阅读说明：本技术 一种敲低cbs基因的方法及其在制备治疗人脑胶质瘤的药物中的应用 (Method for knocking down CBS gene and application of method in preparation of medicine for treating human brain glioma ) 是由 吴东栋 王怡禛 郑蒙 吴海刚 刘媛媛 司伟荣 敬米蓉 张艳霞 蔡春波 王迪 祁慧 于 2021-08-06 设计创作，主要内容包括：本发明公开了一种敲低CBS基因的方法及其在制备治疗人脑胶质瘤的药物中的应用,包含以下步骤：S1:合成siRNA:依据人CBS基因序列,设计引物,用于PCR扩增目的基因,具体序列如下：正义链序列为：5’-CAGACCAAGUUGGCAAAGUTT-3’；反义链序列为：5’-ACUUUGCCAACUUGGUCUGTT-3’；S2:配置高分子聚合物:将PEG-b-P(Gu/Hb)粉末溶于10mM(mmol/L)HEPES(pH 7.2-7.4)缓冲溶液中,配制成10mg/mL的溶液浓度备用。S3:配置siRNA溶液：siRNA粉末溶于HEPES溶液中,配制成200μg/mL的浓度备用,本发明涉及医疗技术领域。将该方法敲低人脑胶质瘤中的CBS基因后,可显著抑制人脑胶质瘤生长、迁移和侵袭。本发明可有针对性地抑制人脑胶质瘤CBS基因表达量；体外实验表明抑制人脑胶质瘤CBS基因表达量能够显著抑制人脑胶质瘤生长、迁移和侵袭,促进人脑胶质瘤凋亡。(The invention discloses a method for knocking down CBS gene and application thereof in preparing medicine for treating human brain glioma, comprising the following steps: s1, synthesizing siRNA, designing a primer according to the human CBS gene sequence for PCR amplification of a target gene, wherein the specific sequence is as follows: the sense strand sequence is: 5'-CAGACCAAGUUGGCAAAGUTT-3', respectively; the antisense strand sequence is: 5'-ACUUUGCCAACUUGGUCUGTT-3', respectively; s2 preparation of high molecular polymer PEG-b-P (Gu/Hb) powder is dissolved in 10mM (mmol/L) HEPES (pH 7.2-7.4) buffer solution to prepare a solution concentration of 10mg/mL for later use. S3 preparation of siRNA solution: the siRNA powder is dissolved in HEPES solution to be prepared into 200 mu g/mL for standby, and the invention relates to the technical field of medical treatment. After the CBS gene in the human brain glioma is knocked down by the method, the growth, migration and invasion of the human brain glioma can be obviously inhibited. The invention can specifically inhibit the expression quantity of the human brain glioma CBS gene; in vitro experiments show that the CBS gene expression level for inhibiting the human brain glioma can obviously inhibit the growth, migration and invasion of the human brain glioma and promote the apoptosis of the human brain glioma.)

1. A method for knocking down a CBS gene, comprising the steps of:

s1, synthesizing siRNA, designing a primer according to the human CBS gene sequence for PCR amplification of a target gene, wherein the specific sequence is as follows:

the sense strand sequence is:

5’-CAGACCAAGUUGGCAAAGUTT-3’；

the antisense strand sequence is:

5’-ACUUUGCCAACUUGGUCUGTT-3’；

s2 preparing high molecular polymer, dissolving PEG-b-P (Gu/Hb) powder in 10mM (mmol/L) HEPES (pH 7.2-7.4) buffer solution to prepare 10mg/mL solution concentration for later use;

s3 preparation of siRNA solution: dissolving siRNA powder in HEPES solution to prepare 200 mug/mL for later use;

s4 preparation of siRNA nano-drug by using NH in polymer₃ ⁺、Gu⁺And PO in siRNA₃ ^4-The molar ratio of (a) to (b) is calculated. Mixing the polymer and the siRNA in the step S2 according to a molar ratio of 1:5, shaking and uniformly mixing the mixed solution, and standing for 30min to form the siRNA nano-drug;

s5 transient transfection: transfecting the nano-drug constructed in the step S4 into human glioma cells; after the prepared nano-drug is transfected into human brain glioma cells, CBS genes in the tumor cells can be knocked down.

