Detection method and application of porcine lincRNA4160 tissue expression
By detecting and analyzing the expression and regulation mechanism of pig lincRNA4160, it was found that it inhibited the proliferation and differentiation of pig muscle satellite cells, promoted apoptosis, solved the problem of unclear mechanism of long-chain non-coding RNA, and provided for muscle development and disease diagnosis. New targets and theoretical foundations.
Patent Information
- Application Number
- CN202411363157.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-27
- Publication Date
- 2024-12-27
AI Technical Summary
The existing technology has not fully analyzed the specific role of long chain non-coding RNA (lncRNA) in pig muscle growth, especially the effect of pig lincRNA4160 on myoblast proliferation, differentiation and apoptosis, which has affected muscle development, livestock and poultry breeding and muscle Diagnosis and treatment of diseases.
Provide a detection method, including sample collection, total RNA extraction and cDNA synthesis, real-time quantitative PCR to detect the expression of lincRNA4160, regulate its expression in cells through siRNA interference technology, analyze its regulatory mechanism in combination with transcriptome sequencing, and study its in-pig Proliferation, differentiation and apoptosis processes in skeletal muscle satellite cells.
By analyzing the expression spectrum and regulatory effects of lincRNA4160, it was found that it inhibited the proliferation and differentiation of pig muscle satellite cells and promoted apoptosis, providing targets for muscle development and muscle-related diseases, helping to analyze the molecular regulatory mechanism of muscle diseases, and improving the Meat production and meat quality provide a theoretical basis.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of molecular biology, and in particular to a method for detecting the effects of porcine lincRNA4160 tissue expression on myoblast proliferation, differentiation and apoptosis and an application thereof. Background Art
[0002] Skeletal muscle is an important component of the animal body, providing structural support, motor control and energy storage for life activities. Skeletal muscle development is a complex and strictly regulated process, involving the proliferation, differentiation and apoptosis of myoblasts. The growth and development of skeletal muscle not only affects the muscle yield and quality of livestock and poultry, but also affects human athletic ability and health status. Analyzing the molecular regulatory mechanism of muscle development can not only guide animal husbandry production, but also help in the diagnosis and treatment of muscle-related diseases. In-depth elucidation of the molecular mechanism of muscle development is of great significance for livestock breeding and the diagnosis and treatment of muscle diseases.
[0003] The development of skeletal muscle is an intricate regulatory network, which is coordinated and orderly regulated by genes, transcription factors, miRNAs, lncRNAs, circRNAs, etc. The specific regulatory mechanism still needs to be explored. Long noncoding RNA (lncRNA) is a type of nucleotide RNA with a length greater than 200 nt, which is mainly transcribed by RNA polymerase II and has stronger spatiotemporal specificity than coding genes. According to its relative physical position to the adjacent protein-coding gene, lncRNA can be divided into sense lncRNA, antisense lncRNA, bidirectional lncRNA, intergenic lncRNA and intragenic lncRNA. In recent years, with the development of high-throughput sequencing technology, a large number of long non-coding RNAs related to muscle development and muscle diseases have been discovered, such as lncMREF interacting with Smarca5, promoting the genomic binding of p300 / CBP / H3K27ac, and upregulating myogenic differentiation; TCONS_00323213 and PKNOX2 relieve the inhibitory effect of PKNOX2 on the MyoG promoter, promoting MyoG expression and thus promoting the differentiation of porcine skeletal muscle satellite cells; lncRNA-MEG3 / miRNA-133a-3p / PRRT2 axis can regulate skeletal muscle regeneration and myogenesis; lncRNA SOX6 AU promotes the proliferation, apoptosis and differentiation of bovine primary skeletal muscle cells through PI3K / AKT and MAPK signaling pathways. However, there are not many lncRNAs that have analyzed the specific mechanism of action, and the functional analysis of a single lncRNA is far from enough, so further research is needed to better understand the role of lncRNAs in porcine muscle growth. Summary of the invention
[0004] In view of the above-mentioned defects and problems, the present invention provides a detection method and application of the effect of porcine lincRNA4160 tissue expression on myoblast proliferation, differentiation and apoptosis. Based on previous high-throughput sequencing data, the present invention screened out lincRNA4160 related to the development of porcine longissimus dorsi muscle, and proved for the first time that lincRNA4160 can inhibit the proliferation and differentiation of porcine muscle satellite cells and promote apoptosis.
