CN114854740B

CN114854740B - Application of miR-483-5p cavernous body in preparation of medicine for inhibiting type 2 diabetes beta cell dedifferentiation

Info

Publication number: CN114854740B
Application number: CN202210322787.8A
Authority: CN
Inventors: 张亚琴; 韩晓; 牛凡弟; 吴涛; 王力冉; 吴倜珺; 孙瞳; 吴婧文
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2022-03-29
Filing date: 2022-03-29
Publication date: 2024-03-15
Anticipated expiration: 2042-03-29
Also published as: CN114854740A

Abstract

The invention discloses miR-483-5p cavernous body and application thereof in preparation of a medicament for inhibiting type 2 diabetes beta cell dedifferentiation. The invention discovers the effect of miR-483-5p cavernous body on inhibiting islet beta cell dedifferentiation for the first time. The miR-483-5p cavernous body serving as the miR-483-5p inhibitor has the advantages of being strong in target site specificity, being mRNA, free of toxic and side effects, easy to degrade in vivo and the like, and is suitable for selecting an effective medicament for preventing and treating type 2 diabetes.

Description

Application of miR-483-5p cavernous body in preparation of medicine for inhibiting type 2 diabetes beta cell dedifferentiation

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an application of miR-483-5p cavernous body in preparation of a medicine for inhibiting type 2 diabetes beta cell dedifferentiation.

Background

At present, traditional hypoglycemic drugs such as insulin, glucosidase inhibitor, insulin secretagogue, insulin sensitizer and the like often bring adverse reactions such as drug resistance, hypoglycemia, lactic acidosis, weight gain and the like while treating. Moreover, it was shown in the uk prospective diabetes study (UKPDS) report that islet beta cell function or progressive loss of islet beta cell function in diabetics is not protected and maintained regardless of the traditional hypoglycemic agent employed. In addition, the current drugs are all used for diagnosing and confirming type 2 diabetes mellitus, and at the moment, islet cells are dysfunctional, irreversible damage is caused to beta cells, and the functions of the beta cells cannot be repaired. Therefore, there is an urgent need to find a hypoglycemic agent which protects the function of the early beta cells of diabetes and has little toxic and side effects. microRNA (miRNA) is a highly conserved non-coding small RNA of about 18-25 nucleotides in length. miRNAs are widely expressed in all stages of beta cell development and mature beta cells, are regulatory factors that play a major role in maintaining beta cell development and function in fetal stage, and are also essential for maintaining mature beta cell function, and expression of miRNAs affects pancreatic development and beta cell function. Diabetes mellitus can be generally divided into three stages of a high-risk group of diabetes mellitus, a sugar regulation impaired stage and a diabetes mellitus stage, and if the characteristics of different stages of the diabetes mellitus can be grasped, early intervention and early treatment are considered to be very beneficial to the prevention and treatment of the diabetes mellitus. Research shows that miR-483-5p is remarkably and highly expressed in serum and primary islets of type 2 diabetics and type 2 diabetes mice, and the highly expressed miR-483-5p promotes islet beta cells to dedifferentiate, so that insulin secretion function of the islet beta cells is lost by half, but most islet beta cells are not dead at the moment. If a novel drug is used for inducing the dedifferentiated beta cells to return differentiation, so that the dedifferentiated beta cells are redifferentiated into mature beta cells with normal functions, which is likely to be an important direction for future diabetes treatment. However, if not paid attention to, under continuous stimulation such as obesity and insulin resistance, inflammatory factor injury, these beta cells gradually fade away from their mature state, eventually losing secretory function and actually "apoptosis", resulting in incurable type 2 diabetes.

Disclosure of Invention

The invention aims to: the invention aims to provide miR-483-5p cavernous body (miR-483-5 p sponge) and application thereof in preparation of medicines for inhibiting type 2 diabetes beta cell dedifferentiation.

