Sirna construct for targeted silencing of EFNA2 gene, and application thereof in preparation of drug for inhibiting lung adenocarcinoma cell growth

Abstract

The present invention relates to the technical field of medicaments for inhibiting lung cancer, and provides an siRNA construct for the targeted silencing of the EFNA2 gene, and an application thereof in the preparation of a drug for inhibiting lung adenocarcinoma cell growth. The present invention comprises four siRNAs for silencing the EFNA2 gene, and the application thereof in the preparation of the drug for inhibiting lung adenocarcinoma cell growth. Specifically, a lipid material is used as a vector for delivering the EFNA2-silencing siRNAs. Among the four siRNA sequences designed for silencing EFNA2, the siRNA-856 sequence exhibits a better silencing effect than siRNA-495, siRNA-547, and siRNA-725, resulting in an apoptosis rate of 28.6% in lung tumor cells. A lipid vector composed of peptide lipids CDO and DOPE efficiently delivers the four EFNA2-siRNA sequences, without cytotoxicity.

Description

siRNA construction for targeting silencing of EFNA2 gene and application thereof in preparation of lung adenocarcinoma cell growth inhibiting drugs TECHNICAL FIELD

[0001] The present application belongs to the technical field of lung cancer inhibiting drugs, and particularly relates to siRNA construction for targeting silencing of EFNA2 gene and application thereof in preparation of lung adenocarcinoma cell growth inhibiting drugs. BACKGROUND

[0002] Cancer is one of the main factors threatening human life and health. In 2020, there were about 19.3 million new cancer cases worldwide, and about 9.96 million deaths. Among them, the incidence of lung cancer was about 2.21 million, and the mortality was about 1.8 million, ranking first, far exceeding other cancers. In 2022, there were about 4.8247 million new cancer cases in China, and about 2.5742 million deaths, of which the incidence (about 22%) and mortality (about 28%) of lung cancer ranked first. According to histopathological classification, lung cancer is mainly divided into non-small cell lung cancer (about 85%) and small cell lung cancer (about 15%). At present, the main treatment methods for lung cancer are surgical treatment, chemotherapy, radiotherapy and targeted therapy. However, because the early symptoms of lung cancer are not obvious, it is not easy to be found and the recurrence and metastasis rate of late-stage surgery is high, so the cure rate of traditional treatment methods for lung cancer is not high. Gene silencing therapy is a post-transcriptional gene silencing triggered by small interfering RNA (siRNA). siRNA can strongly inhibit gene expression and has high sequence specificity. Compared with traditional antisense nucleotides, the efficiency of siRNA silencing genes is tens to thousands of times higher.

[0003] EFNA2 is a gene encoding Ephrin-A2 protein, belonging to the Eph family. Ephrin-A2 is a cell surface molecule that plays an important role in neural system development and angiogenesis. It interacts with its receptor EphA, participates in the regulation of cell migration, adhesion and intercellular signal transduction. In lung adenocarcinoma, EFNA2 gene is highly expressed and becomes an important tumor marker, which participates in the regulation of tumor cell proliferation, invasion and metastasis, and has an important influence on tumor development. Using gene silencing therapy to reduce the expression of EFNA2 gene in tumor tissue will become a new technology for the treatment of lung adenocarcinoma. At present, there is no research on tumor treatment targeting EFNA2 gene.

[0004] SUMMARY

[0005] In order to overcome the prior art, the present application provides siRNA for silencing EFNA2 gene and its application in preparing a drug for inhibiting lung adenocarcinoma cell growth, utilizes high expression of EFNA2 gene in lung adenocarcinoma, participates in regulating proliferation, invasion and metastasis of tumor cells, designs four siRNA sequences capable of silencing the target gene, carries the siRNA for silencing EFNA2 by using lipid material, takes human lung adenocarcinoma cells A549 and H1299 as models, and inhibits growth of tumor cells by silencing expression of EFNA2 gene.

