CN111718891B

CN111718891B - Application of circular RNA in regulation of pancreatic islet beta cell proliferation

Info

Publication number: CN111718891B
Application number: CN202010517832.6A
Authority: CN
Inventors: 贾世奇; 庄景燊; 陶卫华; 邓艳瑞
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2020-06-09
Filing date: 2020-06-09
Publication date: 2021-12-17
Anticipated expiration: 2040-06-09
Also published as: WO2021248408A1; CN111718891A

Abstract

The invention discloses an application of a circular RNA in regulating and controlling pancreatic islet beta cell proliferation, wherein the nucleotide sequence of the circular RNA is shown as SEQ.ID.NO.1 or SEQ.ID.NO. 2. Experiments in the application prove that the over-expression of the circular RNA can effectively promote the proliferation of the beta cells of the islets of Langerhans of mice, and the knockout of the circular RNA can effectively inhibit the proliferation of the beta cells of the islets of Langerhans of mice, so that the circular RNA can be used for preparing the medicines for treating diabetes or the beta-cytosis of the islets of Langerhans. A series of experiments prove that the circular RNA can effectively promote the proliferation of the pancreatic islet beta cells, and the circular RNA has wide application background in treating insulin secretion deficiency caused by reduction of the number of the pancreatic islet beta cells. The invention can provide good candidate drugs for clinically treating diabetes caused by the reduction of the number of islet beta cells, and has very good application prospects.

Description

Application of circular RNA in regulation of pancreatic islet beta cell proliferation

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of a circular RNA in regulation and control of pancreatic beta cell proliferation.

Background

Diabetes Mellitus (DM) is a metabolic disease that seriously threatens human health, and its onset is due to absolute or relative insufficiency of insulin, leading to chronic damage and dysfunction of various tissues (especially eyes, kidneys, heart, blood vessels, nerves) caused by elevated blood sugar.

Diabetes is mainly divided into type 1 diabetes and type 2 diabetes, and the pathogenesis of type 1 diabetes is mainly that islet beta cells are damaged due to various reasons, so that insulin is absolutely absent or remarkably reduced. Type 2 diabetes is the most common in China, and the causes of the diabetes include insulin resistance, insufficient progressive secretion of insulin or both of the insulin resistance and the insulin progressive secretion. In type 1 or more common type 2 diabetes, pancreatic islet beta cells are deficient in numbers, dedifferentiated or dysfunctional, resulting in a shift of glucose metabolism from a compensated to a decompensated state, eventually having to rely on oral hypoglycemic agents or the use of exogenous insulin replacement therapy.

At present, most of medicines only promote insulin secretion or reduce blood sugar concentration to play a role in delaying the progress of diabetes, have little effect on improving and protecting islet beta cells, and have inconstant influence on drug resistance and adverse reaction of various medicines after long-term use. At present, no medicine for promoting the proliferation of the pancreatic islet beta cells is on the market, so the discovery of the medicine for promoting the proliferation of the pancreatic islet beta cells has important biological research significance and practical significance, and a novel candidate medicine molecule can be provided for treating diabetes.

On the other hand, diseases such as hyperinsulinemia and insulinoma are characterized by abnormal proliferation of islet beta cells and excessive insulin secretion, which can cause clinical hypoglycemia symptoms, cause serious metabolic disturbance and even endanger life of serious patients. Surgical removal of a portion of pancreatic tissue is a common clinical treatment, and no specific drug is available for treating this type of disease. Therefore, the development of candidate drug molecules for specifically inhibiting the proliferation of the islet beta cells has important biological research significance and clinical practice significance.

Disclosure of Invention

The invention aims at providing an application of a circular RNA in regulating and controlling the proliferation of islet beta cells.

Another object of the present invention is to provide a plasmid overexpressing the above circular RNA and use thereof.

