A lna leading targeting lncrna inhibitor and application thereof in preparation of hypoglycemic drugs

Abstract

The application discloses a LNA leading targeted LncRNA inhibitor and application thereof in preparation of a hypoglycemic drug, and the nucleotide sequence of the inhibitor is 5'-TGGATAAATGAGCTGT-3'. The LNA leading targeted LncRNA inhibitor can significantly improve the diabetic symptoms by relieving high-fat diet induced obesity, and has the advantages of high stability, good specificity and low immunogenicity as compared with other inhibitors. The raw materials used in the application are scientific and reasonable, the preparation process is simple, the cost is low, and the auxiliary treatment effect is good.

Description

(I) TECHNICAL FIELD

[0001] The present application relates to a LNA leading target LncRNA inhibitor and its application in the preparation of hypoglycemic drugs. (II) BACKGROUND

[0002] In the 21st century, the incidence of type 2 diabetes (T2D) is increasing, and has become a global public health problem. Previously, T2D mainly prevailed in affluent "Western" countries; however, at present, T2D occurs worldwide. According to the latest report of the International Diabetes Federation Atlas of Diabetes, the overall prevalence of adult diabetes is 9.1%, which means that there are 415 million adults worldwide with diabetes. In addition, 318 million adults have impaired glucose regulation, and are at high risk of developing diabetes in the future. It is worth noting that China currently ranks first in the number of diabetic patients.

[0003] Diabetes and related complications, including cardiovascular disease, neuropathy, nephropathy, eye problems, foot disease, and malignancies, adversely affect the quality of human life. Death caused by diabetes and its complications accounts for a large part of non-communicable diseases worldwide, and the high medical costs for treating diabetes and its related complications are a heavy burden on society and individuals.

[0004] Environmental factors, including obesity, aging, diet and physical activity; genetic factors; and epigenetic changes, are all reasons for the accelerated prevalence of diabetes. In the past few decades, the dietary and nutritional patterns of Chinese people have changed rapidly and significantly. The traditional plant-based diet (mainly coarse grains and vegetables) has gradually changed to a Western diet (mainly animal sources, fine grains, high processing, high sugar, high fat foods). Epidemiological studies have shown that a Western diet is one of the main risk factors for inducing diabetes and metabolic syndrome.

[0005] Long non-coding RNAs (LncRNAs) are widely involved in metabolism, immunity and other biological processes. They play an important regulatory role and are a bridge between various physiological and biochemical reactions. The imbalance of lncRNAs in mice or humans can cause metabolic syndrome. It has been reported that some lncRNAs are related to food intake involved in metabolic syndrome.

[0006] Therefore, in-depth exploration of lncRNAs with important potential applications, and evaluation of the application potential of lncRNAs in the clinical diagnosis and treatment of obesity and diabetes, has important clinical guiding significance for intervening in diabetes, thereby reducing the huge burden of diabetes on human health. (III)SUMMARY

[0007] The application aims to provide a LNA leading targeted LncRNA inhibitor and its application in the preparation of a hypoglycemic drug, which can significantly reduce the increase in blood glucose and insulin resistance caused by obesity.

[0008] The technical scheme adopted by the application is:

[0009] The application provides a LNA leading targeted LncRNA inhibitor, and the nucleotide sequence of the inhibitor is 5'-TGGATAAATGAGCTGT-3'(SEQ ID NO.1).

[0010] Further, the inhibitor targets and inhibits the expression of LncRNALINC01139 (denoted as LINK-A), and the nucleotide sequence of the LncRNALINC01139 (LINK-A, NCBI Reference Sequence: NR_015407.1) is shown in SEQ ID NO.2.

[0011] SEQ ID NO.2:

[0012]

[0013] The application also provides a LNA leading targeted LncRNA inhibitor in the preparation of a hypoglycemic drug, and the drug is a nucleotide sequence shown in SEQ ID NO.1 modified by LNA, and is synthesized by a company.

[0014] Further, the drug is an auxiliary treatment drug for obesity.

[0015] Further, the drug is a drug for treating diabetes.

[0016] Further, the drug further comprises a pharmaceutically acceptable carrier, and the carrier is one or more of a lipid nanoparticle, a viral vector, an exosome or an extracellular vesicle, a polymer nanoparticle, an inorganic nanocarrier, and a virus-like nanocarrier.

[0017] The drug is administered by injection, and the injection administration mode includes intramuscular injection, intravenous injection and subcutaneous injection, and the injection dose is 5mg / kg, and the injection is performed every other day.

[0018] High-fat diet-induced obesity is one of the important causes of diabetes. LncRNA LINC01139 (LINK-A) is often up-regulated in high-risk diseases of obesity, and is highly expressed in overweight women's breasts and positively correlated with BMI. The present application finds through experiments that LncRNA LINC01139 (LINK-A) can promote obesity, blood glucose increase and insulin resistance of mice under high-fat diet, and that injection of an LNA-lead targeted LncRNA inhibitor can significantly inhibit blood glucose increase and insulin resistance of mice under high-fat diet. The LNA-lead targeted LncRNA inhibitor provided by the present application can inhibit expression of LncRNA LINC01139 (LINK-A), and significantly improve symptoms of diabetes by relieving high-fat diet-induced obesity.