2. The method for knocking down a CBS gene as claimed in claim 1, wherein the tumor cells are U87 of human brain glioma line.

3. A method for knocking down CBS gene and its application in preparing medicine for treating human brain glioma is characterized by that the knocking down CBS gene in human brain glioma can inhibit growth, migration and invasion of human brain glioma cell and promote apoptosis of human brain glioma cell.

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a method for knocking down a CBS gene and application thereof in preparing a medicament for treating human brain glioma.

Background

Gliomas are one of the most serious malignancies in humans, being tumors of the central nervous system. In this regard, Glioblastoma (GBM) is the most malignant type of glioma. Glioblastoma (GBM) is a glial cell tumor that originates in the brain and spinal cord. Glioblastoma is the most aggressive primary brain tumor derived from glioma, with high incidence and poor prognosis. Gliomas account for 12-15% of all intracranial tumors, accounting for 50-60% of astrocytic tumors. There are also two types of primary gliomas, one that occurs de novo and the other that is secondary gliomas that develop from low-grade astrocytomas. Each subtype has different genetic attributes. Men are usually diagnosed with a higher risk of glioma than women, and both men and women have a highest age of onset of 60-75 years. Despite recent advances in treatment, GBM remains promising, with a median survival time of patients of less than 15 months after diagnosis. Therefore, it is important to find new targets for treating brain glioma and develop new methods for diagnosing brain glioma.

Hydrogen sulfide is a newly discovered gas transmitter with a broad spectrum of regulatory effects on health and disease. Synthesis of H in mammals from three enzymes₂Cystathionine-beta-synthase (CBS), cystathionine gamma-lyase (CSE) and 3-mercaptopyruvic acid (3-MST). Intracellular H compared to adjacent non-cancerous tissue or non-tumor cells₂S concentration and one or more H₂Expression of S synthase is increased in a variety of malignant cells. Increased intracellular CBS protein expression stimulates tumor growth, angiogenesis and peritumoral vascular tone, H₂S concentration, CBS, CSE and 3-MST protein expression also often exhibit an increase in higher tumor grade and stage. In order to understand the potential functions of CBS in the development of glioma, the research and study of the influence and potential molecular mechanism of CBS on the proliferation and metastasis of human glioma cells, and the aim is to provide a new idea for improving the prognosis of glioma and developing a glioma diagnosis and treatment strategy.

Disclosure of Invention

In order to achieve the purpose, the invention is realized by the following technical scheme: comprises the following steps:

s1, synthesizing siRNA, designing a primer according to the human CBS gene sequence for PCR amplification of a target gene, wherein the specific sequence is as follows:

the sense strand sequence is:

5’-CAGACCAAGUUGGCAAAGUTT-3’；

the antisense strand sequence is:

5’-ACUUUGCCAACUUGGUCUGTT-3’；

s2 preparing high molecular polymer, dissolving PEG-b-P (Gu/Hb) powder in 10m M (mmol/L) HEPES (pH 7.2-7.4) buffer solution to prepare 10mg/mL solution concentration for later use.

S3 preparation of siRNA solution: dissolving siRNA powder in HEPES solution to prepare 200 mug/mL for later use;

s4 preparation of siRNA nano-drug by using NH in polymer₃ ⁺、Gu⁺And PO in siRNA₃ ^4-The molar ratio of the siRNA and the polymer in the step S2 is calculated, the polymer and the siRNA are mixed according to the molar ratio of 1:5, the mixed solution is uniformly vibrated, and the siRNA nano-drug can be formed after standing for 30 min;

s5 transient transfection: transfecting the nano-drug constructed in the step S4 into human glioma cells; after the prepared nano-drug is transfected into human brain glioma cells, CBS genes in the tumor cells can be knocked down.

