CN112294835B

CN112294835B - Application of LncRNA-266 in preparation of drug for inducing differentiation of brown adipocytes

Info

Publication number: CN112294835B
Application number: CN201911124258.1A
Authority: CN
Inventors: 孙诚; 汤欣; 马谨瑜; 刘晓宇
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2021-09-17
Anticipated expiration: 2039-11-15
Also published as: AU2020384026B2; WO2021093401A1; AU2020384026A1; CN112294835A

Abstract

The invention relates to a medical application of long-chain non-coding RNA (LncRNA-266), namely the application of the LncRNA-266 in preparing a drug for inducing differentiation of brown fat cells, which can promote energy consumption of organisms and resist obesity. The result of the invention that the LncRNA-266 can promote the beige fat cells of the obese mice to differentiate into brown fat cells and consume the energy of the body, thereby inhibiting the weight increase. The LncRNA-266 can reduce the blood sugar level of obese mice and improve insulin sensitivity.

Description

Application of LncRNA-266 in preparation of drug for inducing differentiation of brown adipocytes

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of LncRNA-266 in preparation of medicines for inducing differentiation of brown adipocytes and promoting energy metabolism of organisms.

Background

Obesity is a physiological state of disorder of energy metabolism of the body, and is represented by a large accumulation of excess energy in the body in the form of fat. Obesity is a serious threat to human health and can induce a variety of metabolic diseases, such as fatty liver, type 2 diabetes, cardiovascular disease, certain types of cancer, and the like. Adipose tissues of the body are mainly classified into two types, i.e., white adipose tissues and brown adipose tissues. In terms of energy metabolism, white adipose cells contain a large amount of triglycerides, and thus white adipose tissues are mainly used as organs for storing body energy; in contrast, brown adipocytes contain a large number of mitochondria and highly express the uncoupling protein UCP1, and can convert stored energy into heat energy by uncoupling to be dissipated, so BAT is considered as an energy-consuming organ. Therefore, it is reasonable to speculate that increasing BAT thermogenesis in humans by certain methods would undoubtedly delay weight gain and reduce the development of obesity.

Long non-coding RNAs (LncRNAs) refer to RNA molecules with a length of more than 200bp and without the function of coding proteins. These LncRNAs were early thought to be only byproducts of gene transcription and have no specific physiological function. However, recent studies have found that many LncRNAs have very important physiological functions, and abnormal levels thereof can cause developmental defects and the occurrence and development of various diseases.

Disclosure of Invention

The invention aims to provide medical application of long non-coding RNA (LncRNA-266), and the specific technical scheme is as follows:

the LncRNA-266 is applied to the preparation of drugs for inducing the differentiation of brown adipocytes, and the cDNA sequence of the LncRNA-266 is shown as SEQ ID No: 1 is shown.

LncRNA-266 can induce beige adipocyte to differentiate into brown adipocyte, thereby promoting the energy metabolism of the organism and inhibiting the weight increase.

Furthermore, LncRNA-266 can reduce the blood sugar level of the body and improve the insulin sensitivity.

Another object of the present invention is to provide an agent for inducing differentiation of adipocytes, which comprises LncRNA-266 or a vector expressing LncRNA-266.

The vector is selected from one or more of plasmids, expression frames, viruses and cells.

The medicine can induce the beige fat cells to differentiate into brown fat cells, consume body energy and inhibit body weight increase.

The medicine can reduce blood sugar level of organism and improve insulin sensitivity.

