Application and method of KRT17 gene in retarding proliferation of pulmonary artery smooth muscle cells

Abstract

The invention belongs to the field of diagnosis and molecular biology, and particularly relates to application and a method of a KRT17 gene in slowing down proliferation of pulmonary artery smooth muscle cells. According to the invention, high expression of KRT17 in PAH and hypoxia-induced PASMCs is found and proved, and proliferation of pulmonary artery smooth muscle cells can be slowed down by knocking out KRT17, so that a foundation is provided for subsequent development of KRT17 genes in cardiovascular diseases.

Description

Technical Field

[0001] The present invention belongs to the field of diagnosis and molecular biology, and specifically relates to the application and method of KRT17 gene in slowing down the proliferation of pulmonary artery smooth muscle cells. Background Art

[0002] Pulmonary artery smooth muscle cells are cells located in the wall of the pulmonary artery, which are mainly responsible for regulating the contraction and relaxation of the pulmonary artery. Excessive proliferation and migration of pulmonary artery smooth muscle cells (PASMC) are the core events of pulmonary hypertension (PH).

[0003] How to slow down the proliferation of pulmonary artery smooth muscle cells has always been a hot topic in academia, so it is crucial to find a gene that can effectively slow down the proliferation of pulmonary artery smooth muscle cells.

[0004] KRT17 is a multipurpose protein involved in controlling proliferation, growth, infiltration, inflammation and other cellular processes. Studies have shown that KRT17 can act as a regulator of epigenetic modifications of nuclear structure, chromatin organization and gene expression in keratinocytes, and it has a clear role in chromatin dynamics, gene expression, RNA processing, nuclear cytoplasmic transport, etc., so KRT17 may regulate gene expression epigenetically. In addition, studies have found that downregulation of KRT17 can reduce cell proliferation by inhibiting the HIF-1α pathway; other studies have confirmed that knocking down KRT17 can weaken ROS levels in cells and reduce cell proliferation, migration and invasion. At present, its role in cardiovascular disease, especially PH, is unknown. Summary of the invention

[0005] In order to solve the technical problems in the above-mentioned background technology, the present invention provides an application and method of KRT17 gene for slowing down the proliferation of pulmonary artery smooth muscle cells.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In one aspect, the present invention provides a gene for slowing down the proliferation of pulmonary artery smooth muscle cells, wherein the gene is the KRT17 gene.

[0008] Based on the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, the forward primer sequence of the KRT17 gene is shown in SEQ ID NO: 1, and the reverse primer sequence of the KRT17 gene is shown in SEQ ID NO: 2.

[0010] Another aspect of the present invention provides the use of the gene for slowing down the proliferation of pulmonary artery smooth muscle cells as described above in the preparation of a kit for treating cardiovascular diseases.

[0011] Another aspect of the present invention provides the use of KRT17 gene in slowing down the proliferation of pulmonary artery smooth muscle cells.

[0012] Another aspect of the present invention provides use of KRT17 gene in treating cardiovascular diseases.

[0013] Another aspect of the present invention provides a method for slowing down the proliferation of pulmonary artery smooth muscle cells, so that the KRT17 gene in the cells is not expressed or its expression is reduced.

[0014] The beneficial effect of the present invention is that the present invention discovers and confirms that KRT17 is highly expressed in both PAH and hypoxia-induced PASMCs, and knocking out KRT17 can slow down the proliferation of pulmonary artery smooth muscle cells, providing a basis for the subsequent development of the KRT17 gene in cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the experimental flow chart of the present invention;

[0016] Figure 2 This is a graph showing the expression of the KRT17 gene in the lung tissue of SuHx-induced PAH mice;

[0017] Figure 3 The figure is the Western Blotting result of KRT17 in lung tissue homogenate of SuHx-induced PAH mice of the present invention;

[0018] Figure 4 This is a Western Blotting result diagram of PAH human lung tissue homogenate KRT17 of the present invention;

[0019] Figure 5 The Western Blotting results of KRT17 in lung tissue homogenate of PAH rats induced by monocrotaline (MCT) of the present invention;

[0020] Figure 6 This is an immunofluorescence staining image of lung tissue of PAH rats induced by SuHx of the present invention;

[0021] Figure 7 This is a Western Blotting result diagram of the interference efficiency of KRT17 in PASMCs of the present invention;

[0022] Figure 8 This is a Western Blotting result diagram of KRT17 gene knockdown in hypoxic PASMCs of the present invention;

[0023] Fig. 9 This is the EDU detection diagram of the hypoxic or KRT17 gene knockout PASMCs of the present invention;

[0024] Fig.10 This is a scratch experiment diagram of the present invention in which PASMCs were subjected to hypoxia or combined with KRT17 gene knockout for 24 hours;

[0025] Fig.11 The figure is the Western Blotting result of cyclinA in PASMCs with hypoxia or combined KRT17 gene knockout of the present invention;

[0026] Fig.12 The figure is the Western Blotting result of cyclinD in PASMCs with hypoxia or combined with KRT17 gene knockout of the present invention;

[0027] Fig.13 This is a diagram showing the Western Blotting results of PCNA in PASMCs subjected to hypoxia or combined with KRT17 gene knockout according to the present invention.

