A siRNA of GILZ gene and its application

Abstract

The present invention discloses a siRNA of a GILZ gene and its application, and belongs to the field of biomedicine technology. The present invention provides a small interfering RNA for the GILZ gene that is lowly expressed in lung cancer cells. Related cytological experiments show that the small interfering RNA provided by the present invention has the important functions of inhibiting the protein expression of GILZ in lung cancer cells and promoting the survival, proliferation and invasion of cancer cells. Therefore, the siRNA molecule targeting the GILZ gene provided by the present invention can be used to establish a lung cancer cell growth-related model, which helps to clarify the role of the GILZ gene and its target gene, helps to clarify the development law of the GILZ transcription factor in malignant tumors, and provides help for studying the pathogenesis of lung cancer at the molecular level.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a siRNA of a GILZ gene and an application thereof. Background Art

[0002] Lung cancer is one of the most common malignancies worldwide, with nearly 2 million new cases and more than 1 million deaths each year, ranking first in cancer-related deaths among men and second in cancer-related deaths among women. Metastasis is a deadly hallmark of cancer, and tumor metastasis is a major challenge in the clinical management of cancer. Metastatic disease accounts for more than 90% of all cancer-related deaths and is often associated with high patient mortality because it is difficult to treat with surgery or conventional chemotherapy and radiation therapy.

[0003] During the progression of lung cancer, the expression of certain genes changes, which are key genes in the occurrence and development of lung cancer and potential targets for lung cancer treatment. GILZ, also known as TSC22D3, is a member of the TGF-β-stimulated clone 22 domain (TCS-22) family. Existing studies have shown that GILZ plays an important role in many cancers, but its impact on the occurrence and development of lung cancer is still not fully understood. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a siRNA molecule of the GILZ gene. Another technical problem to be solved by the present invention is to provide the application of the siRNA molecule of the GILZ gene in establishing a lung cancer growth-related model.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] An siRNA targeting the GILZ gene is a double-stranded siRNA molecule having the following nucleic acid sequence:

[0007] 5′-CCAUAGACAACAAGAUCGAdTdT-3′

[0008] 5′-UCGAUCUUGUUGUCUAUGGdTdT-3′.

[0009] The use of the siRNA targeting the GILZ gene in at least one of the following (a)-(c):

[0010] (a) promoting the proliferation of lung cancer cells, or preparing a product for promoting the proliferation of lung cancer cells;

[0011] (b) promoting lung cancer cell invasion, or preparing a product for promoting lung cancer cell invasion;

[0012] (c) promoting lung cancer cell cloning, or preparing a product for promoting lung cancer cell cloning.

[0013] Furthermore, the product for promoting lung cancer cell proliferation is a lung cancer cell model with enhanced proliferation ability, or a substance for preparing a lung cancer cell model with enhanced proliferation ability.

[0014] Furthermore, the product for promoting lung cancer cell invasion is a lung cancer cell model with enhanced invasion ability, or a substance for preparing a lung cancer cell model with enhanced invasion ability.

[0015] Furthermore, the product for promoting lung cancer cell cloning is a lung cancer cell model with enhanced cloning ability, or a substance for preparing a lung cancer cell model with enhanced cloning ability.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides small interfering RNA for the GILZ gene that is under-expressed in lung cancer cells. Related cytological experiments show that the small interfering RNA provided by the present invention has the important functions of inhibiting the protein expression of GILZ in lung cancer cells and promoting the survival, proliferation and invasion of lung cancer cells. Therefore, the siRNA molecule targeting the GILZ gene provided by the present invention can be used to establish a lung cancer cell growth-related model, which helps to clarify the role of the GILZ gene and its target gene, and helps to clarify the development law of the GILZ transcription factor in malignant tumors, and provides help for studying the pathogenesis of lung cancer at the molecular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the expression detection diagram of GILZ in lung cancer cells and lung cancer tissues;

[0019] Figure 2 This is a diagram of the CCK8 experiment demonstrating the tumor-promoting effect of small interfering fragments at the cellular level;

[0020] Figure 3 This is a diagram of the tumor-promoting effect of the small infected fragment at the cellular level demonstrated by qPCR experiments;

[0021] Figure 4 Experimental diagram for cell clone formation;

[0022] Figure 5 This is a diagram of a cell invasion experiment;

[0023] Figure 6 This is a picture of cell apoptosis experiment. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with specific embodiments.

