CN109628403B

CN109628403B - Regulation and control method for inhibiting proliferation and differentiation of myoblasts

Info

Publication number: CN109628403B
Application number: CN201811548119.7A
Authority: CN
Inventors: 宁越; 昝林森
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2018-12-18
Filing date: 2018-12-18
Publication date: 2022-08-05
Anticipated expiration: 2038-12-18
Also published as: CN109628403A

Abstract

The invention relates to a regulation and control method for inhibiting proliferation and differentiation of myoblasts, which is characterized in that siRNA capable of targeted silencing of SMAD5 genes is designed according to the full length of a CDS region nucleotide sequence of SMAD5 genes, then siRNA capable of targeted silencing of SMAD5 genes with the best effect is selected through online software for synthesis, the synthesized siRNA is named as a siSMAD5 fragment, and the sequence of the synthesized siRNA is as follows: a Sense: CCAUGGAUACAAGCAAUAATT, respectively; anti sense: UUAUUGCUUGUAUCCAUGGTT; when the siSMAD5 fragment is used for transfecting the bovine myoblasts, the mRNA level of a marker gene MyoG for proliferation and differentiation of the myoblasts is obviously reduced, and the myotube fusion process of the myoblasts is also obviously damaged. The method is simple, rapid and accurate, and provides a new reliable research method for regulating and controlling the processes of myocyte proliferation and myogenic differentiation in the future.

Description

Regulation and control method for inhibiting proliferation and differentiation of myoblasts

Technical Field

The invention belongs to the technical field of molecular biology and cell engineering, and particularly relates to a regulation and control method for inhibiting proliferation and differentiation of myoblasts.

Background

Skeletal muscle is the main tissue of vertebrates, the growth and development of the skeletal muscle comprise a plurality of processes of cell proliferation, differentiation, fusion into muscle fibers and the like, and the regulation and control mechanism is disclosed as the basis for understanding the molecular mechanism formed by the skeletal muscle development and the meat production traits of livestock and poultry. In the process of growth and development of muscle tissues, the complex physiological processes of muscle satellite cell activation, proliferation, differentiation and the like are regulated and controlled by various hormones and growth factors. Wherein, the TGF-beta signal pathway is one of important signal pathways which are involved in the regulation and control of muscle growth and development and mainly plays a role in inhibiting myogenic differentiation.

The SMAD family is used as a TGF-beta direct action substrate and plays an important role in the aspects of cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone formation and reconstruction and the like. However, recent studies have shown that the SMAD5 gene can be involved in positive regulation of muscle mass by BMP signaling pathway. It was found that the SMAD5 gene was present not only in muscle tissue, but that two weeks after knock-out of SMAD1/5/8 in mice, the muscle mass was reduced by about 20%. Increasing experiments demonstrated that the SMAD5 gene is likely to be a positive regulatory element of muscle development, and its specific regulatory mechanism in skeletal muscle satellite cells is unclear. Therefore, the study of the SMAD5 gene has important physiological significance on the proliferation and differentiation process of myoblasts.

Disclosure of Invention

The invention aims to provide a regulation method for inhibiting proliferation and differentiation of myoblasts.

In order to realize the task, the invention adopts the following technical solution:

a regulation and control method for inhibiting proliferation and differentiation of myoblasts is characterized in that siRNA capable of targeted silencing of SMAD5 genes is designed according to the full length of a CDS region nucleotide sequence of SMAD5 genes, then siRNA capable of targeted silencing of SMAD5 genes with the best effect is selected through online software for synthesis, and the synthesized siRNA is named as a siSMAD5 fragment; the sequence is as follows:

Sense：CCAUGGAUACAAGCAAUAATT；

Antisense：UUAUUGCUUGUAUCCAUGGTT；

when the siSMAD5 fragment is used for transfecting the bovine myoblasts, the mRNA level of a marker gene MyoG for proliferation and differentiation of the myoblasts is obviously reduced, and the myotube fusion process of the myoblasts is also obviously damaged.

The regulation and control method for inhibiting the proliferation and differentiation of the myoblasts is simple, rapid and accurate, and provides a new reliable research method for regulating the proliferation and differentiation processes of the myoblasts in the future.

