ShRNA interference library for inhibiting ETS2 expression and application thereof

Abstract

The invention discloses a shRNA interference library for inhibiting ETS2 expression and application thereof, and belongs to the technical field of gene drugs. The shRNA interference library for inhibiting ETS2 expression comprises one or more of a sequence shRNA1 as shown in SEQ ID NO. 1, a sequence shRNA2 as shown in SEQ ID NO. 2 and a sequence shRNA3 as shown in SEQ ID NO. 3. The expression of the ETS2 gene in the liver is specifically inhibited by adopting an shRNA interference mode, the liver injury induced by high fat diet can be obviously inhibited, and the liver lipid accumulation of a mouse is also obviously improved, so that the interference of the expression of the ETS2 in the liver can inhibit the lipid accumulation of the mouse and obviously alleviate the liver injury of a fatty liver disease patient.

Description

Technical Field

[0001] The invention belongs to the technical field of gene medicine, and specifically relates to a shRNA interference library for inhibiting ETS2 expression and an application thereof. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) refers to a liver metabolic disease in which lipids, mainly triglycerides, accumulate in hepatocytes as pathological changes, excluding those caused by long-term heavy drinking and other clear liver-damaging factors. In patients with NAFLD, the liver fat metabolism function is impaired, resulting in the accumulation of a large amount of fatty substances in hepatocytes (simple fatty liver), which in turn leads to fatty degeneration of hepatocytes, hepatocyte damage, inflammatory response, and liver fibrosis (non-alcoholic steatohepatitis, NASH). Simple fatty liver is a relatively benign stage of NAFLD and is easily reversed. 10% to 20% of simple fatty liver can progress to NASH. It is currently believed that NASH is an important link in the progression of NAFLD to end-stage liver diseases such as cirrhosis, hepatocellular carcinoma, and liver failure, and may become the primary reason for liver transplantation in the future. NAFLD not only affects the patient's hepatobiliary system, but is also closely related to insulin resistance, dyslipidemia, atherosclerosis, fat embolism, and blood system diseases. NAFLD is considered to be a liver manifestation of metabolic syndrome. NAFLD has become one of the most common liver diseases in clinical practice: epidemiological surveys show that the incidence of NAFLD in my country is about 15%, while the incidence of NAFLD in Europe and the United States is over 20%. Therefore, it is of great significance to explore effective treatments for NAFLD, and in-depth research on the pathogenesis of NAFLD is crucial to developing effective treatment strategies for NAFLD.

[0003] ETS2 is a transcription factor in the ETS family. It is mainly located in the cell nucleus and binds to the conserved DNA binding site (GGAA / T) in a sequence-specific manner to play a regulatory role. The functions of ETS2 are mainly related to the growth and development of stem cells, cell senescence and death, tumor cell development and angiogenesis. The transcription factor ETS2 usually activates the transcription of genes related to pathogen and tumor defense, and mainly exerts its effects through their control over the development of immune cells. In previous studies, ETS2 is often involved in the occurrence and development of tumor diseases. It is highly expressed in tumor diseases of almost all organs in the body, and has also been studied slightly in inflammatory diseases. In addition, many studies have also shown that ETS2 regulates cell damage in tissue damage and lesions. For example, ETS2 can promote the death of neurons in patients with Down syndrome through the mitochondrial apoptosis pathway, accelerate myocardial cell apoptosis and autophagy in the process of heart failure, and directly transcribe and activate the pro-apoptotic protein Bid to promote the death of vascular endothelial cells. ETS2 plays a key role in diabetic complications, tissue inflammation-mediated damage, and liver inflammation caused by macrophages. These studies have shown that abnormal expression and overactivation of ETS2 promote the occurrence of chronic diseases in multiple tissues. However, research on ETS2 as a therapeutic target for fatty liver is still blank. Summary of the invention

[0004] In view of the problems existing in the prior art, the object of the present invention is to provide a shRNA interference library for inhibiting ETS2 expression and its application.

[0005] In a first aspect of the present invention, a shRNA interference library for inhibiting ETS2 expression is provided, wherein the shRNA interference library comprises one or more of shRNA1 shown in SEQ ID NO.1, shRNA2 with a sequence shown in SEQ ID NO.2, and shRNA3 with a sequence shown in SEQ ID NO.3.

[0006] In some embodiments of the present invention, the shRNA interference library includes shRNA1 shown in SEQ ID NO.1, shRNA2 with a sequence shown in SEQ ID NO.2, and shRNA3 with a sequence shown in SEQ ID NO.3.

[0007] The second aspect of the present invention provides a use of the above-mentioned shRNA interference library in the preparation of a drug for inhibiting the expression of ETS2.

[0008] In some embodiments of the present invention, the drug is a drug that inhibits the expression of ETS2 in liver tissue.

[0009] The third aspect of the present invention provides a use of the above-mentioned shRNA interference library in the preparation of a drug for treating liver diseases.

[0010] In some embodiments of the present invention, the drug for treating liver disease is a drug that inhibits lipid accumulation in the liver.

[0011] In some embodiments of the present invention, the drug for treating liver disease is a drug for slowing down liver damage.