[0005] The solution adopted by the present invention to solve the technical problem is: a method for detecting the effect of porcine lincRNA4160 on myoblast proliferation, differentiation and apoptosis, comprising the following steps: (1) Sample collection, total RNA extraction and cDNA synthesis, real-time quantitative PCR, using GAPDH as the internal reference gene, using 2 －△△Ct The relative gene expression was calculated by (2) Cell culture, differentiation induction and transfection, transfecting si-lincRNA4160 and si-NC into cells; (3) Expression profile analysis of lincRNA4160: Real-time fluorescence quantitative PCR was used to detect the expression of lincRNA4160 in different tissues of pigs, and the expression of pig skeletal muscle satellite cells at 30%, 60%, and 90% confluence and at D0, D2, D4, and D6 of induced differentiation; (4) Extract total protein from tissues or cells and analyze protein blots; use EdU to detect cell proliferation; use flow cytometry to detect cell cycle, stain mitochondrial membrane potential, and measure changes in mitochondrial membrane potential; (5) Transfect si-lincRNA4160 and siNC into pig muscle satellite cells, perform transcriptome sequencing analysis, and analyze the specific regulatory mechanism of lincRNA4160; (6) SPSS 22.0 statistical software was used to test for significant differences, and the results were expressed as mean ± SEM.
[0006] Furthermore, in step (1), total RNA is extracted from tissues and cells using AG RNAex Pro reagent; PrimeScript TM The extracted RNA was reverse transcribed into cDNA using the RT Reagent Kit with gDNA Eraser. Taq TM The kit uses a three-step method for real-time fluorescence quantitative PCR. The PCR reaction system is: SYBR Premix Ex Taq TMⅡ 12.5 μL, upstream and downstream primers 1 μL each, cDNA template 2 μL, ddH2O 8.5 μL; PCR reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles.
[0007] Furthermore, in the step (2), the sample tissue is minced, digested with collagenase II and trypsin at 37°C, and then neutralized with DMEM F12 medium containing 20% fetal bovine serum, 1% penicillin-streptomycin, and 1% FGFb, and muscle satellite cells are separated and purified by differential adhesion method; when inducing differentiation, when the cell confluence is 70%-80%, the differentiation medium is replaced; si-lincRNA4160 and si-NC are transfected into cells using DharmaFECT, and si-lincRNA4160 and si-NC are transfected into cells using DharmaFECT.
[0008] Furthermore, in step (3), real-time fluorescence quantitative PCR is used to detect the expression level of lincRNA4160 in the heart, liver, spleen, lung, kidney, small intestine, stomach, longissimus dorsi muscle, and subcutaneous fat.
[0009] Furthermore, in the step (4), total protein of tissues or cells is extracted using RIPA containing protease inhibitors; cell proliferation is detected according to the instructions of the EdU kit; and cell cycle is detected using a cell cycle detection kit.
[0010] Furthermore, in step (5), the transcriptome sequencing analysis steps are as follows: after total RNA extraction, mRNA with polyA is adsorbed by Oligo (dT) magnetic beads for cDNA synthesis, and a cDNA library is constructed after the quality and content of cDNA are detected; sequencing analysis is performed using the PE150 sequencing strategy on the Illumina platform, and adapters and low-quality reads are removed from the raw data; Q20 and Q30 are calculated for Clean Data to detect the sequencing error rate, and the CleanReads are assembled de novo using the HISAT2 software, and gene expression analysis is performed using the FPKM method; subsequently, differential expression analysis between groups is performed using the DESeq2 method; FDR < 0.05, |log2 (Fold Change) | > 1 are used as the criteria for screening differentially expressed genes (DEGs); finally, DEG functional annotation is performed by GO analysis, and DEG enrichment pathways are screened by KEGG analysis.