[0019] Compared with the prior art, the present application has the beneficial effects mainly embodied in that the LNA-lead targeted LncRNA inhibitor can significantly improve symptoms of diabetes by relieving high-fat diet-induced obesity, and has the advantages of strong stability, good specificity and low immunogenicity relative to other inhibitors. The raw materials used in the present application are scientific and reasonable, the preparation process is simple, the cost is low, and the auxiliary treatment effect is good. (IV) DESCRIPTION OF DRAWINGS

[0020] Figure 1 Effects of overexpression of LncRNA LINC01139 (LINK-A) on high-fat diet-induced obesity and fasting blood glucose level and insulin resistance of mice; A represents photos of weight change of mice in each group; B represents photos of subcutaneous tissue of abdomen of mice in each group; C represents a column chart of weight change of mice in each group; D represents fasting blood glucose level of mice in each group; and E represents a curve of insulin resistance level of mice in each group.

[0021] Figure 2 Effects of the LNA-lead targeted LncRNA inhibitor LINK-A LNA on weight (A), fasting blood glucose (B) and insulin resistance level (C) of mice. (V) DETAILED DESCRIPTION

[0022] The present application will be further described below in combination with specific embodiments, but the protection scope of the present application is not limited to this:

[0023] The wild type mouse strain used in the following examples is C57BL / 6; the transgenic mouse is a transgenic mouse constructed by using CRISPR / Cas9 technology to knock in the LncRNALINC01139(LINK-A) gene (NCBI Reference Sequence: NR_015407.1) into the wild type mouse ROSA26 site in the C57BL / 6 background. The amplification enzyme and reverse transcriptase used are purchased from Nanjing Novogene Biotech Co., Ltd. The reagents used are all analytical grade reagents. The fat content in the high-fat feed is 60%, purchased from Research Diets Company, USA.

[0024] Example 1, construction of a transgenic mouse obesity model overexpressing LncRNALINC01139(LINK-A)

[0025] The wild type female mice and the transgenic female mice with LncRNALINC01139(LINK-A) gene knock-in were normally raised until the 8th week, and then divided into four groups (ND-WT, ND-KI, HFD-WT, HFD-KI), 6 in each group. The ND-WT and ND-KI groups were given normal feed, and the HFD-WT and HFD-KI groups were given high-fat feed. After 12 weeks, the mice were photographed and weighed, and the abdominal subcutaneous tissue was taken for photography. It can be observed that the transgenic mice with LINK-A knock-in show the most obvious obesity under high-fat feeding (Fig. 1A), weight gain (Fig. 1B), and increase in size and weight of abdominal subcutaneous tissue (Fig. 1C). Figure 1 Figure 1 Figure 1

[0026] Example 2, detection of fasting blood glucose level in mice

[0027] 1. Preparation of mice: 6 mice from each group after 12 weeks of culture according to the method of Example 1 were changed into clean cages for fasting at 5 pm the day before the experiment. The mice were kept without food for 16 hours until 9 am the next day, and they were allowed to drink water normally during the fasting period. At 9 am the next morning, the glucose tolerance test began.

[0028] 2. Measurement of fasting blood glucose: the mice were taken out of the cages and gently placed on the iron mesh. The tail ends of the mice were cut off 1-2 mm with scissors, and the mouse tail was gently squeezed to make the blood accumulate into a drop. The fasting blood glucose was measured with a blood glucose meter, and the measured value was taken as the fasting blood glucose of the mouse. The operation was as gentle as possible to avoid excessive fright of the mouse.

[0029] 3. After the experiment, the mice in each cage were supplemented with the same feed (high-fat feed or normal feed) according to the grouping of Example 1.

[0030] ​​​Glucose tolerance test confirmed that high-fat diet induced the increase of fasting blood glucose in LINK-A knock-in transgenic mice Figure 1 Middle D).

[0031] Example 3, Insulin resistance test in mice

[0032] 1, Preparation of mice: 6 mice from each group were selected after 12 weeks of culture according to the method of Example 1. On the day of the experiment, the mice were changed into clean cages at 9 am and fasted for 6 hours until 3 pm. During the fasting period, the mice were allowed to drink water normally.

[0033] 2, At 3 pm, the insulin tolerance test began. The mice were taken out of the cages and gently placed on the iron grid. The end of the mouse tail was cut off about 0.5-1 mm with scissors, and the mouse tail was gently squeezed to allow the blood to accumulate into a drop. The blood glucose was measured with a blood glucose meter, and the measured value was defined as the blood glucose value at 0 minutes. The operation was as gentle as possible to avoid excessive fright of the mice.