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

The purpose of the invention is realized by the following technical scheme:

the application of a circular RNA in regulating and controlling the proliferation of islet beta cells;

the nucleotide sequence of the circular RNA is shown as SEQ ID No.1(hsa _ circ _0000885) or SEQ ID No.2(mmu _ circ _0014773), wherein the SEQ ID No.1 codes human circular RNA, the SEQ ID No.2 codes murine circular RNA, the length of the sequences is 552bp, the sequences are highly conserved, only a plurality of bases are different, and the sequences at the protein level are completely the same;

the islet beta cells are mouse islet beta cells SJb;

experiments in the application prove that the over-expression of the circular RNA can effectively promote the proliferation of mouse islet beta cells, and the knockout of the circular RNA can effectively inhibit the mouse islet beta cells, so that the circular RNA can be used for preparing the medicine for treating diabetes.

A plasmid for over-expressing circular RNA, which contains a 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, pK5 ssaAAV-ciR or pK25 ssav-ciR.

After the plasmid for over-expressing the circular RNA is transfected into the islet beta cells, the survival rate of the islet beta cells can be improved, the proliferation of the islet beta cells is promoted, and the plasmid can be used for preparing a medicine for treating diabetes.

An siRNA, wherein the sequence of a sense strand is shown as SEQ.ID.NO.7, and the sequence of an antisense strand is shown as SEQ.ID.NO. 8; the siRNA is designed aiming at the circular RNA, can inhibit the expression of the circular RNA in cells and plays a role in regulating the proliferation of the islet beta cells.

The vector of the plasmid containing the siRNA is pSGLV3/H1/GFP + puro, pYr-1.1-hU6-EGFP, pRI or pLKO-1. The plasmid containing the siRNA can be used for regulating and controlling the proliferation of the pancreatic beta cells, and particularly, can be used for preparing medicines for treating the pancreatic beta cell cytosis such as hyperinsulinemia and insulinoma.

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

a series of experiments prove that the circular RNA (SEQ. ID. NO.1 and SEQ. ID. NO.2) can effectively promote the proliferation of the pancreatic islet beta cells, and essentially proves that the circular RNA has wide application background in treating insulin secretion deficiency caused by reduction of the number of the pancreatic islet beta cells. The invention can provide good candidate drugs for clinically treating diabetes caused by the reduction of the number of islet beta cells, and has very good application prospects.

Drawings

Fig. 1 is a sequencing diagram of the head-to-tail interface sequence at the loop of mmu _ circ _ 0014773.

FIG. 2 is a graph of the effect of overexpression of mmu _ circ _0014773 on pancreatic islet beta cell survival.

FIG. 3 is a graph depicting the effect of knocking out mmu _ circ _0014773 on islet beta cell survival.

FIG. 4 is a graph of the effect of overexpression of mmu _ circ _0014773 on islet beta cell proliferation.

FIG. 5 is a graph depicting the impact of knocking out mmu _ circ _0014773 on islet beta cell proliferation.

Detailed Description

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

Example 1

This example is illustrated with mouse islet beta cells SJb as a model, and therefore, circRNA from mouse was selected, namely circ _ mmu _ circ _0014773(seq. id No. 2).

First, it was verified that there was an endogenously expressed circular RNA sequence in SJb islet β cells with circuit _ mmu _ circuit _ 0014773. PCR amplification is carried out by designing an amplification circular RNA specific primer, an amplification product is subjected to Sanger sequencing to obtain a circular sequence formed by first linking of a linear sequence, and an arrow in figure 1 shows a novel sequence (a circular interface sequence) formed by combining the last base and the first base of the linear sequence.

The over-expression of the circ _ mmu _ circ _0014773 can effectively promote the proliferation of mouse islet beta cells SJb, the knockout of endogenous circ mmu _ circ _0014773 can effectively inhibit the proliferation of mouse islet beta cells SJb, and the over-expression and knockout vectors of the circ mmu _ circ _0014773 are respectively constructed, so that the drug can be further developed into a drug for treating diabetes or hyperinsulinemia, and has wide application prospects.