[0011] Application of porcine lincRNA4160 in regulating myoblast proliferation, differentiation and apoptosis.
[0012] Furthermore, the lincRNA4160 can inhibit the proliferation of pig skeletal muscle satellite cells; lincRNA4160 can inhibit the differentiation of pig skeletal muscle satellite cells; lincRNA4160 can promote the apoptosis process of pig skeletal muscle satellite cells.
[0013] Furthermore, the porcine lincRNA4160 inhibits the proliferation and differentiation of porcine muscle satellite cells and promotes apoptosis by regulating Ankrd23 and CENPK genes via pathways related to muscle diseases and energy metabolism.
[0014] Beneficial effects of the present invention: In the study of the present invention, by analyzing the expression spectrum of lincRNA4160 and its regulatory effects on the proliferation, differentiation and apoptosis of pig muscle satellite cells, and using transcriptome sequencing to screen the differentially expressed genes and pathways interfering with lincRNA4160, the RT-qPCR results showed that lincRNA4160 was most highly expressed in the myocardium and longissimus dorsi muscle. During the proliferation and differentiation of pig muscle satellite cells, the expression of lincRNA4160 was significantly downregulated. In pig muscle satellite cells, inhibiting the expression of lincRNA4160 significantly promoted proliferation and myogenic differentiation, and inhibited apoptosis. It was found that lincRNA4160 had obvious temporal expression specificity, and it inhibited the proliferation and differentiation of pig muscle satellite cells and promoted apoptosis by regulating genes such as Ankrd23 and CENPK, through pathways related to muscle diseases such as Diabeticcardiomyopathy, Parkinson disease, and Huntington disease, and energy metabolism-related pathways such as Oxidative phosphorylation, Thermogenesis, and Metabolic. Studies have shown that lincRNA4160 is involved in regulating the proliferation, differentiation and apoptosis of pig muscle satellite cells, and can be used as a target for muscle development and muscle-related diseases. These findings provide a theoretical basis for further improving meat production and quality and analyzing the molecular regulatory mechanisms of muscle diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 is the expression pattern of lincRNA4160; wherein, a: expression of lincRNA4160 in different tissues of pigs; b: expression of lincRNA4160 in different proliferation stages of pig skeletal muscle satellite cells; c.: expression of lincRNA4160 in different differentiation stages of pig skeletal muscle satellite cells; Figure 2 The interference efficiency of si-linc4160 and si-NC in porcine skeletal muscle satellite cells; Figure 3The effects of lincRNA4160 on the proliferation of porcine skeletal muscle satellite cells; a: the effect of interfering with lincRNA4160 on the mRNA expression of key proliferation genes; b: the effect of interfering with lincRNA4160 on protein expression; c: the results of flow cytometry detection of cell cycle; d: the results of EdU; Figure 4 The effects of lincRNA4160 on the differentiation of porcine skeletal muscle satellite cells; a: the effect of interfering with lincRNA4160 on the mRNA expression of key differentiation genes; b: the effect of interfering with lincRNA4160 on protein expression; c: the effect of interfering with lincRNA4160 on myotube formation; Figure 5 The effects of lincRNA4160 on apoptosis of porcine muscle satellite cells; a: the effect of interfering with lincRNA4160 on the mRNA expression of key apoptosis genes; b: the effect of interfering with lincRNA4160 on protein expression; c: the staining results of TMRE by interfering with lincRNA4160; Figure 6 This is the volcano map of differentially expressed genes in pig skeletal muscle satellite cells interfering with lincRNA4160; Figure 7 This is a hierarchical cluster analysis diagram of differentially expressed genes in pig skeletal muscle satellite cells interfering with lincRNA4160; Figure 8 This is the GO cluster analysis diagram of differentially expressed genes in pig muscle satellite cells interfering with lincRNA4160; Fig. 9 This is the KEGG analysis diagram of differentially expressed genes in pig skeletal muscle satellite cells interfering with lincRNA4160. DETAILED DESCRIPTION
[0016] The present invention is further described below in conjunction with the accompanying drawings and embodiments.
