SiRNA for targeted silencing of GABRD gene expression and application

Abstract

The invention relates to the technical field of medical biology, in particular to siRNA for targeted silencing of GABRD gene expression and application. The siRNA for targeted silencing of the expression of the human GABRD gene has a silencing effect on the expression of the GABRD gene, and can inhibit the multiplication capacity and migration capacity of HGC27 cells after transfecting the HGC27 cells. GABRD is used as a potential gastric cancer treatment target, so that the siRNA sequence and related biological reagents provided by the invention can be used for researching and preparing medicines for treating gastric cancer, and a new thought and a scientific basis are provided for research and development of novel medicines for treating gastric cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, and in particular to a siRNA that targets and silences the expression of the GABRD gene and its applications. Background Technology

[0002] Gastric cancer, as one of the leading malignant digestive system tumors in terms of both incidence and mortality worldwide, carries a particularly heavy disease burden. While clinical treatment for gastric cancer has developed into a comprehensive system centered on surgical resection combined with chemotherapy, radiotherapy, targeted therapy, and immunotherapy, the lack of specific diagnostic markers and the insidious nature of early symptoms mean that over 60% of patients are already in the locally advanced or late-stage stage at the time of diagnosis.

[0003] Current first-line chemotherapy regimens for advanced gastric cancer primarily involve combinations of platinum-based, fluorouracil-based, and taxane-based drugs. While these regimens can prolong patient survival to some extent, the effective rate is only 30%-50%, and tumor cells are prone to developing resistance to chemotherapy drugs. Targeted therapy, as an important means of precision cancer treatment, has made breakthrough progress in the treatment of malignant tumors such as lung cancer and breast cancer. However, targeted therapeutic targets for gastric cancer are relatively scarce, and a large number of gastric cancer patients still cannot benefit from it. Therefore, discovering new gastric cancer-specific therapeutic targets and developing targeted therapy strategies have become key issues that urgently need to be addressed in the current field of gastric cancer research.

[0004] The GABRD gene encodes the delta subunit of the γ-aminobutyric acid (GABA) type A receptor, which is the GABA receptor. A A crucial component of the receptor family. In recent years, with the deepening research into the molecular mechanisms of tumors, scholars have discovered that the GABRD gene exhibits abnormal expression in various malignant tumors, and its expression level is closely related to tumor development, invasion, metastasis, and patient prognosis. In the field of gastric cancer, related studies have shown that the expression level of the GABRD gene in gastric cancer tissues is significantly higher than that in normal gastric mucosa tissues, and gastric cancer patients with high GABRD expression often have adverse clinical characteristics such as poor tumor differentiation, high lymph node metastasis rate, and shortened survival. However, research on targeted therapy for gastric cancer targeting the GABRD gene is still in its early stages. No technology or drug capable of specifically inhibiting GABRD gene expression and being applied to the treatment of gastric cancer has yet entered clinical trials. There is an urgent need to develop efficient and specific GABRD gene intervention methods to provide new directions for targeted therapy of gastric cancer.

[0005] Small interfering RNA (siRNA) technology is a rapidly developing gene silencing technology in recent years, which can specifically bind to the target gene mRNA sequence, induce mRNA degradation or inhibit its translation process, thereby efficiently and specifically inhibit the expression of target gene, with strong targeting, high efficiency, small side effects and other significant advantages. Since the discovery of siRNA technology, it has shown a broad application prospect in the field of gene function research, disease model construction and disease treatment, especially in the field of tumor treatment. Compared with traditional chemotherapy drugs and antibody-based targeted drugs, siRNA drugs can directly act on the gene level, inhibit the expression of tumor-related genes from the source, with higher specificity and pertinence. At the same time, the preparation process of siRNA molecule is relatively mature, which can be mass-produced by chemical synthesis, and its sequence can be designed flexibly according to the target gene sequence, providing convenience for drug research and development targeting different tumor targets.

