CN113604471A - Recombinant miR-218, production method thereof and application thereof in hair regeneration - Google Patents
Recombinant miR-218, production method thereof and application thereof in hair regeneration Download PDFInfo
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Abstract
The invention discloses a recombinant miR-218, wherein the sequence of the recombinant miR-218 is shown in SEQ ID NO: 1 or a sequence similar to SEQ ID NO: 1 with a sequence similarity of more than 90%. In addition, the invention also discloses a production method of the recombinant miR-218 and an application of the recombinant miR-218 in hair regeneration. The invention adopts tRNA as a bracket, is embedded with a miR-218 sequence, and expresses recombinant miR-218 in escherichia coli. The recombinant miR-218 prepared by the invention is externally sprayed on the back of a unhaired C57BL/6 mouse. The results prove that the recombinant miR-218 can remarkably promote hair regeneration. The recombinant miR-218 prepared by the invention is obtained by a bioengineering technology, and has the advantages of high biological activity, convenient equipment, fast production, high yield, low cost, good functionality and the like.
Description
Technical Field
The invention belongs to the technical field of molecular biology and medicine, and particularly relates to recombinant miR-218, a production method thereof and application thereof in hair regeneration.
Background
Hair disorders are clinically common dermatological disorders, especially alopecia. In recent years, the incidence of the disease tends to rise year by year along with the acceleration of the life rhythm of people and the change of the dietary structure. The pathogenesis of hair loss is not completely understood, and it is now believed that it may be the result of the interaction of the genetic background with the external environment, associated with autoimmune disorders, excessive mental stress and physical fatigue, and possibly with disorders in the growth cycle of the hair follicle and the initiation of degenerative changes in the hair follicle, for which no effective prevention or treatment has been available until now. People affected by moderate hair loss are turning to topical treatments such as minoxidil (anti-hypertensive potassium channel opener) and finasteride (treating prostatic hyperplasia), which are not very effective because neither is designed for hair loss treatment, but also require constant re-application by the user to maintain hair growth.
Micro-rna (mirna) is present in all metazoan genomes and plays a role in mRNA degradation and gene regulation, playing a key role in many biological processes. In the biology of skin and hair follicles, researchers are concerned with the role of mirnas in skin morphogenesis and differentiation, Hair Follicle (HF) development, hair cycle, and hair pigmentation. Hair follicles are a complex biological system involving dynamic processes that are genetically regulated. Research shows that miR-218 positively regulates Wnt signal channel by targeting SFRP2, and plays a dynamic regulation role in the development process of skin and hair follicle. SFRP2 plays a central role in the dermal papilla of hair follicles, and Wnt3a can be enhanced by the Wnt/β -catenin signaling pathway to increase the activity of cultured human dermal papilla cells; SFRP2 is also a key factor in maintaining the physiological function of hair follicle development.
The application development of new drugs or cosmetics of the RNA class and the functional study of RNA have been the focus on the acquisition of RNA raw materials. RNA is currently synthesized mainly by chemical or in vitro transcription. The RNA produced by the synthesis methods is expensive and low in yield, and may have more artificial gene modifications for improving stability, so that RNA folding, biological activity and safety are affected. Therefore, the intervention of new technology for biosynthesis of recombinant RNA reduces the cost of RNA raw materials, and simultaneously improves the bioactivity and safety of RNA. The production and expression of small RNA molecules in living cells by utilizing endogenous recombinant tRNA scaffolds are applied to a plurality of research fields and are well developed.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a recombinant miR-218 and a production method and application thereof aiming at the defects of the prior art. The human serine tRNA chimeric has-miR-34a precursor and the human serine tRNA is human source tRNA, so that the development prospect of the recombinant small RNA produced by using the human serine tRNA chimeric has better prospect, and because human cells contain serine tRNA but do not contain bacterially derived methionyl tRNA, the toxicity and immunogenicity of the human serine tRNA used as a scaffold are probably lower. The invention shortens the length of the expressed recombinant tRNA, thereby reducing the cytotoxicity and improving the expression quantity. The sequence in the has-miR-34a precursor is modified, complementary pairing is improved, and the stability of the recombinant miR-218 is improved. The recombinant miR-218 obtained through bioengineering has good biological activity, and the miR-218 activates the effects of skin development, hair follicle growth and hair cycle through the positive regulation of a Wnt/beta-catenin signal channel.
