Use of MicroRNA or inhibitors thereof in regulation of lipid metabolism

Abstract

The present invention relates to use of a microRNA or an inhibitor thereof, and specifically, the present invention relates to use of a microRNA or an inhibitor thereof in preparing a medicament for regulating lipid metabolism or preparing a medicament for preventing or treating a disease related to lipid metabolism. The microRNA is one or more of the following: miRNA-96, miRNA-185, and miRNA-223. The present invention also relates to use of the microRNA or the inhibitor thereof in regulating the expression level of a protein related to lipid metabolism. The present invention also relates to a composition comprising the microRNA or the inhibitor thereof. The microRNA or the inhibitor thereof in the present invention can be used as a pharmaceutical component, and can be applied in preventing or treating a disease caused by lipid metabolism disorders such as hyperlipidemia, atherosclerosis, coronary heart disease or other diseases.

Claims

1. A method for treating a disease related to lipid metabolism, comprising the step of administering an effective amount of an antisense RNA of a microRNA to a subject in need of treating said disease, wherein the microRNA is one or two or three miRNAs selected from the group consisting of miRNA-96, miRNA-185 and miRNA-223, and wherein the disease related to lipid metabolism is atherosclerosis or a cardiovascular disease, and the miRNA-185 has a sequence as set forth in SEQ ID NO:2.

2. The method according to claim 1, wherein said cardiovascular disease is coronary heart disease or myocardial infarction.

3. The method according to claim 1, wherein the antisense RNA of miRNA-185 has a sequence as set forth in SEQ ID NO:5.

4. The method according to claim 1, wherein the subject is human.

5. A method for improving the level of high density lipoprotein (HDL) in a mammal and/or reducing the level of low density lipoprotein (LDL), cholesterol or triglyceride in blood in a mammal, which comprises the step of administering an effective amount of an antisense RNA of a microRNA to a mammal in need of improving the level of high density lipoprotein (HDL) and/or reducing the level of low density lipoprotein (LDL), cholesterol or triglyceride, wherein the microRNA is one or two or three miRNAs selected from the group consisting of miRNA-96, miRNA-185, and miRNA-223, and the miRNA-185 has a sequence as set forth in SEQ ID NO:2.

6. The method according to claim 5, wherein the antisense RNA of miRNA-185 has a sequence as set forth in SEQ ID NO:5.

7. The method according to claim 5, wherein the mammal is human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the effects of gene silencing of Dicer or Drosha on SR-BI mRNA level; wherein the left shows the mRNA level of SR-BI after Dicer or Drosha is silenced; the middle shows the mRNA level of Drosha after Drosha is silenced; the right shows the mRNA level of Dicer after Dicer is silenced; Scr-si represents the control sequence of siRNA (Invitrogen, 12935-200, 12935-300, 12935-400), Drosha-si (invitrogen, HSS120887) represents the group of Drosha gene silencing, and Dicer-si (invitrogen, HSS118719) represents the group of Dicer gene silencing. *P<0.05, **P<0.01, in comparison to scr-si.

(2) FIG. 2 shows the predicted targets of mirR-96, miR-185 and miR223 in 3UTR of SR-BI.

(3) FIG. 3 shows sequence conservation in multiple species of predicted targets of mirR-96, miR-185 and miR223 in 3UTR of SR-BI (Hsa, human; Ptr, chimpanzees; Mml, Macaque; Ssc, pig; Bta cattle; Eca, horse; Cfa, dog; Fca, cat; Mmu, mouse; Cpo, Guinea pig; Rno, rattus norvegicus; OCU, rabbits; Ete, Hedgehog).