The following experiments were used for validation:

(1) construction and identification of pCDH-circ mmu _ circ _0014773 plasmid

The pCDH _ circ mmu _ circ _0014773 vector was constructed using Novozam seamless cloning kit (ref: C112) as follows:

1) preparation of linearized vector

The vector plasmid pCDH is subjected to double enzyme digestion by EcoRI and BamHI, and a target fragment is recovered by glue.

The enzyme digestion system is shown in Table 1:

TABLE 1 vector plasmid pCDH double digestion System

The enzyme was cleaved at 37 ℃ for 4 h. After agarose gel electrophoresis, the linearized plasmid pCDH was recovered from the gel and verified.

2) Obtaining of insert

General principle of primer design: introducing homologous sequences at two ends of a linearized vector into the 5' end of the forward and reverse amplification primers of the insert fragment, so that the 5' and 3' extreme ends of the amplified insert fragment respectively have homologous sequences (15-20bp, excluding enzyme cutting sites) corresponding to the two ends of the linearized cloning vector.

The final amplification insert primers used are shown below:

TABLE 2 use of primers

The insert was amplified using Takara high fidelity enzyme in the following amplification system:

TABLE 3 insert amplification System

The amplification product is identified, concentration determined and purified for subsequent testing.

3) Recombination reactions

The following reaction system was prepared on ice:

TABLE 4 reaction System

Ligation was carried out overnight at 16 ℃. The ligation products were introduced into DH 5. alpha. competent cells, plated on screening medium plates containing ampicillin, and single colonies were picked. Sequencing was carried out 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 is constructed according to earlier verified circ mmu _ circ _0014773siRNA, and the part is completed by committing the company of Biotechnology engineering (Shanghai) GmbH. The vector used was pSGLV3/H1/GFP + puro, and the sequence of the circ mmu _ circ _0014773siRNA used was as follows:

circ mmu_circ_0014773siRNA sence：5’AGAAAGUGUGCCCUGGUAUtt 3’(SEQ.ID.NO.7)

circ mmu_circ_0014773siRNA antisence:5’AUACCAGGGCACACUUUCUga 3’(SEQ.ID.NO.8)

(3) construction and identification of overexpression and knockout of circ mmu _ circ _0014773 stable strain

Islet beta cells SJb were inoculated in advance, gently blown into single cells using pancreatin digestion with 2.5% EDTA, centrifuged at 200g for 5min, and then resuspended. Cells were counted and 4X 10 cells were taken from each transfection group⁶Centrifuging the islet beta cells SJb at 200g for 5min, removing supernatant as much as possible, adding 100 μ l of electrotransformation liquid and 5 μ g plasmid into each well of cells, gently and rapidly mixing, transferring to a dot rotor, and performing electrotransformation by selecting a proper electrotransformation program. After the completion of the electrotransfer, 500. mu.l of 10% FBS-containing RPMI 1640 medium was added, transferred to a 1.5EP tube, incubated at 37 ℃ for 15min, and transferred to 75cm²And (4) normally culturing in a cell culture flask. And (3) after 24h of electric conversion, replacing the screening culture medium, observing GFP expression under a fluoroscope, obtaining stable transfected cells when the cells basically express GFP fluorescence, and then carrying out Qpcr to verify the transfection efficiency.

Example 2

The over-expression of the circ mmu _ circ _0014773 can effectively promote the proliferation of mouse islet beta cells SJb, the deletion of the mmu _ circ _0014773 can effectively inhibit the proliferation of mouse islet beta cells SJb, the over-expression of the circ _ mmu _ circ _0014773 and the stable expression strain of the mouse islet beta cells SJb are respectively constructed, the influence of the proliferation of the mmu _ circ _0014773 mouse islet beta cells SJb is verified, the drug can be further developed into a drug for treating diabetes, and the drug has wide application prospect.