The present invention investigated the induction of beige fat browning by LncRNA-266. The constructed adenovirus for expressing LncRNA-266 (Ad-LncRNA-266) is used for transducing precursor adipocytes, and then the constructed adenovirus for expressing LncRNA is used for directionally inducing the brown adipocytes, and oil red staining and qRT-PCR results show that the LncRNA-266 can remarkably induce the precursor adipocytes to differentiate into the brown adipocytes, and the brown adipocyte specific gene UCP1 also has a high expression tendency. Furthermore, the invention constructs the interference virus (Ad-LncRNA-266shRNA) thereof, and the constructed interference virus is used for transducing the precursor adipocyte, so that the result shows that the reduction of the expression of the LncRNA-266 can inhibit the differentiation of the precursor adipocyte to the brown adipocyte, and the UCP1 gene expression is in a descending trend, thereby confirming the promotion effect of the LncRNA-266 in the process of inducing the differentiation of the precursor adipocyte to the brown adipocyte. In the invention, the adenovirus (Ad-LncRNA-266) for expressing LncRNA-266 is directly injected into the inguinal beige adipose tissue of an obese mouse (a type II diabetes model mouse) on the level of living animals. After 3 weeks of virus injection, the inguinal beige adipose tissue was found to be transformed into brown adipose tissue, and the expression of UCP1 was also significantly increased. In addition, the LncRNA-266 can obviously reduce the blood sugar and serum insulin level of the mice of the type II diabetes mellitus model, and obviously improve the glucose tolerance and the insulin tolerance of obese mice, which shows that the LncRNA-266 can improve the glucose clearance in the mice of the type II diabetes mellitus model and can improve the insulin sensitivity.

Drawings

FIG. 1 shows that LncRNA-266 induces differentiation of precursor adipocytes to brown fat. FIG. 1A shows the results of oil red staining of adipocytes; FIG. 1B shows the quantitative PCR detection of UCP1 gene expression level.^***p<0.001, Student's t test analysis.

FIG. 2 is a graph showing that interference with LncRNA-266 expression inhibits differentiation of precursor adipocytes into brown adipocytes. FIG. 2A shows oil red staining of adipocytes; FIG. 2B shows the quantitative PCR detection of UCP1 gene expression level.^**p<0.01, Student's t test analysis.

FIG. 3 is a graph showing the effect of LncRNA-266 on blood glucose levels in type II diabetic mice.^**p<0.01, Student's t test analysis.

FIG. 4 is a graph showing the effect of LncRNA-266 on serum insulin levels in diabetic mice.^*p<0.05, Student's t test analysis.

FIG. 5 is a graph showing the effect of LncRNA-266 on glucose tolerance in type II diabetic mice.^**p<0.01，^***p<0.001, Student's t test analysis.

FIG. 6 shows the effect of LncRNA-266 on insulin resistance in type II diabetic mice.^*p<0.05，^***p<0.001, Student's t test analysis.

FIG. 7 shows that LncRNA-266 treatment promoted the conversion of mouse inguinal beige adipose tissue to brown adipose tissue. FIG. 7A is a fat morphology map; FIG. 7B shows the quantitative PCR detection of UCP1 gene expression level.^***p<0.001, Student's t test analysis.

Detailed Description

The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The invention is described in further detail below with reference to specific examples and data, it being understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Example 1 LncRNA-266 overexpression and interfering Virus construction

Preparation of over-expressed virus: the overexpression viruses in this study were constructed using the Gateway series kit from Invitrogen. Firstly, the complete sequence of LncRNA is amplified by PCR, then the LncRNA is connected into an intermediate Vector pENTR3C-Entry Vector and sent to a company for sequencing, the Vector with correct sequencing is connected into a target Vector (pAd-CMV-DEST Vector) by a recombination method (LR), after the sequencing is detected to be correct, a positive plasmid is extracted, and the positive plasmid is singly cut by endonuclease Pac I and then transfected into 293A cells by Lipofectamine 2000 liposome of Invitrogen. Observing the formation condition of virus spots for about one week, collecting 293A cells after most virus spots appear, performing repeated freeze thawing at-80 ℃ and 37 ℃ to break the cells, centrifugally collecting supernate to obtain first-generation viruses, continuously infecting the 293A cells with the first-generation viruses to obtain second-generation viruses by the same method, and storing the second-generation viruses in a low-temperature refrigerator at-80 ℃ for subsequent experiments after virus titer detection. The control virus was an adenovirus expressing LacZ (Ad-LacZ). The plasmid for constructing the virus was pAd/CMV/V5-GW/LacZ supplied by Invitrogen corporation, and 293A cells were transfected after Pac I single-excision, and the transfection and virus preparation procedures were the same as described above.