The technical scheme is as follows: aiming at the defects of the existing type 2 diabetes mellitus treatment drugs, the miR-483-5p cavernous body has a nucleotide sequence shown in SEQ ID NO:1, specifically:

application of miR-483-5p sponge (miR-483-5 p cavernous body) or down regulator and derivatives thereof in preparation or screening of medicines for inhibiting type 2 diabetes beta cell dedifferentiation. The miR-483-5p cavernous body is an mRNA, and a 3' untranslated region (UTR) of the mRNA comprises a plurality of miR-483-5p targeting points. More importantly, these RISC cleavage sites have some mismatches with the targeted sites so that the inhibitor miR-483-5p cavernous body is not degraded.

The application of the miR-483-5p cavernous body in preparing a medicine for inhibiting type 2 diabetes beta cell dedifferentiation.

The miR-483-5p cavernous body (miR-483-5 p cavernous body) is an inhibitor of miR-483-5p, and targets to bind miR-483-5p so as to inhibit functions of the cavernous body. The miR-483-5p sponge seals the expression of miR-483-5p in beta cells, and further applies the miR-483-5p to the preparation of medicaments for inhibiting the dedifferentiation of the beta cells of the type 2 diabetes, so as to solve the problems of the lack of the current medicaments for treating the type 2 diabetes and side effects.

The miR-483-5p cavernous body can inhibit the generation of high-fat diet induced type 2 diabetes beta cell dedifferentiation. In particular, when the miR-483-5p sponge is applied, the sponge is effectively connected with an expression vector. By "operably linked" is meant that the ligation of the miR-483-5p sponge of the invention to an expression vector enables the resulting nucleic acid construct to transcribe the miR-483-5p sponge of the invention in a cell or animal.

Further, the expression vector is an adeno-associated virus expression plasmid, preferably, an adeno-associated virus expression plasmid pHBAAV-CMV-MCS-T2Am-zsgreen is selected. The miR-483-5p nucleic acid construct can be operably linked to an expression vector by adding cleavage sites to both ends of the miR-483-5p sponge sequence, which sites are compatible with the expression vector, to synthesize a forward sequence and a reverse sequence.

Further, a miR-483-5p nucleic acid construct (also known as miR-483-5p sponge-adeno-associated virus) can be prepared by:

(1) Selecting an adeno-associated virus vector pHBAAV-CMV-MCS-T2Am-zsgreen; the competent cell is selected from Escherichia coli strain DH5 alpha; resistance: amp;

(2) Synthesizing miR-483-5p sponge aiming at a target gene, then constructing an adeno-associated virus expression vector, and greatly amplifying the adeno-associated virus vector; the adenovirus vector is packaged in large quantity in 293T cells, the adenovirus is concentrated and purified, and finally the titer of the adenovirus is measured (10≡11TU/ml).

The invention observes the dedifferentiation of islet beta cells of mice by tail intravenous injection or intraperitoneal injection or pancreatic duct internal injection of AAV-mmu-miR-483-5 p-spike (miR-483-5 p-spike) or control virus AAV-mmu-NC-GFP (GFP) in high-fat diet mice.

Therefore, the invention provides a clear and effective medicament for inhibiting miR-483-5p expression, and inhibiting beta cell dedifferentiation in early diabetes, so that the quantity and structure of islet beta cells are not changed, and insulin secretion function is normal, so that type 2 diabetes can not occur, and the invention has important significance in controlling the occurrence and development of diabetes.

The beneficial effects are that: the invention discovers the effect of miR-483-5p cavernous body on inhibiting islet beta cell dedifferentiation for the first time. miR-483-5p has been identified to inhibit expression of transcription factors Pdx1 and MafA, which are essential for maintaining beta cell specificity (beta cell identity), so that endocrine progenitor cell markers (Ngn 3) and stem cell markers (OCT 4, nanog) are up-regulated. miR-483-5p sponge (miR-483-5 p corpus cavernosum) or downregulator, derivatives thereof and the like can be used as a specific inhibitor of miR-483-5p, provide a new target point for preparation or screening of effective medicaments for treating type 2 diabetes mellitus, and have important significance for preventing and treating type 2 diabetes mellitus. Meanwhile, the miR-483-5p cavernous body serving as the miR-483-5p inhibitor has the advantages of being strong in target site specificity, being mRNA, free of toxic and side effects, easy to degrade in vivo and the like, and is suitable for selecting an effective medicament for preventing and treating type 2 diabetes.