[0028] The information of the sequences involved in the present invention is described in the following table:

[0029] DETAILED DESCRIPTION

[0030] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0031] Embodiment 1,

[0032] 1.1、Use Figure 1 The experimental process shown in the figure was used to carry out the research, and the experimental information used is shown in Table 1 below:

[0033] Table 1

[0034]

[0035] 1.2 Functional testing experimental data

[0036] Using KRT17 gene as template, RNA interference target sequence was designed to construct target gene SiRNA knockdown model. The experimental materials are shown in Table 2 below:

[0037] Table 2

[0038]

[0039]

[0040]

[0041] 1.3. RNA interference target design:

[0042] 1. Gene information is as shown in Table 3

[0043] Table 3

[0044]

[0045] 2. RNA interference target design

[0046] According to the RNA interference sequence design principle, multiple RNA interference target sequences were designed using the KRT17 gene as a template. After western blotting experiments, the sequences in Table 4 below were selected as interference targets.

[0047] Table 4

[0048]

[0049]

[0050] 1.4. Construction of SiRNA cell interference model

[0051] 1. PASMCs transfection efficiency model

[0052] ①Ultraviolet light should be used to irradiate the cell room and clean bench for 30 minutes in advance;

[0053] ② Observe the cell status under the microscope. When the cell density reaches 60-80%, the transfection efficiency experiment can be performed. Select 15 bottles of cells with similar growth status and randomly divide them into Control, siNC, si-001, si-002, and si-003 groups.

[0054] ③ Prepare RNA oligo reagent according to Table 5:

[0055] Table 5

[0056]

[0057] Note: Control: control; si-NC: negative control; si-001: KRT17 001 interference sequence; si-002: KRT17 002 interference sequence; si-003: KRT17 003 interference sequence.

[0058] ④After the above reagents are prepared, let them stand for 5 minutes; mix tubes A and B and let them stand for 15 minutes. During the standing period, wash the cells 3 times with PBS, aspirate the PBS in the wells, and add 900μL of incomplete low-glucose DMEM to each well; after 15 minutes, add 100μL of the mixed reagent to each well and place it in the incubator for culture.

[0059] ⑤After 6 hours, replace with incomplete low-glucose DMEM containing 1% FBS, culture in an anoxic chamber, and extract protein for 24 hours.

[0060] 1.5. PASMCs KRT17 knockout model

[0061] ①Ultraviolet light should be used to irradiate the cell room and clean bench for 30 minutes in advance;

[0062] ② Observe the cell status under the microscope. When the cell density reaches 60-80%, the transfection experiment can be carried out. Select 15 bottles of cells with similar growth status and randomly divide them into siNC group, Hyp+siNC group, and Hyp+siGPR146 group.

[0063] ③ Prepare RNA oligo reagent according to Table 6 below:

[0064] Table 6

[0065]

[0066] Note: siNC: negative control; siKRT17: KRT17 gene knockout. The nucleic acid sequence of KRT17 is as follows: GCGACTACAGTGCTTATTA.

[0067] ④After the above reagents are prepared, let them stand for 5 minutes; mix tubes A and B and let them stand for 15 minutes. During the standing period, wash the cells 3 times with PBS, aspirate the PBS in the bottle, and add 1.8mL of incomplete low-glucose DMEM to each bottle; after 15 minutes, add 200μL of mixed reagent to each bottle and place it in the incubator for culture;

[0068] ⑤ After 6 hours, the incomplete low-glucose DMEM containing 1% FBS was replaced, the siNC group was placed in a cell culture incubator, and the Hyp+siNC group and the Hyp+siKRT17 group were placed in an oxygen-deficient chamber for culture, and the protein was extracted after 24 hours.