(4) FIG. 4 shows regulatory effects of miR-96, miR-185, miR-223 and its antagonist on the mRNA level and protein expression level of SR-BI, wherein A represents the mRNA and protein level of SR-BI determined at different time after miR-185 and antisense oligodeoxynucleotide inhibitor thereof are transiently transfected into human hepatoma HepG2 and Bel-7402 or human normal hepatocyte HL-7702 using lipofectamine RNAiMAX transfection reagent; B represents changes in the mRNA and protein level of SR-BI after miR-96, miR-223 or their antisense oligonucleotide inhibitors are transiently transfected into hepatoma HepG2 using lipofectamine RNAiMAX transfection reagent. *P<0.05, **P<0.01, ***P<0.001, in comparison to ctl-miR (miR); .sup.#P<0.05, in comparison to ctl-miR (anti-miR). (con miR in Figure A and ctl-miR in Figure B are the same)

(5) FIG. 5 shows verification of direct binding sites of miR-96, miR-185 and miR-223 in 3UTR of SR-BI; wherein A shows that miR-185 can significantly reduce the expression of luciferase reporter gene in the presence of corresponding predicted target of miR-185 in 3UTR fragment of SR-BI, while miR-185 cannot significantly reduce the expression of luciferase reporter gene in the absence of corresponding predicted target in 3UTR fragment of SR-BI; B shows that miR-96 and miR-223 can significantly reduce the expression of luciferase reporter gene in the presence of corresponding predicted targets of miR-96 and miR-223 in 3UTR fragment of SR-BI; C shows the effects of miR-96, miR-185 and miR-223 alone or in combination on the expression of luciferase reporter gene, which confirms that miR-96, miR-185 and miR-223 have synergetic effects; wherein anti-miR represents miRNA antagonists (antisense oligonucleotide inhibitors), ctl-miR represents miRNA control (5-UGGAAUGUAAAGAAGUAUGUAU-3, SEQ ID NO: 7), pc-luc represents a control plasmid with no 3UTR of SR-BI; the nucleotide sequence of U1 is full-length of 3UTR fragment of SR-BI (959 bp), the nucleotide sequence of U2 is the 120.sup.th-959.sup.th bp of 3UTR fragment of SR-BI, the nucleotide sequence of U3 is the 312.sup.th-959.sup.th bp of 3UTR fragment of SR-BI; the nucleotide sequence of U4 is the 498.sup.th-959.sup.th hp of 3UTR fragment of SR-BI; the nucleotide sequence of U5 is the 664.sup.th-959.sup.th bp of 3UTR fragment of SR-BI, the nucleotide sequence of U6 is the 870.sup.th-959.sup.th bp of 3UTR fragment of SR-BI, WT represents wild type of 3 UTR fragment of SR-BI, DEL1 represents 3UTR fragment of SR-BI lacking binding site 1 for miR-185, and DEL2 represents 3UTR fragment of SR-BI lacking binding site 2 for miR-185. *P<0.05, **P<0.01, in comparison to ctl-miR; .sup.#P<0.05, .sup.## P<0.01, in comparison to the wild-type vector.

(6) FIG. 6 shows the effect of miR-96, miR-185 and miR-223 on Dil-HDL uptake in hepatocytes; A: detecting Dil-HDL uptake in hepatocytes by flow cytometry, wherein the hepatocytes are obtained by the following steps: transiently transfecting hepatoma HepG2 and Bel-7402 or normal hepatocyte HL-7702 with miR-185 or antisense oligonucleotide inhibitor thereof using lipofectamine RNAiMAX transfection reagent, and after 72 hours, incubating the cells with 2 g/mL Dil-HDL for 4 hours; B: detecting Dil-HDL uptake in hepatocytes by flow cytometry, wherein the hepatocytes are obtained by the following steps: transiently transfecting hepatoma HepG2 with miR-96, miR-223 or antisense oligonucleotide inhibitors thereof using lipofectamine RNAiMAX transfection reagent, and after 72 hours, incubating the cells with 2 g/mL Dil-HDL for 4 hours; CU-miR represents control miRNA, anti-miR represents miRNA antagonist; *P<0.05, **P<0.01, ***P<0.001, in comparison to ctl-miR (miR); .sup.#P<0.05, in comparison to ctl-miR (anti-miR).

(7) FIG. 7 shows effects of SR-BI on inhibition of Dil-HDL uptake mediated by miR-185 in hepatocyte; wherein ctl-miR represents control miRNA, scr-si represents siRNA control, and SR-BI-si represents the group of SR-BI gene silencing. **P<0.01; ***P<0.001; NS, no significant differences.

(8) FIG. 8 shows predicted targets of miR-96 and miR-223 in 3UTR of ABCA1.

