| FHL2 (Four and a Half LIM protein 2) belongs to FHL protein family, which only has four and a half LIM domain. Recent researchs showed that FHL2 gene regulates the signal transduction of the signal pathway (Wnt/β-catenin、TGFβ) that related with satellite cells’proliferation and differentiation directly or indirectly, the expression of this gene in pectorals at different stages after birth presents significantly diversity, so we speculate that FHL2 gene might be a important factor of regulating satellite cells’ proliferation and differentiation from chicken’s skeletal muscle. Based on this, we conducted a test that transfected FHL2 SiRNA to chicken’s satellite cells, then observed the morphological change of satellite cells at different time after transfected (12h 24h 48h and 72h), and collected three gourps cells after transfected (12h 24h and 48h), extracting total RNA, using RT-PCR method to detect the expression of related myogenic regulatory factors and key genes from related signal pathway, finally we collected three groups cells after transfected (24h 48h and 72h), extracting total protein, using western blot to detect the expression of FHL2 MYH7B protein and NUMB protein.The results show that:(1) The morphological changes of satellite cells:Three groups cells have no obvious morphological differences after transfected 12h; At 24h, treatment group cells’ quantity is less than two control groups which all have a few cells begin to differentiate; At 48h, two control groups cells are overgrowing and a large number of these cells begin to differentiate, at the same time, treatment group only has a small increase in cells and tiny minority cells begin to differentiate; At 72h, two control groups cells are entirely differentiating and appearing muscle tube, but treatment group’s cell quantity has no significant changes and no muscle tube appears, it only has a part of cells begin to differentiate;(2) The mRNA expression of myogenic regulatory factors:The relative expression of target gene after transfected 12h and 24h, it shows that treatment group is significantly lower than two control groups(P<0.05); The relative expression of MyoD and MyoG after transfected 12h, treatment group is significantly lower than two control groups(P<0.05); After transfected 24h, the relative expressions of MyoG and MYHC show that two control groups are significantly higher than treatment group(P<0.05); However, at three points of time, the relative expression of MRF4 has no obvious difference among the three groups(P>0.05);(3) Notch signal pathway:The relative expression of hairyl shows that treatment group is significantly lower than the two control group after transfected 12h (P<0.05); At 24h, the expressions of Notch1、Hey1 and hairy1 reflect that treatment group is highly significantly or significantly lower than two control groups (P<0.05 or P<0.01); At 48h, the expression of NUMB in treatment group is highly significantly lower than the other gourps (P<0.01);(4) TGFβ signal pathway:The key gene ACVR1’s expression in treatment group is significantly lower than two control groups after transfected 12h (P<0.05); The expression of ACVR2 at three points of time both show that treatment goup is significantly lower than the other two groups (P<0.05);(5) Wnt/β-catenin signal pathway:After transfected 12h, the expressions of β-catenin、 Wnt2b and Wnt4 in treatment group is significantly or highly significantly lower than the control groups (P<0.05 or P<0.01); At 24h, Wnt4 and Wnt6 expressed in treatment is highly significantly lower than two control groups (P<0.01); At 48h, the expressions of Gskβ and Wnt5 in treatment group are highly significantly lower than the other groups (P<0.01);(6) Western-Blot:After transfected 48h, FHL2 protein and MYH7B protein in treatment group, their expression are obviously lower than two control groups; At 72h, the expression of NUMB protein in treatment group is obviously lower than free control group and negative control group.To sum up, after transfected FHL2 SiRNA, the proliferation and differentiation of skeletal muscle satellite cells are both suppressed, when the expression of FHL2 reduced, the expression of part myogenic regulatory factors are also reduced, the signal transduction of Wnt/β-catenin、TGFβ、Notch signal pathway recived different degree of inhibition. Hence, our research speculate that the specific influence mechanism of FHL2 probably is taking part in the signal transduction of Wnt/β-catenin、TGFβ、Notch pathway to achive regulating the proliferation and differentiation of skeletal muscle satellite cells, and effecting the growth of muscle. |
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