CN115976030A - Method for promoting bovine skeletal muscle satellite cell myogenic differentiation by interfering HNRNPR expression and application - Google Patents

Abstract

The invention belongs to the technical field of biology, cell and tissue engineering, and discloses a method for promoting the myogenic differentiation of bovine skeletal muscle satellite cells by interfering the expression of HNRNPR, which realizes the promotion of the myogenic differentiation of bovine skeletal muscle satellite cells by reducing the expression of HNRNPR; wherein, the nucleotide sequence of the HNRNPR gene is as follows: SEQ ID NO.1. The method provided by the invention regulates and controls the proliferation and myogenic differentiation process of bovine muscle satellite cells by changing the expression of HNRNPR, can provide inspiration for research on the muscle development and differentiation process, provides reference for exploring a muscle growth and development mechanism and improving the yield and quality of beef, can lay a good foundation for further research on the action mechanism of key hnRNPs protein in the muscle development process, and provides a theoretical foundation for rapid cultivation and meat quality improvement of new high-quality beef cattle varieties.

Description

Method for promoting bovine skeletal muscle satellite cell myogenic differentiation by interfering HNRNPR expression and application

Technical Field

The invention belongs to the technical field of biotechnology, cell and tissue engineering, and particularly relates to a method for promoting bovine skeletal muscle satellite cell myogenic differentiation by interfering HNRNPR expression and application.

Background

The growth and development of muscle involve a series of complex processes such as proliferation, migration, differentiation and fusion of muscle cells, and are regulated by various transcription factors and epigenetic regulation factors in a signal path mode. Skeletal muscle satellite cells (skeletal muscle satellite cells) are cells which exist between skeletal muscle fibers and a basement membrane and are usually in a resting state, are activated when subjected to certain stimuli, proliferate and differentiate into myoblasts, and finally form skeletal muscle cells, and the satellite cells are considered stem cells for normal muscle growth and injury repair, and rupture or interruption of the basement membrane can enable the muscle satellite cells to repair injured muscle cells through proliferation and differentiation in pathological conditions or after the muscle cells are injured. The in vitro myogenic differentiation process of the muscle satellite cells can well simulate the in vivo muscle development process, and is a good cell model for researching cell differentiation and muscle development. The activation, proliferation and myogenic differentiation processes of the muscle satellite cells are regulated and controlled by various factors, the specific action mechanisms of a plurality of key regulating and controlling factors are unknown, and the myogenic differentiation regulating and controlling mode of the skeletal muscle satellite cells is understood, so that the possibility of artificially controlling the formation of the muscle cells can be increased.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a superfamily of proteins involved in a variety of vital activities. About 20 members of the family are different in molecular size and isoelectric point, and the molecular size is from 34 kDa to 120kDa, and the family is named as hnRNPA1-U according to the molecular size. hnRNPs can be combined with pre-mRNA through specific structures to form a complex, and participate in physiological processes such as mRNA transport, splicing, expression and the like. The main structural difference of the members of hnRNPs family lies in the sequence composition of RNA binding region, so that the RNA binding region is the main factor for determining the functional specificity of protein, hnRNPs can be combined with the non-coding region of messenger RNA through the RNA binding region, and play an important role in cellular nucleic acid metabolism, alternative splicing, regulation of telomerase activity, DNA damage repair and mRNA stabilization, HNRNPR is originally determined as a member of hnRNP family, and HNRNPR has multiple functions in organisms, such as regulation of splicing, RNA transport and RNA stability, and can also regulate the transcription process of pre-mRNA and mature mRNA. HNRNPR can promote the proliferative capacity, migratory capacity, and invasive capacity of a variety of cancer cells. When HNRNPR in a mouse model is knocked out, the invasiveness and the metastasis of tumors can be reduced. However, the research of HNRNPR participating in the skeletal muscle development of animals is only rarely reported. Based on the method, the bovine skeletal muscle satellite cell in-vitro induced myogenic differentiation model is utilized, the effect of HNRNPR on the bovine skeletal muscle satellite cell in-vitro myogenic differentiation is researched by interfering the expression of HNRNPR in the bovine skeletal muscle satellite cell, and a certain foundation is laid for further excavating genes influencing the bovine muscle development.