(9) FIG. 9 shows the sequence conservation in multiple species of predicted targets of mirR-96 and miR223 in 3UTR of ABCA1 (Hsa, human; Pt, chimpanzees; Pab, Sumatra chimpanzees; Ame, giant panda; Mmu, mouse).

(10) FIG. 10 shows regulation effects of miR-96, miR-223 and their antagonists on the mRNA level of ABCA1; wherein anti-miR represents miRNA antagonists, and ctl-miR represents miRNA control. **P<0.01, in comparison to ctl-miR (miR); .sup.##P<0.01, in comparison to ctl-miR (anti-miR).

(11) FIG. 11 shows the two targets of miR-185 in 3UTR of LDLR.

(12) FIG. 12 shows the sequence conservation in multiple species of predicted targets of miR-185 in 3UTR of LDLR (Hsa, human; Ptr, chimpanzees; pab, Sumatra chimpanzees; Bta cattle).

(13) FIG. 13 shows regulation effects of miR-185 and its antagonist on the mRNA level of LDLR; wherein anti-miR represents miRNA antagonist, and ctl-miR represents miRNA control. **P<0.01, in comparison to ctl-miR (miR); .sup.# P<0.05, in comparison to ctl-miR (anti-miR).

(14) FIG. 14 shows one predicted target of miR-96 in 3UTR of PPAR-.

(15) FIG. 15 shows sequence conservation of predicted target of miR-96 in 3UTR of PPAR- in multiple species (Hsa, human; Ptr, chimpanzees; pab, Sumatra chimpanzees; Ssc, pig; Bta cattle; Fca, cat; Mmu, mouse; Rno, rattus norvegicus; Gga, chicken).

(16) FIG. 16 shows regulatory effects of miR-96 and its antagonist on the mRNA level of PPAR-; wherein anti-miR represents miRNA antagonist, and ctl-miR represents miRNA control. **P<0.01, in comparison to ctl-miR (miR); .sup.## P<0.01, in comparison to ctl-miR (anti-miR).

(17) FIG. 17 shows expression levels of miR-96 and miR-185 in liver in high fat diet fed mice, wherein the first picture shows body weight of mice, the second picture shows total cholesterol level, the third picture shows LDL level, and the fourth picture shows abundances of miR-96 and miR-185, chow represents the group of normal diet fed mice, HFD represents the group of high fat diet fed mice. *P<0.05, **P<0.01, in comparison to ctl-miR.

(18) FIG. 18 shows effects of miR-96, miR-185 and miR-223 on the mRNA level of SR-BI and Dil-HDL uptake in macrophage, where ctl-miR represents miRNA control, chow represents the group of normal diet, and HFD represents the group of high-fat diet. *P<0.05, **P<0.01, n=6 (chow) and n=5 (HFD).

(19) FIG. 19 shows regulation of HDL on the mRNA level of miR-185 and SR-BI, wherein Veh represents the solvent control. *P<0.05, **P<0.01, in comparison to veh (mir-185); .sup.#P<0.05, in comparison to veh (SR-BI).

(20) FIG. 20 shows distribution of miR-96, miR-185, or miR-223 in different tissues or cells in ApoE-knockout mice; wherein A represents abundances of miR-96 or miR-185 in different tissues (liver, kidney, heart, intestines, lungs, and spleen); B represents abundances of miR-96, miR-185 or miR-223 in different cells (human liver cancer cell line HepG2 and Bel-7402, normal liver parenchyma cells HL-7702 and THP-1 macrophages induced with PMA for 24 hours).

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

(21) The embodiments of the invention are illustrated in detail by referring to the examples, but those skilled in the art would understand that the following examples are merely for illustrating the invention and should not be deemed as restriction of the invention. The examples in which specific conditions are not indicated are performed according to conventional conditions or conditions suggested by manufacturers. The reagents or instruments for which manufacturers are not indicated are all conventional products commercially available.