[0017] In the early stage of this study, the lncRNA differential expression profiles of the longissimus dorsi muscle of 21-day-old Huainan pigs and Large White pigs at different developmental stages were compared. It was found that ALDBSSCT0000004160 (lincRNA4160) was located on chromosome 15, with a total length of 280 nt and 2 exons. Its expression was significantly different between the two breeds ( P <0.05), and the expression level in Large White pigs was three times that in Huainan pigs, suggesting that lincRNA4160 may be related to the development and regulation of the longissimus dorsi muscle. To further explore the role of lincRNA4160 in skeletal muscle development, this study confirmed through a series of experiments that lincRNA4160 can inhibit the proliferation and differentiation of pig muscle satellite cells and promote apoptosis.
[0018] See also Figure 1-9 The present invention provides a method for detecting the effect of porcine lincRNA4160 tissue expression on myoblast proliferation, differentiation and apoptosis and a technical solution for its application: Example 1: Detection method for regulating myoblast proliferation, differentiation and apoptosis by porcine lincRNA4160 1. Sample Collection Three healthy Huainan pigs were selected and raised in Henan Xingrui Agriculture and Animal Husbandry Technology Co., Ltd. according to the NY / 65-2004 feeding standard. They were slaughtered on day 300. Within 30 minutes of slaughter, 9 tissue samples including heart, liver, spleen, lung, kidney, small intestine, stomach, longissimus dorsi muscle, and subcutaneous fat were collected, placed in cryopreservation tubes, and quickly put into liquid nitrogen and transported to the laboratory for standby use.
[0019] 2. Total RNA Extraction, cDNA Synthesis and Real-time Quantitative PCR Total RNA was extracted from tissues and cells using AG RNAex Pro reagent (AG21102, Accurate Biology) according to the instructions. The quality and concentration of the extracted RNA were detected by agarose gel electrophoresis and Nano-Drop2000 UV spectrophotometer. PrimeScript TM The extracted RNA was reverse transcribed into cDNA using the RT Reagent Kit with gDNA Eraser (Perfect Real-time).
[0020] Using SYBR Premix Ex Taq TM The kit uses a three-step method for real-time fluorescence quantitative PCR. PCR reaction system: SYBR Premix Ex Taq TM Ⅱ 12.5 μL, upstream and downstream primers 1 μL each, cDNA template 2 μL, ddH2O 8.5 μL. PCR reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, a total of 40 cycles. GAPDH was used as the internal reference gene, and 2 －△△Ct The relative expression of genes was calculated by the method. GAPDH was selected as the reference gene. The primers used in this study were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequences are shown in Table 1.
[0021] Table 1. Real-time quantitative primer information
[0022] 3. Cell Culture, Differentiation Induction and Transfection Newborn piglets (1 day old) were washed with 75% alcohol and sacrificed by exsanguination to obtain longissimus dorsi muscle samples. The tissues were minced, digested with collagenase II (FBS, Gibco, USA) and trypsin (FBS, Gibco, USA) at 37°C, and then neutralized with DMEM F12 medium (FBS, Gibco, USA) containing 20% fetal bovine serum (FBS, Gibco, USA), 1% penicillin-streptomycin, and 1% FGFb. Muscle satellite cells were isolated and purified by differential adhesion. When inducing differentiation, the differentiation medium (2% horse serum + 1% penicillin-streptomycin + DMEM F12 medium) was replaced when the cell confluence was 70%-80%. Small interfering RNAs (siRNAs) targeting lincRNA4160 and negative controls were designed by RiboBio (Genepharma, China). According to the instructions, si-lincRNA4160 and si-NC were transfected into cells using DharmaFECT.
[0023] 4. Analysis of lincRNA4160 expression profile Real-time fluorescence quantitative PCR was used to detect the expression level of lincRNA4160 in heart, liver, spleen, lung, kidney, small intestine, stomach, longissimus dorsi muscle, and subcutaneous fat, as well as the expression level of porcine skeletal muscle satellite cells at 30%, 60%, and 90% confluence and at D0, D2, D4, and D6 of induced differentiation.