Through searching, no patent publication related to the present patent application has been found.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a method for promoting the myogenic differentiation of bovine skeletal muscle satellite cells by interfering the expression of HNRNPR and application thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for promoting the myogenic differentiation of bovine skeletal muscle satellite cells by interfering with the expression of HNRNPR, by reducing the expression of HNRNPR; wherein the nucleotide sequence of the HNRNPR gene is as follows: SEQ ID NO.1.

Further, the method comprises the following steps:

the siRNA of the HNRNPR is designed and synthesized according to the genome base sequence of the HNRNPR, the bovine skeletal muscle satellite cells are transfected by the siRNA, the expression level of the HNRNPR is reduced, and the myogenic differentiation of the bovine skeletal muscle satellite cells is promoted.

Further, the sequence of the siRNA is: SEQ ID NO.2.

Further, the method for interfering with the expression level of HNRNPR comprises: plasmids, viral vectors or gene knock-outs interfering with HNRNPR expression are used.

The method for promoting the myogenic differentiation of bovine skeletal muscle satellite cells by interfering with the expression of HNRNPR, which is applied to the proliferation and the myogenic differentiation of bovine muscle satellite cells.

The invention has the advantages and positive effects that:

1. the method provided by the invention regulates and controls the proliferation and myogenic differentiation process of the bovine muscle satellite cells by changing the expression of HNRNPR, can provide inspiration for research on the muscle development differentiation process, provides reference for exploring a muscle growth and development mechanism and improving the yield and quality of beef, can lay a good foundation for further research on the action mechanism of key hnRNPs protein in the muscle development process, and provides a theoretical foundation for rapid culture and meat quality improvement of a new high-quality beef cattle variety.

2. The method for promoting the proliferation and myoblast differentiation process of bovine skeletal muscle satellite cells by changing the expression of HNRNPR can effectively regulate and control the muscle development and differentiation process, can provide a certain reference for the utilization of HNRNPR in the muscle development and differentiation and injury repair and provides a new thought and reference for the clinical research and diagnosis and treatment of the muscle development and differentiation and injury repair.

3. In view of the fact that whether the HNRNPR has a certain regulation effect on bovine skeletal muscle satellite cells or not at present and the specific effect is not clear, the invention designs and synthesizes siRNA of the HNRNPR, then adopts lip3000 reagent to transfect the bovine skeletal muscle satellite cells, interferes the expression level of the HNRNPR in the bovine skeletal muscle satellite cells, and promotes the proliferation and myogenic differentiation processes of the bovine skeletal muscle satellite cells by changing the expression of the HNRNPR in the bovine skeletal muscle satellite cells. The method can provide reference for the utilization of HNRNPR in muscle development differentiation and injury repair, and provides a new idea and method for clinical research and diagnosis and treatment of muscle development differentiation and injury repair.

4. The method utilizes HNRNPR to regulate and control the proliferation and myogenic differentiation of the bovine muscle satellite cells, can provide inspiration for the research of the muscle development and differentiation process, and provides reference for exploring a muscle growth and development mechanism and improving the yield and quality of beef.