Preferably, the tumor cell is U87 of human brain glioma line.

Preferably, the knocking down of the CBS gene in the human glioma inhibits the growth, migration and invasion of human glioma cells and promotes the apoptosis of the human glioma cells.

Advantageous effects

The invention provides a method for knocking down a CBS gene and application thereof in preparing a medicament for treating human brain glioma. The method has the beneficial effect that after the CBS gene in the human brain glioma is knocked down, the growth, migration and invasion of the human brain glioma can be obviously inhibited. The invention can specifically inhibit the expression quantity of the human brain glioma CBS gene; in vitro experiments show that the CBS gene expression level for inhibiting the human brain glioma can obviously inhibit the growth, migration and invasion of the human brain glioma and promote the apoptosis of the human brain glioma.

Drawings

FIG. 1 is a result diagram of Western blot detection of protein expression level of CBS in tumor cells after knocking down of CBS genes in human brain glioma cells by using nano-drug complexes according to an embodiment of the present invention;

FIG. 2 is a graph showing the effect of MTT method for detecting CBS knockdown on human glioma cell proliferation in accordance with an embodiment of the present invention;

FIG. 3 is a graph of the effect of EDU method of the present invention on the proliferation of human glioma cells to detect CBS knockdown;

FIG. 4 is a diagram of the TUNEL method of the present invention for detecting the effect of knocking down CBS gene on apoptosis of human glioma cells;

FIG. 5 is a graph of the effect of the Transwell method of the present invention on the detection of CBS knockdown on the migration of human glioma cells;

FIG. 6 is a graph of the effect of Invasion on the detection of CBS knockdown on human glioma cell Invasion in accordance with an embodiment of the present invention.

Detailed Description

All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to FIGS. 1-6, control is wild type cell, HB @ siCBS is negative control cell, Ang-HB @ siNC is negative control cell, and Ang-HB @ siCBS is gene knockdown cell.

The present invention is further illustrated by the following examples. Before describing the specific embodiments, the main instruments and devices and reagents used in the present invention are briefly described as follows.

Main reagents, drugs and samples:

human brain glioma cell U87-MG is present at the university of Henan-McCor university biomedical Union Innovation center.

The transfection reagent nanoparticle complex is present at the biomedical integrated innovation center of Henan university-McCory university;

MTT was purchased from Sigma, USA;

the cell proliferation detection kit is purchased from Ruibo Biotechnology, Inc.;

the apoptosis detection kit is purchased from Biyuntian biotechnology limited company;

transwell chambers were purchased from Corning corporation;

the plasmid is provided by Shanghai Jikai Genencochemistry technologies, Inc.;

the rest unexplained reagents, medicines and the like are common analytical pure products in laboratories and are not described any more.

The main apparatus comprises:

a real-time quantitative PCR instrument (model: QuantStaudio 5) which is a product of Thermo Fisher company in the United states;

fluorescence inverted microscope (model: ECLIPSE Ti) from Nikon.

Example 1

(1) Synthesizing siRNA, namely designing a primer for PCR amplification of a target gene according to a human CBS gene sequence, wherein the specific sequence is as follows:

the sense strand sequence is:

5’-CAGACCAAGUUGGCAAAGUTT-3’；

the antisense strand sequence is:

5’-ACUUUGCCAACUUGGUCUGTT-3’；

(2) preparing high molecular polymer, dissolving PEG-b-P (Gu/Hb) powder in 10m M (mmol/L) HEPES (pH 7.2-7.4) buffer solution to prepare 10mg/mL solution concentration for later use.

(3) Preparing siRNA solution: the siRNA powder was dissolved in HEPES solution and prepared to a concentration of 200. mu.g/mL for use.

