Application of miR-328 to regulation and control of WFIKKN2 expression in pig skeletal muscle cells

Abstract

The invention discloses an application of miR-328 in regulation and control of WFIKKN2 expression in pig skeletal muscle cells. After the ssc-miR-328 simulant is transfected, the expression quantity of the WFIKKN2 gene is remarkably reduced, and a dual luciferase report experiment verifies that the expression of the ssc-miR-328 is regulated and controlled by combining the ssc-miR-328 with the WFIKKN2 gene 3 'UTR (Untranslated Region). Therefore, the miR-328 is taken as a research object, a cell proliferation test is detected through CCK-8, and a result shows that the proliferation rate of the porcine skeletal muscle cells can be inhibited after overexpression of the miR-328, and proves that the miR-328 can regulate and control expression of WFIKKN2, so that proliferation of the porcine skeletal muscle cells is inhibited or promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular genetics, and specifically relates to the application of miR-328 in regulating the expression of WFIKKN2 in porcine skeletal muscle cells. Background Art

[0002] As an organ, muscle can maintain the normal movement and physiological metabolism of animals, and as food, it is the main source for humans to obtain protein. Muscle tissue is mainly composed of muscle cells, and the differences in the morphology and distribution of muscle cells divide muscle tissue into three types: skeletal muscle, cardiac muscle, and smooth muscle. Skeletal muscle accounts for about 40% of the total body weight, has strong activity and plasticity, and is mainly composed of water, protein, and other substances. Skeletal muscle satellite cells can promote muscle growth, repair, and regeneration, and are located between the sarcolemma and the basement membrane. The development of skeletal muscle first involves the differentiation of stem cells into mononuclear myocytes to form multinuclear myotubes, and then differentiates into muscle fibers, and finally forms skeletal muscle. Studies have shown that the number of muscle fibers is determined during pregnancy, and postnatal skeletal muscle growth consists of the hypertrophy development of muscle fibers and the transformation of muscle fiber types. However, the muscle fiber type is also basically fixed at about 210 days of age after birth. The growth and development process of skeletal muscle is also accompanied by a complex gene regulation network, which involves many signaling pathways and complex cytokines, such as the MAPK signaling pathway, the Wnt signaling pathway, and myogenic regulatory factors. Therefore, exploring the molecular regulation mechanism of skeletal muscle is of great significance for the growth and development of pigs.

[0003] The WFIKKN2 gene (Growth and differentiation factor associated serum protein-1, GASP1), also known as growth differentiation factor associated serum protein 1, has 5 different functional domains in its protein and belongs to the multi-domain protein family. The WFIKKN1 gene (Growth and differentiation factor associated serum protein-2, Gasp2), also known as growth differentiation factor associated serum protein 2, belongs to the same family as the WFIKKN2 gene. The WFIKKN2 gene was originally called the WFIKKNP gene and was discovered on human chromosome 17. The WFIKKN with similar exon and intron structures is located on human chromosome 16 and is also called the WFIKKN1 gene. Studies have shown that during embryonic development, the WFIKKN2 gene is highly expressed in skeletal muscle, kidney, thymus, and brain tissues, and the WFIKKN1 gene is highly expressed in skeletal muscle, liver, and kidney tissues. He Jianxiong's research found that the methylation degree of the WFIKKN2 gene promoter region in the longissimus dorsi muscle of pigs at different 10-week-old ages is different, thus affecting the expression of this gene.

[0004] miRNA (MicroRNA) is a small RNA with a length of approximately 20 - 24 nt, which can precisely regulate gene expression levels. The development of high-throughput sequencing technology has led to the discovery of more and more miRNAs, and their effects on skeletal muscle growth and development have been reported more and more frequently. Nakasa et al. found that miR-1, 133, and 206 can interact to promote myotube differentiation and can be used as a new method for treating skeletal muscle injuries. Pengfei Wu et al. discovered two miRNAs related to chicken growth and development (novel_miR_133 and miR-24-3p), and verified the function of miR-24-3p, finding that this miRNA can inhibit the proliferation of primary chicken myoblasts and promote differentiation. Jia B et al. identified age-dependent miRNAs and single genes by measuring miRNAs in the skeletal muscles of sika deer of different ages, providing insights into the molecular mechanisms of muscle development, growth, and maintenance. In this study, overexpression and interference vectors of this gene were constructed and transfected into porcine skeletal muscle cells to detect the expression levels of related genes, and miRNAs binding to the WFIKKN2 gene were predicted by bioinformatics and verified by dual-luciferase reporter assays, providing a preliminary research basis for exploring its mechanism of regulating muscle growth and development. Summary of the Invention