Drawings

FIG. 1 is a diagram showing the results of quantitative PCR detection of the changes in expression levels of HNRNPR in different stages of bovine skeletal muscle satellite cell myoblast differentiation in the present invention; wherein GM is a bovine skeletal muscle satellite cell proliferation period, DM1 is a bovine skeletal muscle satellite cell differentiation first day, DM2 is a bovine skeletal muscle satellite cell differentiation second day, and DM3 is a bovine skeletal muscle satellite cell differentiation third day;

FIG. 2 is a diagram showing the interference effect of the siRNA interfering HNRNPR in the present invention;

FIG. 3 is a diagram showing the result of qRT-PCR detection of the differentiation marker genes MyoG and MyHC in bovine skeletal muscle satellite cells by interfering with HNRNPR expression with siRNA in the present invention;

FIG. 4 is a Western blot detection result diagram of a differentiation marker gene MyHC in bovine skeletal muscle satellite cells expressed by siRNA interfering HNRNPR in the present invention; wherein A is a MyHC protein expression level graph after interference of Western blot detection, and B is a visual schematic diagram of Western blot detection.

Detailed Description

The present invention is described in detail below with reference to the following examples, which are intended to be illustrative and not limiting, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

A method for promoting the myoblast differentiation of bovine skeletal muscle satellite cells by interfering with the expression of HNRNPR, by reducing the expression of HNRNPR; wherein, the nucleotide sequence of the HNRNPR gene is as follows: SEQ ID NO.1.

Preferably, the method comprises the following steps:

and (3) designing and synthesizing the siRNA of the HNRNPR according to the genome base sequence of the HNRNPR, transfecting the bovine skeletal muscle satellite cells with the siRNA, reducing the expression level of the HNRNPR, and realizing the promotion of the myogenic differentiation of the bovine skeletal muscle satellite cells.

Preferably, the sequence of the siRNA is: SEQ ID NO.2.

Preferably, the method of interfering with the expression level of HNRNPR comprises: plasmids, viral vectors or gene knock-outs interfering with HNRNPR expression are used.

The method for promoting the myogenic differentiation of bovine skeletal muscle satellite cells by interfering with the expression of HNRNPR, which is applied to the proliferation and the myogenic differentiation of bovine muscle satellite cells.

Specifically, the preparation and detection examples are as follows:

one design idea of the invention may be:

designing and synthesizing siRNA of the HNRNPR, then adopting a lip3000 reagent to transfect the bovine skeletal muscle satellite cells, interfering the expression level of the HNRNPR in the bovine skeletal muscle satellite cells, and promoting the myogenic differentiation process of the bovine skeletal muscle satellite cells by changing the expression of the HNRNPR in the bovine skeletal muscle satellite cells.

A method of promoting bovine skeletal muscle satellite cell myogenic differentiation by interfering with HNRNPR expression comprising the steps of:

the method comprises the following steps of firstly, bovine skeletal muscle satellite cell separation and culture, establishment of an in vitro myoblast induced differentiation model and detection of HNRNPR in myoblast differentiation timing sequence expression:

separating the bovine skeletal muscle satellite cells by adopting a combined digestion method of pancreatin and collagenase, and establishing a bovine skeletal muscle satellite cell in-vitro myoblast induced differentiation model. Detecting the expression quantity of HNRNPR before and after the differentiation of bovine skeletal muscle satellite cell myoblasts by adopting a quantitative PCR and Westernblot method;

the method comprises the following specific steps:

(1) Bovine skeletal muscle satellite cell isolation culture and establishment of in vitro myogenic induced differentiation model.

The bovine skeletal muscle satellite cells are separated by adopting a combined digestion method of pancreatin and collagenase. Collecting fetal hind limb muscle of cattle under aseptic condition, cutting into suitable size, washing in PBS buffer solution several times, sufficiently cutting with ophthalmic scissors in a culture dish, washing once with preheated PBS buffer solution, centrifuging at 1000r/min for 5min in a centrifuge, discarding supernatant, adding 5ml of 0.2% collagenase II, digesting in 37 deg.C water bath for 1h while vortexing for 10s every 10min, adding 0.25% pancreatin containing EDTA for digestion for 30min while vortexing for 10s every 10min, adding 20% fetal bovine serum culture medium (20 FBS 80 DMEM) to terminate digestion, sieving the above mixture with 100 mesh, 200 mesh and 400 mesh cell sieves, collecting filtrate in 50ml centrifuge tube, centrifuging at 1000r/min in centrifuge tube for 10min, inoculating with culture medium (20 FBS 80 DMEM), resuspending in suitable culture dish, resuspending at 37 deg.C and 5 CO, and re ₂ Culturing in an incubator. Culturing in DMEM growth medium containing 20% fetal calf serum, adding DMEM differentiation medium containing 2% horse serum when the cells grow to 80% fusion to perform in vitro myogenic induced differentiation, observing myosatellite cell differentiation condition and myotube formation state, and establishing a bovine skeletal muscle satellite cell in vitro myogenic induced differentiation model.