[0006] The above object of the present application is achieved by the following technical scheme: siRNA for silencing EFNA2 gene, a total of four siRNAs, named EFNA2-Homo-495, EFNA2-Homo-547, EFNA2-Homo-725 and EFNA2-Homo-856; wherein the forward sequence of EFNA2-Homo-495 is shown as SEQ ID No. 1, and the reverse sequence is shown as SEQ ID No. 2; the forward sequence of EFNA2-Homo-547 is shown as SEQ ID No. 3, and the reverse sequence is shown as SEQ ID No. 4; the forward sequence of EFNA2-Homo-725 is shown as SEQ ID No. 5, and the reverse sequence is shown as SEQ ID No. 6; and the forward sequence of EFNA2-Homo-856 is shown as SEQ ID No. 7, and the reverse sequence is shown as SEQ ID No. 8.

[0007] Another object of the present application is to protect the construction method of the above-mentioned siRNA for silencing EFNA2 gene, specifically: selecting nucleotides of the RNA polymerase gene on the L2 segment of the EFNA2 segmented genome as a target, synthesizing two siRNAs with a length of 21 bp, and modifying the 5' and 3' ends. The 5' end is replaced by a phosphate, and the 3' end remains unchanged. The 3' end is composed of 2 nucleotide bases to form an overhanging end.

[0008] Still another object of the present application is to protect the application of the above-mentioned siRNA for silencing EFNA2 gene in preparing a drug for inhibiting lung adenocarcinoma cell growth.

[0009] Further, the application of the siRNA for silencing EFNA2 gene in preparing a drug for inhibiting lung adenocarcinoma cell growth specifically utilizes lipid material as a carrier to carry the siRNA for silencing EFNA2.

[0010] Further, the lipid material is N,N-ditetradecyloxyacyl ethyl trimer ornithine amide (labeled as CDO) and dioleoyl phosphatidyl ethanolamine (DOPE), and the structure of CDO is as follows:

[0011] Further, the method for using lipid material as a carrier to carry the siRNA for silencing EFNA2 is specifically as follows: lipid CDO and DOPE are mixed and dissolved in chloroform at a mass ratio of 1:1, the chloroform is blown dry under N2 to form a thin and uniform film, and ultrasonic oscillation is alternately performed with the addition of ultrapure water to prepare the lipid carrier CDO-DOPE. In the formula, no DOPE is added, and the lipid carrier CDO is prepared by using lipid CDO alone. The lipid carrier is compounded with siRNA at a mass ratio of 3:1 for delivery.

[0012] Compared with the prior art, the application has the beneficial effects that: the four siRNA sequences for silencing EFNA2 designed in the application, wherein the silencing effect of the siRNA-856 sequence is better than that of siRNA-495, siRNA-547 and siRNA-725, and the apoptosis rate of lung tumor cells is 28.6%. The lipid carrier composed of the peptide lipids CDO and DOPE can efficiently deliver the four sequences of EFNA2-siRNA, and has no cytotoxicity. BRIEF DESCRIPTION OF DRAWINGS

[0013] The application will be further described below in combination with the drawings and specific embodiments

[0014] Fig. 1 is a schematic view of the relative expression amount of EFNA2 genes in A549, H1299 and BEAS-2B cells;

[0015] Fig. 2 is a schematic view of the protein expression level (A) and gray value (B) of EFNA2 in A549, H1299 and BEAS-2B cells;

[0016] Fig. 3 is a schematic view of the cytotoxicity of the lipid carrier to normal lung epithelial cells BEAS-2B (cultured for 48 h after transfection);

[0017] Fig. 4 is a schematic view of the relative expression amount (A), protein expression (B) and protein relative expression amount (C) of EFNA2 genes in H1299 cells 48 h after the cells are transfected with the complex of the lipid carrier and siRNA-495;

[0018] Fig. 5 is a schematic view of the relative expression amount (A), protein expression (B) and protein relative expression amount (C) of EFNA2 genes in H1299 cells 48 h after the cells are transfected with the complex of the lipid carrier and siRNA-547;

[0019] Fig. 6 is a schematic view of the relative expression amount (A), protein expression (B) and protein relative expression amount (C) of EFNA2 genes in H1299 cells 48 h after the cells are transfected with the complex of the lipid carrier and siRNA-725;

[0020] Figure 7 is a schematic diagram of the relative expression (A), protein expression (B) and relative protein expression (C) of the EFNA2 gene in H1299 cells 48 hours after transfection with a complex of a lipid carrier and siRNA-856;

[0021] Figure 8 is a schematic diagram of the apoptosis of H1299 cells 48 hours after transfection with a complex of a lipid carrier and siRNA-856. DETAILED DESCRIPTION

[0022] The application will be described in detail below with specific examples, but the scope of protection of the application is not limited. Unless otherwise specified, the experimental methods used in the application are conventional methods, and the experimental equipment, materials and reagents used can be obtained from commercial sources.