[0069] 1.6 Mouse SuHx Model

[0070] We chose hypoxia + intraperitoneal injection of Sugen5416 to establish the mouse SuHx model, which is a classic PH animal model with similar characteristics to human vascular occlusive pulmonary hypertension. Ten adult male mice weighing 200g±10g were randomly divided into two groups: Control group and SuHx group. Sugen5416 was dissolved in DMSO as a solvent, and SU5416 (20mg / kg) was injected intraperitoneally once into the mice of the SuHx group, and then they were raised under hypoxic conditions (10% O2, humidity 60-70%, temperature 18-22℃) for three weeks. The mice in the Control group were injected intraperitoneally with the same volume of DMSO once and placed under normoxic conditions (21% O2, humidity 60-70%, temperature 18-22℃) for three weeks.

[0071] 1.7. qPCR detection of mRNA expression of KRT17 gene in lung tissue of SuHx-induced PAH mice

[0072] 1. Experimental process: Total RNA was extracted from tissues and reverse transcribed to obtain cDNA. Real-time PCR was used to detect the expression level of KRT17 gene.

[0073] 2. PCR primer information is as shown in Table 7:

[0074] Table 7

[0075]

[0076] 3. Results and conclusions: After successful mouse modeling, the test results are as follows Figure 2 As shown, the expression of KRT17 gene at the mRNA level in the control group Control and the experimental group SuHx was significantly upregulated, indicating that the expression of KRT17 in SuHx was significantly increased.

[0077] 1.8. Detection of KRT17 gene expression in PAH mice, rats and human IPAH lung tissues 1. Experimental process:

[0078] (1) Tissue protein extraction: Cut the lung tissue into pieces the size of soybeans, place the lung tissue in a mortar, add 1 mL of lysis solution (containing 1% 100×Protease Inhibitor Cocktail) and grind until the blocky tissue disappears, let it stand for 30 minutes to lyse. The whole process is carried out on ice. Set the centrifuge at 12000 rpm at 4°C for 15 minutes, add 5 x Loading Buffer at a ratio of 4:1 and mix well, and place in a 100°C water bath for 5 minutes. (2) Electrophoresis: Prepare separation gel and concentration gel, load the sample after solidification, set the voltage to 120V and start electrophoresis. The electrophoresis is completed when bromophenol blue approaches the bottom of the separation gel. Remove the glass plate, rinse the electrophoresis solution outside the plate with deionized water, and prepare for transfer.

[0079] (3) Transfer: Cut the BIO-RAD transfer filter paper, sponge and NC membrane to appropriate sizes, and place them on the transfer clip in the order of sponge, filter paper, glue, NC membrane, filter paper, and sponge from the negative electrode to the positive electrode. After the transfer is completed, take out the NC membrane and dye it in the Ponceau red dye solution. Rinse the Ponceau red dye solution on the surface of the NC membrane with deionized water and wash with TBS-T for 5 minutes. Place the NC membrane in 5% skim milk powder and block it on a shaker for 1 hour. After blocking, wash it with TBS-T for 5 minutes.

[0080] (4) Apply primary and secondary antibodies: Dilute the primary antibody in proportion with universal antibody diluent, and incubate the strips in the primary antibody solution at 4°C overnight. Take out the strips and wash them with TBS-T for 40 min, dilute the secondary antibody in proportion with 5% skim milk powder, and incubate at room temperature for 1 h; after incubation, wash with TBS-T for 40 min.

[0081] (5) Development: Take equal volumes of ECL super-sensitive luminescent liquid A and liquid B and mix them. Add the mixture dropwise onto the NC membrane until the membrane is completely covered. Let it stand at room temperature for 5 min and remove the luminescent liquid. Cut an X-ray film of appropriate size and place it on the strip for 3-5 min. Take out the film and place it in the developer for development. When the development result appears on the film, quickly place the strip in the fixer for fixation. After 2 min, wash off the residual fixer on the film with water and let it dry.

[0082] Scan the film and analyze the results. Figure 3 to Figure 5 As shown in the figure, after the PAH mice and rats were successfully modeled, Western Blotting experiments were performed on the tissue samples of the control group CON and the experimental groups SuHx, MCT, HYP and IPAH, and it was found that the expression of KRT17 gene in these groups of samples was upregulated compared with the control group, proving that KRT17 is involved in the process of PAH.

[0083] 1.9 KRT17 knockout reduces the expression of hypoxia-induced pulmonary artery smooth muscle cells

[0084] 1. Experimental process:

[0085] (1) Equilibrate the lung tissue slices to room temperature, wipe off excess water with absorbent paper, and draw the outline of the lung slices with a fluorescent pen;

[0086] (2) Wash with PBS three times, 5 min each time;

[0087] (3) Prepare 3% BSA with PBS, filter small particles with a 0.45 μm semipermeable membrane, evenly cover the sections with blocking solution, and block at room temperature for 1 h;

[0088] (4) Dilute the primary antibody with PBS at a dilution ratio of 1:200. Evenly cover the sections with the primary antibody solution and incubate in a 4°C refrigerator overnight. Remove the primary antibody the next day and wash with PBS three times, 5 min each time.