Drawings

FIG. 1 is a graph of silencing efficiency of the synthesized siSMAD5 fragment tested by qRT-PCR;

FIG. 2 is a graph showing the comparison of the change in the mRNA level expression of marker genes associated with the cell proliferation process after silencing SMAD5 gene by qRT-PCR;

FIG. 3 is a Western-blot comparison of the expression of marker gene proteins related to the cell proliferation process after SMAD5 gene silencing;

FIG. 4 is an observation picture of myoblast myotube fusion and qRT-PCR comparison of the change of the expression level of mRNA of marker genes related to the cell differentiation process after silencing SMAD5 gene;

FIG. 5 is the myotube formation process upon myoblast fusion differentiation.

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Detailed Description

The design idea of the invention is that a specific silencing gene is designed for inhibiting the proliferation and differentiation process of myoblasts, the function of the SMAD5 gene is silenced in cells by utilizing the functional characteristics of the SMAD5 gene, and the cell proliferation and differentiation process of the SMAD5 gene is further hindered.

The following examples refer to vectors, strains, cells, reagents from the following sources:

the bovine primary myoblasts are obtained by separating longissimus dorsi tissues of Qinchuan cattle in the national beef cattle improvement center by an enzyme digestion method.

siRNA sequences were synthesized by Shanghai Jima pharmaceutical technology, Inc.

The primers were synthesized by Shanghai Bioengineering Co., Ltd.

DMEM/F12, Fetal Bovine Serum (FBS), Horse Serum (HS) were purchased from Hyclone.

Lipofectamine ^TM 3000 transfection reagents, purchased from Promega.

PrimeScript RT Reagent Kit with DNAeraser reverse transcription Kit,

Primix Ex TaqTM II kits were purchased from TaKaRa

Rabbit anti-CDK 1, CDK2 polyclonal antibody (primary antibody) were purchased from wangchi biotechnology, rabbit anti- β -actin polyclonal antibody (primary antibody) and anti-rabbit HRP (secondary antibody) from Abcam, uk. The rest Western-blot related reagents were purchased from Biyunyan.

This example provides a method for regulating and controlling proliferation and differentiation of myoblasts, which includes designing siRNA capable of targeted silencing of SMAD5 gene according to the full length of the nucleotide sequence of CDS region of SMAD5 gene, selecting siRNA with best effect of targeted SMAD5 gene through online software for synthesis (synthesized by shanghai gimar pharmaceutical technology limited), wherein the synthesized siRNA is named as siSMAD5 fragment, transfecting bovine myoblasts with siSMAD5 fragment, significantly reducing mRNA level of myoblast proliferation and differentiation marker gene MyoG, and significantly damaging myoblast myotube fusion process.

The function of the SMAD5 gene is mainly determined by the nucleic acid sequence of a coding region, namely the SMAD5 gene sequence (NM-001077107.3) published by GeneBank, and the CDS region nucleotide sequence of the SMAD5 gene is as follows:

ATGACGTCAATGGCCAGCTTGTTTTCTTTTACTAGTCCAGCAGTAAAACGATTGTTGGGC TGGAAGCAAGGTGATGAAGAGGAGAAATGGGCAGAAAAGGCAGTTGATGCTTTGGTGAAAAAAC TAAAAAAGAAAAAAGGTGCCATGGAGGAACTGGAGAAAGCCTTGAGCAGTCCAGGACAGCCCAG CAAATGTGTCACTATCCCCAGATCTTTAGATGGACGCCTGCAGGTTTCTCACAGAAAAGGCTTA CCCCACGTTATATACTGTCGTGTTTGGCGCTGGCCTGATCTGCAGAGTCATCATGAGCTAAAGC CATTGGATATTTGTGAATTTCCTTTTGGATCTAAGCAAAAAGAAGTTTGTATCAACCCATACCA CTATAAGAGAGTGGAGAGTCCAGTCTTACCTCCAGTATTAGTGCCTCGTCATAATGAATTCAAT CCACAACACAGCCTTCTGGTTCAGTTTAGAAACTTAAGCCACAATGAACCGCACATGCCACAAA ATGCAACGTTTCCTGATTCTTTCCATCAGCCCAACAACACTCCTTTTCCCTTATCTCCAAATAG TCCCTACCCGCCTTCCCCTGCTAGCAGCACATATCCCAACTCCCCAGCAAGTTCTGGACCAGGA AGTCCATTTCAGCTCCCAGCTGATACTCCTCCTCCTGCCTATATGCCACCAGATGATCAGATGG GTCAAGATAATTCCCAGCCCATGGATACAAGCAATAATATGATTCCTCAGATTATGCCAAGTAT ATCCAGCAGAGATGTTCAGCCTGTCGCCTATGAAGAGCCCAAACATTGGTGTTCAATTGTCTAT TATGAATTAAACAATCGTGTTGGGGAAGCTTTTCATGCATCTTCTACTAGTGTGTTAGTAGATG GATTCACAGACCCTTCGAATAACAAAAGTAGATTCTGCTTGGGATTGTTGTCAAATGTTAATCG TAATTCAACAATTGAAAACACTAGGCGACATATTGGAAAAGGTGTTCATCTGTACTATGTTGGT GGAGAGGTATATGCAGAGTGCCTCAGTGACAGCAGCATATTTGTTCAGAGTAGGAACTGCAACT TTCATCATGGCTTTCATCCTACCACTGTCTGTAAGATTCCCAGCAGCTGCAGCCTCAAAATTTT TAACAATCAGGAATTTGCTCAGCTTCTGGCTCAGTCTGTCAACCATGGATTTGAGGCAGTATAT GAGCTCACCAAAATGTGTACCATTCGAATGAGTTTTGTCAAGGGCTGGGGAGCAGAGTATCACC GGCAAGATGTTACCAGCACCCCGTGTTGGATTGAGATTCATCTTCATGGGCCTCTTCAGTGGCT AGATAAAGTCCTCACTCAGATGGGCTCCCCGCTAAACCCCATTTCTTCTGTTTCATAG。

based on the nucleotide sequence of the CDS region of the SMAD5 gene, sirnas capable of targeted binding to the SMAD5 gene were designed as shown in table 1 below:

table 1: siRNA sequences

Through the designed and synthesized siSMAD5 fragment, according to the experimental verification of the applicant, the siSMAD5 fragment can be effectively introduced into bovine myoblasts to function.

Starvation of primary myoblasts with serum-free medium (DMEM/F12), and the siSMAD5 fragment and siRNA-NC fragment (control) were treated with Lipofectamine ^TM 3000 transfection reagent to primary myoblasts. After transfection for 6h, the medium was changed to a differentiation medium containing 2% HS for myogenic differentiation induction, and the medium was changed every other day. Collecting the treated cells two days later and extracting RNA, then reverse transcribing into cDNA with PrimeScriptTM RT Reagent Kit with DNA Eraser reverse transcription Kit, finally reverse transcribing into cDNA with TaKaRa company

The Primix Ex TaqTM II kit was used for real-time fluorescent quantitative PCR assay (primers synthesized from Shanghai Biotechnology, Inc.), and the sequence information is shown in Table 2 below.

TABLE 2 PCR primer sequences

Through the silencing efficiency test of the siSMAD5 fragment and the siRNA-NC fragment (comparison group), the siSMAD5 fragment can maintain better silencing efficiency for at least five days (as shown in figure 1, the silencing efficiency is more than 70 percent, and P is less than 0.01), and can be used for subsequent experiments.

The sipsad 5 fragment and siRNA-NC fragment (comparative group) were transferred into primary myocytes of qinchuan cattle with Lipo3000 transfection reagent, after they fully acted, the mRNA level of proliferation marker gene (as shown in fig. 2) and the protein level expression (rabbit anti-CDK 1, CDK2 polyclonal antibody, anti-beta-actin polyclonal antibody, secondary antibody anti rabbit HRP) were detected, and the results are shown in fig. 3. Analysis of the assay results revealed that the fragment of siSMAD5 was effective in inhibiting cell cycle progression by affecting CDK1 protein, mainly by affecting the G2 to M phase to inhibit primary myoblast proliferation.

When the Lipo3000 transfection reagents siSMAD5 fragment and siRNA-NC fragment (comparison group) are used for artificially inducing differentiation after being transfected into bovine primary myoblasts, mRNA expression quantity detection is carried out on differentiation marker genes of SMAD5 gene cells on the differentiation day 2, and the result shows that the inhibition of the SMAD5 by the siSMAD5 fragment can effectively inhibit the differentiation process of the myoblasts, particularly the terminal differentiation stage of the cells (figure 4).