(2) Genomic base sequence of HNRNPR (2602 bp): SEQ NO.1.

AAATGGCTAATCAGGTGAATGGTAATGCGGTACAGTTAAAAGAAGAGGAAGAGCCAATGGATACTTCCAGTGTAAATCACACAGAGCACTACAAGACACTGATAGAGGCAGGCCTCCCTCAGAAGGTGGCAGAGAGACTTGATGAAATATTTCAGACAGGATTGGTAGCTTATGTCGATCTTGATGAAAGAGCAATTGATGCTCTCAGGGAATTTAATGAAGAAGGAGCTCTGTCTGTACTACAGCAGTTCAAGGAAAGTGACTTATCACATGTTCAGAACAAAAGTGCATTTTTATGTGGAGTTATGAAGACCTACAGGCAAAGGGAGAAACAGGGGAGCAAGGTGCAAGAGTCCACGAAGGGACCTGATGAAGCAAAGATCAAGGCCTTGCTTGAAAGGACCGGTTATACCTTGGATGTAACCACAGGACAGAGGAAGTATGGCGGCCCTCCCCCGGACAGTGTGTACTCTGGTGTGCAGCCTGGAATCGGAACCGAAGTCTTTGTAGGTAAAATACCAAGAGATTTATACGAGGATGAATTGGTGCCCCTTTTTGAAAAGGCTGGTCCCATTTGGGATCTGCGTCTTATGATGGATCCACTGTCTGGTCAGAACAGAGGGTATGCATTTATCACCTTCTGTGGAAAGGAAGCTGCACAGGAAGCTGTTAAACTGTGTGACAGCTATGAAATCCGCCCCGGTAAACACCTTGGAGTGTGCATTTCTGTGGCAAACAACAGGCTTTTTGTTGGATCAATTCCGAAGAATAAGACTAAAGAAAACATTCTGGAAGAATTCAGTAAAGTCACAGAGGGTTTGGTGGACGTTATTCTCTATCATCAACCCGATGACAAAAAGAAGAATCGGGGGTTCTGCTTCCTTGAGTATGAGGATCACAAGTCCGCAGCACAAGCCAGACGCCGGCTGATGAGTGGAAAAGTGAAAGTGTGGGGGAATGTAGTTACAGTTGAATGGGCTGACCCTGTGGAAGAACCAGATCCAGAAGTCATGGCGAAGGTGAAAGTTTTATTTGTGAGAAACTTGGCTACTACAGTGACAGAAGAAATATTGGAAAAGTCATTTTCTGAATTTGGAAAACTTGAAAGAGTGAAGAAGTTGAAAGATTATGCATTTGTTCATTTCGAAGACAGAGGAGCAGCTGTTAAGGCCATGGATGAAATGAATGGCAAAGAAATAGAAGGAGAAGAAATTGAAATTGTCTTAGCCAAGCCACCAGACAAGAAAAGGAAAGAGCGCCAAGCTGCTAGACAGGCCTCGAGGAGTACTGCGTATGAAGATTATTACTATCACCCTCCTCCTCGCATGCCACCTCCAATTAGAGGTCGGGGTCGTGGTGGGGGGAGAGGTGGATATGGCTACCCTCCAGATTACTATGGCTATGAAGATTACTATGATGATTACTATGGTTATGATTATCACGACTATCGTGGAGGCTATGAAGATCCCTACTACGGCTATGATGATGGCTATGCAGTAAGAGGAAGAGGAGGAGGAAGGGGAGGGCGAGGTGCTCCACCACCACCAAGGGGGCGGGGAGCACCACCTCCAAGAGGTAGAGCTGGCTACTCACAGAGGGGGGCACCTTTGGGACCACCAAGAGGCTCTAGGGGTGGCAGAGGGGGTCCTGCACAACAGCAGAGAGGCCGTGGTTCCCGTGGATCTCGGGGCAACCGTGGGGGCAATGTAGGAGGCAAGAGAAAGGCAGACGGGTACAACCAACCTGACTCCAAGCGCCGTCAGACCAACAACCAACAGAACTGGGGTTCCCAACCCATCGCTCAACAGCCGCTTCAGCAAGGTGGTGACTATTCTGGTAACTATGGTTACAATAATGACAACCAGGAATTTTATCAGGATACTTATGGGCAACAGTGGAAATAGACAAGTGAGGGCTTGAAAATGATATTGACAAGATACGATTGGCTCTAGATCTACATCCTTCAAAAAAATTGGCTTATCTGTTTCATCTTTAAGTAGCAATTTGCTGCCATTTGTATTTGGCTGAAGAAATCACTATTGTGTATATACTCAAGTCTTTTTATTTTTTCCTCTTTTCATAAATGCTCTTGGACATTATTGGGCTTGCAGAGTTCCCTTATTCTGGGAGTTACAATGCTTTTATCGTTTCAGGCTTCACTTTAGCTTCAAAACAAGCTGAGCACACTGTTAAAATCATGATTTTGCAGAACCTTTGGTTTTGGACAGTTTCATTTTTTGGATTTGGGACAGCTTACATAGGGGTATGGAGTATGCTGTAAATAAAAATACAAGCTAGTGCTTTGTCTTAGTAGTTTGAAGAAATTAAAAGCAAACAAATTTAAGTTTTCTTGTATTGAAAATAACCTATGATTGTATGTTTTGCATTCCTAGAAGTAGGTCAACTGTGTTTTTAAATTGTTATATCTTCACACCTTTTTGAAACCTGCCCTACAAAATTTGTTTGGCTTAAACGTCAAAGCCGTGACAATTTGTTCTTTGATGTGATTGTATTTCCAATTTCTTGTTCATGTAAGATTTCAATAAAACTCAAAAATCTATTCAAAACATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