(4) Preparation of siRNA Nanoparticulate with NH in Polymer₃ ⁺、Gu⁺And PO in siRNA₃ ^4-The molar ratio of the siRNA and the polymer in the step (2) is calculated, the polymer and the siRNA are mixed according to the molar ratio of 1:5, the mixed solution is uniformly shaken and mixed, and the mixture is kept stand for 30min to form the siRNA nano-drug.

In order to objectively evaluate the over-expression effect of the CBS gene in the tumor cell, the protein expression level of the CBS protein expression in the tumor cell can be tested, and the specific process is briefly described as follows.

Detection of CBS protein expression level:

and respectively extracting the wild type (namely blank control) of the tumor cell, the total protein in the tumor cell after the two negative controls and the CBS gene are knocked down, measuring the concentration of the total protein, and detecting by using Western blot (Western blot) to determine the expression level of the CBS protein in the cell.

The CBS protein expression level detection is shown in figure 1, and it can be seen from the figure that the protein expression level of CBS in cells is also greatly reduced compared with that of a control group, which shows that the siCBS nano-drug prepared by the invention well knocks down CBS genes in tumor cells.

Example 2

To examine the effect of knocking down the CBS gene in tumor cells on tumor cell proliferation in example 1, the inventors conducted further experiments and the related procedures are described below.

(1) Firstly, determining the influence of knocking down CBS on the proliferation of tumor cells by adopting an MTT method, and the specific process is as follows:

the tumor cells of the blank control, the two negative controls and the knockdown CBS gene of example 1 were separately digested and counted, and plated in 96-well plates in an inoculum size of 6X 10³Each hole is provided with 6 auxiliary holes, each hole is added with 100 mu L of culture medium, and the culture medium is incubated at 37 ℃ in an incubator;

after 24h, adding 20L of MTT solution and 5mg/mL of MTT solution into each hole respectively, and continuously culturing for 2h in an incubator;

then, the cells are taken out, the absorbance of each hole is measured at 490nm by using an enzyme-labeling instrument, and the inhibition rate of the knocked-down CBS gene on the cell growth is calculated according to the absorbance.

The cell growth inhibition rate is calculated by the formula:

cell growth inhibition (%) - (control well OD value-experimental well OD value)/control well OD value × 100%.

The experimental result is shown in fig. 2, and it can be seen from fig. 2 that the proliferation and growth of the tumor cells after the CBS gene is knocked down are significantly lower than those of the tumor cells of the negative control group, and it can be considered that knocking down of CBS inhibits the growth of the tumor cells.

(2) The EDU method is adopted to determine the influence of the knocked-down CBS on the proliferation of tumor cells, and the specific process is as follows:

EdU labeling (12 well plate operation): diluting the EdU solution (reagent A) with a cell culture medium at a ratio of 1000:1 to prepare a suitable amount of 50. mu.M EdU medium; adding 300L of 50 mu M EdU culture medium into each hole, incubating for 2h, and removing the culture medium; the cells were washed 2 times with PBS for 5min each.

Cell immobilization: adding 150L cell fixing solution (PBS containing 4% paraformaldehyde) into each well, incubating at room temperature for 30min, and discarding the fixing solution; adding 150L of 2mg/mL glycine into each hole, incubating for 5min by a decoloring shaker, and removing the glycine solution; adding 300L PBS into each hole, washing for 1 time for 5min, and discarding PBS; (boost) 100L of osmotic agent (0.5% Triton-X100 in PBS) was added to each well and incubated on a destaining shaker for 10min, followed by 1 wash in PBS for 5 min.

Apollo staining: adding 300L of 1 XApollo staining reaction solution into each well (prepared in sequence, prepared immediately before use, and used up for 30 min), incubating for 30min in a decolorizing shaker at room temperature in a dark place, and discarding the staining reaction solution;

preparation of Apollo dyeing reaction liquid: 1.5mL

Adding 300L of penetrant (0.5% Triton-X100 PBS), decolorizing and washing with shaker for 2 times, each time for 10min, and discarding the penetrant; (enhancement) Each well was washed 1-2 times with 5min of 300L methanol and 1 time with 5min of PBS.