Drawings

Fig. 1: miR-483-5p is remarkably and highly expressed in serum and primary islets of type 2 diabetics and type 2 diabetes mice: respectively extracting serum of a type 2 diabetic patient and primary islet RNA, and detecting the level of miR-483-5p by fluorescent quantitative PCR; (a and B) graphs show the expression levels (n=10) of type 2 diabetic patients and normal control serum (a) and primary islets (B) miR-483-5 p; (C and D) graphs of serum (C) and primary islet (D) miR-483-5P expression levels (n=10) (< P < 0.01, < P < 0.001) in type 2 diabetic mice and control mice;

fig. 2: the expression of β -cell specific marker molecules (e.g., pdx1, mafA, and ins) was significantly down-regulated in primary islets of type 2 diabetics and type 2 diabetics, while the expression of β -cell dedifferentiating marker molecules (e.g., ngn3, oct4, nanog) was significantly up-regulated: extracting primary islet RNA of a type 2 diabetic patient and a type 2 diabetic mouse respectively, and detecting the levels of Pdx1, mafA, insulin, ngn3, oct4 and Nanog by fluorescent quantitative PCR; (A) The figure shows the expression level of the above molecules in primary islets of type 2 diabetics and normal controls (n=8); (B) The expression level of the above molecules in primary islets of type 2 diabetic mice (n=8) (< 0.05, < 0.01, < P);

fig. 3: overexpression of miR-483-5p significantly reduced β -cell synthesis and insulin secretion function without affecting β -cell proliferation and apoptosis: transfecting MIN6 cells with the synthesized miR-483-5P mimic, carrying out GSIS experiment (A) after 48 hours, detecting insulin content (B) by using a RIP method, analyzing proliferation (C) of MIN6 cells by using MTT, and analyzing apoptosis (D) of MIN6 cells by using Hoechst, (n=2-3; P < 0.05; P < 0.01);

fig. 4: overexpression of miR-483-5p significantly enhances β -cell dedifferentiation: transfecting MIN6 cells with the synthesized miR-483-5P mimic, carrying out quantitative PCR after 48 hours, and detecting the expression of beta cell specific marker molecules Pdx1, mafA, insulin 1 and Insulin2 (A) and dedifferentiated marker molecules Ngn3, oct4 and Nanog (B), (n=2-3; P < 0.05; P < 0.01);

fig. 5: male C57BL/6J mice fed with high fat for 12 weeks are subjected to continuous high fat feeding after being injected with AAV-mmu-miR-483-5 p-spike (miR-483-5 p-spike) or control virus AAV-mmu-NC-GFP (GFP) in pancreatic ducts, and metabolic phenotype of the mice is monitored; wherein (A) is a graph showing the weight change of the experimental mice with time; (B) FIG. is a schematic representation of the results of IPGTT in mice 4 weeks after injection of AAV-mmu-miR-483-5 p-spike or AAV-mmu-NC-GFP; (C) The graph shows insulin secretion amounts at 0, 5, 30min of intraperitoneal injection of glucose after 6 weeks of injection of AAV-mmu-miR-483-5P-spike or AAV-mmu-NC-GFP, (n=6;: < 0.05: < 0.01: P);

fig. 6: after 6 weeks of injection of AAV-mmu-miR-483-5P-spine or AAV-mmu-NC-GFP in pancreatic ducts, islets of mice were extracted, and expression of Pdx1, mafA, instulin 1 and instulin 2 (a) was detected by real-time fluorescent quantitative PCR, while expression of dedifferentiating marker molecules Ngn3, oct4 and Nanog (B) was detected (n=6; P < 0.05; P < 0.01).