(22) Materials and Methods

(23) 1. Material

(24) 1.1 The plasmids, cell lines and animals: eukaryotic cell expression vector pcDNA3.1 and pGL3-Basic are products from Promega in United States. The human hepatoma cell line HepG2, Bel-7402 and HL-7702 are kept in our laboratory (HepG2 is purchased from ATCC, HB-8065, Bel-7402 and HL-7702 are purchased from institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) and the ApoE-knockout mice are purchased from Peking University Health Science Center.
1.2 Primary Reagent: A cell lysate for DNA extraction and a luciferase assay system and a RNA extraction Kit (SV Total RNA Isolation System) are purchased from Promega in United States. A transfection reagent (Lipofectamine 2000) and a reverse transcription Kit (SuperScript III First-Strand) are purchased from Invitrogen in United States. A real-time fluorescent quantitative PCR kit (FastStart Universal SYBR Green PCR Master) is purchased from Roche. A RNA extraction Mini Kit (miRNeasy Mini Kit), A small RNA RT Kit (miScript II RT Kit) and a fluoresence Quantitative assay Kit (miScript SYBR Green PCR Kit) are purchased from Qiagen in United States. cell culture medium MEM is purchased from Thermo in United States. Fetal bovine serum, sodium pyruvate, non-essential amino acid and antibiotic G418 are purchased from Gibco in United States. Dimethyl sulfoxide (DMSO) is purchased from Sigma in United States.
1.3 Apparatus: MiniCycle PTC-200 PCR is the product from MJ Research in United States; EnVision multi-functional microplate detector is the product from PerkinElmer in United States; and Real-time fluorescence quantitative PCR instrument-iQ5 Multicolor Real-Time PCR is the product from Bio-Rad.
2 Methods
2.1 Construction of recombinant plasmid pc-luc-3UTR: 3UTR sequence of CLA-1 (NM_005505) is obtained by PCR and is cloned into downstream of luciferase reporter gene of plasmid pc-luc (inserting luciferase reporter gene into multiple cloning site of plasmid vector pcDNA3.1 to construct plasmid pc-luc) to obtain the recombinant plasmid pc-luc-3UTR, which are confirmed by sequencing.
2.2 Cell culture and transfection: HepG2 cells and the like are cultured at 37, in 5% CO.sub.2 in MEM medium containing 10% fetal bovine serum, 1 mmol/L sodium pyruvate and 1% non-essential amino acids. The plasmids used for transfection are extracted according to instructions of PureYield Plasmid Midiprep System kit. 110.sup.5 HepG2 cells are seeded on 30 mm Petri dishes. When the cells grow adhering to the wall and the convergence degree is about 80%, pc-luc-3UTR or miRNA is transiently transfected into HepG2 cells using liposome Lipofectamine 2000 or Lipofectamine RNAiMAX. The detection is carried out a certain time post transfection.
2.3 Real-Time fluorescent quantitative PCR: Total RNA is extracted from cells, cDNA is synthesized using a kit of SuperScript III First-Strand (Invitrogen) or miScript II RT Kit, the fluorescence quantitative PCR reaction is performed using 2TaqMan Gene Expression Master Mix or miScript SYBR Green PCR Kit, and the analysis is performed with data analysis software and the Ct value is calculated. GAPDH or U6 is used as an internal control, and the transcription level of luciferase gene is quantified using the relatively Ct method.
2.4 Detection of expression levels of the proteins on cell surface: cells are seeded at density of 110.sup.5/ml on 24-well cell culture plates, digested and collected after treatment, fixed with 4% paraformaldehyde for 4 h, and are resuspended in 1 ml PBS containing 5% FBS and left to stand for 15 min at 4 C. The primary antibody (1:500) and the FITC-labeled secondary antibody (1:1000) are added respectively and each is incubated for 50 min. After rinsed with PBS, filtration is carried out through the 300 mesh of nylon membrane and the detection is performed using flow cytometry. The average number of cells detected in each of sample groups is 10,000. The relative fluorescence intensity of cells in each group is shown as the figure of logarithmic integral.
2.5 Determination of cellular function for Dil-HDL uptake: Effect of compound on cellular Dil-HDL uptake is determined using flow cytometry. After HepG2 cells and the like are passaged, the cells are seeded at density of 110.sup.5 cells/ml on 24-well cell culture plate. After treatment, 2 g/ml of Dil-HDL is added and incubation is carried out at 37 C. for 4 h. The cells are digested and collected, resuspended in 700 l PBS and filtered through 300 mesh of nylon membrane. The cellular fluorescence is determined using flow cytometry. The average 10,000 cells are determined for each sample. The relative fluorescence intensity of cell in each group is shown as the figure of logarithmic integral.
Process for Screening miRNA

(25) Predicted miRNAs are transfected into HepG2 cells to determine effects of miRNA on the expression of SR-BI using real-time quantitative PCR.

