WO2021248408A1

WO2021248408A1 - APPLICATION OF CIRCULAR RNA TO REGULATION AND CONTROL OF PANCREATIC ISLET β CELL PROLIFERATION

Info

Publication number: WO2021248408A1
Application number: PCT/CN2020/095609
Authority: WO
Inventors: 贾世奇; 庄景燊; 陶卫华; 邓艳瑞
Original assignee: 暨南大学
Priority date: 2020-06-09
Filing date: 2020-06-11
Publication date: 2021-12-16
Also published as: CN111718891B; CN111718891A

Abstract

An application of circular RNA to regulation and control of pancreatic islet β cell proliferation. The nucleotide sequence of the circular RNA is as shown in SEQ.ID.NO. 1 or SEQ.ID.NO. 2. Overexpression of the circular RNA can effectively promote proliferation of mouse pancreatic islet β cells, and knockout of the circular RNA can effectively inhibit proliferation of the mouse pancreatic islet β cells, so that the circular RNA can be used for preparing drugs for treating diabetes or pancreatic islet β cytosis. A series of experiments prove that the circular RNA can effectively promote proliferation of pancreatic islet β cells, and essentially proves that the circular RNA has a wide application background in treatment of insufficiency in insulin secretion caused by reduction of the number of pancreatic islet β cells. The present invention can provide a good candidate drug for clinical treatment of diabetes caused by reduction of the number of islet β cells, and has a very good application prospect.

Description

Application of a circular RNA in regulating the proliferation of pancreatic β-cells

Technical field

The invention belongs to the field of biomedicine, and specifically relates to an application of circular RNA in regulating the proliferation of pancreatic β-cells.

Background technique

Diabetes (diabetes mellitus, DM) is a metabolic disease that seriously threatens human health. Its onset is due to absolute or relative lack of insulin, which causes increased blood sugar and leads to various tissues (especially eyes, kidneys, heart, blood vessels, nerves) Chronic damage and dysfunction.

Diabetes is mainly divided into type 1 and type 2 diabetes. The pathogenesis of type 1 diabetes is mainly the destruction of pancreatic islet β cells caused by various reasons, resulting in absolute deficiency or significant reduction of insulin. Type 2 diabetes is the most common in my country, and its causes include insulin resistance, insufficient insulin secretion, or both. Whether it is type 1 or the more common type 2 diabetes, insufficient number of islet β cells, dedifferentiation or dysfunction, leading to glucose metabolism from compensatory to decompensated state, and ultimately have to rely on oral hypoglycemic drugs or the use of exogenous Insulin replacement therapy.

At present, most of the drugs only promote insulin secretion or lower blood sugar concentration, play a role in delaying the process of diabetes, and have little effect on improving and protecting pancreatic β cells, and long-term use of various drugs is caused by drug resistance and adverse reactions. The impact cannot be underestimated. At present, there are no drugs that promote the proliferation of pancreatic β-cells on the market. Therefore, the discovery of drugs that promote the proliferation of pancreatic β-cells has important biological research significance and practical significance, and can provide new drug candidates for the treatment of diabetes.

On the other hand, diseases such as hyperinsulinemia and insulinoma are manifested by the abnormal proliferation of pancreatic β-cells and excessive insulin secretion, which can lead to clinical symptoms of hypoglycemia and cause severe metabolic disorders. In severe cases, it can even be life-threatening. Surgical removal of part of the pancreatic tissue is a common clinical treatment method, and there is no specific medicine for this type of disease. Therefore, the development of candidate drug molecules that specifically inhibit the proliferation of pancreatic β-cells has important biological research significance and clinical practice significance.

Summary of the invention

The primary purpose of the present invention is to provide an application of circular RNA in regulating the proliferation of pancreatic β-cells.

Another object of the present invention is to provide a plasmid that overexpresses the above circular RNA and its use.

Another object of the present invention is to provide an siRNA that inhibits the expression of the above-mentioned circular RNA.