In addition, the myotube formation process during myoblast fusion differentiation was also significantly impaired after silencing SMAD5 gene (fig. 5).

Nucleotide sequence listing

<110> northwest agriculture and forestry science and technology university

<120> a method for controlling proliferation and differentiation of myoblasts

<160>

<210> 1

<211>1398

<212> CDS region nucleotide sequence of SMAD5 gene

<213> DNA

<220>

<400>

ATGACGTCAATGGCCAGCTTGTTTTCTTTTACTAGTCCAGCAGTAAAACGATTGTTGGGCTGGAAGCAAGGTGATGAAGAGGAGAAATGGGCAGAAAAGGCAGTTGATGCTTTGGTGAAAAAACTAAAAAAGAAAAAAGGTGCCATGGAGGAACTGGAGAAAGCCTTGAGCAGTCCAGGACAGCCCAGCAAATGTGTCACTATCCCCAGATCTTTAGATGGACGCCTGCAGGTTTCTCACAGAAAAGGCTTACCCCACGTTATATACTGTCGTGTTTGGCGCTGGCCTGATCTGCAGAGTCATCATGAGCTAAAGCCATTGGATATTTGTGAATTTCCTTTTGGATCTAAGCAAAAAGAAGTTTGTATCAACCCATACCACTATAAGAGAGTGGAGAGTCCAGTCTTACCTCCAGTATTAGTGCCTCGTCATAATGAATTCAATCCACAACACAGCCTTCTGGTTCAGTTTAGAAACTTAAGCCACAATGAACCGCACATGCCACAAAATGCAACGTTTCCTGATTCTTTCCATCAGCCCAACAACACTCCTTTTCCCTTATCTCCAAATAGTCCCTACCCGCCTTCCCCTGCTAGCAGCACATATCCCAACTCCCCAGCAAGTTCTGGACCAGGAAGTCCATTTCAGCTCCCAGCTGATACTCCTCCTCCTGCCTATATGCCACCAGATGATCAGATGGGTCAAGATAATTCCCAGCCCATGGATACAAGCAATAATATGATTCCTCAGATTATGCCAAGTATATCCAGCAGAGATGTTCAGCCTGTCGCCTATGAAGAGCCCAAACATTGGTGTTCAATTGTCTATTATGAATTAAACAATCGTGTTGGGGAAGCTTTTCATGCATCTTCTACTAGTGTGTTAGTAGATGGATTCACAGACCCTTCGAATAACAAAAGTAGATTCTGCTTGGGATTGTTGTCAAATGTTAATCGTAATTCAACAATTGAAAACACTAGGCGACATATTGGAAAAGGTGTTCATCTGTACTATGTTGGTGGAGAGGTATATGCAGAGTGCCTCAGTGACAGCAGCATATTTGTTCAGAGTAGGAACTGCAACTTTCATCATGGCTTTCATCCTACCACTGTCTGTAAGATTCCCAGCAGCTGCAGCCTCAAAATTTTTAACAATCAGGAATTTGCTCAGCTTCTGGCTCAGTCTGTCAACCATGGATTTGAGGCAGTATATGAGCTCACCAAAATGTGTACCATTCGAATGAGTTTTGTCAAGGGCTGGGGAGCAGAGTATCACCGGCAAGATGTTACCAGCACCCCGTGTTGGATTGAGATTCATCTTCATGGGCCTCTTCAGTGGCTAGATAAAGTCCTCACTCAGATGGGCTCCCCGCTAAACCCCATTTCTTCTGTTTCATAG