(26) The selected miRNAs are miR-96, miR-185 and miR-223, of which sequences are as follows:

(27) TABLE-US-00001 hsa-miR-96 (SEQIDNO:1) 5-uuuggcacuagcacauuuuugcu-3 hsa-miR-185 (SEQIDNO:2) 5-uggagagaaaggcaguuccuga-3 hsa-miR-223 (SEQIDNO:3) 5-ugucaguuugucaaauacccca-3

(28) These miRNAs of the present invention are commercially available from QIAGEN, wherein hsa-miR-96 with Cat. No. MSY0000095, hsa-miR-185 with Cat. No. MSY0000455, and hsa-miR-223 with Cat. No. MSY0004570.

Example 1

Regulatory Effects of miR-96, miR-185 and miR-223 on SR-BI which is an Important Target in Reverse Cholesterol Transport

(29) 1.1, Effects of Dicer and Drosha Gene Silencing on the mRNA Level of SR-BI

(30) Two key enzyme, Dicer and Drosha, in microRNA biosynthesis are knocked out by siRNA gene silencing, and then the mRNA level of SR-BI is detected by Real-time RT-PCR.

(31) From experimental results, it is shown that the mRNA level of SR-BI is up-regulated significantly after Dicer and Drosha genes are silenced, indicating microRNAs are involved in regulation of SR-BI (as shown in FIG. 1).

(32) 1.2, Regulatory Effects of miR-96, miR-185, miR-223 and their Antagonists Thereof on the mRNA and Protein Expression Level of SR-BI.

(33) The miRNA antagonists (anti-miR) in the present invention are purchased from QIAGEN, and their sequences and catalogue numbers are as follows:

(34) TABLE-US-00002 anti-miR-96(Cat.No.MIN0000095) (SEQIDNO:4) 5-agcaaaaaugugcuagugccaaa-3 anti-miR-185(Cat.No.MIN0000455) (SEQIDNO:5) 5-ucaggaacugccuuucucucca-3 anti-miR-223(Cat.No.MIN0004570) (SEQIDNO:6) 5-ugggguauuugacaaacugaca-3

(35) The control of miRNA antagonists is available from QIAGEN, with Cat. No. 1027271.

(36) With lipofectamine RNAi MAX transfection reagent, miR-96, miR-185, miR-223 and antisense oligonucleotide inhibitors thereof are transiently transfected into human hepatoma cells HepG2 or Bel-7402 and human normal heptical cell HL-7702. The total cellular RNA is extracted using RNA extraction Mini Kit (Promega) 48 or 72 hours after transfection, and the reverse transcript is performed using cDNA synthesis Kit (Invitrogen), and SR-BI mRNA level is determined by Real-time RT-PCR. With lipofectamine RNAi MAX transfection reagent, miR-96, miR-185, miR-223 and antisense oligonucleotide inhibitors thereof are transiently transfected into hepatocarcinoma cells HepG2 or Bel-7402 and normal heptical cell HL-7702. The change in expression level of SR-BI protein is determined by flow cytometry 48 or 72 hours after transfection.

(37) From experimental results, it is shown that expression levels of mRNA and protein of SR-BI are down-regulated by miR-96, miR-185 and miR-223 in hepatocyte (as shown in FIG. 5).

(38) 1.3, Prediction of miRNA Targeting 3UTR of SR-BI Gene and Homology Analysis of Target Sites

(39) It is predicted that miRNAs such as miR-96, miR-185 and miR-223 directly bind on 3UTR of SR-BI (NM_005505) using MicroRNA.org (www.microrna.org) and TargetScan (www.targetscan.org) and other online software. Using software Clustal X2, 3UTR of SR-BI of multiple species are aligned.