[0089] (5) Dilute the secondary antibody with PBS at a dilution ratio of Mouse / Rabbit = 1:200. Evenly cover the slices with the secondary antibody solution and incubate at room temperature in the dark for 1 h. Remove the secondary antibody and wash with PBS three times, 5 min each time.

[0090] (6) Take photos and analyze them under a fluorescence microscope.

[0091] (7) A PASMCs transfection efficiency model was established, and the optimal transfection efficiency among the three groups of interference sequences was confirmed by Western Blotting experiments.

[0092] (8) Select the optimal interference sequence for the experiment and establish the KRT17 knockout model of PASMCs.

[0093] (9) Western Blotting experiments were performed to verify the expression of KRT17 under hypoxia and after the addition of small molecule RNA interfering with KRT17. The results are as follows Figures 6 to 8 As shown, first, we confirmed through immunofluorescence staining that the increase of KRT17 was mainly concentrated in the media layer of the reconstructed blood vessels, that is, the smooth muscle cell layer. Secondly, to explore the role of KRT17 in PH, we used a set of siRNA to knock down the expression of KRT17 in hypoxia-induced PASMCs. After detecting the transfection efficiency, we selected the si-002 interference sequence to continue the experiment. Western Blotting results showed that hypoxia promoted the expression of KRT17. When the small molecule RNA interfering with KRT17 was added, the increase of KRT17 stimulated by hypoxia was successfully inhibited.

[0094] 2.0, EdU, and scratch assays to detect the effects of KRT17 gene knockout on cell proliferation

[0095] EDU Experiment:

[0096] (1) Experimental process: edu scratch

[0097] (2) Inoculate an appropriate amount of PASMCs suspension into a 24-well plate and place it in a cell culture incubator for culture;

[0098] (3) When the cell density reaches 60-80%, PASMCs are modeled according to experimental requirements;

[0099] (4) Dilute the EDU solution with incomplete high-glucose DMEM at a ratio of 1000:1 to prepare 50 μM EDU culture medium; add 200 μL of 50 μM EDU culture medium to each well and incubate in a cell culture incubator for 2 h;

[0100] (5) Wash the cells three times with PBS, 5 min each time; add 200 μL of 4% paraformaldehyde to each well to fix the cells; 30 min later, add 200 μL of 2 mg / mL glycine solution to each well and incubate on a shaker for 5 min;

[0101] (6) Wash the cells three times with PBS, 5 min each time; add 200 μL of PBS containing 0.5% TritonX-100 to each well and incubate on a shaker for 10 min;

[0102] (7) Add 200 μL 1× The staining reaction solution was incubated on a shaker in the dark for 30 min;

[0103] (8) Wash the cells three times with PBS containing 0.5% TritonX-100, each time for 10 min; wash the cells three times with PBS;

[0104] (9) Add 200 μL DAPI staining solution to each well and incubate in the dark for 15 min; wash the cells three times with PBS, 5 min each time;

[0105] (10) Photograph and analyze under a fluorescence microscope.

[0106] Scratch test

[0107] (1) Use a marker to draw evenly spaced horizontal lines of 0.5-1 cm on the back of the 6-well plate;

[0108] (2) Inoculate an appropriate amount of PASMCs suspension into a 6-well plate and place it in a cell culture incubator for culture;

[0109] (3) When the cell density reaches 60-80%, PASMCs are modeled according to experimental requirements;

[0110] (4) Use the tip of the gun to compare with the ruler and scratch along the line of the marker pen, wash the cells three times with PBS, and take pictures at 0 h;

[0111] (5) After placing the cells in an anaerobic chamber for 24 hours, wash the cells three times with PBS.

[0112] (6) Take photos and analyze them under a fluorescence microscope.

[0113] The results are as follows Fig. 9 and Fig.10 As shown, the results of EDU and scratch experiments showed that under hypoxic conditions, the rate of cell proliferation was significantly increased; and after KRT17 gene knockout in PASMCs, the abnormal proliferation of PASMCs caused by hypoxia could be alleviated.