In order to solve the technical problems, the invention adopts the technical scheme that: the recombinant miR-218 is characterized in that the sequence of the recombinant miR-218 is shown in SEQ ID NO: 1 or a sequence similar to SEQ ID NO: 1 with a sequence similarity of more than 90%.
In addition, the invention also provides a production method of the recombinant miR-218, which is characterized by comprising the steps of embedding the designed recombinant miR-218 sequence into a tRNA stent and performing recombinant expression in Escherichia coli.
The production method is characterized in that the sequence of the tRNA scaffold is a sequence with similarity of more than 90% with the sequence of human serine tRNA, and the sequence of the human serine tRNA is shown in SEQ ID NO: 2, respectively.
The production method is characterized by comprising the following specific steps:
step one, designing and synthesizing hsa-miR-34a recombination sequence of a chimeric miR-218 sequence;
step two, inserting the recombination sequence in the step one into pBSMrnaSeph plasmid by utilizing the enzyme cutting site of the pBSMrnaSeph plasmid at the tRNA anticodon ring to construct an expression vector;
step three, transforming the expression vector of the chimeric target sequence into competent escherichia coli;
and step four, after escherichia coli is cultured and amplified, extracting total RNA in the bacteria, and separating and purifying the target recombinant miR-218 by FPLC.
Further, the invention provides an application of the recombinant miR-218 in preparation of a prodrug, a medicine, a raw material medicine or a medicine combination for promoting hair regeneration and/or treating alopecia, and an application in preparation of a product for nourishing, growing or caring hair.
Compared with the prior art, the invention has the following advantages:
1. the sequence in the has-miR-34a precursor is modified, complementary pairing is improved, and the stability of the recombinant miR-218 is improved.
2. The human serine tRNA chimeric has-miR-34a precursor and the human serine tRNA is human source tRNA, and the development prospect of the recombinant small RNA produced by using the human serine tRNA chimeric has better prospect, because human cells contain serine tRNA, but no methionine tRNA derived from bacteria, the human serine tRNA used as the scaffold has lower toxicity and immunogenicity.
3. The miR-218 with biological activity, which is expressed by using a tRNA (ribonucleic acid) bracket (OnRS), has the advantages of simple production process, high yield, high cost performance and high safety.
4. The recombinant miR-218 designed and prepared by the invention has better biological activity, and the miR-218 activates the functions of skin development, hair follicle growth and hair cycle through the positive regulation of a Wnt/beta-catenin signal channel, can promote the regeneration of hair follicle stem cells, promotes the growth of hair and plays a role in hair growth.
The technical solution of the present invention is further described in detail with reference to the accompanying drawings and embodiments.
Drawings
FIG. 1 shows the identification and detection of the expression of recombinant mir-218 in Escherichia coli by denaturing polyacrylamide gel electrophoresis in example 1 of the present invention.
FIG. 2 is a diagram showing the identification of the purity of the collected fraction by denaturing polyacrylamide gel electrophoresis in this example
FIG. 3 is a FPLC chromatogram of the present invention for the purification of recombinant miR-218 using the Bio-Rad NGCTM Chromatography System.
Fig. 4 is a graph showing the results of experiments in which the hair growth on the back of mice was continuously monitored for 20 days, which was performed in the experiment of treating C57BL6 mice with depilatory on the back by means of the spray method for external use in example 3 of the present invention.
Fig. 5 is a statistical chart of data analysis of the results of a back hair growth experiment of mice photographed 20 days continuously, which was performed in a treatment experiment of a back depilated C57BL6 mouse using an external spray method in inventive example 3.
Detailed Description
Embodiments of the present invention are illustrated below by specific examples, and unless otherwise indicated, the experimental methods disclosed in the present invention are all performed by conventional techniques in the art.
