lncRNA DC624545 and its application, siRNA for inhibiting the expression of lncRNA DC624545 sequence and its conjugate and application

Abstract

The present invention relates to the field of biomedicine technology. The present invention provides a lncRNA DC624545 and an application thereof, wherein the nucleotide sequence of the lncRNA DC624545 is shown in SEQ ID NO.1. Also provided are siRNA for inhibiting the expression of the lncRNA DC624545 sequence, a conjugate containing the siRNA sequence and an application thereof. The present invention uses lncRNA DC624545 as a target, and achieves prediction and / or treatment of hyperlipidemia by inhibiting the expression of the lncRNA DC624545 sequence, which can simultaneously meet the targeting of two different species, humans and cynomolgus monkeys, and has higher reliability in the subsequent drug-making process; at the same time, the siRNA silencing method is adopted to reduce the number of injections required by the patient, and reduce the patient's time cost and money cost. This provides a feasible solution for developing hyperlipidemia drugs and conducting preclinical research, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to lncRNADC624545 and applications thereof, siRNA for inhibiting the expression of the lncRNADC624545 sequence and conjugates thereof and applications thereof. Background Art

[0002] Hyperlipidemia is a common disease, with 13% of adults nationwide reported to suffer from it in 2023. Evidence from many prospective epidemiological studies has recognized hyperlipidemia, and genome-wide association studies have provided repeated evidence of associations between blood lipid profiles and variants in two other genes (PPP1R3B (8p23.1) and TTC39B (9p22.3)) with potential roles in lipid metabolism. Genome-wide significant associations of PPP1R3B variants; PPP1R3B is a gene involved in the regulation of blood fatty acid metabolism (high-density lipoprotein and non-high-density lipoprotein cholesterol) and has genome-wide significant characteristics of hepatic steatosis. PPP1R3B deficiency and reduced glucogenesis shift the storage of exogenous glucose to hepatic triglycerides (TG), and when PPP1R3B expression is increased, SNP carriers associated with increased plasma liver fat and lower blood lipids.

[0003] lncRNA is a non-coding RNA with a length of more than 200 nucleotides. Among the RNA contained in cells, the proportion of lncRNA far exceeds the well-known mRNA, miRNA, etc., accounting for more than 90% of lncRNA, while mRNA only accounts for 2%. Studies in recent years have shown that lncRNA plays an important role in many life activities such as dosage compensation effect, epigenetic regulation, and post-transcriptional regulation, and has become a hot topic of research. However, there is currently no information on the application of relevant lncRNA as a therapeutic target for hyperlipidemia.

[0004] Currently, LOC157273 is a potential therapeutic target. When LOC157273 is downregulated, the expression of PPP1R3B will be upregulated, which will help control the blood fat (lipid) content and the accumulation of liver fat. However, it only exists in the human genome. Although most of the genes of humans and primates are the same, there are still a large part of them that are different. At the same time, there are also large differences in the location of exons and introns. In the process of preclinical experiments in humans, different animals are needed to verify safety and effectiveness, so LOC157273 cannot meet the requirements of such experiments.

[0005] Based on the above, it is very necessary to obtain a technical solution that is not only effective for humans but can also be used to treat hyperlipidemia. Summary of the invention

[0006] The purpose of the present invention is to provide a lncRNA DC624545 and its application, taking lncRNA DC624545 as a target, and realizing the prediction and / or treatment of hyperlipidemia by inhibiting the expression of lncRNA DC624545 sequence, which can simultaneously meet the targeting of two different species, human and cynomolgus monkey, and has higher reliability in the subsequent drug-making process; at the same time, the siRNA silencing method of the present invention is adopted to reduce the number of injections required by the patient, and reduce the patient's time cost and money cost.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a lncRNA DC624545, and the nucleotide sequence of the lncRNA DC624545 is shown as SEQ ID NO.1.

[0009] The present invention provides the use of the lncRNA DC624545 as a target in screening, predicting and / or treating hyperlipidemia products.

[0010] Preferably, a substance that inhibits the expression of the lncRNADC624545 sequence is screened.

[0011] The present invention also provides the use of a preparation for detecting the expression level of the lncRNA DC624545 in the preparation of a product for predicting hyperlipidemia.

[0012] The present invention also provides a siRNA for inhibiting the expression of the lncRNA DC624545 sequence, wherein the siRNA consists of a sense chain and an antisense chain, the nucleotide sequence of the sense chain is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.3.

[0013] The present invention also provides a conjugate comprising the siRNA, wherein the conjugate comprises Galnac and the siRNA.

[0014] The present invention also provides the use of the siRNA or the siRNA conjugate in preparing a drug for predicting and / or treating hyperlipidemia.