Preparing interfering virus: the method comprises the steps of designing shRNA by using online software of Invitrogen, synthesizing single-stranded DNA by using the shRNA, annealing to form double-stranded DNA, connecting the double-stranded DNA to an intermediate Vector BLOCK-iT U6 RNAi Entry Vector, then sending the double-stranded DNA to a company for sequencing, connecting the Vector with correct sequencing to a target Vector (pAd/BLOCK-iT-DEST Vector) by a recombination method (LR), preparing positive plasmids after correct sequencing detection, performing single cutting by using endonuclease Pac I, and transfecting 293A cells by using Lipofectamine 2000 liposome of the Invitrogen. Virus collection and amplification was as described previously. The control virus is Ad-NC shRNA, and the construction method is the same as that of the control virus.

Example 2 Induction of differentiation of precursor adipocytes by LncRNA-266

Primary preadipocyte culture: 8 week old Male C57BL/6J mice, inguinal white adipose tissue was collected and placed in a centrifuge tube, collagenase digest (collagenase 1mg/ml dissolved in separation buffer- -containing 0.123M NaCl,5mM KCl,1.3mM CaCl)₂5nM glucose, 100mM Hepes, 4% BSA) was digested in a water bath at 37 ℃ for 30min, shaken once at 5min intervals, filtered through a 100 μm nylon mesh, centrifuged, 200g × 5 min. Remove the supernatant, add 2ml separation buffer to fully suspend the cells, centrifuge, 200g × 5 min. Removing supernatant, adding 2ml culture solution (DMEM high sugar medium, 20% fetal calf serum, 20mM Hepes,100U/ml penicillin/streptomycin), suspending cells thoroughly, plating in a culture dish, and standing at 37 deg.C and 5% CO₂Culturing in an incubator.

Directionally inducing and differentiating brown fat cells: to examine the transformation of LncRNA-266 into brown adipocytes with adipogenic precursor cells, 1X 10 cells were added to the precursor adipocytes before the induction medium was added, respectively⁸PFU overexpression virus (Ad-LncRNA-266) or interference virus (Ad-LncRNA-266shRNA) treatment, and after 24 hours, the culture medium is sucked up and changed to brown adipocyte induction medium. The induction medium comprises DMEM high-sugar medium, 10% fetal bovine serum, 20mM insulin, 1nM T3, 0.5mM IBMX, 125. mu.M indomethacin, and 1. mu.M dexamethasone. Rosiglitazone was added 2 days after induction. After 2 days, the cells were induced with basal induction medium (DMEM high-glucose medium, 10% fetal bovine serum, 20mM insulin, 1nM T3) for 3-4 days, and fresh medium was changed every other day.

(1) Oil red dyeing

After the cell treatment is finished, removing the supernatant culture medium, adding PBS for washing once, removing the PBS, adding 4% paraformaldehyde for fixing, and keeping the room temperature for 20-30 min. Subsequently, the cells were incubated on a low speed shaker for 50-60min (room temperature) after washing 1-2 times with PBS, PBS was completely removed, oil red working solution was added and washed 3 times with PBS, and the results were photographed by an inverted microscope, as shown in fig. 1A and 2A, where the results of fig. 1A indicate that LncRNA-266 significantly increased the accumulation of lipid droplets in the cells, indicating the induced differentiation of precursor adipocytes into brown adipocytes. Fig. 2A shows that down-regulation of LncRNA-266 reduces lipid droplet accumulation in cells, indicating that induced differentiation of precursor adipocytes into brown adipocytes is inhibited.

(2) Quantitative PCR detection of gene expression level

Total cellular RNA was extracted using Trizol reagent (Invitrogen) and transcribed into cDNA using a cDNA synthesis kit (Bio-Rad). Using this cDNA as a template, the gene expression level was analyzed by SYBR Green Supermix (Bio-Rad). The Detection apparatus is an iQ5Multicolor Real-Time PCR Detection System (Bio-Rad). Results the 2- Δ Ct method calculates mRNA levels. mRNA levels were calibrated with the 18S housekeeping gene. The primer sequences used were as follows:

18S rRNA forward primer 5'-AGTCCCTGCCCTTTGTACACA-3' (SEQ ID NO: 2);

18S rRNA negative primer 5'-CGTTCCGAGGGCCTCACT-3' (SEQ ID NO: 3);

UCP1 forward primer 5'-AGGCTTCCAGTACCATTAGGT-3' (SEQ ID NO: 4);

UCP1 negative primer: 5'-CTGAGTGAGGCAAAGCTGATTT-3' (SEQ ID No: 5).