(3) And (3) detecting the expression change of HNRNPR in different periods of skeletal muscle satellite cell myoblast differentiation.

And extracting the total RNA of the proliferated and differentiated cells by adopting a cell RNA rapid extraction kit. The extraction step comprises: 600ML of cell lysate was added to a 24-well cell culture plate and the pipetting was repeated until the cells were fully lysed. After observing the cells to be completely lysed under a microscope, the above lysate was collected in a 1.5ml RNase-free tube. The lysis mixture was applied to the DNA clean-up column in its entirety. Centrifuge at 13000rpm for 60s at once. The volume of the filtrate was accurately estimated using a micropipette, an equal volume of 70% ethanol was added, and the mixture was immediately shaken and mixed without centrifugation. The mixture was immediately added to an adsorption column RA, centrifuged at 13000rpm for 30s and the waste liquid discarded. Add 700. Mu.L of deproteinizing solution RW1, leave at room temperature for 1 minute, centrifuge at 12000rpm for 30 seconds, and discard the waste liquid. 500ML of the rinsing solution RW was added and centrifuged at 12000rpm for 30s, and the waste solution was discarded. Add 500ML of rinse RW and repeat. The adsorption column RA was returned to empty and collected, and centrifuged at 13000rpm for 2 minutes to remove the rinse as much as possible so as to prevent the residual ethanol in the rinse from inhibiting the downstream reaction. Taking out the adsorption column RA, placing into an enzyme-free centrifuge tube, adding 30-50ML enzyme-free water at the middle part of the adsorption membrane according to the expected RNA yield, standing at room temperature for 1 minute, and centrifuging at 12000rpm for 1 minute.