[0023] Example 1

[0024] The method for silencing the EFNA2 gene is as follows: H1299 cells are inoculated in a 6-well cell culture plate, 0.5 x 10 6 ~ 1 x 10 6 cells are inoculated in each well, and the cells are incubated in a 37°C, 5% CO2 incubator to achieve a cell density of about 80%-90%. 2.4 μg of siRNA is diluted with serum-free medium to 200 μL, 7.2 μg of lipid carrier is diluted with serum-free medium to 200 μL, the diluted siRNA is mixed with the lipid carrier in a mass ratio of 1:3, and the mixture is incubated at room temperature for 20 min. Then 400 μL of the above complex is added to each well. The control group does not add siRNA complex, and is incubated in the incubator for 4 h. The original culture medium is aspirated and 2 mL of RPMI 1640 medium containing 10% serum and 1% double antibody is added. The culture is continued, and after 48 h of cell culture, total RNA and total protein are extracted, and the expression of the EFNA2 gene is detected by RT-qPCR and Western Blot experiments, respectively.

[0025] Compared with human lung epithelial cells BEAS-2B, the expression of the EFNA2 gene in human lung adenocarcinoma cells H1299 and A549 is significantly increased, about 3-4 times that of BEAS-2B cells.

[0026] Compared with human lung epithelial cells BEAS-2B, the expression of the EFNA2 gene in human lung adenocarcinoma cells H1299 and A549 is significantly increased, about 3-4 times that of BEAS-2B cells.

[0027] After transfection of normal lung epithelial cells BEAS-2B with a lipid carrier, the cell survival rate was greater than 90% after 48 h of culture, indicating that the three lipid carriers had no significant toxicity to normal lung epithelial cells BEAS-2B.

[0028] The siRNA-495 was carried by the lipid carrier CDO14-DOPE to transfect the human lung adenocarcinoma cells H1299, and the cells were cultured for 48 hours after being transfected for 4 hours. The silencing efficiency of the EFNA2 gene in the cells was 58.1%, the silencing efficiency of the EFNA2 gene in the cells of the lipid carrier CDO14 group was 23.4%, and the silencing efficiency of the EFNA2 gene in the cells of the commercial transfection reagent DOTAP group was 51.4%.

[0029] The siRNA-547 sequence was carried by the lipid carrier CDO14-DOPE to transfect the human lung adenocarcinoma cells H1299, and the cells were cultured for 48 hours after being transfected for 4 hours. The silencing efficiency of the EFNA2 gene in the cells was 70.2%, the silencing efficiency of the EFNA2 gene in the cells of the lipid carrier CDO14 group was 29.7%, and the silencing efficiency of the EFNA2 gene in the cells of the commercial transfection reagent DOTAP group was 56%.

[0030] The siRNA-725 was carried by the lipid carrier CDO14-DOPE to transfect the human lung adenocarcinoma cells H1299, and the cells were cultured for 48 hours after being transfected for 4 hours. The silencing efficiency of the EFNA2 gene in the cells was 70%, the silencing efficiency of the EFNA2 gene in the cells of the lipid carrier CDO14 group was 31.8%, and the silencing efficiency of the EFNA2 gene in the cells of the commercial transfection reagent DOTAP group was 62.2%. The silencing effect of the siRNA-725 sequence was equivalent to that of the siRNA-547 and superior to that of the siRNA-495.