[0005] The object of the present invention is as follows: In view of the above problems, the present invention provides the application of miR-328 in regulating WFIKKN2 expression in porcine skeletal muscle cells, and the specific scheme is as follows:

[0006] One object of the present invention is to provide the application of miR-328 in regulating WFIKKN2 expression in porcine skeletal muscle cells, and the specific application is in regulating the proliferation of porcine skeletal muscle cells;

[0007] For any one of the following applications (1) - (2);

[0008] (1) In an in vitro environment, by increasing miR-328 to inhibit WFIKKN2 expression, the proliferation of porcine skeletal muscle cells can be inhibited;

[0009] (2) In an in vitro environment, by reducing miR-328 to promote WFIKKN2 expression, the proliferation of porcine skeletal muscle cells can be promoted.

[0010] Further explanation, the down-regulation of WFIKKN2 gene expression caused by increasing miR-328 inhibits the proliferation rate of porcine skeletal muscle cells.

[0011] Further explanation, the up-regulation of WFIKKN2 gene expression caused by reducing miR-328 promotes the proliferation rate of porcine skeletal muscle cells.

[0012] Further explanation: The increase of miR-328 is achieved by the following method: Transfecting miR-328 mimics into porcine skeletal muscle cells.

[0013] Further explanation: The down-regulation of the WFIKKN2 gene is achieved by transfecting siRNA that inhibits the expression of WFIKKN2 into porcine skeletal muscle cells.

[0014] The siRNA has the following sequences:

[0015] WFIKKN2-siRNA-1: 5'-GCAACCACTTTGAGACCTA-3';

[0016] WFIKKN2-siRNA-2: 5'-GGAGTGACTTCGTCATCTT-3';

[0017] WFIKKN2-siRNA-3: 5'-CCCAGCTGGTCATCTATAA-3'.

[0018] Further explanation: The miR-328 mimics have the following sequences:

[0019] ssc-miR-328: 5'-CUGGCCCUCUCUGCCCUUCCGU-3'.

[0020] The present invention also provides a method for promoting or inhibiting the proliferation of porcine skeletal muscle cells. The method is that promoting the expression of the WFIKKN2 gene can promote the proliferation of porcine skeletal muscle cells; inhibiting the expression of the WFIKKN2 gene can inhibit the proliferation of porcine skeletal muscle cells.

[0021] The up-regulation of the WFIKKN2 gene is achieved by transfecting an overexpression vector containing the sequence shown in SEQ ID NO:1;

[0022] The down-regulation of the WFIKKN2 gene is achieved by transfecting siRNA;

[0023] The siRNA is siRNA-1, siRNA-2 or siRNA-3;

[0024] siRNA-1: 5'-GCAACCACTTTGAGACCTA-3';

[0025] siRNA-2: 5'-GGAGTGACTTCGTCATCTT-3';

[0026] siRNA-3: 5'-CCCAGCTGGTCATCTATAA-3'.

[0027] The present invention also provides a method for regulating the expression of the WFIKKN2 gene in porcine skeletal muscle cells, which is achieved by using an ssc-miR-328 mimic targeting the 3'UTR of the WFIKKN2 gene; the ssc-miR-328 mimic can bind to the 3'UTR of the WFIKKN2 gene, thereby affecting its expression, and the binding site is predicted by the online websites miRBase and RNAhybrid and verified by a dual-luciferase reporter assay; the sequence of the ssc-miR-328 mimic is 5'-CUGGCCCUCUCUGCCCUUCCGU-3'.

[0028] In summary, the beneficial effects of the technical solution of the present invention are as follows:

[0029] 1. The experimental research results of the present invention show that: the agarose gel electrophoresis results show a clear single band at 1500 bp to 2000 bp, which is consistent with the length of the target fragment, proving that the overexpression vector construction is successful; the online website RNAhybrid results show a high degree of binding between ssc-miR-328 and the 3'UTR of the WFIKKN2 gene; the MegAlign software comparison results show that the wild-type and mutant vector constructions are successful; the fluorescence activity of the cells co-transfected with the wild-type plasmid and the ssc-miR-328 mimic group is extremely significantly lower (P<0.01) than that of the co-transfected mutant and ssc-miR-328 mimic group, proving that the binding site is correct; the real-time fluorescence quantitative results show that the interference effect of siRNA2 is the best and is used for subsequent experiments; after overexpressing the WFIKKN2 gene, its expression level increases extremely significantly (P<0.01), and the expression levels of the CyclinD, CyclinE, and PCNA genes also increase extremely significantly (P<0.01); after interfering with the WFIKKN2 gene, its expression level decreases extremely significantly (P<0.01), and the expression levels of the CyclinD, CyclinE, and PCNA genes also decrease extremely significantly (P<0.01); after transfecting the ssc-miR-328 mimic, the expression level of the WFIKKN2 gene decreases extremely significantly (P<0.01). Therefore, it can be concluded that the WFIKKN2 gene can promote the proliferation of porcine skeletal muscle cells, and ssc-miR-328 can bind to the 3'UTR of the WFIKKN2 gene to affect its expression, providing a theoretical basis for exploring skeletal muscle growth and development.

[0030] 2. Synthesize 3 pairs of small interfering fragments / controls (WFIKKN2-siRNA / siRNA-NC) for interfering with WFIKKN2, and screen and detect their interference efficiency. From Figure 4 the results, it can be seen that after transfecting the gene interfering small fragments into porcine skeletal muscle cells, by means of qRT-PCR, the WFIKKN2-siRNA-2 small fragment with better interference effect is finally screened for subsequent experiments.

[0031] WFIKKN2-siRNA-2: 5'-CCACAGGGCAGCAGCACTA-3'.

[0032] 3. Through the CCK-8 assay for cell proliferation detection, it was found that after interfering with miR-328, the proliferation rate of porcine skeletal muscle cells could be promoted.

[0033] In summary, it is proved that miR-328 can regulate the expression of WFIKKN2, thereby inhibiting or promoting the proliferation of porcine skeletal muscle cells. Brief Description of the Drawings

[0034] Figure 1 This is the double digestion identification of the recombinant plasmid pEGFP-N1-WFIKKN2 of the present invention. Among them, M, DL10000 DNA Marker; 1, recombinant plasmid pEGFP-N1-WFIKKN2.

[0035] Figure 2 This is the predicted map of the binding site of ssc-miR-328 of the present invention.

[0036] Figure 3 This is the comparison map of the sequencing results of the wild-type and mutant sites of the 3'UTR of the WFIKKN2 gene of the present invention.

[0037] Figure 4 This is the screening map of the small interfering RNA of the WFIKKN2 gene of the present invention.

[0038] Figure 5 This is the transfection result of the recombinant plasmid of the present invention (10 × ) map.

[0039] Figure 6 This is the comparison map of the expression levels of the WFIKKN2 gene and other genes of the present invention.

[0040] Figure 7 This is the expression change map of the reporter genes in the WFIKKN2-WT and WFIKKN2-MUT groups of the present invention.

[0041] Figure 8 This is the schematic diagram of the binding site between ssc-miR-328 and WFIKKN2 of the present invention.

[0042] Figure 9 This is the schematic diagram of the expression levels of ssc-miR-328 and the WFIKKN2 gene of the present invention.

[0043] Figure 10 This is the detection of the effect of miR-328 on the proliferation ability of porcine skeletal muscle cells by the CCK-8 method. ** indicates P < 0.01, with extremely significant differences. Detailed Embodiments

[0044] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following will describe the specific embodiments of the present invention in detail with reference to the accompanying drawings. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0045] Example:

[0046] 1 Materials and Methods

[0047] 1.1 Samples

[0048] The porcine skeletal muscle cells, 293T cells, pEGFP-N1 vector, and psiCHECK-2 vector used in this experiment were all stored in the Animal Husbandry Research Institute of Guangxi Agricultural Vocational and Technical University.

[0049] 1.2 Reagents

[0050] Lipofectamine 3000 (L3000015), Opti-MEM (31985070), HindⅢ (SM0102), BamHI (FD0054) / XhoI (ER0691), and NotI (ER0591) restriction endonucleases were purchased from Thermo Fisher Scientific; Premix Taq enzyme (R004A), reverse transcription kit (RR037A), SYBR enzyme (RR820A), and competent cells (DH5a) (9057) were purchased from Takara Biotechnology Co., Ltd.; 1×PBS buffer (P1020) and trypsin digestion solution (0.25%) (T1300) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; Gel Extraction Kit (DP214-03), Endotoxin-Free Plasmid Mini Kit (DP118-02), miRcute Enhanced miRNA cDNA First Strand Synthesis Kit (KR211-02), and miRcute Enhanced miRNA Fluorescent Quantitative Detection Kit (FP411-02) were all purchased from Tiangen Biochemical Technology Co., Ltd.