(40) The predicted results are shown in FIG. 2. There are one predicted target of miR-96 and one predicted target of miR-223 in 3UTR of SR-BI and there are two predicted targets of miR-185 in 3UTR of SR-BI. The results of sequence alignment are shown in FIG. 3. The sequence of the predicted targets of miR-96, miR-185 and miR-223 in 3UTR of SR-BI are conserved in multiple species (Hsa, human; Ptr, chimpanzees; Mml, Macaque; Ssc, pig; Bta, cattle; Eca, horse; Cfa, dog; Fca, cat; Mmu, mouse; Cpo, Guinea pig; Rno, rattus norvegicus; Ocu, rabbit; Ete, Hedgehog).

(41) 1.4, Verification of Direct Binding Sites of miR-96, miR-185 and miR-223 in 3UTR of SR-BI

(42) The luciferase reporter gene is inserted into multiple cloning site of the plasmid vector pcDNA3.1 to construct the plasmid pc-luc. According to the predicted targets of miR-96, miR-185 and miR-223, different length of 3UTR fragments of SR-BI are amplified by PCR method, or prediction targets of miR-185 are removed from 3UTR of SR-BI using overlap PCR method to obtain a series of different length 3UTR fragments of SR-BI. The different length of 3UTR fragments of SR-BI are inserted into downstream of luciferase reporter gene of plasmid vector pc-luc. By transient transfection method and using Lipofectamine RNAiMAX transfection reagent, the constructed plasmid pc-luc containing a series of different length of 3UTR fragments of SR-BI, miR-96, and miR-185, miR-223 or their antisense oligonucleotides acid inhibitors are transfected into HepG2 cells, and after 48 hours, the expression activities of the luciferase reporter genes are determined by luciferase reporter gene analysis.

(43) The results shown in FIG. 4 show that if there are corresponding predicted targets of miR-96, miR-185 and miR-223 in the 3UTR fragment of SR-BI, miR-96, miR-185 and miR-223 can significantly reduced the expression activities of luciferase reporter genes; if there are not, miR-96, miR-185 and miR-223 cannot significantly reduced the expression activities of luciferase reporter genes. Experimental results verify the targets of miR-96, miR-185 and miR-223 in 3UTR of SR-BI, and further experiments confirm that there are synergistic effects between these miRNAs.

(44) 1.5, Effects of miR-96, miR-185 and miR-223 on Dil-HDL (Dil Fluorescein-Labeled HDL) Uptake in Hepatocytes.

(45) miR-96, miR-185, miR-223, or antisense oligonucleotide inhibitors thereof are transiently transfected into hepatocarcinoma cells HepG2, Bel-7402, and normal hepatocytes HL-7702 by Lipofectamine RNAiMAX reagent (Invitrogen), and after 72 hours, the cells are incubated with 2 g/mL Dil-HDL for 4 hours. Then Dil-HDL uptake by hepatocytes is determined by flow cytometric.

(46) The results show that Dil-HDL uptake decreases significantly in hepatocytes after transfected with miR-96, miR-185, miR-223, and Dil-HDL uptake increases significantly in three kinds of hepatocytes after transfected with antisense oligonucleotide inhibitors of miR-96, miR-185 and miR-223, which shows that miR-96, miR-185, miR-223 can inhibit HDL uptake in hepatocytes, and the antisense oligonucleotide inhibitors of miR-96, miR-185, miR-223 can increase HDL uptake in hepatocyte (as shown in FIG. 6).

(47) 1.6, Effect of SR-BI Gene on Inhibition of Dil-HDL Uptake in Hepatocyte Mediated by miR-185

(48) After SR-BI gene is knocked out using siRNA gene silencing, the expression of SR-BI protein is significantly reduced in HepG2 cells. After transient transfection of miR-185 for 72 hours, the cells are incubated with 2 g/mL Dil-HDL for 4 hours. Then the Dil-HDL uptake is determined by flow cytometry in hepatocyte.

(49) The results shown in FIG. 7 show that the effect of down-regulating Dil-HDL uptake by miR-185 disappears after SR-BI gene is silenced, which proves that miR-185 inhibits Dil-HDL uptake in hepatocyte through SR-BI gene.

