Application of MAFG as homeostatic regulation target of cholesterol and regulation agent of MAFG
A technology of cholesterol and modulators, applied in the biological field, can solve problems such as difficulty in finding alternative drugs
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Embodiment 1
[0087] Example 1. MAFG is the target gene of miR-378
[0088] In order to explore the molecular mechanism by which miR-378 regulates cholesterol and bile acid metabolism, the inventors screened the genes involved in cholesterol and bile acid metabolism targeted by miR-378 and found that the 3'UTR of MAFG contains a highly conserved gene of miR-378. binding site ( figure 1 a). Thus, miR-378 targets binding to the 3'UTR of MAFG.
[0089] The inventors cloned the 3'UTR sequence of MAFG containing the miR-378 binding site ( Figure 5 a) onto a fluorescent reporter gene (pRL-TK plasmid), and co-transfected with AgomiR-378 into 293T cells, the inventors found that overexpression of miR-378 can inhibit the fluorescence intensity of MAFG-3'UTR ( figure 1 b). However, when the binding site of miR-378 on MAFG-3'UTR was mutated, the inhibitory effect of miR-378 on MAFG-3'UTR fluorescence intensity disappeared ( figure 1 c).
[0090] Mut1: put the 5'-AGUC C AG-3' segment was mutate...
Embodiment 2
[0094] Example 2. Inhibition of MAFG in the liver can reduce serum cholesterol levels and promote the synthesis and excretion of bile acids
[0095] Although MAFG was found to inhibit bile acid synthesis, whether MAFG can affect cholesterol metabolism still needs further exploration. The inventors constructed a shRNA plasmid against MAFG and further constructed an adenovirus ( figure 2 a).
[0096] Taking pENTR-U6 as the backbone plasmid, the shRNA sequence inserted into the plasmid is:
[0097] 5'-GCCACCAGCGTCATCACAATACGAATATTGTGATGATGACGCTGGTGGC-3' (SEQ ID NO: 5).
[0098] The inventors found that after inhibiting the expression of MAFG in the liver, the serum cholesterol level was significantly decreased ( figure 2 b), and both serum low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were significantly reduced ( figure 2 c), indicating that MAFG may mediate the regulatory effect of miR-378 on cholesterol. The inventor...
Embodiment 3
[0101] Example 3. Overexpression of MAFG in the liver inhibits bile acid synthesis and increases serum cholesterol levels
[0102] The inventors constructed an adenovirus ( Figure 4 a), it was found that after overexpression of MAFG in mouse liver, the cholesterol level in mouse serum was significantly increased ( Figure 4 b).
[0103] The inventors further found that after overexpression of MAFG in the liver, the RNAs of CYP7B1, CYP8B1 and CYP27A1 ( Figure 4 c) and protein ( Figure 4 d) The expression levels were significantly decreased, but the expression levels of CYP7A1 did not change significantly ( Figure 4 c-d). Consistent with this, the inventors also found in primary hepatocytes that the RNAs of CYP7B1, CYP8B1 and CYP27A1 ( Figure 4 e) and protein ( Figure 4 f) The expression levels were significantly decreased, but the expression levels of CYP7A1 did not change significantly ( Figure 4 e-f).
[0104] These findings suggest that overexpression of MAFG ...
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