CN111411112B - Targeting inhibitor of MAPK6P2 gene and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a targeted inhibitor of MAPK6P2 gene and application thereof. A targeted inhibitor of the MAPK6P2 gene, which has the targeting sequence as follows: 5'-GCGAGTGAAGTTAACCATTAC-3' (SEQ ID No. 1). The MAPK6P2 gene targeted inhibitor is applied to the preparation of medicaments for treating human glioblastoma multiforme. The invention develops a targeting inhibitor of MAPK6P2 gene by using RNA interference technology, the inhibitor can be specifically combined with MAPK6P2 gene to silence MAPK6P2 gene, thereby inhibiting the proliferation, migration and invasion capacity of glioblastoma cells and achieving the purpose of treating glioblastoma. The MAPK6P2 gene targeted inhibitor plays an important role in the field of glioblastoma gene therapy, and provides a new targeted therapeutic drug for clinical therapy of glioblastoma.

Description

Targeting inhibitor of MAPK6P2 gene and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a targeted inhibitor of MAPK6P2 gene and application thereof.

Background

Glioma is the most common primary central nervous system tumor, the annual incidence rate is 3-8 people/10 ten thousand people, and the composition accounts for 45.2% of all malignant central nervous system tumors and 80% of all primary malignant central nervous system tumors. According to the current treatment standard, firstly, the operation resection with the maximum safety range is carried out, then, the operation radiotherapy is carried out, and meanwhile, chemotherapy such as temozolomide and the like are supplemented, and the treatment of anti-angiogenesis medicines is carried out. However, patients with low differentiation and high grade in histopathological diagnosis in glioma have poorer prognosis, and the median survival time of patients with stage IV glioma is only 12-18 months. In recent years, in research on the pathogenesis of glioma, the regulation and control effect of pseudogenes on the biological behavior of glioma cells at the level of transcription, posttranscription and the like is highly emphasized by more researchers. Therefore, the role and mechanism of pseudogene regulation of glioma cell biological behavior are deeply researched, and a new target can be provided for glioma molecular therapy.

Pseudogenes are also called pseudogenes, DNA sequences which are similar to normal genes but lose gene function. It has been thought that it is a non-functional residue formed during the evolution of gene families, and is not generally transcribed, so it has no clear physiological significance and is often present in multigene families of eukaryotes. Recent studies indicate that pseudogenes have functions similar to non-coding RNA, play important molecular regulation and control roles at the level of transcription, posttranscription and the like, participate in the regulation of the occurrence and development of various malignant tumors, and play an important regulation role in tumors of various systems such as the respiratory system, the digestive system, the nervous system and the like of human beings.

The MAPK6P2 gene maps to 21q21.1, and its parent MAPK6 maps to human chromosome 15q 21.2. The coding nucleotide sequence is 4036bp in length. No study report on the relevance of MAPK6P2 in glioma has been retrieved. The principle of RNA interference technology is that Dicer enzyme is utilized to cut RNA molecules to form an RNA silencing complex, and the process of target RNA molecule combination and RNA molecule degradation is targeted, so that the RNA interference technology can play a therapeutic role in medical application. The shRNA is an artificial RNA consisting of two short reverse complementary sequences with a stem-loop interval, and is used as a common RNA interference tool, and the shRNA is combined with target gene mRNA to play a role. Because siRNA has off-target effect or influences normal gene expression, the prior shRNA technology is safer and more reliable.

At present, the role and the related mechanism of MAPK6P2 in the development of tumors are not reported, and the application of MAPK6P2 in glioma gene therapy is not clear at present. Therefore, the development of a MAPK6P 2-related drug is a problem to be solved.

Disclosure of Invention

In view of the problems in the prior art, the invention aims to provide a targeted inhibitor of MAPK6P2 gene and application thereof. Experiments of the invention prove that the MAPK6P2 gene is highly expressed in glioma tissues, and the proliferation, migration and invasion capacities of glioma cells are inhibited by inhibiting the expression of the MAPK6P2 gene. The invention develops a targeting inhibitor of MAPK6P2 gene by RNA interference technology, the inhibitor can be specifically combined with MAPK6P2 gene to silence MAPK6P2 gene, thereby inhibiting the proliferation, migration and invasion capacity of glioma cells and achieving the purpose of treating glioma. The targeted inhibitor of the MAPK6P2 gene plays an important role in the fields of glioma gene therapy and molecular targeted therapy, and provides a new targeted therapeutic drug for clinical treatment of glioma.

In order to achieve the above object, the present invention adopts the following technical solutions.