Example 1: and (3) constructing a recombinant miR-218 plasmid by using a pBSKrnaSeph/has-miR-34a expression vector, and expressing the recombinant miR-218.
(1) A primer is designed according to a recombinant miR-218 sequence (SEQ ID NO: 1) and a sequence on a pBSKrnaSeph/has-miR-34a expression vector, is named miR-34 a/recombinant miR-218, and homologous sequences on two sides of a 1-15nt vector insertion site are added to two ends of the primer. Designing and synthesizing hsa-miR-34a recombination sequence of the chimeric miR-218 sequence.
TABLE 1miR-34a/miR-218 primer
(2) Synthesis of the insert
Two primers (SEQ ID NO: 3 and SEQ ID NO: 4) are mutually used as templates, a recombination sequence is inserted into the pBSMrnaSeph plasmid by utilizing the enzyme cutting site of the pBSMrnaSeph plasmid at the tRNA anticodon loop, and an expression vector is constructed; the reaction system is shown in table 2, and the reaction process is shown in table 3:
TABLE 2 polymerase in vitro amplification chain reaction System (50. mu.L)
TABLE 3 polymerase in vitro amplification Strand reaction Process
(3) Double enzyme digestion of pBSKrnaSeph/has-mir-34a vector
The vector was digested with Eag I-HFTM, Sac II restriction enzymes at 37 ℃ in the reaction system shown in Table 4.
TABLE 450 μ L double enzyme digestion System
(4) Recovery and purification of enzyme digestion plasmid and PCR fragment
The PCR product and the digested plasmid were identified by agarose Gel electrophoresis, and recovered and purified using an OMEGA Gel Extraction Kit (OMEGA).
(5) Ligation of the insert to the vector
For recovering fragments from glue
Ligation was performed by Ligation using the Ligation-Free Cloning System, and the reaction System is shown in Table 5.
TABLE 5 seamless ligation reaction System (20. mu.L)
Mixing, and incubating at 37 deg.C for 30 min; transforming escherichia coli HST08 competent bacteria; ampicillin resistance screening was performed on the cloned colonies.
(6) DNA sequencing identification recombinant miR-218 expression vector
Single colonies were picked and cultured in LB medium containing ampicillin for about 3 h. 100 mu L of the bacterial solution is delivered to the department of Ongjingke Biotech Co., Ltd, and DNA sequencing identification is carried out by using a sequencing primer.
Example 2: expression of recombinant miR-218
(1) After 200ng of the recombinant miR-218 expression plasmid is transformed into HST08 competent bacteria, 5mL of LB medium is added, and shaking culture is carried out at 37 ℃ and 200rpm overnight. The bacterial liquid is centrifuged for 2min at 10000g, and then the precipitate is collected. The precipitate was resuspended in 180. mu.L of 10mM magnesium acetate-Tris-HCl solution, followed by addition of 200. mu.L of saturated phenol and shaking at room temperature for 20-60 min. Centrifuging at 10000g for 10min, collecting water phase, and adding 5M NaCl with 0.1 time of the volume of the water phase to precipitate macromolecular impurities. Adding 2 times volume of anhydrous ethanol into the supernatant, centrifuging at 10000g for 10min, and removing the supernatant. Absorbing residual ethanol with absorbent paper, adding DEPC water to dissolve RNA after the RNA is dried, measuring the concentration, and storing in a refrigerator at-80 ℃.
(2) Modified polyacrylamide gel electrophoresis identification
Mu.g of RNA sample was mixed with 2 XRNA loading buffer and added to the denatured gel sample wells. And (3) after electrophoresis at 120-150V for 40-60 min, putting the mixture into a solution containing 0.5 mu g/mL ethidium bromide, slightly shaking for 20-30 min, observing the mixture under a gel imaging system, and taking a picture for storage.