[0006] However, in the field of gastric cancer GABRD gene targeted therapy, there is no GABRD gene intervention scheme based on siRNA technology reported. The lack of siRNA design and application research targeting GABRD gene leads to the inability to specifically inhibit the expression of GABRD gene by siRNA technology to achieve gastric cancer treatment, and also cannot provide an efficient tool for the functional research of GABRD gene in gastric cancer. Therefore, the development of siRNA sequence that can specifically target and silence the expression of human GABRD gene not only can fill the gap of current GABRD gene targeted therapy research for gastric cancer, provide new treatment options for gastric cancer patients, but also can provide key technical support for the molecular mechanism research of GABRD gene in the occurrence and development of gastric cancer, which has important scientific significance and clinical application value. SUMMARY

[0007] The application provides a siRNA for targeting and silencing the expression of GABRD gene and application, which can significantly inhibit the cell proliferation and migration of gastric cancer HGC27 cells by transfecting the siRNA molecule to target and silence the expression of GABRD gene, has very important significance for the treatment of gastric cancer, and can be used for the preparation of gastric cancer treatment drugs, solving the problems in the prior art.

[0008] One of the technical solutions adopted by the application is: The siRNA for targeting and silencing GABRD is provided, wherein the sequence of the sense strand of the siRNA is shown as SEQ ID NO. 1, and the sequence of the antisense strand is shown as SEQ ID NO. 2.

[0009] The second technical solution adopted by the application is: The application of GABRD gene inhibitor in the preparation of drugs for treating / preventing gastric cancer is provided, and the GABRD gene inhibitor is the siRNA with the sequence as described above.

[0010] The third technical solution adopted by the present application is: A pharmaceutical preparation for treating / preventing gastric cancer is provided, which comprises the siRNA as described above.

[0011] Further, the pharmaceutical preparation further comprises a pharmaceutically acceptable pharmaceutical carrier or excipient.

[0012] Further, the above-mentioned "pharmaceutically acceptable" means that when the molecular entity and the composition are properly administered to animals or humans, they do not produce adverse, allergic or other untoward reactions. The "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, i.e., it can be blended with the active ingredient without substantially reducing the effect of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help the stability of the preparation or to help improve the activity or its bioavailability or to produce an acceptable taste or smell in the case of oral administration.

[0013] Further, the above-mentioned pharmaceutical preparation can be prepared into a needle injection, an oral liquid, a tablet, a capsule, a dripping pill, a spray.

[0014] The beneficial effects of the present application are: 1. The siGABRD-3 of the present application is designed based on the mRNA sequence of GABRD. The detection results by qRT-PCR technology confirm that it has a silencing effect on the expression of GABRD gene. After transfection of HGC27 cells, it can significantly inhibit the proliferation ability and migration ability of HGC27 cells, and has a very important significance for the treatment of gastric cancer. It provides a theoretical basis for the new strategy of gastric cancer treatment with GABRD related preparations as the core, and is expected to be applied to the preparation of high-efficiency, high-specificity, small-side-effect anticancer gene drugs, especially for the research and development of gastric cancer gene drug preparations, which has great social and economic benefits.

[0015] 2、The application specifically designs three pairs of primer sequences: siGABRD-1, siGABRD-2 and siGABRD-3 through the mRNA sequence of the GABRD (NM_000815.5) gene, and the experimental results can obviously show that, compared with other siRNAs and the control group si-NC, siGABRD-3 is particularly significant in inhibiting the activity of HGC27 cells, and through the investigation of the influence on the migration ability of cells, it can significantly inhibit the migration ability of HGC27 cells, and is a potential drug with important treatment value for gastric cancer. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The expression of the GABRD gene in the gastric cancer cell line HGC27 cells is shown in the following table: Figure 2 The GABRD expression level in HGC27 cells after transfection of siGABRD-1, siGABRD-2, siGABRD-3 and si-NC in HGC27 cells is shown in the following table: Figure 3 The influence of siGABRD-1, siGABRD-2, siGABRD-3 and si-NC on the activity of HGC27 cells in the CCK8 proliferation experiment is shown in the following table: Figure 4 The influence of siGABRD-3 and si-NC on the migration ability of HGC27 cells in the cell migration experiment is shown in the following table. DETAILED DESCRIPTION

[0017] In order to clearly illustrate the technical features of the present application, the following will describe the present application in detail through specific embodiments combined with the drawings.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0019] The present embodiment provides the application of the GABRD gene inhibitor in the preparation of a drug for treating / preventing gastric cancer, wherein the GABRD gene inhibitor is siRNA for silencing GABRD, the sequence of the sense strand of the siRNA is shown as SEQ ID NO. 1, and the sequence of the antisense strand is shown as SEQ ID NO. 2.