A targeted inhibitor of the MAPK6P2 gene, which has the targeting sequence as follows: 5'-GCGAGTGAAGTTAACCATTAC-3' (SEQ ID No. 1).

Further, the targeted inhibitor of the MAPK6P2 gene can inhibit shRNA sequence expressed by the MAPK6P2 gene, the shRNA template sequence comprises a sense strand and an antisense strand, and the sense strand and the antisense strand are respectively:

sense strand:

5’-CACCGCGAGTGAAGTTAACCATTACTTCAAGAGAGTAATGGTTAACTTCACTCGCTTTTTTG-3’ (SEQ ID No.2)。

antisense strand:

5’-GATCCAAAAAAGCGAGTGAAGTTAACCATTACTCTCTTGAAGTAATGGTTAACTTCACTCGC-3’ (SEQ ID No.3)。

further, a transcription product for transcribing the shRNA is obtained, and the sequence is as follows:

5’-GCGAGTGAAGTTAACCATTACTTCAAGAGAGTAATGGTTAACTTCACTCGCTT-3’ (SEQ ID No.4)。

preferably, the inhibitor is in the form of an injectable formulation.

Preferably, the inhibitor is in any pharmaceutically therapeutically acceptable dosage form.

Preferably, the inhibitor is in any pharmaceutically therapeutically acceptable dose.

The MAPK6P2 gene targeted inhibitor is applied to the preparation of the drugs for treating human brain glioma.

Compared with the prior art, the invention has the following beneficial effects.

1) The MAPK6P2 gene targeted inhibitor provided by the invention has strong specificity, high silencing efficiency and low off-target rate, and inhibits the expression of the MAPK6P2 gene.

2) The invention relates to a method for inhibiting the expression of human MAPK6P2 gene by an inhibitor of MAPK6P2 gene, thereby inhibiting the proliferation, migration and invasion capabilities of glioma cells and providing a brand-new treatment method for glioma gene therapy.

3) The inhibitor of the MAPK6P2 gene provided by the invention can obviously reduce the drug resistance problem of the traditional therapeutic drugs by targeted therapy.

4) The inhibitor of the MAPK6P2 gene provided by the invention is applied and researched on the in vitro cytology level, and experiments prove that the targeted treatment effect of the inhibitor of the MAPK6P2 gene is exact and no adverse reaction occurs.

Drawings

Fig. 1 is a bar graph of the significant increase in expression levels of the MAPK6P2 gene in glioma tissues and cells using realtome PCR.

Figure 2 is a bar graph of the significant down-regulation of MAPK6P2 expression levels in glioma cells following the application of MAPK6P2 gene inhibitor using realtome PCR.

FIG. 3 is a statistical chart of the ability of CCK-8 cell viability assay to inhibit glioma cell proliferation using MAPK6P2 gene inhibitors.

FIG. 4 is a photograph and a statistical chart of the ability of a Transwell cell migration assay to detect inhibition of glioma cell migration using an inhibitor of the MAPK6P2 gene.

FIG. 5 is a photograph and a statistical chart of the ability of a Transwell cell invasion assay to detect inhibition of glioma cell invasion by the application of MAPK6P2 gene inhibitor.

Detailed Description

The technical solutions and effects of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. The following examples are only preferred embodiments of the present invention and are not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art without departing from the spirit and the principle of the present invention, and any modifications, equivalents, improvements, etc. made within the scope of the present invention are intended to be covered by the present invention.

Example design of shRNA and preparation of interference vector and application of MAPK6P2 gene inhibitor.

Firstly, cell culture.

Human glioma cell lines U87, U251, U373 and Human Astrocytes (HA) were purchased from the Shanghai institute bioscience cell resource center. The cells of U87, U251, U373, and HA were cultured in a 100mm cell culture dish in DMEM high-glucose medium (astrocyte medium AM) containing 10% fetal bovine serum, and the medium was changed every 2 days, and the cells grew to a monolayer in about 2-3 days.

Secondly, real-time quantitative PCR is used for detecting the expression of MAPK6P 2.

1. Total RNA was extracted from the cells by Trizol method.

Washing the collected cells with cold PBS, adding 1ml Trizol reagent, blowing for several times, observing the cells under a mirror to form oil drops (fully cracking), transferring into a 1.5ml EP tube, and standing for 5 minutes to fully crack the cells; adding 0.2ml of chloroform into the sample, manually and violently shaking the sample, and standing the sample for 3 minutes at room temperature; centrifuging at 12000g at 4 deg.C for 15 min, adding the upper water phase into a new EP tube, adding 0.5ml isopropanol, turning upside down, mixing, and standing at room temperature for 10 min; centrifuging at 12000g at 4 deg.C for 15 min, discarding supernatant, and adding 1ml 75% ethanol; after centrifuging at 7500g for 5 minutes at 4 ℃ and drying for 15 minutes, 40. mu.l of DEPC water is added, and the sample can be frozen in a refrigerator at-80 ℃.