The purpose of the present invention is achieved through the following technical solutions:

Application of a circular RNA in regulating the proliferation of pancreatic β-cells;

The nucleotide sequence of the circular RNA is as shown in SEQ.ID.NO.1 (hsa_circ_0000885) or SEQ.ID.NO.2 (mmu_circ_0014773), wherein SEQ.ID.NO.1 encodes human cyclic RNA, SEQ.ID.NO.2 encodes murine circular RNA, both have a sequence length of 552bp, and the sequence is highly conservative, with only a few base differences, and the sequence at the protein level is exactly the same;

The pancreatic islet β cell is mouse pancreatic islet β cell SJb;

The experiment in this application confirms that overexpression of the above-mentioned circular RNA can effectively promote the proliferation of mouse pancreatic islet β-cells, and knocking out the above-mentioned circular RNA can effectively inhibit mouse pancreatic islet β-cells, so the above-mentioned circular RNA can be used for preparation of therapeutics Diabetes medication.

A plasmid overexpressing circular RNA, containing the nucleotide sequence shown in SEQ.ID.NO.1 or SEQ.ID.NO.2;

The vector of the plasmid is pCDH, pCD5-ciR, pCD25-ciR, pLO5-ciR, pLC5-ciR, pK5ssAAV-ciR or pK25ssAAV-ciR.

After transfecting pancreatic β cells with the plasmid overexpressing circular RNA, it can increase the survival rate of pancreatic β cells and promote the proliferation of pancreatic β cells, and can be used to prepare drugs for the treatment of diabetes.

A siRNA whose sense strand sequence is shown in SEQ.ID.NO.7 and antisense strand sequence is shown in SEQ.ID.NO.8; the siRNA shown is designed for the above-mentioned circular RNA and can inhibit the above-mentioned loop The expression of shape RNA in cells plays a role in regulating the proliferation of pancreatic β-cells.

A plasmid containing the above-mentioned siRNA, and its vector is pSGLV3/H1/GFP+puro, pYr-1.1-hU6-EGFP, pRI or pLKO-1. The plasmid containing the above-mentioned siRNA can be used to regulate the proliferation of pancreatic β-cells, in particular, for the preparation of drugs for the treatment of hyperinsulinemia, insulinoma and other pancreatic β-cell hyperplasia.

Compared with the prior art, the present invention has the following advantages and effects:

The present invention has confirmed through a series of experiments that circular RNA (SEQ.ID.NO.1 and SEQ.ID.NO.2) can effectively promote the proliferation of pancreatic β-cells, and essentially proves that the circular RNA is effective in treating pancreatic islets. Insufficient insulin secretion caused by the decrease in the number of β cells has a wide range of application backgrounds. The invention can provide a good candidate drug for the clinical treatment of diabetes caused by the decrease in the number of islet β cells, and has very good application prospects.

Description of the drawings

Figure 1 is a sequence diagram of the head-to-tail interface sequence at the loop of mmu_circ_0014773.

Figure 2 shows the effect of overexpression of mmu_circ_0014773 on the survival rate of pancreatic β-cells.

Figure 3 shows the effect of knocking out mmu_circ_0014773 on the survival rate of pancreatic β-cells.

Figure 4 shows the effect of overexpression of mmu_circ_0014773 on the proliferation of pancreatic β-cells.

Figure 5 shows the effect of knocking out mmu_circ_0014773 on the proliferation of pancreatic β-cells.

detailed description

Hereinafter, the present invention will be further described in detail with reference to the examples and drawings, but the implementation of the present invention is not limited thereto.

Example 1

In this example, mouse pancreatic islet β-cell SJb is used as a model for illustration, so circRNA from mouse, namely circ_mmu_circ_0014773 (SEQ.ID.NO.2), is selected.

First, it was verified that circ_mmu_circ_0014773 has an endogenously expressed circular RNA sequence in SJb pancreatic islet β cells. PCR amplification was carried out by designing specific primers for the amplified circular RNA. The amplified products were sequenced by Sanger, and the circular sequence formed by the first link of the linear sequence was obtained. The arrow in Figure 1 shows the last base and the first of the linear sequence. A new sequence of bases combined (looping interface sequence).

Overexpression of circ_mmu_circ_0014773 can effectively promote the proliferation of mouse pancreatic β-cell SJb, and knocking out endogenous circ mmu_circ_0014773 can effectively inhibit the proliferation of mouse pancreatic β-cell SJb. The circ mmu_circ_0014773 overexpression and knock-out vectors are constructed separately, which can be further developed for treatment Drugs for diabetes or hyperinsulinemia have broad application prospects.

Use the following experiment to verify:

(1) Construction and identification of pCDH-circ mmu_circ_0014773 plasmid

The pCDH_circ mmu_circ_0014773 vector was constructed using the Novavizin Seamless Cloning Kit (ref: C112), and the specific steps are as follows:

1) Preparation of linearized vector

The vector plasmid pCDH was digested with EcoRI and BamHI, and the target fragment was recovered by gelatinization.