[0051] 1.3 Methods

[0052] 1.3.1 Design and Synthesis of WFIKKKN2 Gene Primers

[0053] According to the porcine WFIKKN2 gene sequence on NCBI (accession number: XM_005668923.3), specific primer sequences were designed with an expected amplification length of 1760 bp, including a full-length coding region sequence of 1743 bp; referring to the wild boar WFIKKN2 gene, CyclinD gene, CyclinE gene, PCNA gene, and GAPDH gene mRNA published by NCBI, primers for real-time quantitative PCR were designed, and the specific information is shown in Table 1.

[0054] Table 1 Primer Information

[0055]

[0056] 1.3.2 Cloning of the CDS Region of the WFIKKN2 Gene

[0057] Using the longissimus dorsi cDNA of Luchuan pigs as a template to amplify the WFIKKN2 gene, the PCR amplification system was 10 μL: 1 μL of cDNA template, 5 μL of Premix Taq enzyme, 0.4 μL each of WFIKKN2-clone-F and WFIKKN2-clone-R, and 3.2 μL of RNase-free ddH2O. The PCR reaction program was: 94°C for 30 s, 59°C for 100 s, 72°C for 30 s, for a total of 30 cycles, and cooled to 16°C. The PCR products were identified by 1% agarose gel electrophoresis, and the target fragments were recovered according to the instructions of the gel recovery kit, ligated to the pMD18-T vector, the ligation products were transformed into Escherichia coli DH5α competent cells, spread on LA plates, and several single colonies were picked for colony PCR identification. Positive colonies with the same size as the target fragment and clear bands were selected for sequencing.

[0058] 1.3.3 Construction and Identification of Eukaryotic Expression Vectors

[0059] The pMD18-T-WFIKKN2 recombinant plasmid after sequencing identification was selected for amplification and plasmid extraction. The pMD18-T-WFIKKN2 recombinant plasmid and the pEGFP-N1 empty vector were double-digested with the restriction endonucleases HindⅢ and BamHⅠ respectively, detected by agarose gel electrophoresis, and the target fragments and the enzyme digestion products of the pEGFP-N1 empty vector were ligated overnight with T4 ligase. The ligation products were transformed into Escherichia coli DH5α competent cells, transformed, plated, picked, amplified, and sequenced. After sequencing and PCR verification, the correct recombinant plasmid was selected for amplification and small-scale plasmid extraction, and the plasmid was extracted after endotoxin removal by double digestion verification and stored at -80°C for later use.

[0060] 1.3.4 Prediction of miRNAs Targeting the 3'UTR of the WFIKKN2 Gene

[0061] Predict miRNAs that can bind to the 3' UTR of the WFIKKN2 gene using the online websites miRBase (http: / / www.mirbase.org / search.shtml) and RNAhybrid (https: / / bibiserv.cebitec.uni-bielefeld.de / rnahybrid / ).

[0062] 1.3.5 Construction of dual-luciferase vectors

[0063] Select a 500-bp fragment containing the binding site and construct wild-type and mutant vectors, namely Psicheck2-WFIKKN2-WT and Psicheck2-WFIKKN2-MUT, by Sangon Biotech (Shanghai) Co., Ltd.

[0064] 1.3.6 Synthesis of interfering RNA and miRNA

[0065] The interfering RNA (siRNA) of the WFIKKN2 gene was constructed by Guangzhou Ribobio Co., Ltd., and the mimic of the target miR-328 was synthesized by Guangzhou Ribobio Co., Ltd.

[0066] 1.3.7 Cell culture and transfection

[0067] Take out the cryopreserved porcine skeletal muscle cells and 293T cells from liquid nitrogen and resuscitate them in a 37°C water bath. Inoculate them into cell culture dishes and place them in a cell culture incubator for culture. Passage when the cell confluence reaches 90%. After 3 passages, transfer them to 6-well plates for culture. When the cell confluence reaches 70%, perform transfection experiments according to the instructions of the Lipofectamine 3000 kit. After successful transfection, culture the cells for 48 h and collect the cells to detect the expression levels of related genes.