Example 2

Regulatory Effects of miR-96 and miR-223 on ABCA1, an Important Target of Reverse Cholesterol Transport

(50) 2.1, Prediction of Targets of miR-96 and miR-223 in 3UTR of ABCA1 and Homology Analysis

(51) The binding sites of miR-96 and miR-223 in 3UTR of ABCA1 are predicted using MicroRNA.org and TargetScan. Sequences of predicted targets of miR-96 and miR-223 in 3UTR of ABCA1 are aligned in multiple species using Clustal X2 software.

(52) The predicted results are shown in FIG. 8. There are one target of miR-96 and one target of miR-223 in 3UTR of ABCA1. The results of sequence alignment are shown in FIG. 9. The sequence of the predicted targets of miR-96 and miR-223 in 3UTR of ABCA1 are conserved in multiple species (Has, human; Ptr, chimpanzee: Pab, Sumatra chimpanzees; Ame, giant panda; Mmu, mouse).

(53) 2.2, Regulatory Effects of miR-96, miR-223 and their Antagonist on the mRNA Level of ABCA1

(54) Hepatoma cells HepG2 are transiently transfected with miR-96, miR-223 or their antisense oligonucleotide inhibitors using Lipofectamine RNAiMAX Transfection reagent (Invitrogen), and after 72 hours, the total RNA is extracted using RNA extraction mini kit (Promega) and is reverse transcribed using cDNA synthesis Kit (Invitrogen), and mRNA level is determined by real-time RT-PCR assay.

(55) The results are shown in FIG. 10. MiR-96 and miR-223 can significantly reduce mRNA levels of ABCA1 in HepG2 cells, and their antisense oligonucleotide inhibitors can significantly increase the mRNA levels of ABCA1 in HepG2 cells.

Example 3

Regulatory Effects of miR-185 and its Antagonists on LDLR

(56) 3.1, Prediction of Targets of miR-185 in 3UTR of LDLR and Homology Analysis

(57) The binding sites of miR-185 in 3UTR of LDLR are predicted using MicroRNA.org and TargetScan. Sequences of predicted targets of miR-185 in 3UTR of LDLR are aligned in multiple species using Clustal X2 software.

(58) The predicted results are as shown in FIG. 11. There are two predicted targets of miR-185 in 3UTR of LDLR. The results of sequence alignment are shown in FIG. 12. The sequence of the predicted targets of miR-185 in 3UTR of LDLR are conserved in multiple species (Has, human; Ptr, chimpanzee; Pab, Sumatra chimpanzees; Bta, cattle).

(59) 3.2, Regulation Effects of miR-185 and its Antagonist on the mRNA Level of LDLR

(60) Hepatoma cells HepG2 are transiently transfected with miR-185 or its antisense oligonucleotide inhibitors using Lipofectamine RNAiMAX Transfection reagent (Invitrogen), and after 72 hours, the total RNA is extracted using RNA extraction mini kit (Promega) and is reverse transcribed using cDNA synthesis Kit (Invitrogen), and mRNA level is determined by real-time RT-PCR assay.

(61) The results are shown in FIG. 13. MiR-185 can significantly reduce LDLR mRNA levels in cells HepG2, and their antisense oligonucleotide inhibitors can significantly increase the mRNA level of LDLR in cells HepG2.

Example 4

Regulation Effects of miR-96 and its Antagonists on PPAR-

(62) 4.1, Prediction of Targets of miR-96 in 3UTR of PPAR- and Homology Analysis

(63) The binding sites of miR-96 in 3UTR of PPAR- are predicted using MicroRNA.org and TargetScan. Sequences of predicted targets of miR-96 in 3UTR of PPAR- are aligned in multiple species using Clustal X2 software.

(64) The predicted results are as shown in FIG. 14. There is one target of miR-96 in 3UTR of PPAR-. The results of sequence alignment are shown in FIG. 15. The sequence of the predicted targets of miR-96 in 3UTR of PPAR- are conserved in multiple species (Has, human; Ptr, chimpanzee; Pab, Sumatra chimpanzees; Ssc, pig; Bta, cattle; Fca, cat; Mmu, mouse; Rno, Rattus norvegicus; Gga, chicken).