<210> 2

<211>21

<212> Sense sequence of sMAD5 fragment

<213> DNA

<220>

<400>

CCAUGGAUACAAGCAAUAATT

<210> 3

<211>21

<212> Antisense sequence of sMAD5 fragment

<213> DNA

<220>

<400>

UUAUUGCUUGUAUCCAUGGTT

<210> 4

<211>21

<212> Sense sequence of siRNA-NC fragment

<213> DNA

<220>

<400>

UUCUCCGAACGUGUCACGUTT

<210> 5

<211>21

<212> Antisense sequence of siRNA-NC fragment

<213> DNA

<220>

<400>

ACGUGACACGUUCGGAGAATT

<210> 6

<211>20

<212> RT-SMAD5 primer F sequence

<213> DNA

<220>

<400>

AGAAAGCCTTGAGCAGTCCA

<210> 7

<211>20

<212> RT-SMAD5 primer R sequence

<213> DNA

<220>

<400>

ACGTGGGGTAAGCCTTTTCT

<210> 8

<211>25

<212> PCNA-RTF/R primer F sequence

<213> DNA

<220>

<400>

TGAACCTCACCAGCATGTCCAAAAT

<210> 9

<211>26

<212> PCNA-RTF/R primer R sequence

<213> DNA

<220>

<400>

TTCAAATACTAGTGCCAACGTGTCCG

<210>10

<211>22

<212> Mcm6-RTF/R primer F sequence

<213> DNA

<220>

<400>

AGTCAGTGGTGTTGATGGATAC

<210>11

<211>19

<212> Mcm6-RTF/R primer R sequence

<213> DNA

<220>

<400>

ACACAGCAGGCAAGAAAGA

<210>12

<211>22

<212> CCNE1-RTF/R primer F sequence

<213> DNA

<220>

<400>

CTGGCTATGCTGGATGAAGTAA

<210>13

<211>21

<212> CCNE1-RTF/R primer R sequence

<213> DNA

<220>

<400>

GCTCTTCGGTGGTGTCATAAT

<210>14

<211>23

<212> CCNA2-RTF/R primer F sequence

<213> DNA

<220>

<400>

AGAGATATACCCGCCAGAAGTAG

<210>15

<211>22

<212> CCNA2-RTF/R primer R sequence

<213> DNA

<220>

<400>

GACTAGGTGCTCCATCCTTAGA

<210>16

<211>21

<212> CCNB 1-RTF/R-RTF/R primer F sequence

<213> DNA

<220>

<400>

TGATGGAACTAACTATGCTGG

<210>17

<211>21

<212> CCNB 1-RTF/R-RTF/R primer R sequence

<213> DNA

<220>

<400>

GCATAACAACGAGAAGGGATT

<210>18

<211>18

<212> CCN D2-RTF/R-RTF/R primer F sequence

<213> DNA

<220>

<400>

GGGCAAGTTGAAATGGAA

<210>19

<211>17

<212> CCN D2-RTF/R-RTF/R primer R sequence

<213> DNA

<220>

<400>

TCATCGACGGCGGGTAC

<210>20

<211>21

<212> CDK 1-RTF/R-RTF/R primer F sequence

<213> DNA

<220>

<400>

AGTGGAAACCAGGAAGCTTAG

<210>21

<211>22

<212> CDK 1-RTF/R-RTF/R primer R sequence

<213> DNA

<220>

<400>

ATTCGTTTGGCAGGATCATAGA

<210>22

<211>22

<212> CDK 2-RTF/R-RTF/R primer F sequence

<213> DNA

<220>

<400>

GGGTCCCTGTTCGTACTTATAC

<210>23

<211>22

<212> CDK 2-RTF/R-RTF/R primer R sequence

<213> DNA

<220>

<400>

CCACTGCTGTGGAGTAGTATTT

<210>24

<211>18

<212> RT-MyoD primer F sequence

<213> DNA

<220>

<400>

TCCGCGACGTAGACTTGA

<210>25

<211>22

<212> RT-MyoD primer R sequence

<213> DNA

<220>

<400>

CGAAACACGGGTCATCATAGAA

<210>26

<211>19

<212> RT-MyoG primer F sequence

<213> DNA

<220>

<400>

GGCGTGTAAGGTGTGTAAG

<210>27

<211>20

<212> RT-MyoG primer R sequence

<213> DNA

<220>

<400>

CTTCTTGAGTCTGCGCTTCT

<210>28

<211>22

<212> RT-Myf5 primer F sequence

<213> DNA

<220>

<400>

CCTCTAGTTCCAGGCTCATCTA

<210>29

<211>22

<212> RT-Myf5 primer R sequence

<213> DNA

<220>

<400>

ACCTCCTTCCTCCTGTGTAATA

<210>30

<211>20

<212> RT-GAPDH primer F sequence

<213> DNA

<220>

<400>

5'-CCAACGTGTCTGTTGTGGAT-3'