[0015] By adopting the above technical solution, the present invention has the following beneficial effects:

[0016] 1. The technical solution of the present invention uses lncRNA DC624545 as a target, and achieves prediction and / or treatment of hyperlipidemia by inhibiting the expression of the lncRNA DC624545 sequence. It can simultaneously meet the targeting requirements of two different species, humans and cynomolgus monkeys, and has a wider range of preclinical applications and higher reliability in the subsequent drug development process.

[0017] 2. The technical solution of the present invention adopts the siRNA silencing method and the corresponding siRNA sequence, which reduces the number of injections required by the patient and reduces the patient's time and money costs.

[0018] 3. The location of the silencing of the Galnac-siRNA in the present invention is located upstream of the protein PPP1R3B that affects glycogen. Therefore, compared with the direct administration of chemical drugs, the therapeutic effect achieved by reducing the gene expression level will inevitably have fewer side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The expression of blood glucose regulating protein PPP1R3B in different treatment groups ( Figure 1 A in the figure is the group treated with cynomolgus monkey primary hepatocytes, and B is the group treated with human normal hepatocytes);

[0020] Figure 2 The expression of DC624545, the upstream gene of glucose regulating protein, in different treatment groups ( Figure 2 A in the figure is the group treated with cynomolgus monkey primary hepatocytes, and B is the group treated with human normal hepatocytes);

[0021] Figure 3 is the content of high density lipoprotein in different treatment groups ( Figure 3 A in the figure is the group treated with cynomolgus monkey primary hepatocytes, and B is the group treated with human normal hepatocytes). DETAILED DESCRIPTION

[0022]

[0023] The present invention also provides the use of the lncRNA DC624545 as a target in screening, predicting and / or treating hyperlipidemia products.

[0024] In the present invention, a substance that inhibits the expression of the lncRNA DC624545 sequence is screened.

[0025] The present invention also provides the use of a preparation for detecting the expression level of the lncRNA DC624545 in the preparation of a product for predicting hyperlipidemia.

[0026] The present invention also provides a siRNA for inhibiting the expression of the lncRNA DC624545 sequence.

[0027] In the present invention, the siRNA consists of a sense strand and an antisense strand. The nucleotide sequence of the sense strand is shown in SEQ ID NO.2, and the specific sequence is 5'-CCAGGGGCCACUAGCAAUUGC-3'; the nucleotide sequence of the antisense strand is shown in SEQ ID NO.3, and the specific sequence is 5'-GCAAUUGCUAGUGGCCCCUGG-3'.

[0028] The present invention also provides a conjugate comprising the siRNA, wherein the conjugate comprises Galnac and the siRNA.

[0029] The present invention also provides the use of the siRNA or the siRNA conjugate in preparing a drug for predicting and / or treating hyperlipidemia.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1 Screening target lncRNA DC624545

[0032]

[0033] Example 2 Design of siRNA

[0034] According to the obtained gene sequence of lncRNA DC624545, siDirect version 2.0 siRNA design software was used, and reference was made to “siDirect 2.0: updated software for designing functional siRNA with reduced seed-dependent off-target effect” (Naito Y, Yoshimura J, Ui-Tei MK. siDirect 2.0: updated software for designing functional siRNA with reduced seed-dependent off-target effect [J]. BMC Bioinformatics, 2009. DOI: 10.1186 / 1471-2105-10-392.), siRNA was screened as a candidate, and then siRNA with a GC content of 50% was selected therefrom, and then the siRNA was compared with the cynomolgus monkey DC624545 sequence included in the Genebank database through the NCBI BLAST function, and the following siRNA was designed, wherein the sense chain sequence was the same as the target sequence designed on the target gene (U replaced T), and the antisense chain sequence was completely complementary to the target sequence (U replaced T).

[0035] The designed siRNA consists of a sense strand and an antisense strand. The nucleotide sequence of the sense strand is shown in SEQ ID NO.2, and the specific sequence is 5'-CCAGGGGCCACUAGCAAUUGC-3'; the nucleotide sequence of the antisense strand is shown in SEQ ID NO.3, and the specific sequence is 5'-GCAAUUGCUAGUGGCCCCUGG-3'.

[0036] Example 3

[0037] Primary hepatocytes of cynomolgus monkeys (purchased from Liwo Biotechnology Shenzhen Co., Ltd.) and normal hepatocytes of humans (purchased from IMMOCELL) were cultured at a rate of 2×10 5 / well were inoculated into six-well cell culture plates, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. The siRNA designed above was transfected into primary cynomolgus monkey hepatocytes and normal human hepatocytes respectively using transfection reagent Lipo8000 (purchased from Beyotime / Biyuntian Biotechnology).