[0025] Embodiment 1:

[0026] Step 1: Detection of GILZ expression in lung cancer cells and lung cancer tissues

[0027] The total protein of each group of cells was extracted and collected, and the protein content was determined by BCA protein quantification kit (purchased from Thermo Scientific). 50 μg of total protein from each group was separated by 12% SDS-PAGE gel electrophoresis, and the protein was transferred to a nitrocellulose membrane by semi-dry electrotransfer, and then blocked with a blocking solution containing 5% skim milk powder at room temperature for 2 hours. Anti-GILZ antibody (purchased from Invitrogen) was used as the primary antibody, incubated at room temperature for 2 hours, and then HRP-labeled goat anti-rabbit IgG (purchased from Santa Cruz) was used as the secondary antibody, and incubated at room temperature for 2 hours. Finally, the bands were developed by chemiluminescence (ECL) and GAPDH was used as the internal reference. The results are shown in Figure 1 , GILZ expression in H1299 lung cancer cells and tissues was lower than that in normal tissues and cells.

[0028] Step 2: Construction and identification of siGILZ small interfering fragment

[0029] siGILZ was synthesized by Heyuan Biotechnology Co., Ltd. The specific sequence is as follows:

[0030] 5′-CCAUAGACAACAAGAUCGAdTdT-3′

[0031] 5′-UCGAUCUUGUUGUCUAUGGdTdT-3′

[0032] Step 3: Detection of the function of siGILZ small interfering fragment in promoting lung cancer proliferation

[0033] Cytological experiments

[0034] 1: CCK8 experiments demonstrate the tumor-promoting effect of small interfering fragments at the cellular level

[0035] Lung cancer cells H1299 were seeded in a 96-well culture plate at a density of 2000 cells / well, and the GILZ small interfering fragment was transfected with a transfection reagent, and the upstream and downstream siRNAs were used together. H1299 cells were transiently transfected with siGILZ and the control group, and 10μL CCK-8 solution was added to each well after 0h, 24h, 72h, and 96h, respectively. After 2 hours, the wavelength was selected, and the light absorption value of each well at different time points was measured on an enzyme-linked immunosorbent assay, and the results were recorded. The size of the absorption value reflects the activity of the cells. The larger the light value, the higher the cell viability. According to the experimental data, a graph was drawn with time as the horizontal axis and the absorption value as the vertical axis. The CCK-8 results showed that compared with the control group, the small interfering experimental group significantly increased the proliferation of H1299 cells, as shown in Figure 2. Figure 2 shown.

[0036] 2: qPCR experiments demonstrate the tumor-promoting effect of small infected fragments at the cellular level

[0037] Lung cancer cells H1299 were inoculated in 6-well culture plates at a density of 20,000 cells / well. GILZ small interfering fragments and other control groups were transiently transfected into H1299 cells using transfection reagents, and cells were collected after 48 hours. After washing the cell samples in the culture plate twice with 1*PBS, the PBS was sucked clean with a 1mL gun, and 1mL trizol reagent was added to extract total cell RNA. According to the instructions for use of the reagent: collect the cells by centrifugation, pour off the cell solution, mix to room temperature for 5 minutes, and repeatedly blow with a 1mL pipette until the cells are completely lysed into a uniform solution and are not viscous. Then add 200μL of chloroform, shake vigorously for 15 seconds, leave at room temperature for 5 minutes, then centrifuge at 40C, 12000rpm for 5 minutes, transfer the upper aqueous phase to an Eppendorf tube, add 500μL of isopropanol and mix. Centrifuge at 4℃, 15000rpm for 15 minutes, and pour out the isopropanol. Wash the precipitate once with 70% ethanol, place on ice, and let the residual ethanol evaporate. Dissolve in an appropriate amount of DEPC water, measure the purity and content of RNA by UV spectrometer, and store in aliquots and freeze. In a 20μL system, add 1μg total RNA, 1μL oligo dT (500μg / mL). RNase-Free water, incubate at 70℃ for 10 minutes, then immediately cool in an ice bath, add 4μL 5×Buffer, 2μL DTT, 1μL dNTPs, mix well, and keep warm at 42℃ for 2 minutes, add 1μL (200U) reverse transcriptase, and continue incubation at 42℃ for 50 minutes. Perform qPCR reaction on the machine. The results are as follows: Figure 3 As shown, the small interfering fragment transfection group effectively reduced the mRNA level of GILZ in lung cancer cells.