The following experiments were used for validation:

1) effect of overexpression of mmu _ circ _0014773 on survival of pancreatic islet beta cells

The overexpression circular mmu _ circ _0014773(circ _ mmu _ circ _0014773), overexpression Linear mmu _ circ _0014773(Linear _ mmu _ circ _0014773) and control group plasmid (pCDH) cells constructed above were inoculated into a 96-well plate containing 100. mu.l of medium per well and 10. mu.l of culture medium⁴The cell suspension is pre-cultured in a cell culture box for 24 hours. The next day (this counts as day 0) complete medium containing 10. mu.l CCK-8 solution was added to each well (care was taken not to generate air bubbles, mix gently). And incubating the culture plate in an incubator for 2h, and measuring the light absorption value at 450nm by using an enzyme-labeling instrument. After the measurement, the normal medium was changed, and the survival rate of the cells at that time was measured on day 2 and day 6 as described above.

From the results in FIG. 2, it can be seen that cell proliferation was significantly increased in the circ _ mmu _ circ _0014773 and Linear _ mmu _ circ _0014773 groups at day 2, and the circ _ mmu _ circ _0014773 group could still maintain a higher growth rate at day 6.

2) Knocking out influence of circ _ mmu _ circ _0014773 on survival rate of pancreatic islet beta cells

The knockout circular circ _ mmu _ circ _0014773 and control group plasmid (sh NC) cells constructed above were inoculated into a 96-well plate containing 100. mu.l of medium per well, 10⁴The cell suspension is pre-cultured in a cell culture box for 24 hours. The rest steps are the same as above.

From the results of FIG. 3, it can be seen that the growth rate of the cells of the sh _ circ _ mmu _ circ _0014773 group was significantly lower than that of the cells of the control group at day 6.

3) Effect of overexpression of circ _ mmu _ circ _0014773 on promoting proliferation of islet beta cells

10 cells of the overexpression circ _ mmu _ circ _0014773, the overexpression Linear Linear-mmu _ circ _0014773 and the control group plasmid (pCDH) which are constructed as above were taken respectively⁶200g of the supernatant was centrifuged for 5min, the supernatant was discarded, 4% precooled PFA was fixed for 15min, and precooled PBS was washed three times. The supernatant was discarded, 10. mu.l of PBX (PBS + 1% Tween-20) was added, and the mixture was incubated at room temperature for 20 min. The pre-cooled PBS was washed three times. 1ml of Ki67 dye (2. mu.l of anti-Ki67+ PBS + 0.02% BSA +5mM EDTA) was prepared at a ratio of 1:500, incubated at room temperature for 40min, and the corresponding fluorescent secondary antibody was prepared at a ratio of 1: 500. After incubation at room temperature for 40min, the cells were washed three times with pre-cooled PBS. And (4) detecting by using a flow cytometer.

According to the results of fig. 4, it can be seen that the Ki67 positive cells in the circ _ mmu _ circ _0014773 group are significantly more than those in the control group, suggesting that the overexpression of the circ _ mmu _ circ _0014773 can significantly promote the proliferation of the mouse islet beta cell SJb.

4) Knocking out the effect of circ _ mmu _ circ _0014773 on inhibiting the proliferation of islet beta cells

The thus-constructed knockout circular circ _ mmu _ circ _0014773 cells were taken and 10 each of the control plasmid (sh NC) cells was used⁶The rest steps are the same as above. And (4) detecting by using a flow cytometer.

From the results of fig. 5, it can be seen that Ki67 positive cells in the sh _ circ _ mmu _ circ _0014773 group were significantly less than those in the control group. It was suggested that the knockout of circ _ mmu _ circ _0014773 could inhibit proliferation of mouse islet beta cell SJb.

In summary, the following steps: during the in vitro promotion process, the over-expression of the circ _ mmu _ circ _0014773 can significantly promote the proliferation of the beta cell SJb, and conversely, the knockout of the circ _ mmu _ circ _0014773 can inhibit the proliferation of the mouse islet beta cell SJb. According to the experimental result, the molecular sequence of circ _ mmu _ circ _0014773 has great potential and application prospect when being used for regulating beta cells.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

<120> application of circular RNA in regulation and control of pancreatic islet beta cell proliferation

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 552

<212> DNA

<213> human (Homo sapiens)