[0012] In some embodiments of the present invention, the drug is a drug for alleviating liver damage induced by a high-fat diet.

[0013] Compared with the prior art, the present invention has found through research that ETS2 is significantly highly expressed in the liver tissue of patients with non-alcoholic fatty liver disease, and its high expression is mainly stimulated by saturated fatty acids. Mouse experiments have confirmed that the use of shRNA interference to specifically inhibit the expression of the ETS2 gene in the liver can significantly inhibit liver damage induced by a high-fat diet, and the lipid accumulation in the liver of mice has also been significantly improved, thus proving that interfering with the expression of ETS2 in the liver can inhibit lipid accumulation in mice and significantly slow down liver damage in patients with fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] These and / or other aspects and advantages of the present invention will become apparent and readily understood from the following description of the preferred embodiments in conjunction with the accompanying drawings, in which:

[0015] Figure 1 This is the relative expression result of ETS2 in liver tissue of mice fed a normal diet and a high-fat diet;

[0016] Figure 2 The figure is the weight change results of mice fed with normal diet and high-fat diet (**, P<0.01);

[0017] Figure 3 This is a graph showing the difference in expression of Ets2 in control and palmitic acid (PA)-stimulated HepG2 cells;

[0018] Figure 4 This is the relative expression result of Ets2 in the liver of mice in the control shRNA group and the Ets2 interference library group;

[0019] Figure 5 This is a comparison chart of the changes in alanine aminotransferase (ALT) levels in the serum of mice in the control shRNA group and the Ets2 interference library group, as well as the relative fat accumulation levels in the liver of mice. DETAILED DESCRIPTION

[0020] In order to facilitate understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Some embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0022] Example 1

[0023] Twenty 4-week-old C57BL / 6J male mice were randomly divided into a normal diet group (NCD) of 10 mice and a high-fat diet group (HFD) of 10 mice.

[0024] Normal diet group (NCD): mice were given a normal standard feed, in which the proportion of various ingredients in calories were: 10% fat, 22% protein, and 68% carbohydrates.

[0025] High-fat diet group (HFD): The mice were given a high-fat diet in which the caloric ratios of various components were as follows: 49% fat, 21% protein, and 30% carbohydrates.

[0026] At the 12th week of feeding, the mice in the two groups were anesthetized and killed, and fresh liver tissues were obtained from the mice. The tissues were lysed with an appropriate amount of protein homogenate and immunoblot analysis was performed to detect the expression of ETS2 protein. GADPH was used as the internal reference protein. The results are shown in Figure 1 The left figure shows the results of Ets2 immunoblotting in the liver of mice fed a normal diet and a high-fat diet; the right figure shows the relative expression of Ets2 in the liver of mice fed a normal diet and a high-fat diet (**, P<0.01). The results showed that ETS2 was significantly overexpressed in the liver tissue of non-alcoholic fatty liver disease, and its high expression was mainly stimulated by saturated fatty acids.

[0027] A high-fat diet was used to simulate the process of diet-induced hepatic fatty lesions. The body weights of the two groups of mice were continuously monitored (body weight was recorded once a week). The body weight changes of the mice in the normal diet group (NCD) and the high-fat diet group (HFD) during 12 weeks of feeding were as follows: Figure 2 As shown (**, P<0.01). It can be found that since the fourth week of diet feeding, mice fed with a high-fat diet showed a significant increase in weight compared with mice fed a normal diet, suggesting that the mice had metabolic disorders and were potentially accompanied by abnormal liver metabolism.

[0028] Example 2

[0029] In the human liver cell line HepG2, cells were stimulated with the saturated fatty acid palmitic acid (PA) and the expression of ETS2 was analyzed. Figure 3 shown. Figure 3 The left figure shows the results of immunoblotting of Ets2 in control (HepG2 cells without PA stimulation) and HepG2 cells after PA stimulation; the right figure shows the results of quantitative analysis of the relative expression of Ets2 in control and HepG2 cells after PA stimulation (**, P<0.01). The results suggest that saturated fatty acids can induce significant expression of ETS2, which indicates that the expression of ETS2 is mainly regulated by high fat, indicating that it is involved in liver lipid accumulation and inflammatory response, thereby promoting the occurrence of liver damage.

[0030] Example 3

[0031] Preparation of ETS2 interference adenovirus.

[0032] The following three shRNAs were constructed into Ad-Easy adenovirus vectors, and mixed in equal amounts to make shRNA interference libraries and package adenoviruses. The adenovirus packaging vector was transfected into 293 cells with a confluency of 50-70%, and 6 μg of plasmid was transfected per 10 cm culture dish. The medium containing the complex was discarded 8 hours after transfection and replaced with new medium. 7-10 days after transfection, the cells were scraped from the culture dish with a cell scraper and transferred to a 50 ml centrifuge tube. After centrifugation, the supernatant was discarded and the cells were resuspended in 2 ml of PBS solution. The cells were quickly moved in liquid nitrogen, then dissolved in a 37°C water bath and shaken vigorously. Repeat this step 4 times. The above virus-containing supernatant was added to 50-70% of the 293 cells, and the volume of the virus supernatant was 30-50% of the total culture medium volume. After 3-5 days of infection, the virus supernatant was collected repeatedly. Repeat this virus infection-virus collection 3 times to increase the virus titer to 5*107 / ml each time.