The digestion system is shown in Table 1:

Table 1 Vector plasmid pCDH double restriction digestion system

Digestion at 37°C for 4h. After agarose gel electrophoresis, the linearized plasmid pCDH was recovered and verified by the gel.

2) Insert fragments are obtained

The general principle of primer design: Introduce homologous sequences at both ends of the linearized vector at the 5'end of the insert's forward and reverse amplification primers, so that the 5'and 3'ends of the amplified insert have and linearized cloning vector respectively The two ends correspond to identical homologous sequences (15-20bp, excluding restriction sites).

The final amplified insert primers used are as follows:

Table 2 Primers used

Use Takara high-fidelity enzyme to amplify the insert, the amplification system is as follows:

Table 3 Insert amplification system

The amplified product was identified, determined and purified for concentration and used in subsequent experiments.

3) Recombination reaction

Prepare the following reaction system on ice:

Table 4 Reaction System

Connect overnight at 16°C. The ligation product was introduced into DH5α competent cells, spread on a selection medium plate containing ampicillin, and a single colony was picked. Send sequencing to identify the correctly constructed pCDH-circ mmu_circ_0014773 plasmid.

(2) Construction and identification of sh circ mmu_circ_0014773 vector

The LV-shRNA was constructed based on the previously verified circ mmu_circ_0014773 siRNA, and this part was commissioned by Shenggong Bioengineering (Shanghai) Co., Ltd. to complete. The vector used is pSGLV3/H1/GFP+puro, and the circ mmu_circ_0014773 siRNA sequence used is as follows:

circ mmu_circ_0014773 siRNA sequence: 5’AGAAAGUGUGCCCUGGUAUtt 3’ (SEQ.ID.NO.7)

circ mmu_circ_0014773 siRNA antisence: 5’AUACCAGGGCACACUUUCUga 3’ (SEQ.ID.NO.8)

(3) Construction and identification of stable strains with overexpression and knockout of circ mmu_circ_0014773

Inoculate pancreatic β-cell SJb in advance, use trypsin containing 2.5% EDTA to gently pipette into single cells, and then resuspend the cells after centrifugation at 200g for 5 minutes. Count the cells. Take 4×10 ⁶ pancreatic islet β cells SJb from each transfection group, centrifuge at 200g for 5 minutes and remove the supernatant as much as possible. Add 100 μl electroporation solution + 5 μg plasmid to each well of cells, mix gently and quickly, and transfer to a spot rotor cup. Choose appropriate The electric transfer procedure is carried out. After the electroporation is completed, add 500μl of RPMI 1640 medium containing 10% FBS, transfer to a 1.5EP tube, incubate at 37°C for 15min, and transfer to a 75cm ² cell culture flask for normal culture. After 24 hours of electroporation, change the selection medium and observe the expression of GFP under a fluoroscope. Once the cells basically express GFP fluorescence, they are stably transfected cells. Follow-up QPCR to verify the transfection efficiency.

Example 2

Overexpression of circ mmu_circ_0014773 can effectively promote the proliferation of mouse pancreatic islet β-cell SJb, knocking out mmu_circ_0014773 can effectively inhibit the proliferation of mouse pancreatic β-cell SJb, and constructing circ_mmu_circ_0014773 overexpression and knock-out mouse pancreatic β-cell SJb stable expression strains respectively, verifying The effect of mmu_circ_0014773 mouse pancreatic β-cell SJb proliferation can be further developed as a drug for the treatment of diabetes, which has a wide range of application prospects.

Use the following experiment to verify:

1) The effect of overexpression of mmu_circ_0014773 on the survival rate of pancreatic β-cells

Take the above constructed overexpression cyclic mmu_circ_0014773 (circ_mmu_circ_0014773), overexpression linear mmu_circ_0014773 (Linear_mmu_circ_0014773), the control plasmid group (PCDH) cells were seeded in 96-well plates, each well containing 100μl culture medium, 10 ⁴ cell suspension , Pre-culture for 24h in a cell incubator. On the next day (this is counted as day 0), add a complete medium containing 10 μl of CCK-8 solution to each well (be careful not to generate bubbles and mix gently). After the culture plate was incubated in the incubator for 2 hours, the absorbance value at 450 nm was measured by the microplate reader. After the determination, the normal medium was replaced. On the second and sixth days, the survival rate of the cells at this time was determined according to the above method.