2. The expression of MAPK6P2 is detected by a one-step dye method qRT-PCR.

The CT value was determined and the relative expression level of MAPK6P2 was calculated using B-ACTIN as the internal reference and 2^ (Δ Ct). Statistical plots of significantly increased expression levels of the MAPK6P2 gene in glioma tissues and cells were examined, as shown in figure 1.

Thirdly, preparation and application of MAPK6P2 gene inhibitor.

Designing an interference sequence of the MAPK6P2 gene, and selecting an inhibitor targeting sequence which targets the human MAPK6P2 gene and specifically inhibits the expression of the MAPK6P2 gene as follows: 5'-GCGAGTGAAGTTAACCATTAC-3' are provided.

The GCGAGTGAAGTTAACCATTAC sequence is input in the homologous sequence alignment analysis nucleotide blast of NCBI for alignment analysis, and the result shows that the sequence has no high homology with other human mRNA genes and can be used as a specific sequence for specifically interfering MAPK6P2 gene. The shRNA sequence of the targeted inhibitor was further designed to include the sense and antisense strands as follows.

The sense strand sequence of the shRNA is as follows:

5’-CACCGCGAGTGAAGTTAACCATTACTTCAAGAGAGTAATGGTTAACTTCACTCGCTTTTTTG-3’。

the sequence of the shRNA antisense strand is as follows:

5’-GATCCAAAAAAGCGAGTGAAGTTAACCATTACTCTCTTGAAGTAATGGTTAACTTCACTCGC-3’。

the sequence of the transcription product of the shRNA is as follows:

5’-GCGAGTGAAGTTAACCATTACTTCAAGAGAGTAATGGTTAACTTCACTCGCTT-3’。

sequence information corresponding plasmids were designed and synthesized as MAPK6P2 gene inhibitors.

Transfection with MAPK6P2 gene inhibitor: plasmid U6/GFP/Neo of sh-NC and sh-MAPK6P2 enables expression of MAPK6P2 to be silent, and an empty plasmid without MAPK6P2 sequence or shRNA is used as an experimental negative control; culturing glioma cells by using a 24-hole culture plate, and performing transfection when the cell growth reaches about 80%; plasmid, Opti-MEM, required for the preparation of transfections^®I and LTX and Plus reagent (Life Technologies) transfection reagents. Tube A: dissolving a hole according to 1 mu g plasmid DNA in 50 mu l Opti-MEM I +1 mu l p3000 for 5min, placing a tube B: the wells were dissolved in 50 μ l Opti-MEM according to 1 μ l LTX and Plus^®In the step I; evenly mixing A, B two tubes, and standing for 5 min; sucking out the culture solution, adding 100 mul of transfection mixed solution into each hole, and adding 400 mul of EBM-2 culture solution; after 48 h, the selection was carried out in a medium containing the antibiotic G418 at a concentration of 0.4 mg/ml, and the concentration of G418 was increased continuously, so that a glioma cell line capable of stably silencing MAPK6P2 was obtained after about 4 weeks. In subsequent experiments, the groups were divided into 3 groups, each of which was: blank Control (Control) without any treatment; negative control group (sh-NC) transfected with MAPK6P2 silent empty plasmid; experimental group transfected with MAPK6P2 silencing plasmid (sh-MAPK6P 2). Statistical plots of significant down-regulation of MAPK6P2 expression levels in glioma cells following MAPK6P2 gene inhibitor, as shown in figure 2.

Example 2 the use of MAPK6P2 gene inhibitors significantly inhibited the malignant biological behavior of gliomas.

Firstly, a CCK-8 cell viability method is used for detecting the influence of the MAPK6P2 gene inhibitor on the proliferation capacity of glioma cells.

U251 cells were trypsinized and single cell suspensions were prepared separately in normal medium. Cells were counted and seeded at a concentration of 2000 cells/well in 96-well cell culture plates. Each group is provided with 5 multiple holes, and each hole accounts for 200 mu l. After 24h, 10 mul CCK-8 was added. After 2 hours, the light absorption value of each well at a wavelength of 450nm was measured by a microplate reader. A statistical chart of inhibition of U251 cell proliferation by the CCK-8 cell viability assay using MAPK6P2 gene inhibitors is shown in FIG. 3.