[0024] 5. Western Blot Analysis Total protein from tissues or cells was extracted using radio-immunoprecipitation assay (RIPA) containing protease inhibitors. Equal amounts of protein were separated by SDS-PAGE electrophoresis, transferred to PVDF membranes, and the membranes were incubated with antibodies against PCNA, CDK2, CyclinD, MyoD, MyoG, Bcl2, Bax, P21, P53, and GapDH, and then incubated with secondary antibodies. Image-Pro Plus 6.0 software was used to quantify band intensity.
[0025] 6. EdU detection of cell proliferation Cell proliferation was detected according to the EdU kit instructions (RiboBio, Guangzhou, China), 50 μM EdU (5-Ethynyl-2'-deoxyuridine) solution was added to the cells, incubated for 4 h, and then the cell nuclei were stained with Hoechst 33342 (RiboBio, Guangzhou, China). An inverted fluorescence microscope was used to observe and photograph.
[0026] 7. Flow cytometry detection of cell cycle The cell cycle was detected using a cell cycle detection kit (Multisciences, Hangzhou, China). The cells were washed with PBS, and staining solution and permeabilization reagent were added. After incubation for 30 min, the cell cycle was analyzed using a flow cytometer (FACS Canto II, BD BioSciences, USA), with 3 replicates for each treatment group.
[0027] 8. Mitochondrial Membrane Potential Staining The cells were washed with PBS, incubated with 1×TMRE staining solution (Solarbio, China) and Hoechst33342 working solution (Solarbio, China) in sequence, observed and photographed using an inverted fluorescence microscope, and the changes in mitochondrial membrane potential were measured based on the fluorescence intensity.
[0028] 9. Transcriptome Analysis In order to analyze the specific regulatory mechanism of lincRNA4160, si-lincRNA4160 and siNC were transfected into porcine muscle satellite cells, and the cells were collected 48 h after transfection and sent to Guangzhou Kidio Biotechnology Co., Ltd. for transcriptome sequencing analysis. The analysis steps are briefly described as follows: After total RNA extraction, Oligo (dT) magnetic beads were used to adsorb mRNA with polyA for cDNA synthesis. After the quality and content of cDNA were detected, a cDNA library was constructed. Sequencing analysis was performed using the PE150 sequencing strategy on the Illumina platform. Adapters and low-quality reads were removed from the raw data. Clean Data was Q20 and Q30 calculated to detect the sequencing error rate. Clean Reads were assembled de novo using HISAT2 software, and gene expression analysis was performed using the FPKM (Fragments Per Kil obase Per Million Fragments) method. Subsequently, the DESeq2 method was used for differential expression analysis between groups. FDR < 0.05, |log2 (Fold Change) | > 1 were used as the criteria for screening differentially expressed genes (DEGs). Finally, GO (Gene Ontology) analysis was used for DEG functional annotation, and KEGG (Kyoto Encyclopedia Of Genes And Genomes) analysis was used to screen DEG-enriched pathways.
[0029] 10. Statistical Analysis SPSS 22.0 statistical software was used to test the significance of the difference, and the results were expressed as mean ± SEM. t Test. Statistical significance is indicated as follows:* P < 0.05, ** P < 0.01.
[0030] Example 2: Application of porcine lincRNA4160 in regulating myoblast proliferation, differentiation and apoptosis 1. Expression profile of lincRNA4160 qRT-PCR analysis showed that lincRNA4160 was expressed in 8 tissues except kidney, with the highest expression in myocardium and longissimus dorsi muscle, followed by subcutaneous fat and liver (e.g. Figure 1 a). During the proliferation of pig skeletal muscle satellite cells, the expression level of lincRNA4160 gradually decreased, with significant differences ( P <0.05) (e.g. Figure 1 b). During the differentiation of porcine skeletal muscle satellite cells, the expression level of lincRNA4160 was always lower than that before differentiation induction. Although the expression level of lincRNA4160 increased slightly on D4, it was still significantly lower than that on D0 ( P <0.05) (e.g. Figure 1 c).