<400> 1

tgtgtcccgg catggatatc cggaacaacc tcactaggtt gcatgagctg gagaattgct 60

ctgtcatcga aggacacttg cagatactct tgatgttcaa aacgaggccc gaagatttcc 120

gagacctcag tttccccaaa ctcatcatga tcactgatta cttgctgctc ttccgggtct 180

atgggctcga gagcctgaag gacctgttcc ccaacctcac ggtcatccgg ggatcacgac 240

tgttctttaa ctacgcgctg gtcatcttcg agatggttca cctcaaggaa ctcggcctct 300

acaacctgat gaacatcacc cggggttctg tccgcatcga gaagaacaat gagctctgtt 360

acttggccac tatcgactgg tcccgtatcc tggattccgt ggaggataat tacatcgtgt 420

tgaacaaaga tgacaacgag gagtgtggag acatctgtcc gggtaccgcg aagggcaaga 480

ccaactgccc cgccaccgtc atcaacgggc agtttgtcga acgatgttgg actcatagtc 540

actgccagaa ag 552

<210> 2

<211> 552

<212> DNA

<213> mouse (Mus musculus)

<400> 2

tgtgccctgg tatggacatc cggaacaacc tgaccaggct acatgagctg gagaactgct 60

cagtcattga gggccatctg cagatcctcc tgatgttcaa gaccagaccc gaagatttcc 120

gagacctcag tttccccaaa ctcatcatga tcacagatta cctgcttctc ttccgtgtct 180

atggtctgga aagtctgaaa gacctcttcc caaatctcac agtcatccga ggctcccgtc 240

tcttcttcaa ctatgccctg gttatcttcg agatggtcca cctgaaggag ctggggcttt 300

ataacctcat gaacatcacc cggggctctg tccgcatcga gaagaataat gagctctgct 360

acctggccac tatcgactgg tcccgtatcc tggattctgt ggaggacaac tacattgtac 420

tgaacaaaga tgacaacgag gaatgtgggg atgtctgtcc aggcaccgcc aagggcaaga 480

ccaactgtcc tgccactgtc atcaatgggc agtttgtgga acggtgctgg acacacagtc 540

attgtcagaa ag 552

<210> 3

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> circ mmu_circ_0014773-Fw

<400> 3

atctgttcaa ttaacgaatt ctgaaatatg ctatcttaca gtgtgccctg gtatgga 57

<210> 4

<211> 65

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> circ mmu_circ_0014773-Rv

<400> 4

tcatcccaaa ttagtggatc ctcaagaaaa aatatattca cctttctgac aatgactgtg 60

tgtcc 65

<210> 5

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Linear mmu_circ_0014773-Fw

<400> 5

atctgttcaa ttaacgaatt ctgtgccctg gtatggacat c 41

<210> 6

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Linear mmu_circ_0014773-Rv

<400> 6

tcatcccaaa ttagtggatc cctttctgac aatgactgtg tgtcc 45

<210> 7

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> circ mmu_circ_0014773 siRNA sence

<400> 7

agaaagugug cccugguau 19

<210> 8

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> circ mmu_circ_0014773 siRNA antisence

<400> 8

auaccagggc acacuuucu 19

Claims

1. The application of the circular RNA in preparing the medicine for treating diabetes is characterized in that: the nucleotide sequence of the circular RNA is shown as SEQ No. ID. NO.1 or SEQ No. ID. NO. 2.

2. The application of a plasmid for over-expressing circular RNA in the preparation of a medicament for treating diabetes is characterized in that: the plasmid contains a nucleotide sequence shown in SEQ No. ID. No.1 or SEQ No. ID. No. 2.

3. The application of a circular RNA, siRNA or plasmid expressing siRNA in preparing medicine for treating hyperinsulinemia and insulinoma is characterized in that:

the nucleotide sequence of the circular RNA is shown as SEQ No. ID. No.1 or SEQ No. ID. No. 2;

the sense strand sequence of the siRNA is shown as SEQ No. ID. NO.7, and the antisense strand sequence is shown as SEQ No. ID. NO. 8.