(65) 4.2, Regulatory Effects of miR-96 and its Antagonist on the mRNA Level of PPAR-

(66) Hepatoma cells HepG2 are transiently transfected with miR-96 or its antisense oligonucleotide inhibitor using Lipofectamine RNAiMAX Transfection reagent (Invitrogen), and after 72 hours, the total RNA is extracted using RNA extraction mini kit (Promega) and is reverse transcribed using cDNA synthesis Kit (Invitrogen), and mRNA level is determined by real-time RT-PCR assay.

(67) The results are shown in FIG. 16, miR-96 can significantly reduce the mRNA level of PPAR- in HepG2 cells, and its antisense oligonucleotide inhibitors can significantly increase the mRNA level of PPAR- in HepG2 cells.

Example 5

Effects of miR-96, miR-185 and miR-223 on Regulation of Lipid Metabolism

(68) 5.1, Regulatory Effects of HDL on mRNA Levels of miR-185 and SR-BI.

(69) After hepatoma cells HepG2 are incubated with HDL, the abundance of miR-185 and the mRNA level of SR-BI are determined by Real-time RT-PCR assay.

(70) Experimental results show that the abundance of miR-185 is significantly down-regulated in a dosage-dependent manner, and the mRNA levels of SR-BI are significantly up-regulated in a dosage-dependent manner (FIG. 19), which suggests that miR-185 and SR-BI are both involved in lipid regulation mediated by HDL.

(71) 5.2, Distributions of miR-96, miR-185 or miR-223 in Different Tissues of ApoE-Knockout Mice or Cell Lines.

(72) Tissues from liver, kidney, heart, intestine, lung, spleen and other organs from ApoE-knockout mice, and cell lines such as human hepatoma cell HepG2 and Bel-7402, normal hepatocyte HL-7702 and macrophage THP-1 induced by PMA for 24 hours are taken as the samples, and the abundances of miR-96, miR-185 and miR-223 in different tissues or cells are determined by real-time RT-PCR assay.

(73) The results show that miR-185 and miR-96 present high expression in the tissues from liver, kidney, heart and other organs from mice, and human hepatocytes and macrophages. MiR-223 presents high expression in human macrophages (FIG. 20).

(74) 5.3, Expression Levels of miR-96 and miR-185 in Liver in High Fat Diet Fed Mice.

(75) ApoE knockout mice are fed on high-fat diet for 8 weeks, blood was taken from orbit and the total cholesterol level and LDL cholesterol level in serum are determined. Meanwhile, the liver is taken as sample, and the abundances of miR-96 and miR-185 are determined by real-time RT-PCR assay (Qiagen).

(76) After ApoE knockout mice are fed on high-fat diet for 8 weeks, the total cholesterol level and LDL cholesterol level in serum significantly increase, and the expression of miR-96 and miR-185 significantly decrease in hepatocytes, and the expression level of SR-BI significantly increase (As shown in FIG. 17).

(77) 5.4 Effects of miR-96, miR-185 and miR-223 on the mRNA Level of SR-BI in Macrophage and Dil-HDL Uptake.

(78) Macrophage THP-1 induced by PMA for 24 hours is transiently transfected with miR-96, miR-185 or miR-223 using Lipofectamine RNAiMAX Transfection reagent (Invitrogen), and after 72 hours, The mRNA level of SR-BI is detected by real-time RT-PCR, or Dil-HDL uptake by macrophage is detected by flow cytometry after incubation of the transfected macrophage with 2 g/ml Dil-HDL for 4 hours.

(79) From experimental results, it is shown that the mRNA level of SR-BI in macrophage is down-regulated after transfection with miR-96, miR-185 and miR-223. The Dil-HDL uptake significantly decreases after transfection with miR-185, while the uptake of Dil-HDL significantly increases after transfection with miR-96 (as shown in FIG. 18).

(80) Although the specific models for carrying out the invention have been described in detail, those skilled in the art will understand these details can be modified and changed according to all teachings in the art, and these changes are within the protection scope of the invention. The whole scope of the invention is given by the attached claims and any equivalents thereof.