Detailed Description

Example 12 expression of miR-483-5p in the serum and islets of type 12 diabetes mellitus mice is significantly upregulated.

1) Preparation of serum:

a: extracting 3ml of peripheral venous blood of each experimental object, adding the peripheral venous blood into a separation gel and coagulant vacuum blood collection tube, and standing for 30-60min at normal temperature or in a 37 ℃ water bath box;

b: centrifuging at room temperature or 4deg.C for 5-10min, separating into three layers, wherein the upper layer is yellowish clear serum layer, the middle separating gel layer, and the bottom layer is dark red blood cell layer; moving to an ultra clean bench for further operation, thereby avoiding the contamination of samples by saliva, external enzymes and the like.

C: transferring the taken serum into an enzyme-free Epp tube, centrifuging 12000g for 5min, and further discarding residual cells or cell fragments, wherein the centrifuging temperature is 4 ℃; transferring to a new enzyme-free 1.5ml freezing tube, and preserving at-80deg.C for use.

2) Primary islet extraction:

primary islets of type 2 diabetics and normal control groups were given away by the university of Tianjin medical university Wang Shusen, only the extraction of mouse primary islets being described herein.

A: separating: mice were fasted overnight and anesthetized by intraperitoneal injection with 3.5% (w/v) chloral hydrate; fixing the animal on an operating table; opening the abdominal and thoracic cavities and exposing the heart; finding the common bile duct at the opening of the duodenum, and ligating with a thread; cutting the right auricle to exsanguinate; finding out the junction of the hepatic common pipe and the common bile pipe, and preparing an intubation tube; catheterization with an intravenous infusion tube (venting prior to catheterization), collagenase V (1.5-2 mL/mouse) was injected, and the pancreas was seen to be in a transparent bleb; the filled pancreatic tissue is sheared and stripped along the intestinal canal and rapidly placed in 50mL sterilized plastic centrifuge tubes (2 mouse pancreas can be placed in each tube) pre-chilled on ice; adding 2-5mL collagenase V into a 50mL centrifuge tube for external digestion, and standing and digesting for 28min in a water bath at 37 ℃; taking out the 50mL centrifuge tube from the water bath, and immediately placing the centrifuge tube on ice to terminate digestion; vortex vibration for 3×5sec until the tissue is broken into silt; adding 2 times of HBSS (precooling on ice) containing 10% FBS, mixing, further stopping digestion, filtering with 30 mesh stainless steel screen, and removing undigested tissue block; centrifugation (acceleration: 9, deceleration: 9) at 350g at 4℃for 2min; removing supernatant, adding ice HBSS to resuspend cell sediment, centrifuging at 4 ℃ for 2min at 350g (acceleration: 9; deceleration: 9); the supernatant was discarded, 5mL of Histopaque-1077 was added to resuspend the cell pellet, and transferred to a 10mL glass centrifuge tube; carefully add 5mL HBSS slowly along the tube wall of the glass centrifuge tube with a bara tube to maintain delamination between HBSS and Histopaque-1077; centrifuging at 4deg.C for 20min at 500g (acceleration: 9; deceleration: 1); after centrifugation, islets were placed in the sandwich between HBSS and Histopaque-1077, and this sandwich was transferred with a 200 μl pipette into 6 well plates pre-loaded with serum-containing HBSS, and whole islets were picked under a split microscope with a 10 μl pipette.

B: purity identification: dissolving 100mg of islet Dithizone (DTZ) in 30mLDMSO, adding 500 μl of 25% ammonia water, dissolving thoroughly, filtering to remove insoluble substances, packaging into EP tube, and preserving at-20deg.C to obtain stock solution; after the obtained islets are washed twice by using physiological saline, 1mL of physiological saline and 10 mu L of dithizone storage solution are added, the cells are incubated for 30min at 37 ℃, the cell staining condition is observed under an inverted microscope, the islets are stained with DTZ to be scarlet, and the purity of the islets is identified to be more than 90%.