[0020] SEQ ID NO. 1: 5'-GCUACGGUUACUCAUCGGA-3' SEQ ID NO. 2: 5'-UCCGAUGAGUAACCGUAGC-3'.

[0021] The U in the gene sequence of the present example is replaced by T in the corresponding sequence in the sequence listing.

[0022] The GABRD gene inhibitor comprising the above sequence can be used as a pharmaceutical preparation for treating / preventing gastric cancer. The pharmaceutical preparation can further comprise a pharmaceutically acceptable pharmaceutical carrier.

[0023] As an embodiment, the above-mentioned drug can be used in combination with other drugs for treating gastric cancer, or can be used alone.

[0024] The term "treatment" in the present example refers to reducing symptoms, eliminating symptoms, or preventing or slowing the appearance of symptoms of a specified disease or condition on a temporary or permanent basis. This term is intended to encompass therapeutic and prophylactic or suppressive measures that cause any clinically desirable or beneficial effect on a disease or condition, including but not limited to alleviating or reducing one or more symptoms, regression, slowing down or stopping the progression of a disease or condition.

[0025] The term "pharmaceutically acceptable" means that it is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes that it is acceptable for human pharmaceutical use as well as veterinary use.

[0026] The siRNA for targeting and silencing the expression of GABRD gene and the application scheme thereof provided by the present application will be described in detail below through specific examples.

[0027] In the following examples, the specific conditions not specified are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are conventional products that can be obtained by market purchase. Unless otherwise specified, the present application uses the existing technology in the field.

[0028] The following examples are only used to illustrate the present application and do not limit the scope of the present application. In the following examples, the experimental methods not specified with specific conditions are preferred to refer to the guidance given in the present application, and can also be carried out according to the experimental manual or conventional conditions in the field, or by referring to the experimental methods known in the art. Unless otherwise specified, they are the conventional methods in the field.

[0029] In the following specific examples, the measurement parameters of the raw material components may, unless otherwise specified, have slight deviations within the weighing accuracy range. With respect to temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed. The equipment and raw materials used can be purchased from the market or are commonly used in the field.

[0030] Example 1 qRT-PCR experiment for detecting the expression of GABRD in gastric mucosa epithelial cells GES-1 and gastric cancer cell line HGC27 cells The human gastric mucosa epithelial cells GES-1 and the human gastric cancer cells HGC27 used in the present embodiment were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The human gastric mucosa epithelial cells GES-1 and the human gastric cancer cells HGC27 were cultured in 1640 medium containing 10% fetal bovine serum and 10 U / ml penicillin and 0.1 mg / ml streptomycin in a 5% CO2, 37°C cell incubator. The cell growth state was observed once a day. When the cells reached 70% or more of the surface area of the culture dish, they were passaged in time. When the cells were in good condition and in the logarithmic growth phase, they were used for subsequent experiments.

[0031] The 24-well plate was taken, and the human gastric mucosa epithelial cells GES-1 and the human gastric cancer cells HGC27 were seeded at 7x10 4 The cells were seeded in 500 uL of 1640 medium, and when the cell confluence was 80%, the total RNA in the cells was extracted and separated by the Trizol method, and then reverse transcribed into cDNA using the whole type gold reverse transcription kit. The expression level of GABRD in GES-1 and HGC27 was detected by using the automatic medical PCR analysis system Gentier 96R of Xi'an Tianlong Science and Technology Co., Ltd. with GABRD and GAPDH as primers. The nucleotide upstream sequence of GABRD is shown in SEQ ID NO. 7, i.e. 5'-AGAGCTACGGTTACTCATCGG-3', and the downstream sequence is shown in SEQ ID NO. 8, i.e. 5'-GGCCAGCGGACTTGAAGTT-3'. The nucleotide upstream sequence of GAPDH is shown in SEQ ID NO. 9, i.e. 5'-GGAGTCCACTGGCGTCTTCA-3', and the downstream sequence is shown in SEQ ID NO. 10, i.e. 5'-GTCATGAGTCCTTCCACGATACC-3'. The PCR reaction conditions were pre-denaturation: 95°C, 10 min; denaturation: 95°C, 15 s; annealing / extension: 60°C, 60 s; 40 cycles, and finally 2 -△△CT The expression level was determined by the method.