FIG. 1 shows the detection of the expression of recombinant miR-218 in Escherichia coli by denaturing polyacrylamide gel electrophoresis in this example. In the figure, M represents RNA marker; 1 represents wild type HST08 e.coli total RNA; 2 represents total RNA after HST08 E.coli transformed by recombinant miR-218 expression plasmid. Compared with the total bacterial RNA of the untransformed recombinant miR-218 expression plasmid, the total bacterial RNA after transformation has one more band between 150 and 300 nt. The result shows that the recombinant miR-218 expression plasmid can highly express the recombinant miR-218 in escherichia coli.
Example 3: HPLC purification of recombinant miR-218
(1) Recombinant miR-218 was purified on an ion exchange Column (ENrichTMQ 10X 100Column) using a Bio-Rad NGCTM Chromatography System.
Mobile phase A: 10mM NaH2PO4 solution, pH 7.0. Mobile phase B: 10mM NaH2PO4Solution, 1M NaCl solution, pH 7.0.
The flow rate was 2.0 mL/min. The column was washed alternately with DEPC water, mobile phase A, and mobile phase B for about 1 h. 5 column volumes were washed each time.
The RNA was detected by absorbance at 260nm and the peak corresponding to the recombinant RNA was collected. The purity was verified by denaturing polyacrylamide gel electrophoresis.
(2) RNA sample processing method
Total RNA extraction procedure was as above. The extracted total RNA is centrifuged at 13000rpm at 4 ℃ for 10min, and the supernatant is filtered by a 0.22 mu m microporous filter membrane, and then 5-10mg of the total RNA is injected each time.
(3) HPLC fraction collection and concentration desalination
The purity of the collected fractions was verified by denaturing polyacrylamide gel electrophoresis. The mixed components were subjected to 2-fold volume of absolute ethanol to precipitate RNA, and the mixture was stored in a refrigerator at-80 ℃ for about 1 hour. The RNA was collected by centrifugation at 10000g for 10min at 4 ℃. The resulting RNA pellet was dissolved in DEPC water, centrifuged at 7500g at 4 ℃ for 10min with tra-2mL Centrifugal Filters, the filtrate was removed, the procedure was repeated until all solutions were centrifuged, Filters were inverted and centrifuged at 2000g for 2min, the resulting solution was collected, the concentration was determined and stored at-80 ℃.
FIG. 2 shows the purification of recombinant miR-218 by means of Bio-Rad NGCTM Chromatography System in this example, and the purity of the collected fractions was verified by denaturing polyacrylamide gel electrophoresis. The result shows that the high-purity recombinant miR-218 can be obtained after HPLC purification.
FIG. 3 is an HPLC chromatogram for the purification of recombinant miR-218 using the Bio-Rad NGCTM Chromatography System of the present invention. The target RNA was well separated from other endogenous RNAs in the total bacterial RNA and the expression of the target RNA was shown to be greater than 15% in the total bacterial RNA by peak area integration.
Example 4: experiment of recombinant miR-218 on hair growth of back epilation C57BL6 mouse
The experiment is divided into: blank control group (PBS solution) and treatment group (recombinant miR-218). 30 healthy C57BL6 male mice of 3 months of age were purchased as experimental animals, and 10 mice were randomly selected per group. Selecting skin parts on two sides of the back spine to remove hairs under the condition of not damaging epidermis. After the molding is finished, the medicine is administrated according to different groups. The blank control group is sprayed with PBS, the treatment group is sprayed with recombinant miR-218 solution, the dosage of each mouse is 1mL, the solution is sprayed on the back unhaired skin of the experimental animal and then massaged for 1 minute to promote skin absorption, the concentration is 10 mug/mL for 1 time every day, the treatment period lasts for 20 days, and a camera is used for taking pictures every day.
As shown in FIG. 4, compared with the PBS blank control group, the treatment group of the recombinant miR-218 promotes hair regeneration to different degrees with the increase of time, and the effect is very obvious at 20 d.
Quantification of hair coverage on days 10, 15 and 20, as shown in figure 5. The coverage rate of new hairs of the treatment group of the recombinant mir-218 is more than 60% at 20d, and is obviously different from the coverage rate of the new hairs of the control group of mice. P <0.05, P <0.01, and P < 0.001.