3) Extraction of RNA from serum or islets

A: taking out the frozen sample at-80 ℃ to be melted on ice, sucking 200 mu l of the frozen sample, adding the frozen sample into 600 mu l of Trizol, fully and uniformly mixing the frozen sample and the Trizol on an oscillator, and standing the obtained homogenate at room temperature (15-30 ℃) for 10min to fully separate nucleic acid protein complexes;

b: adding 1/5 volume of chloroform, namely 120 μl of chloroform, mixing for 15sec with vigorous vortex, and standing at room temperature for 10min;

c: the centrifuge was conditioned at 4℃and centrifuged at 12000g for 15min, and the samples were seen to be three layers: the upper layer is colorless water phase (in which RNA is dissolved), the middle layer is red layer (in which DNA is dissolved), the lower layer is organic layer (organic substance such as protein, etc.) (the tube can not be swayed or reversed when sampling after centrifugation is finished, and three layers are prevented from mixing again after centrifugation);

d: pipetting the upper aqueous phase (taking care to avoid pipetting the middle layer) with a 200 μl pipette into a new 1.5ml enzyme-free Epp tube, adding equal volume of isopropanol, mixing well, and standing at room temperature for 25min;

e: after completion of the standing, the mixture was centrifuged at 12000g for 10min at 4℃to discard the supernatant.

F: 600 μl of 75% ethanol prepared in advance was added, and the mixture was centrifuged at 7500g for 5min at 4 ℃. Discarding the supernatant, airing at room temperature for about 5min, adding 20 μl RNAase-free ddH2O, gently stirring and mixing, and fully dissolving RNA;

4) RNA concentration and purity detection

Taking 1 μl of the extracted RNA, opening an ultraviolet spectrophotometer to obtain absorbance at wavelengths of 260nm and 280nm, calculating the ratio of the absorbance to the absorbance, and if the value of 260/280 OD is in the range of 1.8-2.1, the concentration of the RNA is about 25-50 ng/. Mu.l, and then carrying out the next experiment.

5) cDNA synthesis and real-time fluorescent quantitative PCR

cDNA was synthesized by reverse transcription using a reverse Tra Ace-alpha-reverse transcription kit manufactured by TOYOBO Co.

A: reverse transcription: 20 μl of the system and all manipulations were performed on ice. A0.2 ml RNAase-free centrifuge tube was taken and the following reagents were added:

reverse transcription reaction system

B: reverse transcription procedure

37℃60min；

85℃5min；

4℃60min。

C: the reverse transcribed cDNA was stored at-80℃or RNAase-free H2O was added thereto to dilute it to 100. Mu.l for the next step.

D: 10 μl of PCR reaction system

Real-time fluorescent quantitative PCR reaction system

E: the system was added to an octant tube and PCR amplification was performed according to the following procedure:

real-time fluorescent quantitative PCR reaction program

Three auxiliary holes are set for each sample, and CT values of internal reference and sample reactions are recorded respectively.

6) Statistical analysis of data

The ratio of the miRNA expression levels of the two groups of serum samples was calculated using a 2- Δct method, wherein ΔΔct= [ Ct1 (miRNA) -Ct1 (internal reference) ] -Ct2 (miRNA) -Ct2 (internal reference) ], ct (miRNA) is the Ct value of the sample miR-483-5p amplification, ct (internal reference) is the Ct value of the sample internal reference gene amplification, ct1 is the Ct value of the type 2 diabetes group sample amplification, and Ct2 is the Ct value of the healthy control group amplification. All data in this experiment are expressed as mean ± standard deviation (±s), the inter-group variability analysis is performed using t-test, P < 0.05 as a statistical variability reference.