[0031] 2. lincRNA4160 interference efficiency detection To study the function of lincRNA4160, si-linc4160 and si-NC were transfected into porcine skeletal muscle satellite cells, and the cells were collected 24 h after transfection. Figure 2 As shown, the RT-qPCR results showed that compared with si-NC, the expression level of lincRNA4160 in the si-linc4160 group was reduced by about 60%, and the difference was extremely significant (p < 0.01), indicating that the interference efficiency was obvious and could be used for the next functional verification experiment.
[0032] 3. Effect of interfering with lincRNA4160 on the proliferation of porcine skeletal muscle satellite cells Interference of lincRNA4160 in porcine skeletal muscle satellite cells, such as Figure 3As shown in the figure, RT-qPCR results showed that as the expression of lincRNA4160 decreased, the mRNA expression levels of PCNA, CDK2, and CyclinD increased significantly compared with the control group (P < 0.01), and the Western blot results were consistent with the RT-qPCR results. EdU detection indicated that interfering with lincRNA4160 could significantly promote cell proliferation (P < 0.01). Annexin V-PE / 7-AAD staining and flow cytometry results were consistent with the above results. After interfering with lincRNA4160, the number of cells in the G0 / G1 phase and S phase decreased by 10.62% and 8.64%, respectively, and the number of cells in the G2 / M phase increased by 19.26%, indicating that the decrease in lincRNA4160 expression relieved the G1 phase arrest and promoted cell proliferation. These results all indicate that lincRNA4160 can inhibit the proliferation of porcine skeletal muscle satellite cells.
[0033] 4. Effect of interfering with lincRNA4160 on differentiation of porcine skeletal muscle satellite cells To investigate whether lincRNA4160 regulates myogenic differentiation, after interfering with lincRNA4160, Figure 4 As shown, RT-qPCR results showed that the expression level of lincRNA4160 decreased, the expression level of MyoG increased significantly (P < 0.01), and the expression level of MyoD increased significantly (P < 0.05) ( Figure 4 a), WB results are consistent with this ( Figure 4 b). Figure 4 As shown in c, the myotubes of the cells in the interference group became thicker and longer than those in the control group. These results indicate that lincRNA4160 can inhibit the differentiation of pig skeletal muscle satellite cells.
[0034] 5. Effect of interfering with lincRNA4160 on apoptosis of porcine skeletal muscle satellite cells When studying the effect of lincRNA4160 on cell apoptosis, it was found that the expression of lincRNA4160 decreased and the expression of Bcl-2 increased significantly ( P <0.01), the expression levels of Bax, p21 and p53 were significantly decreased ( P <0.01), Western blot results were consistent with RT-qPCR results. TMRE immunofluorescence staining results showed that the addition of lincRNA4160 siRNA increased mitochondrial membrane potential, indicating a decrease in the number of apoptotic cells. These results indicate that lincRNA4160 can promote the apoptosis of porcine skeletal muscle satellite cells.
[0035] 6. Screening and functional enrichment of differentially expressed genes by interfering with lincRNA4160 To further analyze the molecular regulatory mechanism of lincRNA4160, transcriptome sequencing technology was used to analyze the changes in gene expression after interfering with lincRNA4160. The results showed that compared with si-NC, si-linc4160 upregulated the expression of 8 genes and downregulated the expression of 11 genes ( Figure 6-Figure 7 ).
[0036] like Figure 8 As shown, GO enrichment analysis of differentially expressed genes found that these 19 differentially expressed genes were mainly involved in cellular, development, and metabolic process, and their molecular functions were mainly related to binding, catalytic activity, and transcriptional regulation.
[0037] like Fig. 9 As shown, the results of KEGG enrichment analysis suggested that these differentially expressed genes were mainly enriched in muscle development-related pathways, such as Diabetic cardiomyopathy, Parkinson disease, Huntington disease, Amyotrophic lateral sclerosis, Alzheimer disease, and energy metabolism-related pathways, such as Oxidative phosphorylation, Thermogenesis, and Metabolic pathways.
