocytes with cumulus cells removed. In vitro culture of fertilized eggs and observation of early embryonic development: Fertilized oocytes were transferred into CM solution (TCM199 + 10% FBS), and cumulus cells were removed by repeated blowing with a pipette, and then washed once with CM solution, and transferred into granulosa cell monolayer droplets. Granular cell monolayers were divided into Wnt2 overexpression group, Wnt2 interference group and control group, and placed in CO 2 Incubator, 5% CO 2 , cultured at 39°C and saturated humidity. The medium was replaced by half every 48 hours. The cleavage rate was recorded 48 hours after fertilization, and the blastocyst development was recorded 5 to 9 days after fertilization.

[0060] The data of cell cleavage rate and blastocyst rate are shown in the table below.

[0061]

[0062] The maturation rate, cleavage rate and blastocyst rate were significantly increased in the Wnt2 overexpression group, while the opposite was true in the interference group.

[0063] 7. Immunofluorescence Detection

[0064] Embryos or cells that developed to 7 days old were washed 3 times with PBS, fixed in 4% paraformaldehyde solution at room temperature for 30 minutes, washed 3 times with PBST (PBS containing 0.1% Tween-20), 5 minutes each time, and then transferred to a permeabilization solution containing 0.1% TritonX-100 for 30 minutes, washed with PBST, and then transferred to a blocking solution (PBST+5% BSA) for blocking at room temperature for 1 hour, and then transferred to the primary antibody after washing with PBST. The negative control group was not incubated with the primary antibody and incubated in a humidified box at 4°C overnight; after washing with PBST, the cells were transferred to the secondary antibody (goat anti-rabbit IgG H&L, 1:1000, goat anti-mouse IgG H&L, 1:1000) and incubated at room temperature for 2 hours, washed with PBST, and then transferred to 10μg / mL Hoechst33342 for nuclear counterstaining for 15 minutes, and then washed with PBST, and then transferred to a small amount of antifade droplets on a glass slide, covered with a cover glass, fixed, and observed and photographed under a laser confocal microscope. The fluorescence intensity was analyzed using ImageJv1.8.0 software, and the mean fluorescence intensity was used to semi-quantitatively characterize the specific protein expression.

[0065] like Figure 6 As shown, compared with the control group, the Wnt2 group promoted the expression of Oct4 protein, and the si-Wnt2 group reduced the expression of Oct4 protein.

[0066] 8. Real-time fluorescence quantitative PCR detection

[0067] After collecting the cells of the experimental group and the control group 24 hours after interference, RNA was extracted using an RNA extraction kit and reverse transcribed, and cDNA was diluted to a concentration of 100 ng / μL. Primers were designed using PrimerPremier5.0, and the primers were synthesized by Wuhan Qingke Innovation Biotechnology Co., Ltd. Real-time fluorescence quantitative PCR reaction system 20 μL: cDNA 100 ng, upstream and downstream primers 0.5 μL each, 2×PowerUpSYBR Green MasterMix 10 μL, ddH 2 O to 20 μL. PCR reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 60 s, a total of 40 cycles. Each group of experiments was repeated 3 times. -△△Ct The expression level of each gene was calculated relative to the control group, and Graph PadPrism was used for significance analysis and plotting.

[0068] like Figure 2 As shown, overexpression of Wnt2 in buffalo granulosa cells promotes the expression of genes such as Axin2, CTNNB1, Gsk3B, Caspase3, Bax, Bcl2, Cyp19a1, CCND2, Cart, DVL1, and Wnt10, and inhibits the expression of genes such as C-myc and SFRP1.

[0069] 9.CCK8 detection of cell proliferation

[0070] Inoculate the cell suspension in a 96-well plate, about 100 μl per well, and repeat 3 times for the same sample; add 10 μl of CCK-8 solution to each well at 24, 48, and 72 h, mix well, and place the culture plate in an incubator for 1-4 h; measure the absorbance (OD) at 450 nm with an enzyme-labeled instrument.

[0071] like Figure 4 As shown, overexpression of Wnt2 promoted the proliferation of buffalo granulosa cells, but the difference was not significant.

[0072] 10.E2 and P4 hormone determination

[0073] The collected cell culture fluid, E2 (estrogen) and P4 (progesterone) were determined using ELISA kits. First, the standard curve was drawn, and blank wells, standard wells and sample wells were set. 50 μL of standard diluted with standard diluent (0, 3.75, 7.5, 15, 30, 60, 120 pmol / mL) was added to the standard wells, and 50 μL of cell culture fluid diluted with sample diluent at 1:5 was added to the sample wells. After sealing, the membrane was incubated at 37°C for 30 minutes, the liquid was discarded, and it was washed with PBS 4 to 5 times. After patting dry, 50 μL of enzyme labeling reagent was added to continue sealing the membrane, incubated at 37°C for 30 minutes, and after repeating the washing steps, 50 μL of color developer A and B were added, and the color was developed at 37°C for 15 minutes. Then 50 μL of stop solution was added, and the OD value at 450nm was measured with an enzyme labeler.

[0074] like Figure 5 As shown, overexpression of Wnt2 in buffalo granulosa cells decreased the expression of E2 and promoted the expression of progesterone.

