HMGCR: A key enzyme in hepatic cholesterol synthesis.

• Gene Information: The HMGCR gene, located on chromosome 5 (5q13.3), encodes 3-hydroxy-3-methylglutaryl-CoA reductase. This enzyme serves as the primary rate-limiting enzyme in the mevalonate pathway, responsible for endogenous cholesterol biosynthesis.
• Protein Expression: HMGCR is an integral membrane protein predominantly expressed in the endoplasmic reticulum (ER) of hepatocytes (liver cells) and enterocytes.
• Signaling Pathway: HMGCR catalyzes the conversion of (3S)-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid. This reaction represents the rate-limiting step for the biosynthesis of cholesterol and other isoprenoids, rendering HMGCR critical for cellular cholesterol homeostasis. HMGCR serves as the primary molecular target for statins, a class of cholesterol-lowering therapeutics.
• Therapeutic Inhibition: HMG-CoA reductase inhibitors are a class of drugs commonly known as statins. From a biomedical perspective, these drugs are used to lower cholesterol levels in the blood.

HMGCR

• The exons 1 20 of the mouse Hmgcr gene that encode the whole molecule, including promoter, 5’UTR, and 3’UTR, were replaced by the exons 1 20 of the humancounterparts.
• The promoter, 5’UTR and 3’UTR of the mouse gene were also replaced by human promoter, 5’UTR, and human 3’UTR.
• The human HMGCR expression is driven by the human HMGCR promoter, while the mouse Hmgcr gene transcription and translation will be disrupted.

Species specific analysis of HMGCR mRNA expression in wild-type C57BL/6JNifdc mice and humanized B-hHMGCR mice by RT-PCR. Liver was collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hHMGCR mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human HMGCR primers. Mouse Hmgcr mRNA was exclusively detectable in wild-type mice but not in B-hHMGCR mice. Human HMGCR mRNA was detectable only in homozygous B-hHMGCR mice, but not in wild-type C57BL/6JNifdc mice. Human sequences were confirmed via Sanger sequencing.

Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hHMGCR mice (H/H), and then analyzed by western blot with anti-HMGCR antibody (Novus, NBP2-66888). 40 μg total proteins were loaded for western blot analysis. HMGCR was detected in the liver, lung, kidney, brain and stomach of wild-type mice and homozygous B-hHMGCR mice, as the antibody cross-recognized both human and mouse HMGCR.

Mouse LDLR expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hHMCGR mice by ELISA. Liver was collected from wild-type C57BL/6JNifdc mice (+/+) (male and female, n=3, 6~8 weeks old) and homozygous B-hHMCGR mice (H/H) (male and female, n=3, 6~8 weeks old). Expression level of mouse LDLR were analyzed by ELISA (Mouse LDLR Quantikine ELISA Kit: R&D, MLDLR0). Mouse LDLR in wild-type mice was similar with the LDLR level in homozygous B-hHMGCR mice. Values are expressed as mean ± SEM.

The inhibitory efficiency of the HMGCR-targeted small nucleic acid drug in B-hHMGCR mice. B-hHMGCR mice were randomly divided into 3 groups (n=2-3, male). The human HMGCR-targeted nucleic acid drug (From a client) and Saline were administered to the mice individually. The mice were sacrificed on day 14, and the liver tissues were collected to detect the human HMGCR expression by RT-qPCR. (A) The schematic diagram of experimental processing. (B) The expression of human HMGCR mRNA in liver. The human HMGCR mRNA in the treatment group was significantly reduced compared to the control group. Values are expressed as mean ± SEM. (Note: Data are shared upon the client's approval.)