[0032] Figure 1 The expression of GABRD in the gastric cancer cell line HGC27 cells is shown in Figure 1 Compared with the human gastric mucosa epithelial cells GES-1, the expression level of GABRD in the human gastric cancer cells HGC27 was significantly increased. (* indicates P<0.05, and the number of experiments is ≥3) Example 2 Design and synthesis of siRNA for silencing GABRD expression Three siRNA sequences were designed and screened based on the mRNA sequence of the GABRD (NM_000815.5) gene by the BLOCK-iT™ RNAi Designer of Thermo Fisher Scientific, and were designed and named as siGABRD-1, siGABRD-2, and siGABRD-3.

[0033] The sequence of the siGABRD-3 sense strand is shown in SEQ ID NO. 1, i.e., 5’ - GCUACGGUUACUCAUCGGA - 3’, and the sequence of the antisense strand is shown in SEQ ID NO. 2, i.e., 5’ - UCCGAUGAGUAACCGUAGC - 3’. The sequence of the siGABRD-1 sense strand is shown in SEQ ID NO. 3, i.e., 5’ - CAUCGACGCAGACACCAUU - 3’, and the sequence of the antisense strand is shown in SEQ ID NO. 4, i.e., 5’ - AAUGGUGUCUGCGUCGAUG - 3’; the sequence of the siGABRD-2 sense strand is shown in SEQ ID NO. 5, i.e., 5’ - GAGUACGCCUUUGCUCAUU - 3’, and the sequence of the antisense strand is shown in SEQ ID NO. 6, i.e., 5’ - AAUGAGCAAAGGCGUACUC - 3’; The siGABRD-1 powder, the siGABRD-2 powder, and the siGABRD-3 powder were synthesized according to the primer sequences by Beijing Chengke Biological Technology Co., Ltd. The synthesized siGABRD-1 powder, the siGABRD-2 powder, and the siGABRD-3 powder were configured into a concentration of 20 µM with DNase / RNase-free distilled water (DNase / RNase-Free Distilled Water) according to the instructions, and were stored in a -20 °C refrigerator for standby use. Meanwhile, si-NC with a concentration of 20 µM was configured as a control.

[0034] Example 3 qRT-PCR experiment for detecting siGABRD transfected cells 24 h before transfection, a 24-well plate was taken, 7 × 10 4HGC27 cells in 400 uL of anti-culture medium, when the cell state is good, in the logarithmic growth phase, randomly divide the cells into siGABRD-1 group, siGABRD-2 group, siGABRD-3 group and siNC group, when the cell fusion degree is 60-80%, transfection. The siRNA dilution concentration is 20 μM, and the final concentration is 50 nM. Dilute 1.25 μL siRNA with 50 μL Opti-MEM, mix gently for 3-5 times; invert the mixing transfection reagent, dilute 1.0 μL transfection reagent with 50 μL Opti-MEM, mix gently for 3-5 times; room temperature for 5 min; mix siRNA and transfection reagent, mix gently for 3-5 times, room temperature for 20 min; add the transfection complex to the 24-well cell plate, 100 μL / well, shake well, and incubate the cell plate in a 37°C, 5% CO2 incubator. Replace the fresh culture medium after 4-6 h of transfection.

[0035] The total RNA in the cells was extracted and separated by Trizol method, and then reverse transcribed to synthesize cDNA using a whole type gold reverse transcription kit. The expression level of GABRD in siGABRD-1 group, siGABRD-2 group, siGABRD-3 group and si-NC group was detected by using GABRD and GAPDH as primers. The nucleotide upstream and downstream sequences of GABRD are shown in SEQ ID NO. 7 and SEQ ID NO. 8; the nucleotide upstream and downstream sequences of GAPDH are shown in SEQ ID NO. 9 and SEQ ID NO. 10; the PCR reaction conditions are pre-denaturation: 95°C, 10 min; denaturation: 95°C, 15 s; annealing / extension: 60°C, 60 s; 40 cycles, finally use 2 -△△CT The expression level was determined by the method.

[0036] Reference Figure 2 is the comparison chart of the expression level of GABRD in siGABRD-1, siGABRD-2, siGABRD-3 and si-NC in HGC27 cells. Figure 2 Compared with the control group si-NC, siGABRD-1, siGABRD-2 and siGABRD-3 can significantly inhibit the expression level of GABRD in HGC27 cells (** represents P<0.01, *** represents P<0.001, and the test number is ≥3).