Example 5: therapeutic effect of recombinant miR-218 on alopecia patients
In order to verify the effect of recombinant miR-218 on hair growth, 8 volunteers with alopecia between 30 and 60 years of age were selected. Before use, the scalp of the patient suffering from alopecia is cleaned and dried. According to the area of the alopecia skin, about 1mL of hair restorer is sprayed on the exposed part of the scalp of the alopecia patient, and the hair restorer is dried in the air for 1 time every day. In the process, the sample is absorbed by being matched with massage or other scalp stimulating methods, and the sample stays on the surface of the scalp for more than 8 hours for washing the hair. Spraying is continued for 28 days. Photographs were taken daily under the same conditions and hair changes were recorded.
Hair changes were analyzed using a computer:
the first step is as follows: the areas with similar sizes of the same parts are manually selected (the shooting angles are kept consistent as much as possible) for the next calculation.
The second step is that: the selected area is converted into a grayscale image to avoid color-induced interference.
The third step: the images are normalized to reduce the effect of global illumination. The specific formula is as follows:
the fourth step: the ratio of black pixels (representing hair) in the image is calculated as follows:
result＝1-mean(Xnew)
as shown in the table, 8 subjects, 6 subjects, increased black pixels after use. The effective rate reaches 75 percent.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, changes and equivalent structural changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.
Sequence listing
<110> northwest university of industry
<120> recombinant miR-218, production method thereof and application thereof in hair regeneration
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Claims (5)
1. The recombinant miR-218 is characterized in that the sequence of the recombinant miR-218 is shown in SEQ ID NO: 1 or a sequence similar to SEQ ID NO: 1 with a sequence similarity of more than 90%.
2. A method for producing the recombinant miR-218 of claim 1, which comprises the step of chimeric design of the recombinant miR-218 sequence into a tRNA scaffold, and the recombinant miR-218 sequence is recombinantly expressed in Escherichia coli.
3. The method of claim 2, wherein the tRNA scaffold has a sequence that has a degree of similarity of greater than 90% to a human serine tRNA sequence as set forth in SEQ ID NO: 2, respectively.
4. The production method according to any one of claims 2 to 3, characterized in that the specific steps of the production method comprise:
step one, designing and synthesizing hsa-miR-34a recombination sequence of a chimeric miR-218 sequence;
step two, inserting the recombination sequence in the step one into pBSMrnaSeph plasmid by utilizing the enzyme cutting site of the pBSMrnaSeph plasmid at the tRNA anticodon ring to construct an expression vector;
step three, transforming the expression vector of the chimeric target sequence into competent escherichia coli;
and step four, after escherichia coli is cultured and amplified, extracting total RNA in the bacteria, and separating and purifying the target recombinant miR-218 by HPLC.
5. The use of the recombinant miR-218 of claim 1 in the preparation of a prodrug, a drug substance or a drug combination for promoting hair regeneration and/or treating alopecia, or in the preparation of a hair nourishing, growing or caring product.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180237772A1 (en) * | 2014-05-28 | 2018-08-23 | The Regents Of The University Of California | HYBRID tRNA/pre-miRNA MOLECULES AND METHODS OF USE |
| CN108753780A (en) * | 2018-06-11 | 2018-11-06 | 西安荣清畅生物科技有限公司 | It is a kind of recombination tiny RNA production method and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180237772A1 (en) * | 2014-05-28 | 2018-08-23 | The Regents Of The University Of California | HYBRID tRNA/pre-miRNA MOLECULES AND METHODS OF USE |
| CN108753780A (en) * | 2018-06-11 | 2018-11-06 | 西安荣清畅生物科技有限公司 | It is a kind of recombination tiny RNA production method and application |
Non-Patent Citations (1)
| Title |
|---|
| BOHAO ZHAO ET AL: "miR-218-5p regulates skin and hair follicle development through Wnt/β-catenin signaling pathway by targeting SFRP2", 《J CELL PHYSIOL.》, pages 11 * |
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