Secondly, a Transwell cell migration experiment detects the influence of the MAPK6P2 gene inhibitor on the migration capacity of glioma cells.

Detecting the migration capacity of the cells: adding 600 μ l of culture solution containing serum to each well of a 24-well plate, and then placing a Transwell chamber; digesting different groups of U251 cells by pancreatin, slightly blowing the cells apart, centrifuging the cells, adding a serum-free culture solution to resuspend the cells, adding 100 mu l of cell suspension into each small chamber, uniformly paving the cells in an upper chamber, wherein the cell suspension is about 10 mu l⁵And (4) cells. PlacingCulturing in a cell culture box at 37 ℃ for 24 h; after 24h, the chamber was removed, the upper chamber was wiped down gently with a cotton swab, the chamber was washed with PBS and dried. Then preparing a stationary liquid according to the ratio of methanol to glacial acetic acid = 3: 1, namely blowing and uniformly mixing 750 mul of methanol and 250 mul of glacial acetic acid; immersing the chamber into a fixing solution to fix the cells for 30 min; washing the small chamber by using PBS and drying, spreading 200 mul of Sa dye of Jimu at the bottom of the inverted small chamber, and dyeing for at least 30 min; the chamber was washed twice with PBS, placed under an inverted microscope, observed under a 400 Xmicroscope, and 5 fields were randomly selected for cell count to demonstrate cell migration ability. Transwell cell migration experiments detected a significant reduction in the number of U251 cells migrated after the application of MAPK6P2 gene inhibitor, as shown in figure 4.

And thirdly, detecting the influence of the MAPK6P2 gene inhibitor on the invasion capacity of glioma cells by a Transwell cell invasion experiment.

And (3) detecting the invasion capacity of the cells: adding 600 mu l of culture solution containing serum into each well of a 24-well plate, then placing a Transwell chamber, adding 50 mu l of Matrigel collagen solution into the chamber, and culturing for 30 mins in a cell culture box at 37 ℃; digesting different groups of U251 cells by pancreatin, slightly blowing the cells apart, centrifuging the cells, adding a serum-free culture solution to resuspend the cells, adding 100 mu l of cell suspension into each small chamber, uniformly paving the cells in an upper chamber, wherein the cell suspension is about 10 mu l⁵And (4) cells. Culturing in a cell culture box at 37 deg.C for 24 hr; after 24h the chamber was removed, washed with PBS and dried. Then preparing a stationary liquid according to the ratio of methanol to glacial acetic acid = 3: 1, namely blowing and uniformly mixing 750 mul of methanol and 250 mul of glacial acetic acid; immersing the chamber into a fixing solution to fix the cells for 30 min; washing the small chamber by using PBS and drying, spreading 200 mul of Sa dye of Jimu at the bottom of the inverted small chamber, and dyeing for at least 30 min; the chamber was washed twice with PBS, placed under an inverted microscope, observed under a 400 Xmicroscope, and 5 fields were randomly selected for cell count to demonstrate the ability to invade cells. Transwell cell invasion assay detection the number of U251 cell migration inhibition was significantly reduced after application of MAPK6P2 gene inhibitor, as shown in fig. 5.

The statistical method comprises the following steps: all the above experimentsAll were performed in triplicate individually and the data are presented as mean ± sd. The statistical software SPSS 19.0 is used for analyzing the data of the plurality of groups by using a one-way analysis of variance (ANOVA) method, whether the difference exists between the groups is determined, and when the difference exists, the difference is determinedPValue of<0.05 was considered statistically significant. IC50 was calculated using Graphpad software.

Sequence listing

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Claims (4)

1. The application of a targeted inhibitor of MAPK6P2 gene in the preparation of a medicament for treating human glioblastoma multiforme is characterized in that the targeted inhibitor of the MAPK6P2 gene is a shRNA sequence capable of inhibiting the expression of the MAPK6P2 gene, the shRNA template consists of a sense strand and an antisense strand, and the sense strand and the antisense strand are respectively:

sense strand:

5’-CACCGCGAGTGAAGTTAACCATTACTTCAAGAGAGTAATGGTTAACTTCACTCGCTTTTTTG-3’；

antisense strand:

5’-GATCCAAAAAAGCGAGTGAAGTTAACCATTACTCTCTTGAAGTAATGGTTAACTTCACTCGC-3’。

2. the use of claim 1, wherein the inhibitor is in any pharmaceutically acceptable dosage form.

3. The use of claim 2, wherein the inhibitor is in the form of an injectable formulation.

4. The use of claim 1, wherein the inhibitor is in any pharmacotherapeutically acceptable dose.

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