[0038] 3: Cell clone formation experiment

[0039] Take lung cancer H1299 cells in the logarithmic growth phase and culture them in 6-well plates. The NC group and the experimental group siGILZ are digested with trypsin and blown into a single cell suspension in DMEM with 10% fetal bovine serum for use. Inoculate 2mL of 6-well plates with a density of 50-100 cells in each group and gently rotate to disperse the cells evenly. Culture them in a 37-degree 5% CO2 incubator for 2 to 3 weeks. Observe every 2-3 days. When visible clones appear in the culture dish, terminate the culture and discard the supernatant. Wash carefully twice with PBS. Figure 4 As shown in the results, compared with the control group, the clone proliferation in the siGILZ experimental group was significantly greater than that in the control group.

[0040] 4: Cell invasion assay

[0041] Take 300μL serum-free culture medium, add 60μL Matrigel, and mix well. Operate on ice, add 100μL to the upper chamber, put it in a 37-degree incubator, incubate for 4-5h, and solidify. Serum-free culture medium and matrix gel are diluted at 1:5, and 50μL is added to each well. In a 37-degree incubator, make NC control group and siGILZ experimental group, respectively. Digest lung cancer H1299 cells, count, and prepare cell suspension. Add 100μL of cell suspension to each well, add 500μL of culture medium containing 20% ​​FBS to the lower chamber, culture for 24h, remove the transwell, wash twice with PBS, fix with methanol, add 0.1% crystal violet for 5-10min, keep at room temperature for half an hour, wash twice with PBS, wipe off the cells on the surface with cotton balls, and observe under a microscope. The results are as follows Figure 5 As shown, the invasion ability of the siGILZ experimental group was increased compared with the control group.

[0042] 5: Cell apoptosis experiment

[0043] Extract H1299 cells, control group NC and experimental group siGILZ protein, prepare protein gel, measure protein concentration by BCA, perform NC and siGILZ protein electrophoresis, then transfer the membrane for about 2 hours, then block for 1-2 hours, incubate with primary antibodies: Bcl2 anti-apoptotic protein; Bax and Beclin pro-apoptotic proteins, the results are as follows Figure 6 As shown, compared with the control group, the siGILZ experimental group inhibited apoptosis. Sequence Listing <110> Nantong Cancer Hospital <120> A siRNA of GILZ gene and its application <130> 100 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> RNA <213> siGILZ-F(Artificial) <400> 1 ccauagacaa caagaucga 19 <210> 2 <211> 19 <212> RNA <213> siGILZ-R(Artificial) <400> 2 ucgaucuugu ugucuaugg 19

Claims

1. Use of siRNA targeting the GILZ gene in at least one of the following (a)-(c): (a) preparing a product for promoting lung cancer cell proliferation, wherein the product for promoting lung cancer cell proliferation is a lung cancer cell model with enhanced proliferation ability, or a substance for preparing a lung cancer cell model with enhanced proliferation ability; (b) preparing a product for promoting lung cancer cell invasion, wherein the product for promoting lung cancer cell invasion is a lung cancer cell model with enhanced invasion ability, or a substance for preparing a lung cancer cell model with enhanced invasion ability; (c) preparing a product for promoting lung cancer cell cloning, wherein the product for promoting lung cancer cell cloning is a lung cancer cell model with enhanced cloning ability, or a substance for preparing a lung cancer cell model with enhanced cloning ability; The siRNA is a double-stranded siRNA molecule with the following nucleic acid sequence: 5′-CCAUAGACAACAAGAUCGAdTdT-3′ 5′-UCGAUCUUGUUGUCUAUGGdTdT-3′; The lung cancer cells are H1299 lung cancer cells.

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