[0068] 1.3.8 Real-time fluorescence quantitative PCR

[0069] Using the cDNA of each tissue and the cDNA of transfected cells as templates, and GAPDH as an internal reference, detect the expression of the WFIKKN2 gene by real-time fluorescence quantitative PCR. The PCR reaction system is 10 μL: 5.0 μL of TBGreen Ⅱ, 0.25 μL of each upstream and downstream primer, 2.5 μL of cDNA template, and 2.0 μL of RNase-free ddH2O. The PCR reaction program: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, for a total of 37 cycles. Use 3 samples for the experiment, and each sample is repeated 3 times.

[0070] 1.3.9 Data analysis and processing

[0071] The quantitative results were statistically sorted out by Excel, and one-way ANOVA was performed using SPSS Statistics 26.0 software. The experimental results were expressed as mean ± standard deviation. P < 0.05 indicated significant differences; P < 0.01 indicated extremely significant differences.

[0072] 2 Results

[0073] 2.1 Construction of eukaryotic expression vector of WFIKKN2 gene

[0074] Total RNA was extracted from the longissimus dorsi muscle of Luchuan pigs and reversely transcribed into cDNA by ordinary reverse transcription. The full-length 1743 bp of the WFIKKN2 gene was cloned for constructing the overexpression vector (the sequence shown in SEQ ID NO: 1). The recombinant plasmid pEGFP-N1-WFIKKN2 was double-digested with restriction endonucleases HindⅢ and BamHI, and the results of agarose gel electrophoresis are shown in Figure 1 It shows that there is a clear single band at 1500 bp - 2000 bp, which is consistent with the length of the target fragment. The sequencing results were compared with the CDS region of the WFIKKN2 gene published on NCBI using MegAlign software, and the results all proved that the vector construction was successful.

[0075] 2.2 Prediction of miRNA targeting the 3’UTR of WFIKKN2 gene

[0076] The 3’UTR sequence of the WFIKKN2 gene was copied on NCBI, and it was predicted by the online website miRBase that ssc-miR-328 could bind to it. Then RNAhybrid was used to predict its binding site, and the result showed that there was a binding site at 295 bp. The results are shown in Figure 2 as follows.

[0077] 2.3 Construction of wild-type and mutant vectors of the 3’UTR of WFIKKN2 gene

[0078] The constructed Psicheck2-WFIKKN2-WT and Psicheck2-WFIKKN2-MUT vectors were double-digested with restriction endonucleases XhoI and NotI, and the gel-extracted products were sent for sequencing and compared using MegAlign software. The results are shown in Figure 3 as follows, proving that the wild-type and mutant vectors were successfully constructed.

[0079] 2.4 Synthesis and screening of interfering RNA of WFIKKN2 gene

[0080] Three interfering RNAs of the WFIKKN2 gene, namely siRNA1 (5'-GCAACCACTTTGAGACCTA-3'), siRNA2 (5'-GGAGTGACTTCGTCATCTT-3'), and siRNA3 (5'-CCCAGCTGGTCATCTATAA-3'), were transfected into porcine skeletal muscle cells respectively. The cells were collected after 48 h to detect the expression level of the WFIKKN2 gene. The results were as Figure 4 shown. The interference efficiency of siRNA2 was the highest, and siRNA2 was selected for subsequent experiments.

[0081] 2.5 Cell transfection experiment

[0082] The recombinant plasmid pEGFP-N1-WFIKKN2 and the empty pEGFP-N1 were transfected into porcine skeletal muscle cells respectively according to the instructions of the Lipofectamine 3000 kit. After 48 h, the cells were observed under a microscope. The results were as Figure 5 shown. Both the empty vector and the recombinant plasmid had green fluorescence, indicating successful transfection. siRNA2 and miR-328 mimics were transfected into porcine skeletal muscle cells according to the above method; the wild-type and mutant vectors of the 3'UTR of the WFIKKN2 gene were transfected into 293T cells.

[0083] 2.6 Real-time fluorescence quantitative PCR

[0084] After transfection with the overexpression and interference vectors for 48 h, the cells were harvested to extract RNA and reverse transcribed to detect the expression of related genes. As Figure 6 shown, after overexpression, the expression level of the WFIKKN2 gene increased extremely significantly (P < 0.01), and the expression levels of the CyclinD, CyclinE, and PCNA genes also increased extremely significantly (P < 0.01); the interference results were opposite. It was initially speculated that the WFIKKN2 gene had the effect of promoting the proliferation of skeletal muscle cells.