DNA staining: diluting the reagent F with deionized water according to the proportion of 100:1, preparing a proper amount of 1 × Hoechst33342 reaction solution, and storing in the dark; adding 300L of 1 Xhoechst 33342 reaction solution into each well, keeping out of the sun, incubating for 30min at room temperature by a decolorization shaking table, and removing the staining reaction solution; adding 300LPBS to each hole, washing for 3 times (5 min each time); the cells were preserved by adding 300LPBS per well, photographed, and counted for cell proliferation rate.

The results are shown in fig. 3, and it can be seen from the figure that the cell proliferation rate of the tumor cells with the knockdown CBS gene is significantly lower than that of the tumor cells of the control group and the blank group, which indicates that the growth of the tumor cells can be inhibited by knocking down the CBS gene in the tumor cells.

Example 3

To examine the effect of knocking down the CBS gene on tumor cell apoptosis in example 1, the inventors performed TUNEL assay, and the related procedures are described below.

The method for determining the influence of the knocked-down CBS gene on the tumor cell apoptosis comprises the following specific steps:

the cells were digested and counted, plated in 96-well plates, and inoculated at 2X 10⁴Per well at 5% CO₂Incubating in an incubator at 37 ℃;

after the cells adhere to the wall for 12 hours, the cells are washed for 1 time by PBS; fixing with 4% paraformaldehyde for 30 min; washing with PBS for 1 time;

adding PBS containing 0.1% Triton-X100, and incubating in ice bath for 2 min; PBS washing 2 times, preparing 250L Tunel detection liquid: 10L of TdT enzyme +240L of fluorescent labeling solution; adding 50L Tunel detection solution to the sample, and incubating at 37 deg.C in dark for 60 min; PBS washing for 3 times;

DAPI staining: 5% BSA 1:1000 dilution, 3 min; adding 200LPBS into each well for storage, observing under a fluorescence microscope, photographing, and counting the apoptosis rate.

The results are shown in FIG. 4. As can be seen from the figure, the apoptosis rate of the tumor cells with the knocked-down CBS gene is obviously higher than that of the tumor cells in the control group, which indicates that the knocking-down CBS gene in the tumor cells can promote the apoptosis of the tumor cells.

Example 4

To examine the effect of knocking down the CBS gene on tumor cell migration and Invasion in example 2, the inventors performed Transwell and invade assay experiments, and the related procedures are described below.

(1) The Transwell method is adopted to determine the influence of the knocked-down CBS gene on the migration of tumor cells, and the specific process is as follows:

the chamber was placed in a 24-well plate, 600L of medium containing 20% serum was added to the lower layer of the chamber, and 200L of medium containing no serum was added to the upper layer, at 1X 10 per well⁴Incubating the cells in an incubator at 37 ℃ for 24 hours;

taking out the culture plate, removing the culture medium, adding 75% alcohol into each hole, fixing for 15min, and washing with PBS for 2 times;

the crystal violet was stained for 10min, rinsed with tap water, the crystal violet was washed off, the upper layer of the cell was wiped clean with a cotton swab, the film was gently scraped off with a razor blade onto a glass slide, fixed with neutral gum, and photographed under a 100 Xlens.

(2) The influence of the knocked-down CBS gene on the Invasion of tumor cells is determined by adopting an Invasion method, and the specific process is as follows:

the specific operation is the same as (1), except that the cells are provided with matrigel, and each hole is 1 multiplied by 10⁴And (4) cells.

The migration result is shown in fig. 5, compared with the control group, the tumor cells with the knocked-down CBS gene have obviously reduced migration ability; the invasion results are shown in fig. 6, and the invasion capacity of the cells is obviously reduced after the CBS gene is knocked down. Thus, it was shown that the knockdown of CBS gene in glioblastoma cells could inhibit cell migration and invasion.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

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