Silencing of mouse hepatic lanosterol 14-alpha demethylase down-regulated plasma low-density lipoprotein cholesterol levels by short-term treatment of siRNA.
Cytochrome P450 lanosterol 14-alpha demethylase (CYP51), participated in keeping serum cholesterol homeostasis, is a key enzyme to synthesize cholesterol from lanosterol. Here we focused on investigating the mechanism of CYP51 in modulating serum cholesterol levels in mouse through RNA interference (RNAi). Mice fed on normal or high fat high cholesterol (HFHC) diets were individually treated with small interference RNA (siRNA) of CYP51 gene by tail vein injection. The results showed that administrated single dose of 10 microg CYP51-siRNAs for 48 h resulted in significantly depletion of CYP51 mRNA in liver of mice fed on normal diet (from 40 to 60%, p<0.05). CYP51-siRNAs exerted the inhibition in a dose dependent manner (from 26% in 5 microg to 40% in 20 microg, p<0.05) and most inhibitive effect from day 3 to day 6 (over 50%, p<0.05) after the treatment. Six days after administration of 30 microg CYP51-siRNAs (20 microg on day 0 and 10 microg on day 3), CYP51 mRNA (normal: 50%; HFHC: 70%, p<0.05) and protein levels (normal and HFHC: over 40%, p<0.05) were significantly knocked down in mice liver. Interestingly, low-density lipoprotein receptor (LDLR) expression was significantly elevated compared with controls in hepatic cells after CYP51-siRNAs (mRNA: about 2 times; protein: about 1.6 times, p<0.05). As a consequence, about 50% of sera low-density lipoprotein cholesterol (LDL-ch) were significantly reduced (p<0.05). The effect on LDLR increase and LDL-ch reduction lasted 8 d after a single 20 microg CYP51-siRNAs injection. In addition, CYP51-siRNAs could not cause any fatty liver compared with Buffer-group and did not interfere with mice ovulation. In conclusion, these data demonstrated that CYP51-siRNAs silenced CYP51 in mouse liver and down-regulated plasma LDL-ch levels. The potential mechanism of LDL-ch reduction may be related to up-regulated LDLR expression of hepatic cells. It indicated that there was a cholesterol levels link-modulation system between cholesterol synthetic pathway through CYP51 and cholesterol transport pathway through LDLR in vivo.
State Key Laboratories for AgroBiotechnology and Department of Animal Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, P.R. China.
Baoshan
B
Xu
Chao
C
Wang
Jie
J
Yang
Guanping
G
Mao
Cheng
C
Zhang
Dan
D
Liu
Ping
P
Tai
Bo
B
Zhou
Guoliang
G
Xia
Meijia
M
Zhang
D000199:Actins / Q000096:biosynthesis; D000818:Animals; D015153:Blotting, Western; D001835:Body Weight / Q000187:drug effects; D002791:Cholesterol, Dietary / Q000378:metabolism; D008078:Cholesterol, LDL / Q000097:blood*; D054885:Cumulus Cells / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004032:Diet; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D005234:Fatty Liver / Q000139:chemically induced / Q000473:pathology; D005260:Female; D020868:Gene Silencing; D008099:Liver / Q000201:enzymology*; D051379:Mice; D010060:Ovulation / Q000187:drug effects / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D011973:Receptors, LDL / Q000187:drug effects / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D058886:Sterol 14-Demethylase; D014158:Transcription, Genetic
D000199:Actins; D002791:Cholesterol, Dietary; D008078:Cholesterol, LDL; D034741:RNA, Small Interfering; D011973:Receptors, LDL; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C548415:Cyp51 protein, mouse; D058886:Sterol 14-Demethylase