Reverse transcription of RNA and quantitative PCR were performed according to the kit instructions. The primer sequences used are shown in Table 1, GAPDH as internal reference. The reverse transcription of RNA and the quantitative PCR conditions were as follows:

(1) RNA reverse transcription system and conditions:

to prepare 20. Mu.L of a mixed solution.

Vortex, shake and mix evenly, centrifuge briefly, collect the solution on the tube wall to the bottom of the PCR tube.

Placing the prepared mixed solution on a PCR instrument, and setting a program: the incubation was performed for 15min at 42 ℃ and then for 5min at 85 ℃. After the above procedure was completed, the mixture was centrifuged briefly, and the reverse transcribed cDNA mixture was taken out and cooled on ice.

(2) Quantitative PCR detection system:

preparation of reaction solution: and (3) preparing reaction liquid on ice, carrying out a multi-hole experiment on detection indexes in each sample, carrying out a single-hole NTC experiment, and carrying out quantitative PCR detection by adopting a white quantitative plate.

The reaction solution in each single well of the quantitative plate was configured as follows:

total 20. Mu.L of reaction system.

Reaction conditions for qRT-PCR:

and (3) after the reaction solution is fully and uniformly mixed, centrifuging to enable the reaction solution to reach the bottom of the reaction tube, and placing on a real-time fluorescent quantitative PCR instrument. The specific reaction design procedure is as follows:

pre-denaturation: 60s at 95 DEG C

Denaturation: at 95 deg.C for 10s

And (3) annealing: at 61 deg.C for 20s

Extension: at 72 deg.C for 15s

The denaturation to annealing step was repeated 35 times.

After the reaction is finished, a dissolution curve is drawn, and the fluorescence temperature range detected in the experiment is as follows: 65-95 ℃, the heating rate is 0.5 ℃/time cycle, and the constant temperature time is 10 s/time cycle.

After all reactions were completed, the temperature was maintained at 37 ℃ for 30 seconds to prevent the reaction tube from being scalded when it was removed after the reaction was completed.

TABLE 1 HNRNPR real-time quantitative PCR primer sequences

The detection result of the quantitative PCR is shown in figure 1, and the experimental result shows that the mRNA expression level of the HNRNPR shows a trend of increasing firstly and then decreasing along with the extension of the differentiation time of bovine skeletal muscle satellite cells, the mRNA expression level of the HNRNPR is the highest on the first day of induced differentiation, and the mRNA expression level of the HNRNPR on the second day of differentiation (DM 2) and the third day of differentiation (DM 3) is obviously reduced compared with the proliferation period. Indicating that HNRNPR may have the function of regulating bovine skeletal muscle satellite cell myogenic differentiation.

The second step: establishing a bovine skeletal muscle satellite cell inhibition expression model and detecting an interference effect:

designing and synthesizing siRNA according to the HNRNPR base sequence, transfecting the siRNA into bovine skeletal muscle satellite cells, detecting the expression level of the HNRNPR by adopting a quantitative PCR method, and detecting the interference effect of the siRNA on the HNRNPR;

the method comprises the following specific steps:

(1) HNRNPR interfering RNA (siRNA) is designed and synthesized.

siRNA was designed and synthesized by Bio-Inc based on the HNRNPR base sequence (SEQ NO.2: GGAAGAACCAGATCCAGAA).

(2) And (3) establishing a bovine skeletal muscle satellite cell inhibition expression model.