[0038] Skeletal muscle development is regulated by a series of coding genes and non-coding genes. Among the differentially expressed genes screened, the Ankyrin repeat domain-containing protein 23 (Ankrd23) gene is a nuclear protein-encoding gene highly expressed in muscle. It is associated with the actin cytoskeleton and can inhibit myoblast differentiation. In this study, the expression of lincRNA4160 was inhibited in porcine muscle satellite cells, and Ankrd23 was also reduced, and myogenic differentiation was enhanced, suggesting that lincRNA4160 may participate in the regulation of myogenic differentiation through Ankrd23. Another differentially expressed gene in this study, Centromeric protein K (CENPK), is involved in regulating the Cell Cycle signaling pathway, which can promote cell proliferation and inhibit apoptosis. In this study, the expression of CENPK decreased with the decrease of lincRNA4160 expression, suggesting that lincRNA4160 has the effect of inhibiting proliferation and promoting apoptosis, which is consistent with the results of EDU staining, flow cytometry staining, and TMRE staining.
[0039] Skeletal muscle dysplasia may be caused by causes such as muscle hypertrophy, muscle atrophy, and mitochondrial myopathy. Studies have shown that noncoding RNA is involved in regulating muscle development and muscle-related diseases. LncRNA ZNF593-AS alleviates diabetic cardiomyopathy by inhibiting the IRF3 signaling pathway. lncRNA-DNM3OS regulates GAPDH by adsorbing miR-196b-5p to affect the onset of Huntington's disease. In this study, the differentially expressed gene mt-ND3 was interfered with by lincRNA4160, and its 10158T>C mutation caused mitochondrial myopathy and lactic acidosis, while the 10191T>C mutation caused Leigh-like syndrome, the classic feature of which is the progressive loss of motor ability. In addition, the differentially expressed genes in this study were enriched in multiple muscle disease-related pathways such as diabetic cardiomyopathy, Parkinson disease, Huntington disease, and amyotrophic lateral sclerosis, suggesting that lincRNA4160 is of great significance to the normal development of muscle and can be used as a biomarker and therapeutic target for muscle diseases.
[0040] In this study, after interfering with lincRNA4160 in porcine skeletal muscle satellite cells, the differentially expressed genes Ankrd23 and mt-Nd3 were also involved in regulating energy metabolism. Studies have shown that Ankrd23 can inhibit LKB1 protein expression (the main upstream kinase of AMPK) and promote AMPK phosphorylation, thereby upregulating glucose uptake and oxidation levels in myotubes; mt-Nd3 mutations can cause mitochondrial complex I functional deficiency, leading to mitochondrial respiratory chain disorders and ultimately causing mitochondrial diseases. In addition, the differentially expressed genes in this study were enriched in the oxidative phosphorylation pathway, which is a way for muscle cells to generate ATP, which can couple the tricarboxylic acid cycle and fatty acid oxidation, not only providing energy for cell survival and proliferation, but also playing an important role in cell anabolism, signal transduction, etc.
[0041] In the study of the present invention, it was found that lincRNA4160 has obvious temporal expression specificity. It inhibits the proliferation and differentiation of pig muscle satellite cells and promotes apoptosis by regulating genes such as Ankrd23 and CENPK, through muscle disease-related pathways such as Diabetic cardiomyopathy, Parkinson disease, and Huntingtondisease, and energy metabolism-related pathways such as Oxidative phosphorylation, Thermogenesis, and Metabolic. These findings provide a theoretical basis for further improving meat production and meat quality and analyzing the molecular regulatory mechanism of muscle diseases.
[0042] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for detecting porcine lincRNA4160 tissue expression, characterized in that: The following steps are involved: (1) Sample collection, total RNA extraction and cDNA synthesis, real-time quantitative PCR, using GAPDH as the internal reference gene, using 2 －△△Ct The relative gene expression was calculated by (2) Cell culture, differentiation induction and transfection, transfecting si-lincRNA4160 and si-NC into cells; (3) Expression profile analysis of lincRNA4160: Real-time fluorescence quantitative PCR was used to detect the expression of lincRNA4160 in different tissues of pigs, and the expression of pig skeletal muscle satellite cells at 30%, 60%, and 90% confluence and at D0, D2, D4, and D6 of induced differentiation; (4) Extract total protein from tissues or cells and analyze protein blots; use EdU to detect cell proliferation; use flow cytometry to detect cell cycle, stain mitochondrial membrane potential, and measure changes in mitochondrial membrane potential; (5) Transfect si-lincRNA4160 and siNC into pig muscle satellite cells, perform transcriptome sequencing analysis, and analyze the specific regulatory mechanism of lincRNA4160; (6) SPSS 22.0 statistical software was used to test for significant differences, and the results were expressed as mean ± SEM.