[0034] 3, After 10 minutes of adaptation, the preparation of intraperitoneal injection of insulin solution began. The amount of insulin used in the insulin tolerance test in mice was generally 0.5-1.2 U / kg, and the insulin was diluted to the appropriate concentration according to the specific experimental requirements. If the amount of insulin used is 0.5 U / kg, then 0.05 U / ml of insulin solution is prepared with normal saline.

[0035] 4, The mice were gently picked up and injected with insulin solution using a 1 ml syringe according to the standard intraperitoneal injection operation. The volume of injection was determined according to the body weight of the mouse, with 0.01 ml (0.05 U / ml) per gram of body weight. The timing started from the completion of injection. Generally, the operation interval of each mouse was 1 minute, so that the blood glucose measurement of each mouse could be accurately completed according to the specified time.

[0036] 5, At 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 90 minutes after the timing started in step 4, the blood glucose values at each time point were measured according to the operation of step 2, and the blood glucose values at different time points were compared with the initial blood glucose value (0 min) to obtain the blood glucose change rate. This ratio can indicate the insulin resistance of the mice through the sensitivity of the mice to insulin.

[0037] 6, After the experiment, the mice in each cage were supplemented with the same feed as before (high-fat feed or normal feed) according to Example 1.

[0038] Insulin resistance test confirmed that high-fat diet induced the decrease of insulin resistance in LINK-A knock-in transgenic mice Figure 1 Middle E).

[0039] Example 4: LINK-A, an LNA lead targeting LncRNA inhibitor, suppresses fasting blood glucose and mouse weight and increases mouse insulin resistance level.

[0040] This example takes LINK-A gene knock-in transgenic mice fed with high-fat feed for 12 weeks in Example 1 as experimental objects, and divides them into two groups, each group of 6. One group is the control group, and the drug Scr LNAs injection is injected; the other group is the experimental group, and the drug LINK-A LNAs injection is injected.

[0041] LINK-A LNAs injection is an inhibitor of the nucleotide sequence shown in SEQ ID NO. 1 modified by LNA, entrusted to Ribo Company for synthesis, and made into an injection with water as the solvent.

[0042] Scr LNAs injection is a non-inhibitor of the nucleotide sequence shown in SEQ ID NO. 3 modified by LNA, entrusted to Ribo Company for synthesis, and made into an injection with water as the solvent. SEQ ID NO. 3: AACACGTCTATACGC.

[0043] 1, Mouse preparation: the number of experimental mice in each group is 6, and each mouse is weighed first.

[0044] 2, Injection administration: gently pick up the mouse, and operate a 1 mL syringe to inject the drug at the fat pad site. The control group is administered with Scr LNAs injection; the experimental group is administered with LINK-A LNAs injection. Both groups are administered at a dose of 5 mg / kg.

[0045] 3, gently put the mouse back into the cage, and supplement the mouse with mouse feed (high-fat) and water.

[0046] 4, administer again after every other day, and repeat steps 1, 2, and 3. A total of 14 days, 7 times of administration.

[0047] After the administration is completed, the mice in each group are weighed, the fasting blood glucose is measured by the method of Example 2, and the blood glucose values of the mice at different time points are tested by the method of Example 2, and the results are shown in Figure 2 .

[0048] Figure 2 Figure A shows the change in mouse weight after injection of the LNA lead targeting LncRNA inhibitor and the control drug, and the results show that LNA lead targeting LncRNA inhibitor treatment can significantly inhibit the increase in mouse weight.

[0049] Figure 2 Figure B shows the level of fasting blood glucose in mice after injection of the LNA lead targeting LncRNA inhibitor and the control drug, and the results show that LNA lead targeting LncRNA inhibitor treatment can significantly inhibit hyperglycemia in mice.

[0050] Figure 2 The blood glucose values of mice after injection of the LNA leading inhibitor targeting LncRNA and the control drug at different time points are statistically analyzed, and the results show that the LNA leading inhibitor targeting LncRNA can significantly enhance the insulin resistance of mice.

[0051] The LNA leading inhibitor targeting LncRNA can significantly improve the symptoms of diabetes by alleviating high-fat diet-induced obesity, and has the advantages of strong stability, good specificity and low immunogenicity compared with other inhibitors.

[0052] The above examples are used to understand the method and main idea of the present application. It should be noted that those skilled in the art can make some improvements to the present application without departing from the principles of the present application, and these improvements are also within the protection scope of the claims of the present application.

Claims

1. The application of an LNA-modified inhibitor targeting LncRNA in the preparation of hypoglycemic drugs, characterized in that, The nucleotide sequence of the inhibitor is 5'-TGGATAAATGAGCTGT-3'; the inhibitor targets and inhibits the expression of LncRNALINC01139, and the nucleotide sequence of LncRNA LINC01139 is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, The drug is used as an adjunct treatment for obesity.

3. The application as described in claim 1, characterized in that, The drug in question is a medication for treating diabetes.

4. The application as described in claim 1, characterized in that, The drug includes a pharmaceutically usable carrier, which is one or more of lipid nanoparticles, viral vectors, exosomes, polymer nanoparticles, and inorganic nanocarriers.

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