As a result, as shown in fig. 1, miR-483-5P showed significantly increased expression levels in serum and primary islets of type 2 diabetes patients (fig. 1A and 1B) and type 2 diabetes mice (fig. 1C and 1D), with significant differences (< 0.01, < 0.001). This suggests that miR-483-5p may be involved in the development and progression of type 2 diabetes.

Example 2 2 beta cell marker molecules were significantly down-regulated in islets of type 2 2 diabetes and type 2 diabetes mice, while expression of beta cell dedifferentiation marker molecules was significantly up-regulated.

In order to analyze whether the pancreatic islet highly expressing miR-483-5p has dedifferentiation under the diabetic state, the expression of the beta cell marker molecules and the beta cell dedifferentiation marker molecules in the pancreatic islet of type 2 diabetes patients and type 2 diabetes mice is detected.

The specific steps are shown in the real-time fluorescence quantitative PCR method of the primary islets described in the example 1. Except that the reverse primer of the miRNA neck ring in the reverse transcription system of example 1 was replaced with Oligo (dT) (note: for mature mRNA with polyA tail); the PCR system of example 1 was replaced with mRNA primers for each of the genes tested (sequences shown in Table 1).

TABLE 1

The results are shown in FIG. 2, in which the expression of the beta cell-specific marker molecules was significantly down-regulated in islets of type 2 diabetics and type 2 diabetics, while the expression of the beta cell dedifferentiation marker molecules was significantly up-regulated.

Example 3 miR-483-5p significantly inhibited islet beta cell synthesis and insulin secretion without affecting islet beta cell proliferation and apoptosis.

In order to explore the possible regulation and control effect of miR-483-5p on islet beta cells, the influence of miR-483-5p on islet beta cell synthesis and secretion of insulin, proliferation and apoptosis is studied.

1) Insulin secretion experimental detection of islet beta cells

GSIS experiments were performed 48h after MIN6 cells cultured in 12-well plates were transfected with negative control mimics or miR-483-5p mimics (see table 2 for sequence). The specific operation is as follows: the culture supernatant was discarded from the treated MIN6 cells, rinsed once with PBS, and incubated for 1h with 1mL of sugarless Kerbs-Ringer-biscarbonate-HEPES (KRBH) buffer; the supernatant was discarded, 1mL of KRBH buffer containing 2mmol/L (low sugar) or 20mmol/L (high sugar) glucose was added to MIN6 cells, incubation was continued for 1h, and after that, the supernatant was collected into 2mL EP tube and frozen at-80℃for measurement. The results are shown in FIG. 3A, where the miR-483-5p mimetic significantly inhibited the MIN6 cell high-glucose stimulated insulin secretion function as compared to the negative control mimetic. (. Times.P < 0.05)

TABLE 2

2) Intracellular insulin extraction experiments

MIN6 cells were seeded in 48-well plates, and after 48h of transfection of cells with negative control mimics or miR-483-5p mimics (see table 2 for sequence), the culture supernatant was discarded and rinsed once in PBS; cells were then incubated with 200. Mu.l of an acid-ethanol extract (acid-ethanol solution:74% ethanol,1.4% HCl) at 4℃for about 12 hours, and the supernatant was taken in a 2ml Epp tube. The sample can be frozen in an ultralow temperature refrigerator at the temperature of-80 ℃ to be tested; insulin levels were detected using the insulin radioimmunoassay kit, radioimmunoassay (radio immuno assay, RIA). The results are shown in FIG. 3B, and the miR-483-5p mimic also significantly inhibited MIN6 cell insulin synthesis function, compared to the negative control mimic.

3) MTT detection of cell viability

Cells were seeded in 96-well plates, and after 48h transfection of cells with negative control or miR-483-5p mimics (see Table 2 for sequence), 10. Mu.l of MTT (MTT in sterile PBS (phosphate buffered saline, phosphate buffer saline) was added to each well, at a concentration of 0.5% (m/v), the 96-well plates were placed in a 37℃incubator for 4h, after completion of the reaction, 100. Mu.l of dissolved purple formazan crystals were added to each well for 10MIN in a 37℃incubator, absorbance was read at 570nm using an enzyme-labeled instrument, and the viability of the treated cells was proportional to the absorbance.