2. The method for detecting tissue expression of porcine lincRNA4160 according to claim 1, characterized in that: In step (1), total RNA was extracted from tissues and cells using AG RNAex Pro reagent; PrimeScript TM The extracted RNA was reverse transcribed into cDNA using RT Reagent Kit with gDNA Eraser; SYBR Premix Ex Taq TM The kit uses a three-step method for real-time fluorescence quantitative PCR. The PCR reaction system is: SYBR Premix Ex Taq TM Ⅱ 12.5 μL, upstream and downstream primers 1 μL each, cDNA template 2 μL, ddH2O 8.5 μL; PCR reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles.
3. The method for detecting tissue expression of porcine lincRNA4160 according to claim 1, characterized in that: In the step (2), the sample tissue is minced, digested with collagenase II and trypsin at 37°C, and then neutralized with DMEM F12 medium containing 20% fetal bovine serum, 1% penicillin-streptomycin, and 1% FGFb, and muscle satellite cells are separated and purified by differential adhesion method; when inducing differentiation, when the cell confluence is 70%-80%, the differentiation medium is replaced; si-lincRNA4160 and si-NC are transfected into cells using DharmaFECT, and si-lincRNA4160 and si-NC are transfected into cells using DharmaFECT.
4. The method for detecting tissue expression of porcine lincRNA4160 according to claim 1, characterized in that: In the step (3), real-time fluorescence quantitative PCR is used to detect the expression level of lincRNA4160 in the heart, liver, spleen, lung, kidney, small intestine, stomach, longissimus dorsi muscle, and subcutaneous fat.
5. The method for detecting tissue expression of porcine lincRNA4160 according to claim 1, characterized in that: In the step (4), total protein of tissue or cells is extracted using RIPA containing protease inhibitors; cell proliferation is detected according to the instructions of the EdU kit; and cell cycle is detected using a cell cycle detection kit.
6. The method for detecting tissue expression of porcine lincRNA4160 according to claim 1, characterized in that: In step (5), the transcriptome sequencing analysis steps are as follows: after total RNA extraction, mRNA with polyA is adsorbed by Oligo (dT) magnetic beads for cDNA synthesis, and a cDNA library is constructed after the quality and content of the cDNA are detected; Sequencing analysis was performed using the PE150 sequencing strategy on the Illumina platform, and adapters and low-quality reads were removed from the raw data. Q20 and Q30 were calculated for Clean Data to detect sequencing error rates, and Clean Reads were assembled de novo using HISAT2 software. Gene expression analysis was performed using the FPKM method. Subsequently, differential expression analysis between groups was performed using the DESeq2 method. FDR < 0.05, |log2(Fold Change)| > 1 were used as criteria for screening differentially expressed genes (DEGs). Finally, GO analysis was used to annotate DEG functions, and KEGG analysis was used to screen DEG-enriched pathways.
7. Application of porcine lincRNA4160 in regulating myoblast proliferation, differentiation and apoptosis.
8. The use of porcine lincRNA4160 in regulating myoblast proliferation, differentiation and apoptosis according to claim 7, characterized in that: The lincRNA4160 can inhibit the proliferation of pig skeletal muscle satellite cells; lincRNA4160 can inhibit the differentiation of pig skeletal muscle satellite cells; lincRNA4160 can promote the apoptosis process of pig skeletal muscle satellite cells.
9. The use of porcine lincRNA4160 in regulating myoblast proliferation, differentiation and apoptosis according to claim 8, characterized in that: The porcine lincRNA4160 inhibits the proliferation and differentiation of porcine muscle satellite cells and promotes apoptosis by regulating Ankrd23 and CENPK genes via pathways related to muscle diseases and pathways related to energy metabolism.