[0075] 11. Western blot detection

[0076] After collecting the cells, centrifuge and discard the supernatant, add RIPA lysis buffer (add 1mmol / LPMSF) to extract the total cell protein, and determine the total protein concentration by BCA method. After the total protein was denatured at 98℃ for 10min, it was loaded for SDS-PAGE electrophoresis, and the gel was transferred to the PVDF membrane by wet transfer method. After blocking for 10min, the PVDF membrane was washed 3 times with TBST. Add the primary antibody (1:1000 dilution), incubate at 4℃ overnight, wash 3 times with TBST, add the secondary antibody (1:1000 dilution), incubate at room temperature for 1h, and wash 3 times with TBST. Prepare the color development solution, add the PVDF membrane and the color development solution to the color development box, react for 3min in the dark, and expose and take pictures in the imager.

[0077] like Figure 3 As shown in the figure, overexpression of Wnt2 in buffalo granulosa cells inhibits p-GSK3β and promotes the expression of β-catenin, p-AKT, LEF1 and other proteins, but the difference is not significant.

[0078] 1.4 Statistical analysis

[0079] Each experiment was repeated 3 times, and GraphPad Prism 7.0 was used for significance analysis and drawing. The experimental data were expressed as mean ± standard error, and the two groups of data were compared using t-test. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0080] 2. Result analysis:

[0081] Overexpression of Wnt2 in buffalo granulosa cells promotes the expression of genes such as Axin2, CTNNB1, Gsk3B, Caspase3, Bax, Bcl2, Cyp19a1, CCND2, Cart, DVL1, and Wnt10, and inhibits the expression of genes such as C-myc and SFRP1. Overexpression of Wnt2 in buffalo granulosa cells inhibits p-GSK3β and promotes the expression of proteins such as β-catenin, p-AKT, and LEF1. Overexpression of Wnt2 in buffalo granulosa cells promotes granulosa cell proliferation, reduces the expression of E2, and promotes the expression of progesterone. When oocytes were matured in granulosa cells of three groups, namely, Wnt2 overexpression group, Wnt2 interference group, and control group, the maturation rate, cleavage rate, blastocyst rate, and blastocyst quality (OCT4 expression was positively correlated with embryo quality) were significantly increased in the Wnt2 overexpression group, while the effect of the Wnt2 interference group was the opposite. It shows that overexpressing the Wnt2 gene can promote the proliferation of granulosa cells and the expression of E2, thereby inhibiting follicular atresia and increasing the synchronous estrus ovulation rate; on the other hand, making a single layer of granulosa cells that overexpress the Wnt2 gene can promote the maturity rate, cleavage rate, blastocyst rate and blastocyst quality, thereby improving the efficiency of embryo culture in vitro.

[0082] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. Application of Wnt2 gene in buffalo ovarian granulosa cells, characterized in that: In vitro, the application of Wnt2 gene in ovarian granulosa cell proliferation, apoptosis, E2 secretion and / or progesterone secretion.

2. The use according to claim 1, characterized in that: In vitro, increasing the Wnt2 gene can promote the proliferation of ovarian granulosa cells, and / or decreasing the Wnt2 gene can inhibit the proliferation of ovarian granulosa cells; In vitro, increasing the Wnt2 gene can inhibit apoptosis of ovarian granulosa cells, and / or decreasing the Wnt2 gene can promote apoptosis of ovarian granulosa cells; In vitro, increasing the Wnt2 gene can inhibit E2 secretion in ovarian granulosa cells, and / or decreasing the Wnt2 gene can promote E2 secretion in ovarian granulosa cells; In vitro, increasing the Wnt2 gene can promote the secretion of progesterone by ovarian granulosa cells, and / or decreasing the Wnt2 gene can inhibit the secretion of progesterone by ovarian granulosa cells; the progesterone is progesterone.

3. Use of the Wnt2 gene in the preparation of a drug for promoting the secretion of progesterone by ovarian granulosa cells, characterized in that: Specifically, it is by increasing the exogenous Wnt2 gene.

4. The use of the Wnt2 gene according to claim 3 in the preparation of a drug for promoting the secretion of progesterone by ovarian granulosa cells, characterized in that: The increase of exogenous Wnt2 gene is achieved by the following method: connecting Wnt2 gene to a vector to construct an overexpression vector containing Wnt2 gene; and then transfecting the overexpression vector containing Wnt2 gene into ovarian granulosa cells.

5. Application of Wnt2 gene in buffalo ovarian granulosa cells. In vitro, increasing the Wnt2 gene can improve the maturation rate, cleavage rate, blastocyst rate and blastocyst quality of buffalo ovarian granulosa cells.

6. The use according to claim 2 or 5, characterized in that: The increase of Wnt2 is achieved by constructing an overexpression vector containing the Wnt2 gene and transfecting the overexpression vector into buffalo ovarian granulosa cells; the reduction of Wnt2 is achieved by transfecting buffalo ovarian granulosa cells with siRNA that inhibits the expression of the Wnt2 gene.

7. Use of siRNA for inhibiting Wnt2 gene in buffalo ovarian granulosa cells, characterized in that: The application environment is an in vitro environment; siRNA can inhibit apoptosis of ovarian granulosa cells, promote proliferation of ovarian granulosa cells, inhibit E2 secretion in ovarian granulosa cells, and promote progesterone secretion in ovarian granulosa cells; The sequence of siRNA that inhibits Wnt2 gene is as follows: siRNA-Wnt2-3: si Wnt2-3-sense(5'-3'):CAGUUUGGAAUCUGUGCAA, si Wnt2-3-antisense(5'-3'):UUGCACAGAUUCCAAACUG.

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