[0037] Example 4 CCK8 experiment to detect the effect of siGABRD on cell activity The control group si-NC cells after transfection for 24 h were re-counted after trypsin digestion, and a standard curve was prepared. The 1640 medium containing 2% FBS was diluted by equal proportion to obtain a cell concentration gradient, which was 2 x 10 4 , 1 x 10 4 , 5 x 10 3 , 2.5 x 10 3 , 1.25 x 10 3 , respectively. Each concentration had 6 duplicate wells. After inoculation, the cells were cultured for 4 h to adhere, and then CCK-8 reagent was added for 1 h before the OD value was measured to prepare a standard curve with cell number as the abscissa (X axis) and OD value as the ordinate (Y axis). According to the standard curve, the cell number of the sample was measured, and the siGABRD-1, siGABRD-2, siGABRD-3 and si-NC HGC27 after transfection for 24 h were counted by trypsin digestion, then re-plated in a 96 plate, 5 x 10 3 cells per well, with a volume of 100 μL. Each set had 6 duplicate wells, and the setting time was 24, 49 and 72 h. The 96-well plate was placed in the incubator, and at 24, 48 and 72 h, 10 μL of CCK-8 solution was added to each well, and after 1 h of culture, the absorbance at 450 nm was measured by an enzyme marker.

[0038] Figure 3 In the CCK8 proliferation experiment, the effects of siGABRD-1, siGABRD-2, siGABRD-3 and si-NC on HGC27 cell activity were as shown in FIG. 5. Compared with the control group si-NC, siGABRD-1, siGABRD-2 and siGABRD-3 could significantly inhibit the cell activity of HGC27 cells. Among them, siGABRD-3 had the most significant effect on cell activity, and siGABRD-3 was selected for further experiments. Figure 3

[0039] Example 5 Transwell experiment to detect the effect of siGABRD on cell migration ability The siGABRD-3 and si-NC HGC27 after transfection for 24 h were counted by trypsin digestion, and then resuspended in 200 μL of FBS-free 1640 medium to a concentration of 7 x 10 4 ​The cells were inoculated into a permeable cell culture upper chamber (chamber aperture 8 μm), 700 μL of 1640 culture medium containing 10% FBS was added into the lower culture hole, the chamber was carefully placed into the lower culture medium, and the incubator was continuously cultured for 48 h. The upper chamber was taken out, the residual liquid in the chamber was spun dry, and PBS solution was washed three times. The residual cells on the chamber membrane were gently wiped with a cotton swab, 700 μL of 4% polyformaldehyde was added into each lower hole for fixation at room temperature for 20 min. The liquid components in the chamber were spun dry, 700 μL of crystal violet was added into each lower hole for dyeing for 15 min. The crystal violet solution in the chamber was spun dry, and the chamber was washed with running water to wash away the excess dyeing liquid, and four fields of view were randomly selected under a microscope for observation and recording.

[0040] Figure 4 Figure 2 is a cell migration experiment showing the effect of siGABRD-3 and si-NC on the migration ability of HGC27 cells; as shown in the figure, compared with the control group si-NC, siGABRD-3 can significantly inhibit the migration ability of HGC27 cells. Figure 4

[0041] The above research shows that siGABRD-3 can target GABRD gene as a target, and by specifically inhibiting the expression of GABRD gene, it is a potential drug preparation for targeted treatment of gastric cancer.

[0042] The above provides a kind of siRNA for silencing GABRD gene expression and application provided by the present application. The above specific embodiments cannot be regarded as the limitation to the protection scope of the present application, and any alternative improvement or change made by the person skilled in the art to the embodiments of the present application falls within the protection scope of the present application.

[0043] The details of the present application are not described, which are well known to those skilled in the art.​

Claims

1. A siRNA that targets and silences the expression of the GABRD gene, characterized in that, The sense strand sequence of the siRNA is shown in SEQ ID NO.1, and the antisense strand sequence is shown in SEQ ID NO.

2.

2. The application of GABRD gene inhibitors in the preparation of drugs for the treatment / prevention of gastric cancer, characterized in that, The GABRD gene inhibitor is the siRNA as described in claim 1.

3. A pharmaceutical preparation for treating / preventing gastric cancer, characterized in that, Includes the siRNA as described in claim 1.

4. The pharmaceutical preparation according to claim 3, characterized in that, It also includes pharmaceutically acceptable drug carriers.

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