4) Detection of apoptosis by Hoechst

Cells were inoculated in 96 well plates, and after 48h of transfection of cells respectively with negative control mimics or miR-483-5p mimics (see Table 2 for sequence), 100. Mu.l Hoechst 33258 staining solution was added to each well, and cultured at a temperature suitable for cell culture for 20-30min; discarding the staining solution, and washing with PBS or culture solution for 2-3 times, each time for 3-5min; and observed under a fluorescence microscope. When the cells undergo apoptosis, the nuclei of the apoptotic cells are densely stained or densely stained in a fragment shape. The results are shown in FIG. 3D, where the miR-483-5p mimetic did not affect apoptosis of MIN6 cells compared to the negative control mimetic.

The results show that miR-483-5p obviously inhibits synthesis and secretion of MIN6 cell insulin without affecting proliferation and apoptosis.

Example 4 Effect of miR-483-5p on islet beta cell dedifferentiation

To further investigate the effect of miR-483-5p on islet beta cell dedifferentiation, mRNA (messenger ribonucleic acid) levels of islet beta cell marker molecules Pdx1, mafA, instein 1 and instein 2 in MIN6 cells transfected with miR-483-5p mimics were examined using qRT-PCR (Real-time fluorescence quantification, real-time PCR) method, while the expression of islet beta cell dedifferentiation marker molecules Ngn3, oct4 and Nanog was analyzed. The primer sequences are shown in Table 1. For specific steps, see the real-time fluorescent quantitative PCR methods described in example 1 and example 2. And will not be described in detail herein.

As a result, as shown in FIG. 4, MIN6 cells transfected with miR-483-5p significantly inhibited the expression of beta cell-specific marker molecules (e.g., pdx1, mafA, insulin 1 and Insulin 2) (FIG. 4A), but significantly promoted the expression of beta cell dedifferentiation marker molecules Ngn3, oct4 and Nanog (FIG. 4B).

EXAMPLE 5 investigation of the role of miR-483-5 p-front in the treatment of type 2 diabetes islet beta cell dedifferentiation

In order to investigate whether the expression of miR-483-5p can relieve the dedifferentiation of type 2 diabetes islet beta cells in vivo, miR-483-5p front was designed, injected into high-fat diet mice, and the phenotype thereof was monitored.

Experimental animals: male C57BL/6J mice fed with high fat for 12 weeks were purchased from Nanjing university model animal house, and all experimental animals were housed in Nanjing university laboratory animal center barrier facility.

The experimental method comprises the following steps:

1) Male C57BL/6J mice fed with high fat for 12 weeks were randomly divided into three groups of 6 mice each. AAV-mmu-miR-483-5 p-spike (miR-483-5 p-spike group) or AAV-mmu-NC-GFP (GFP group) is injected into mice by tail vein or intraperitoneal injection or pancreatic in situ injection (pancreatic in situ injection method is mainly described herein), 100 μl/dose is 2 x 10≡8TU/ml, and the other group is a sham operation group (Blank group). After the mice recover, the mice continue to be fed with high-fat feed.

2) After 4 weeks of high fat feeding, mice were subjected to IPGTT assay;

3) After 6 weeks of high fat feeding, mice were tested for insulin secretion 0min after intraperitoneal glucose injection. And the mice were sacrificed to extract islets of the mice, and the expression of Pdx1, mafA, instrin 1 and instrin 2 was detected by fluorescent quantitative PCR, while the expression of dedifferentiating marker molecules Ngn3, oct4 and Nanog was detected. The specific primer sequences are shown in Table 1.