The qRT-PCR result is shown in figures 1B and 2B, the result in figure 1B shows that LncRNA-266 can remarkably induce the expression increase of UCP1 specific gene of brown adipocyte, and the result in figure 2B shows that the expression of LncRNA-266 can be remarkably reduced to reduce the expression of UCP1 gene. These results suggest that LncRNA-266 can significantly induce differentiation of precursor adipocytes to brown adipocytes.

Example 3 Effect of LncRNA-266 on sugar metabolism in type II diabetic mice

Male C57BL/6J mice, 6 weeks old, were fed on a high fat Diet (Research Diet, 45% carbohydrates from fat) for 90 days to induce obese model mice (i.e., type II diabetes model mice). The LncRNA-266-expressing adenovirus (Ad-LncRNA-266) was directly injected into the beige adipose tissue of the groin of type II diabetes model mice at a dose of 40. mu.l virus (1X 10) per mouse at the unilateral groin¹²PFU). Control mice were injected with the same dose of control virus(Ad-LacZ). After 3 weeks of virus injection, various physiological indexes are detected.

After fasting for 12 hours, the mice were bled using the tail vein, and the blood glucose level was quantitatively measured with a glucometer (Bayer, Mishawaka, IN), and the results are shown IN fig. 3. The results in FIG. 3 show that LncRNA-266 treatment decreased blood glucose levels in type II diabetes model mice.

After fasting the mice for 12 hours, blood was taken from the tail vein, and serum insulin levels were measured using a mouse serum insulin level kit (Rat/mouse insulin ELISA kit, Mercodia,2758702), and the results are shown in fig. 4. The results in FIG. 4 show that LncRNA-266 decreased serum insulin levels and improved hyperinsulinemia in type II diabetic mice.

Mice were fasted overnight (from 8pm to 8am the next day) and were injected intraperitoneally with D-glucose (0.5 g/kg). The blood glucose levels at the time points were measured with a glucometer (Bayer) at 0, 15, 30, 60, and 90min after the glucose injection, and the glucose tolerance was examined, and the results are shown in fig. 5. The results in FIG. 5 show that LncRNA-266 treatment significantly promoted the ability of type II diabetic model mice to eliminate glucose from peripheral blood.

Mice were fasted for 6 hours (from 8am to 2pm) and were injected intraperitoneally with recombinant human insulin (0.75IU/kg) (purchased from Eli Lilly, Indianapolis, IN). The blood glucose levels at the time points were measured with a glucometer (Bayer) at 0, 15, 30, 60, and 90min after the glucose injection, and the insulin resistance test was examined, and the results are shown in fig. 6. The results in FIG. 6 show that LncRNA-266 treatment enhances the insulin response and insulin sensitivity in type II diabetes model mice.

At the end of the experiment, the mice were sacrificed after anaesthesia, the mice were dissected rapidly and inguinal fat was extracted for the following tests. The general morphology of the abdominal femoral adipose tissue is shown in fig. 7A. The results in FIG. 7A show that LncRNA-266 treatment significantly darkens the inguinal adipose tissue; fig. 7B is a quantitative PCR assay, and the results indicate that LncRNA-266 significantly increased the level of UCP1 gene expression in abdominal femoral adipose tissue. Taken together, the results in FIG. 7 show that LncRNA-266 treatment promotes the conversion of mouse inguinal beige adipose tissue to brown adipose tissue.

Sequence listing

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Claims

The LncRNA-266 is applied to the preparation of drugs for inducing the differentiation of brown adipocytes, and the cDNA sequence of the LncRNA-266 is shown as SEQ ID No: 1 is shown.
2. The use of claim 1, wherein the LncRNA-266 induces differentiation of beige adipocytes to brown adipocytes, thereby promoting energy metabolism in the body.
3. The use of claim 1, wherein the LncRNA-266 inhibits weight gain.
4. The use of claim 1, wherein the LncRNA-266 decreases blood glucose levels in the body and increases insulin sensitivity.
5. An agent for inducing differentiation of brown adipocytes, comprising LncRNA-266 or a vector expressing LncRNA-266, wherein the cDNA sequence of LncRNA-266 is as set forth in SEQ ID No: 1 is shown.
6. The medicament according to claim 5, wherein the vector is selected from one or more of a plasmid, an expression cassette, a virus and a cell.