[0085] 2.7 Dual-luciferase activity detection

[0086] ssc-miR-328 and WFIKKN2 were co-transfected into 293T cells. After 48 h, luciferase detection was performed using an enzyme-labeled instrument. The results were as Figure 7 shown, indicating that the expression of the luciferase reporter gene was regulated by ssc-miR-328, and after mutating the binding site, the expression of the reporter gene could be restored. It was shown that ssc-miR-328 bound to the WFIKKN2 gene, and the verified binding site was correct. The binding site was as Figure 8 shown.

[0087] 2.8 Effect of overexpression of ssc-miR-328 on the expression of the WFIKKN2 gene

[0088] The ssc-miR-328 mimics were transfected into porcine skeletal muscle cells, and the expression of the WFIKKN2 gene was detected 48 h later. The results are as Figure 9 shown, and the expression level of the WFIKKN2 gene decreased extremely significantly (P < 0.01).

[0089] In summary, the WFIKKN2 gene can promote the proliferation of porcine skeletal muscle cells, and ssc-miR-328 can bind to the 3'UTR of the WFIKKN2 gene to affect the expression of this gene.

[0090] Example 2: Effect of transfection with miR-328 mimics on the proliferation of porcine skeletal muscle cells

[0091] Cell Counting Kit-8 (CCK8) was used to detect cell proliferation:

[0092] Porcine skeletal muscle cells were seeded in 96-well plates. When the cell density reached about 50%, miR-328 mimics were transfected. After 48 h of transfection, 10 μL of CCK8 solution was added, and the mixture was incubated in a CO2 incubator at 37 °C for 3 h. The absorbance (OD) at 450 nm was measured using a microplate reader. Wells containing the corresponding amount of cell culture medium, drug, and CCK-8 solution but no cells were selected as blank controls. The results showed that the OD value of the miR-328 mimic transfection group decreased significantly, indicating a decrease in cell proliferation ability. The results are as Figure 10 shown.

[0093] In summary, it is proved that interfering with miR-328 can inhibit / promote the proliferation rate of porcine skeletal muscle cells.

[0094] The above-described embodiments merely represent several embodiments of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. Application of miR-328 in regulating WFIKKN2 expression in porcine skeletal muscle cells, characterized in that: The application described is specifically for the application in regulating the proliferation of porcine skeletal muscle cells; It is any one of the following applications (1) to (2); (1) In an in vitro environment, by increasing miR-328 to inhibit WFIKKN2 expression, the proliferation of porcine skeletal muscle cells can be inhibited; (2) In an in vitro environment, by reducing miR-328 to promote WFIKKN2 expression, the proliferation of porcine skeletal muscle cells can be promoted.

2. The application according to claim 1, wherein: The down-regulation of WFIKKN2 gene expression caused by increasing miR-328 thereby inhibits the proliferation rate of porcine skeletal muscle cells.

3. The application according to claim 2, wherein: The increase in miR-328 is achieved by the following method: transfecting miR-328 mimics into porcine skeletal muscle cells.

4. The application according to claim 2, characterized in that: The sequence of the miR-328 mimics is as follows: ssc-miR-328: 5'-CUGGCCCUCUCUGCCCUUCCGU-3'.

5. A method for promoting or inhibiting the proliferation of porcine skeletal muscle cells, characterized in that, The method is that promoting the expression of the WFIKKN2 gene can promote the proliferation of porcine skeletal muscle cells; inhibiting the expression of the WFIKKN2 gene can inhibit the proliferation of porcine skeletal muscle cells; The up-regulation of the WFIKKN2 gene is achieved by transfecting an overexpression vector containing the sequence shown in SEQ ID NO:1; The down-regulation of the WFIKKN2 gene is achieved by transfecting siRNA; The siRNA is siRNA-1, siRNA-2 or siRNA-3; siRNA-1: 5'-GCAACCACTTTGAGACCTA-3'; siRNA-2: 5'-GGAGTGACTTCGTCATCTT-3'; siRNA-3: 5'-CCCAGCTGGTCATCTATAA-3'.

6. A method for regulating the expression of the WFIKKN2 gene in porcine skeletal muscle cells, characterized in that, It is achieved by ssc-miR-328 mimics targeting the 3'UTR of the WFIKKN2 gene; the ssc-miR-328 mimics can bind to the 3'UTR of the WFIKKN2 gene, thereby affecting its expression, and the binding site is predicted by the online website miRBase and RNAhybrid and verified by a dual-luciferase reporter assay; the sequence of the ssc-miR-328 mimics is 5'-CUGGCCCUCUCUGCCCUUCCGU-3'.

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