The results are shown in FIG. 5 and FIG. 6, and there was no significant difference in body weight between mice injected with GFP and miR-483-5p spike groups (FIG. 5A); however, compared to GFP group, mice injected with miR-483-5p spike group had significantly improved glucose tolerance at 15, 30, 60min (fig. 5B); also, insulin secretion was significantly increased at 30min after glucose stimulation in mice of miR-483-5p spike group compared to GFP group (fig. 5C). In agreement with this, expression of Pdx1, mafA, instein 1 and instein 2 was significantly upregulated in islets of mice injected with miR-483-5D spike group (fig. 6A), while expression of dedifferentiating marker molecules Ngn3, oct4 and Nanog was significantly decreased (fig. 6B) compared to GFP group.

In conclusion, the invention discovers that the expression of miR-483-5p in serum and islets of type 2 diabetes patients and type 2 diabetes mice is obviously increased. And miR-483-5p in pancreatic islet of a type 2 diabetes mouse is inversely related to the expression of a pancreatic islet beta cell specific marker molecule, and is positively related to the expression of a beta cell dedifferentiation marker molecule. Overexpression of miR-483-5p promotes dedifferentiation of islet beta cells. Meanwhile, by using high fat to feed mice, through pancreas in-situ injection technology, AAV-mmu-miR-483-5 p-spike (miR-483-5 p spike group) or AAV-mmu-NC-GFP is injected and high fat feeding is continued, and through continuous monitoring of metabolic phenotype of the mice, the miR-483-5p spike is found to be capable of effectively relieving symptoms of high fat diet induced impaired glucose tolerance of the mice, up-regulating expression of Pdx1, mafA, insulin 1 and Insulin2 in islet beta cells of the mice, and down-regulating expression of dedifferentiation marker molecules Ngn3, oct4 and Nanog.

Sequence listing

<110> university of Nanjing medical science

<120> miR-483-5p cavernous body and application thereof in preparation of drug for inhibiting type 2 diabetes beta cell dedifferentiation

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 152

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

ctcccttctc ttctcccgtc ttcttcctcc cttctcttct cccgtcttct tcctcccttc 60

tcttctcccg tcttcttcct cccttctctt ctcccgtctt cttcctccct tctcttctcc 120

cgtcttcttc ctcccttctc ttctcccgtc tt 152

Claims

The application of miR-483-5p cavernous body in preparing a medicament for inhibiting type 2 diabetes beta cell dedifferentiation is characterized in that the nucleotide sequence of miR-483-5p cavernous body is shown in SEQ ID NO: 1.
2. Use according to claim 1, characterized in that: the miR-483-5p cavernous body is effectively connected with an expression vector.
3. Use according to claim 2, characterized in that: the effective connection refers to the connection of miR-483-5p cavernous body and an expression vector, so that the generated nucleic acid construct can transcribe the miR-483-5p cavernous body in a cell or an animal body.
4. Use according to claim 2, characterized in that: the expression vector is an adeno-associated virus expression plasmid.
5. Use according to claim 4, characterized in that: the adeno-associated virus expression plasmid is pHBAAV-CMV-MCS-T2Am-zsgreen.
6. Use according to claim 3, characterized in that: the nucleic acid construct synthesizes a forward sequence and a reverse sequence by adding enzyme cutting sites matched with an expression vector at two ends of a miR-483-5p cavernous sequence so as to be effectively connected with the expression vector.
7. Use according to claim 3, characterized in that: the preparation method of the nucleic acid construct comprises the following steps:

(1) Selecting an adeno-associated virus vector pHBAAV-CMV-MCS-T2Am-zsgreen; the competent cell is selected from Escherichia coli strain DH5 alpha; resistance: amp;

(2) Synthesizing miR-483-5p cavernous body aiming at a target gene, constructing an adeno-associated virus expression vector, and amplifying the adeno-associated virus vector; and (3) carrying out mass packaging of adeno-associated virus vectors in 293T cells, concentrating and purifying adeno-associated viruses, and finally measuring the titer of the adeno-associated viruses.