According to the results in Figure 2, it can be seen that on the second day, the cell proliferation of the circ_mmu_circ_0014773 group and the Linear_mmu_circ_0014773 group increased significantly. On the sixth day, the circ_mmu_circ_0014773 group could still maintain a higher growth rate.

2) The effect of knocking out circ_mmu_circ_0014773 on the survival rate of pancreatic β-cells

Take the above-described knockout construct cyclic circ_mmu_circ_0014773, control plasmid (sh NC) cells were seeded in 96-well plates, each well containing 100μl culture medium, 10 ⁴ cell suspension, in a cell culture incubator pre 24h. The remaining steps are the same as above.

According to the results in Figure 3, it can be seen that on the 6th day, the growth rate of the cells in the sh_circ_mmu_circ_0014773 group was significantly lower than that in the control group.

3) The effect of overexpression of circ_mmu_circ_0014773 on promoting the proliferation of pancreatic β-cells

Take the above constructed overexpression circ_mmu_circ_0014773, overexpression linear Linear_mmu_circ_0014773, control plasmid (PCDH) of each cell ^106, 200 g of 5min centrifugation the supernatant was discarded, chilled 4% PFA fixed 15min, washed three times with pre-cooled PBS. Discard the supernatant, add 10 μl of PBX (PBS + 1% Tween-20), and incubate at room temperature for 20 min. Wash three times with pre-cooled PBS. Prepare 1ml Ki67 dye solution (2μl anti-Ki67+PBS+0.02%BSA+5mM EDTA) at a ratio of 1:500 and incubate at room temperature for 40min. Prepare the corresponding fluorescent secondary antibody at a ratio of 1:500. After incubating at room temperature for 40 minutes, pre-cooled PBS washed three times. Flow cytometry detection.

According to the results in Figure 4, it can be seen that Ki67 positive cells in the circ_mmu_circ_0014773 group were significantly more than those in the control group, suggesting that overexpression of circ_mmu_circ_0014773 can significantly promote the proliferation of mouse pancreatic β-cell SJb.

4) The effect of knocking out circ_mmu_circ_0014773 to inhibit the proliferation of pancreatic β-cells

Take the above-described knockout construct cyclic circ_mmu_circ_0014773, control plasmid (sh NC) of each cell ^106, step I above. Flow cytometry detection.

According to the results in Figure 5, it can be seen that the Ki67 positive cells in the sh_circ_mmu_circ_0014773 group were significantly less than those in the control group. It is suggested that knocking out circ_mmu_circ_0014773 can inhibit the proliferation of mouse pancreatic β-cell SJb.

To sum up: in the process of promoting in vitro, overexpression of circ_mmu_circ_0014773 can significantly promote the proliferation of β-cell SJb, on the contrary, knocking out circ_mmu_circ_0014773 can inhibit the proliferation of mouse pancreatic β-cell SJb. According to the experimental results, it can be seen that the molecular sequence of circ_mmu_circ_0014773 has great potential, potential and application prospects as a regulatory β cell.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, etc. made without departing from the spirit and principle of the present invention Simplified, all should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims

An application of circular RNA in regulating the proliferation of pancreatic β-cells, characterized in that: the nucleotide sequence of the circular RNA is shown in SEQ.ID.NO.1 or SEQ.ID.NO.2.
The application according to claim 1, wherein the pancreatic islet β cell is a mouse pancreatic islet β cell SJb.
The application according to claim 1, characterized in that: the application of the circular RNA in the preparation of medicines for the treatment of diabetes.
A plasmid for overexpressing circular RNA, which is characterized in that it contains the nucleotide sequence shown in SEQ.ID.NO.1 or SEQ.ID.NO.2.
The use of the plasmid of claim 4 in regulating the proliferation of pancreatic β-cells.
The use of the plasmid of claim 4 in the preparation of medicines for the treatment of diabetes.
A siRNA, characterized in that the sense strand sequence is shown in SEQ.ID.NO.7, and the antisense strand sequence is shown in SEQ.ID.NO.8.
A plasmid containing the siRNA of claim 7.
The use of the plasmid of claim 8 in regulating the proliferation of pancreatic β-cells.
The use of the plasmid of claim 8 in the preparation of drugs for treating hyperinsulinemia and insulinoma.