Transfecting bovine skeletal muscle satellite cells by siRNA of HNRNPR by using a lip3000 reagent, inoculating the bovine skeletal muscle satellite cells into a 24-hole cell culture plate, culturing by using a growth medium, preparing transfection when the cell growth density is 70-80%, and replacing the medium by the growth medium without antibiotics one day before transfection. Transfection was performed with si-RNA and a negative control at a final concentration of 50nM, according to the recommended concentrations in the reagent instructions. When transfection is performed, the culture medium is replaced by a serum-free Opti-MEM culture medium, the si-RNA or the corresponding negative control is mixed in 50 μ L of the Opti-MEM culture medium, meanwhile, 0.75 μ L of lip3000 transfection reagent is mixed in 50 μ L of the Opti-MEM culture medium, standing is performed for 5min respectively, the Opti-MEM culture medium containing siRNA or the corresponding negative control is added into the Opti-MEM culture medium containing Roche transfection reagent, the mixed solution is gently mixed and then stands for 15min, the mixed solution is uniformly dripped into a 24-well cell culture plate, and the volume of the mixed solution is dripped so as to reach the final concentration of siRNA or the corresponding negative control.

(3) And (5) detecting the interference effect of the siRNA.

After 24h of transfection, the culture medium is replaced by a muscle-induced differentiation culture medium for continuous culture, and induced differentiation is carried out on the muscle satellite cells. When the cells are transfected for 24 hours and induced to differentiate for 48 hours, a cell RNA rapid extraction kit is adopted to extract total RNA of the transfected cells in a 24-well plate, a quantitative PCR method is adopted to detect the mRNA expression level of the HNRNPR, the total RNA extraction, the RNA reverse transcription and the quantitative PCR operation are the same as those in the first step, the used primer sequences are shown in the table 1, the detection result is shown in the figure 2, and the experimental result shows that the siRNA has obvious interference effect on the expression of the HNRNPR, the expression level of the HNRNPR is remarkably reduced (p is less than 0.01 compared with the control NC), and the method can be used for the subsequent expression regulation and control research of the HNRNPR.

The third step: interference with HNRNPR on bovine skeletal muscle satellite cell myogenic differentiation:

transfecting bovine skeletal muscle satellite cells by using siRNA to interfere the expression of HNRNPR, then carrying out myogenic induced differentiation on the muscle satellite cells, and judging the influence of interfering the expression of HNRNPR on the myogenic differentiation of the bovine muscle satellite cells by detecting the expression conditions of differentiation marker genes MyoG and MyHC through quantitative PCR and Western blot;

the method comprises the following specific steps:

the siRNA of HNRNPR is used for transfecting bovine skeletal muscle satellite cells by using lip3000 reagent, and the specific operation is the same as the siRNA transfection method in the second step. After 24h of transfection, the culture medium is replaced by a muscle-induced differentiation culture medium for continuous culture, and induced differentiation is carried out on the muscle satellite cells. The mRNA expression of the differentiation marker genes MyoG and MyHC is detected by quantitative PCR, the sequences of the primers are shown in Table 2, GAPDH is used as an internal reference, and the sequences are as shown in Table 1. The results show that the mRNA expression levels of differentiation marker factors MyoG and MyHC after the interference of HNRNPR are extremely obviously increased, and the detection results are shown in figure 3 (. Beta., p is less than 0.01 compared with the control NC).

TABLE 2 differentiation marker genes MyoG, myHC real-time quantitative PCR primer sequences