[0038] The experiment was divided into a control group (no transfection treatment), a 25pmol Lipo8000-siRNA group, a 30pmol Lipo8000-siRNA group, and a 50pmol Lipo8000-siRNA group. (one)

[0040] RNA was extracted (TRIZOL extraction method) and cDNA was synthesized from different groups of cynomolgus monkey primary hepatocytes and normal human hepatocytes using the kits provided by Guangzhou Ruibo Company. The obtained cDNA was diluted once and then used for qPCR amplification reaction using TransStart Top Green qPCR SuperMix (purchased from TAKARA). The qPCR amplification system was: 50 μg total RNA, 5 μL 10xDNase I Buffer, 4 μL RNase Inhibitor, 2 μL DNase I (RNase-free), and RNase Free dH2O was added to 50 μL.

[0041] The qPCR amplification reaction conditions were as follows: pre-deformation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. The above steps were repeated for 35 cycles, and a final extension was performed at 72°C for 5 min.

[0042] Using ACTIN as the internal reference, Table 1 shows the primer sequence information of ACTIN and lncRNA DC624545 used in the qPCR reaction:

[0043] Table 1 Primer sequence information

[0044]

[0045] Changes of blood glucose regulatory protein PPP1R3B and upstream gene DC624545 in primary hepatocytes of cynomolgus monkeys and human hepatocytes after administration of different treatment groups Figure 1 and Figure 2 As shown. Among them, the expression level of PPP1R3B in the Lipo8000-siRNA group was significantly higher than that in the control group, and the expression level in the 30pmol Lipo8000-siRNA group was relatively the best; the expression level of the upstream gene DC624545 in the Lipo8000-siRNA group was significantly lower than that in the control group, and the expression level in the 25pmol Lipo8000-siRNA group was relatively the lowest. (two)

[0047] Cynomolgus monkey primary hepatocytes were treated with 20 μg / mL 125 I-HDL or 125 I / 3 H-CE-HDL(125 I is ~600 cpm / ng, 3 H-CE is ~800 cpm / ng) in MEM containing 2 mg / mL faf-BSA for 1 hour at 37°C. A 40-fold excess of unlabeled high-density lipoprotein (HDL) was added for every four data points. The medium was recovered and the cell monolayer was washed twice with cold Tris HCl (pH=7.4), 0.9% NaCl and 0.2% BSA, and twice with BSA-free medium. The cells were lysed with 0.1 M NaOH. Use 125 I-HDL gamma counter or 125 I / 3 Radioactivity was measured with a beta counter of H-CE-HDL. Specific cell association was calculated by subtracting the amount of radioactivity detected in 40-fold excess from the total activity measured. Cellular protein was quantified using the Bradford method (HDL uptake was expressed as ng HDL per mg cellular protein). 3 H-CE-HDL uptake was subtracted 125 Selective cholesterol ester uptake was calculated by I-HDL uptake.

[0048] To distinguish HDL binding from uptake, cell surface-bound HDL was replaced with a 100-fold excess of unlabeled HDL in medium containing 2 mg / ml fafBSA and 10 mM Hepes at 4°C for 2 h.

[0049] For HDL degradation analysis, 125 After incubation with I-HDL, the culture medium was collected and protein was precipitated using 50% TCA. The supernatant was extracted with chloroform, oxidized with 30% hydrogen peroxide and counted to determine the amount of acid-soluble material formed by the cells.

[0050] The quantitative analysis results of HDL in primary hepatocytes of cynomolgus monkeys after administration of different treatment groups are shown in Figure 3 , it can be seen that the HDL content in the Lipo8000-siRNA group was significantly lower than that in the control group.

[0051] In summary, it can be seen that by using the technical solution of the present invention to take lncRNADC624545 as the target and adopting the siRNA silencing method and sequence, the expression of the lncRNA DC624545 sequence can be effectively inhibited to achieve the prediction and / or treatment of hyperlipidemia; it can also simultaneously meet the targeting of two different species, humans and crab-eating macaques, and has higher reliability in the subsequent drug development process.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A lncRNA DC624545, characterized in that The nucleotide sequence of the lncRNADC624545 is shown in SEQ ID NO.

1.

2. Use of the lncRNA DC624545 described in claim 1 as a target in screening, predicting and / or treating products for hyperlipidemia.

3. The use according to claim 2, characterized in that: Screening of substances that inhibit the expression of lncRNADC624545 sequence.

4. Use of a preparation for detecting the expression level of lncRNA DC624545 according to claim 1 in the preparation of a product for predicting hyperlipidemia.

5. A siRNA for inhibiting the expression of lncRNADC624545 sequence, characterized in that: The siRNA consists of a sense strand and an antisense strand. The nucleotide sequence of the sense strand is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

3.

6. A conjugate comprising the siRNA according to claim 5, characterized in that: The conjugate includes Galnac and the siRNA.

7. Use of the siRNA according to claim 5 or the siRNA conjugate according to claim 6 in the preparation of a drug for treating hyperlipidemia.

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