[0114] 2.1. Western Blotting to detect the effect of KRT17 gene knockout on cell cycle

[0115] Experimental procedure: Cell protein extraction

[0116] (1) Pour off the culture medium in the cell bottle, wash with PBS three times, aspirate the excess PBS in the bottle, and add lysis buffer (containing 1% 100× Protease Inhibitor Cocktail) to cover the cell surface.

[0117] (2) Use a scraper to scrape the cells off the flask wall to fully lyse them. Repeat this process three times for 10 minutes each time. The entire process should be carried out on ice.

[0118] (3) After lysis, transfer the lysate to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 15 min at 4 °C.

[0119] (4) Collect the supernatant and add 5 x Loading Buffer in a 4:1 ratio, mix well, and incubate in a 100°C water bath for 5 min.

[0120] (5) Electrophoresis: Prepare the separation gel and the concentrated gel, load the sample after solidification, set the voltage to 120V and start electrophoresis. The electrophoresis is completed when the bromophenol blue approaches the bottom of the separation gel. Remove the glass plate and rinse the electrophoresis solution outside the plate with deionized water before preparing for transfer.

[0121] (6) Transfer: Cut the BIO-RAD transfer filter paper, sponge and NC membrane to appropriate sizes, and place them on the transfer clip in the order of sponge, filter paper, glue, NC membrane, filter paper, and sponge from the negative electrode to the positive electrode. After the transfer is completed, take out the NC membrane and stain it in Ponceau red dye solution. Rinse the Ponceau red dye solution on the surface of the NC membrane with deionized water and wash with TBS-T for 5 minutes. Place the NC membrane in 5% skim milk powder and block it on a shaker for 1 hour. After blocking, wash it with TBS-T for 5 minutes.

[0122] (7) Apply primary and secondary antibodies: Dilute the primary antibody in proportion with universal antibody diluent, and incubate the strips in the primary antibody solution at 4°C overnight. Take out the strips and wash them with TBS-T for 40 min, dilute the secondary antibody in proportion with 5% skim milk powder, and incubate at room temperature for 1 h; after incubation, wash with TBS-T for 40 min.

[0123] (8) Development: Take equal volumes of ECL super-sensitive luminescent liquid A and liquid B and mix them. Add the mixture dropwise onto the NC membrane until the membrane is completely covered. Let it stand at room temperature for 5 min and remove the luminescent liquid. Cut an X-ray film of appropriate size and place it on the strip for 3-5 min. Take out the film and place it in the developer for development. When the development result appears on the film, quickly place the strip in the fixer for fixation. After 2 min, wash off the residual fixer on the film with water and let it dry.

[0124] (9) Scan the film and analyze the results.

[0125] Western Blotting results of cyclin A in PASMCs induced by hypoxia or combined with KRT17 gene knockout are shown in Figure 2 Figures 11 to 13 shown.

[0126] In the experimental results of Example 1:

[0127] n=3, *P<0.05, ** P<0.01, *** P<0.001

[0128] CON: control group; SuHx: hypoxia + Sugen5416 group; IPAH: patients with pulmonary arterial hypertension; MCT: hypoxia + monocrotaline group

[0129] SiNC: normoxia + negative control; HYP+SiNC: hypoxia + negative control; HYP+SiKRT17: hypoxia + KRT17 gene knockout.

[0130] Based on the above experimental results, it was confirmed that KRT17 was highly expressed in both PAH- and hypoxia-induced PASMCs, and knocking out KRT17 could slow down the proliferation of pulmonary artery smooth muscle cells.

[0131] In the description of the present invention, it is necessary to understand that the directions or positional relationships indicated by the terms "upper", "lower", "front", "rear", etc. are based on the directions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or unit referred to must have a specific direction, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0132] In the description of this specification, the description of the terms "one embodiment", "some embodiments", "specific embodiments", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A gene for slowing down the proliferation of pulmonary artery smooth muscle cells, characterized in that The gene is the KRT17 gene.

2. The gene for slowing down the proliferation of pulmonary artery smooth muscle cells according to claim 1, characterized in that: The forward primer sequence of the KRT17 gene is shown in SEQ ID NO: 1, and the reverse primer sequence of the KRT17 gene is shown in SEQ ID NO:

2.

3. Use of the gene for slowing down the proliferation of pulmonary artery smooth muscle cells as claimed in claim 1 in preparing a kit for treating cardiovascular diseases.

4. Application of KRT17 gene in slowing down the proliferation of pulmonary artery smooth muscle cells.

5. Application of KRT17 gene in the treatment of cardiovascular diseases.

6. A method for slowing the proliferation of pulmonary artery smooth muscle cells, characterized in that: The KRT17 gene in the cell is not expressed or its expression is reduced.

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