Extracting the protein of the bovine skeletal muscle satellite cells in the proliferation and differentiation states, wherein the extraction step comprises the following steps: the medium was aspirated off, cells were washed with pre-chilled PBS, and 200. Mu.L of bovine skeletal muscle satellite cells containing 1%P per well, for example, in a 6-well plateThe RIPA lysate of MSF was lysed at a dilution ratio of 100 at 1,4 ℃ for 15min and collected in a 1.5mL enzyme-free centrifuge tube. At 4 ℃, the high-speed centrifuge 12000 Xg 10min, the supernatant is transferred to a 1.5mL enzyme-free centrifuge tube, and the protein sample is preserved at-20 ℃. Protein concentration was measured by BCA assay, and proteins were diluted with 4 Xprotein loading buffer to ensure that all sample protein loads were identical and denatured in boiling water at 100 ℃ for 10min. And then carrying out Western blot detection, preparing 10% concentrated gel and separation gel, then carrying out spotting, carrying out electrophoresis for 40min at a constant voltage of 80V, and carrying out electrophoresis for 90min at a constant voltage of 120V after the protein reaches the separation gel. Cutting off concentrated gel, clamping the gel and PVDF membrane, transferring to membrane at 300mA constant current for 2h, sealing with 5% skimmed milk powder for 2h after the membrane transfer is finished, and washing the membrane with TBST for 3 times, each time for 10min. HNRNPR primary antibody final dilution was 1:500, myHC, myoG, pax7 primary antibody final dilution to 0.5. Mu.g/mL; tubulin primary anti-dilution factor is 1:3000, the membrane was sealed with primary antibody to the hybridization bag and incubated overnight at 4 ℃. The membrane was washed 3 times with TBST for 10min each. The dilution of the goat anti-mouse secondary antibody and the goat anti-rabbit secondary antibody was 1. Adding 200 μ L ECL hypersensitive luminescent solution into PVDF membrane, and adopting ChemiDoc ^TM Imaging System images memory map.

The Westernblot is adopted to test the protein expression condition of the differentiation marker gene MyHC, the result shows that the protein expression level of the differentiation marker gene MyHC is remarkably increased after the HNRNPR is interfered, the detection result is shown in figure 4 (p is less than 0.05 compared with the control NC), the reduction of the HNRNPR is shown to promote the myogenic differentiation of the muscle satellite cells, and the research result indicates that the expression of the HNRNPR related to the muscle development and differentiation in the bovine skeletal muscle satellite cells is changed, so that the myogenic differentiation process of the bovine skeletal muscle satellite cells can be influenced.

Of course, the method for modifying the expression level of HNRNPR comprises designing synthetic interfering RNA (siRNA), and also comprises other means capable of modifying the expression level of HNRNPR, such as plasmids, viral vectors and gene knockout for interfering the expression of HNRNPR.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.

Claims (5)

1. A method for promoting bovine skeletal muscle satellite cell myogenic differentiation by interfering with HNRNPR expression, comprising: the method is realized by reducing the expression of HNRNPR to promote the myogenic differentiation of bovine skeletal muscle satellite cells; wherein, the nucleotide sequence of the HNRNPR gene is as follows: SEQ ID NO.1.

2. The method of promoting bovine skeletal muscle satellite cell myogenic differentiation by interfering with HNRNPR expression according to claim 1, wherein: the method comprises the following steps:

the siRNA of the HNRNPR is designed and synthesized according to the genome base sequence of the HNRNPR, the bovine skeletal muscle satellite cells are transfected by the siRNA, the expression level of the HNRNPR is reduced, and the myogenic differentiation of the bovine skeletal muscle satellite cells is promoted.

3. The method of promoting bovine skeletal muscle satellite cell myogenic differentiation by interfering with HNRNPR expression according to claim 1, wherein: the sequence of the siRNA is as follows: SEQ ID NO.2.

4. The method of promoting bovine skeletal muscle satellite cell myogenic differentiation by interfering with HNRNPR expression according to any of claims 1 to 3, wherein: methods of interfering with the expression level of HNRNPR comprise: plasmids, viral vectors or gene knock-outs interfering with HNRNPR expression are used.

5. Use of a method of promoting bovine skeletal muscle satellite cell myogenic differentiation by interfering with HNRNPR expression according to any one of claims 1 to 4 for the proliferation and myogenic differentiation of bovine muscle satellite cells.

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