[0031] After the H1299 cells were transfected with the siRNA-856 delivered by the lipid carrier CDO14-DOPE for 48 hours, the silencing efficiency of the EFNA2 gene in the cells was 80.7%. After the H1299 cells were transfected with the siRNA-856 delivered by the commercial transfection reagent DOTAP for 48 hours, the silencing efficiency of the EFNA2 gene in the cells was 70%. After the H1299 cells were transfected with the siRNA-856 delivered by the lipid carrier CDO14 for 48 hours, the silencing efficiency of the EFNA2 gene in the cells was 33.8%.

[0032] The apoptosis rate of the lung tumor cells induced by the EFNA2-siRNA-856 delivered by the lipid carrier CDO14-DOPE was 28.6%, the apoptosis rate of the lung tumor cells induced by the EFNA2-siRNA-856 delivered by the commercial transfection reagent DOTAP was 24.13%, and the apoptosis rate of the lung tumor cells induced by the EFNA2-siRNA-856 delivered by the lipid carrier CDO14 group was 11.39%, which was consistent with the experimental results of RT-qPCR and Western Blot.

[0033] In summary, four siRNA sequences designed to silence EFNA2 were designed, wherein the silencing effect of siRNA-856 sequence was superior to siRNA-495, siRNA-547 and siRNA-725, causing the apoptosis rate of lung tumor cells to be 28.6%. Lipid carriers composed of the peptide lipids CDO and DOPE can efficiently deliver the four sequences of EFNA2-siRNA, and have no cytotoxicity.

[0034] The above-described embodiments are merely preferred embodiments of the present application, but are not all the embodiments that can be implemented by the present application. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present application should be considered to fall within the scope of the claims of the present application.

Claims

1. An siRNA silencing the EFNA2 gene, characterized in that, There are four siRNAs, named EFNA2-Homo-495, EFNA2-Homo-547, EFNA2-Homo-725 and EFNA2-Homo-856. The forward sequence of EFNA2-Homo-495 is shown as SEQ ID No. 1, and the reverse sequence is shown as SEQ ID No.

2. The forward sequence of EFNA2-Homo-547 is shown as SEQ ID No. 3, and the reverse sequence is shown as SEQ ID No.

4. The forward sequence of EFNA2-Homo-725 is shown as SEQ ID No. 5, and the reverse sequence is shown as SEQ ID No.

6. The forward sequence of EFNA2-Homo-856 is shown as SEQ ID No. 7, and the reverse sequence is shown as SEQ ID No.

8.

2. The method for constructing siRNA silencing the EFNA2 gene according to claim 1, wherein, The steps are: selecting the nucleotides of the RNA polymerase gene on the EFNA2 segmented genomic L2 fragment as the target, synthesizing two siRNAs with a length of 21 bp, and modifying the 5' and 3' ends. The 5' end is replaced with a phosphate, and the 3' end remains unchanged. The 3' end is composed of a 2-nucleotide base overhanging end.

3. The siRNA for silencing the EFNA2 gene according to claim 1 for use in the preparation of a medicament for inhibiting the growth of lung adenocarcinoma cells.

4. The use of the siRNA silencing the EFNA2 gene according to claim 3 for the preparation of a drug for inhibiting the growth of lung adenocarcinoma cells, characterized in that, Specifically, lipid material is used as a carrier to carry siRNA for silencing EFNA2.

5. The use of the siRNA silencing the EFNA2 gene according to claim 4 for the preparation of a medicament for inhibiting the growth of lung adenocarcinoma cells, characterized in that, The lipid material is CDO and DOPE, the structure of CDO is:

6. The use of the siRNA silencing the EFNA2 gene according to claim 4 for the preparation of a medicament for inhibiting the growth of lung adenocarcinoma cells, characterized in that, The method of using lipid material as a carrier to carry siRNA for silencing EFNA2 is as follows: lipid CDO and DOPE are mixed and dissolved in chloroform at a mass ratio of 1:1, the chloroform is blown dry under N2 to form a thin and uniform film, and ultrapure water is added and ultrasonically oscillated alternately to prepare the lipid carrier CDO-DOPE. In the formula, no DOPE is added, and the lipid carrier CDO is prepared alone. The lipid carrier and siRNA are compounded at a mass ratio of 3:1 for delivery.

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