<210>31

<211>20

<212> RT-GAPDH primer R sequence

<213> DNA

<220>

<400>

5'-CTGCTTCACCACCTTCTTGA-3'

Claims

1. A regulation and control method for inhibiting proliferation and differentiation of myoblasts is characterized in that siRNA capable of targeted silencing of SMAD5 genes is designed according to the full length of a CDS region nucleotide sequence of SMAD5 genes, then siRNA with the best effect of targeted silencing of SMAD5 genes is selected and synthesized through online software, the synthesized siRNA is named as a siSMAD5 fragment, and the sequence of the siRNA is as follows:

Sense：CCAUGGAUACAAGCAAUAATT；

Antisense：UUAUUGCUUGUAUCCAUGGTT；

the siSMAD5 is used for transfecting the bovine myoblasts, the mRNA level of the marker gene MyoG for proliferation and differentiation of the myoblasts is obviously reduced, and the myotube fusion process of the myoblasts is also obviously damaged;

the nucleotide sequence of the CDS region of the SMAD5 gene is as follows:

ATGACGTCAATGGCCAGCTTGTTTTCTTTTACTAGTCCAGCAGTAAAACGATTGTTGGGCTGGAAGCAAGGTGATGAAGAGGAGAAATGGGCAGAAAAGGCAGTTGATGCTTTGGTGAAAAAACTAAAAAAGAAAAAAGGTGCCATGGAGGAACTGGAGAAAGCCTTGAGCAGTCCAGGACAGCCCAGCAAATGTGTCACTATCCCCAGATCTTTAGATGGACGCCTGCAGGTTTCTCACAGAAAAGGCTTACCCCACGTTATATACTGTCGTGTTTGGCGCTGGCCTGATCTGCAGAGTCATCATGAGCTAAAGCCATTGGATATTTGTGAATTTCCTTTTGGATCTAAGCAAAAAGAAGTTTGTATCAACCCATACCACTATAAGAGAGTGGAGAGTCCAGTCTTACCTCCAGTATTAGTGCCTCGTCATAATGAATTCAATCCACAACACAGCCTTCTGGTTCAGTTTAGAAACTTAAGCCACAATGAACCGCACATGCCACAAAATGCAACGTTTCCTGATTCTTTCCATCAGCCCAACAACACTCCTTTTCCCTTATCTCCAAATAGTCCCTACCCGCCTTCCCCTGCTAGCAGCACATATCCCAACTCCCCAGCAAGTTCTGGACCAGGAAGTCCATTTCAGCTCCCAGCTGATACTCCTCCTCCTGCCTATATGCCACCAGATGATCAGATGGGTCAAGATAATTCCCAGCCCATGGATACAAGCAATAATATGATTCCTCAGATTATGCCAAGTATATCCAGCAGAGATGTTCAGCCTGTCGCCTATGAAGAGCCCAAACATTGGTGTTCAATTGTCTATTATGAATTAAACAATCGTGTTGGGGAAGCTTTTCATGCATCTTCTACTAGTGTGTTAGTAGATGGATTCACAGACCCTTCGAATAACAAAAGTAGATTCTGCTTGGGATTGTTGTCAAATGTTAATCGTAATTCAACAATTGAAAACACTAGGCGACATATTGGAAAAGGTGTTCATCTGTACTATGTTGGTGGAGAGGTATATGCAGAGTGCCTCAGTGACAGCAGCATATTTGTTCAGAGTAGGAACTGCAACTTTCATCATGGCTTTCATCCTACCACTGTCTGTAAGATTCCCAGCAGCTGCAGCCTCAAAATTTTTAACAATCAGGAATTTGCTCAGCTTCTGGCTCAGTCTGTCAACCATGGATTTGAGGCAGTATATGAGCTCACCAAAATGTGTACCATTCGAATGAGTTTTGTCAAGGGCTGGGGAGCAGAGTATCACCGGCAAGATGTTACCAGCACCCCGTGTTGGATTGAGATTCATCTTCATGGGCCTCTTCAGTGGCTAGATAAAGTCCTCACTCAGATGGGCTCCCCGCTAAACCCCATTTCTTCTGTTTCATAG。