[0033] The target sequences of the three shRNAs are as follows:

[0034] shRNA1: 5'-TTTAACAGAAATTGTATATTG-3',

[0035] shRNA2: 5'-ACAAGAACATCATCCACAAGA-3',

[0036] shRNA3: 5'-GATGAACTACGAGAAGCTGAG-3';

[0037] Example 4

[0038] Preparation of control shRNA adenovirus

[0039] Control shRNA sequence: 5'-GAATCCCTTAACAGTTGTATT-3'.

[0040] The control shRNA was constructed into the Ad-Easy adenovirus vector and packaged into adenovirus to obtain the control shRNA adenovirus.

[0041] Example 5

[0042] The expression of ETS2 gene in the liver was specifically inhibited by shRNA interference.

[0043] After the mice were fed a high-fat diet (49% fat, 21% protein, 30% carbohydrate) for 4 weeks, they were randomly divided into two groups: (1) control shRNA group: mice were injected with control shRNA adenovirus (1*10 8 PFU / mouse); (2) ETS2 interference library group: mice were injected with ETS2 interference adenovirus (1*10 8 PFU / mouse). After the injection, the mice were fed with high-fat diet for 8 weeks. The mice were anesthetized according to the anesthetic dose calculated according to their weight. 100 mg of mouse liver tissue was taken and quickly added with 1 ml of pre-cooled tissue homogenate. The tissue was homogenized for 30 seconds using an electric mechanical homogenizer. After the homogenization, the cells were centrifuged at 13000 rpm and 4°C for 10 minutes. The supernatant was collected for BCA protein quantitative analysis. 20 μg of total protein from the control and ETS2 interference library treatments were loaded onto 10% SDS-PAGE for separation. The expression of ETS2 was verified by immunoblotting. The results are shown in Figure 4 As shown, the left figure shows the relative expression of Ets2 in the liver of mice in the control shRNA group and the Ets2 interference library group; the right figure shows the relative expression of Ets2 in the liver of mice in the control shRNA group and the Ets2 interference library group (**, P<0.01). It can be seen that the infection of mice with the ETS2 shRNA adenovirus interference library can significantly inhibit the expression of ETS2 in mouse liver tissue, and the interference efficiency is about 95%, which is significantly better than the interference efficiency of conventional single shRNA (about 30%), and has the potential to significantly inhibit the molecular pathological changes of hepatocytes mediated by EST2.

[0044] The mice in the control shRNA group and the ETS2 interference library group were anesthetized and one eyeball was fully exposed for blood collection. After the whole blood was collected, it was centrifuged at 3000 rpm at 4°C for 15 minutes, and the supernatant was aspirated for transaminase and triglyceride determination. The results are as follows Figure 5 The left figure shows the changes in serum alanine aminotransferase (ALT) levels in mice in the control shRNA group and the Ets2 interference library group; the right figure shows the relative fat accumulation levels in the liver of mice in the control shRNA group and the Ets2 interference library group (**, P<0.01). Figure 5These results suggest that ETS2 disruption can significantly inhibit lipid accumulation and liver damage induced by a high-fat diet.

[0045] The specific ETS2 interference library according to the embodiment of the present invention has at least one of the following advantages:

[0046] 1. Specifically inhibit the upregulation of ETS2 expression induced by a high-fat diet, and promote its expression to return to normal levels;

[0047] 2. Specifically antagonize the induction of hepatocyte damage by up-regulation of ETS2 expression and inhibit the level of ALT, a marker related to liver damage;

[0048] 3. Alleviate the metabolic pressure of the liver by inhibiting the expression of ETS2, reduce lipid accumulation in hepatocytes, and inhibit fatty lesions and damage process of the liver.

Claims

1. A shRNA interference library for inhibiting ETS2 expression, characterized in that: The shRNA interference library includes one or more of shRNA1 with a sequence as shown in SEQ ID NO.1, shRNA2 with a sequence as shown in SEQ ID NO.2, and shRNA3 with a sequence as shown in SEQ ID NO.

3.

2. The shRNA interference library according to claim 1, characterized in that The shRNA interference library includes shRNA1 shown in SEQ ID NO.1, shRNA2 with a sequence shown in SEQ ID NO.2, and shRNA3 with a sequence shown in SEQ ID NO.

3.

3. Use of the shRNA interference library as described in claim 1 or 2 in the preparation of a drug for inhibiting ETS2 expression.

4. The use according to claim 3, characterized in that The drug is a drug that inhibits the expression of ETS2 in liver tissue.

5. Use of the shRNA interference library as claimed in claim 1 or 2 in the preparation of drugs for treating liver diseases.

6. The use according to claim 5, characterized in that The drug for treating liver disease is a drug for inhibiting the accumulation of lipids in the liver.

7. The use according to claim 5, characterized in that The drug for treating liver disease is a drug for slowing down liver damage.

8. The use according to claim 7, characterized in that The drug is a drug for slowing down liver damage induced by a high-fat diet.

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