SiRNA for inhibiting expression of BNIP3L protein and application of siRNA in preparation of medicine for resisting myocardial cell hypoxia injury

Abstract

The invention belongs to the technical field of biology, and discloses siRNA for inhibiting BNIP3L protein expression and application of the siRNA in preparation of a medicine for resisting myocardial cell hypoxia injury. The siRNA for inhibiting BNIP3L protein expression is composed of a positive-sense strand and an antisense strand, the nucleotide sequence of the positive-sense strand is as shown in SEQ ID No. 1, and the nucleotide sequence of the antisense strand is as shown in SEQ ID No. 2. The siRNA for inhibiting the expression of the BNIP3L protein can obviously inhibit the expression of the BNIP3L of myocardial cells H9c2, down-regulate the autophagy level of mitochondria and obviously enhance the cell activity and mitochondrial function of the myocardial cells under hypoxia, and has the effect of resisting hypoxia-induced myocardial injury.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to siRNA for inhibiting the expression of BNIP3L protein and application thereof in preparing a drug for resisting myocardial cell hypoxia injury. Background Art

[0002] Hypoxia refers to a pathological process in which the metabolism, function and morphological structure of tissues undergo abnormal changes due to insufficient oxygen supply or oxygen use disorders. Studies have shown that hypoxia in the body can cause imbalance in autophagy and mitochondrial autophagy, leading to the occurrence of a variety of diseases on a macro scale. As a blood pumping organ, the heart is responsible for providing oxygen required for life activities to tissues and organs throughout the body, but at the same time, the heart is also an organ in the body that is highly oxygen-demanding and highly dependent on mitochondria, requiring high levels of oxygen and energy to maintain normal physiological activities of the body. The heart is extremely sensitive to hypoxia, and ischemic and hypoxic factors can easily cause myocardial damage, which in turn causes heart-related diseases. Autophagy and mitochondrial autophagy of cardiomyocytes are closely related to myocardial damage.

[0003] BNIP3L is one of the main mitophagy receptors, which exists on mitochondria and mediates mitophagy by interacting with LC3. BNIP3L is closely related to the occurrence and development of cancer, nervous system diseases, and cardiovascular diseases. For example, BNIP3L-mediated mitophagy has a protective effect on cerebral ischemia-reperfusion injury and is a potential target for stroke treatment. Studies have confirmed that increased HIF expression in a hypoxic environment activates BNIP3L-mediated mitophagy. In addition, in some diseases where mitophagy is downregulated, such as chronic obstructive pulmonary disease, brain trauma, and acute myeloid leukemia, the expression of BNIP3L is also significantly downregulated. However, there are currently no reports on the relevance of BNIP3L in hypoxia-induced cardiomyocyte damage, and the molecular mechanism of BNIP3L regulating mitophagy has not been fully elucidated; at the same time, no relevant technology has been found to combat hypoxic injury to cardiomyocytes with BNIP3L.

[0004] The siRNA that inhibits the expression of BNIP3L protein is abbreviated as: si-BNIP3L. Summary of the invention

[0005] The purpose of the present invention is to provide siRNA for inhibiting the expression of BNIP3L protein and its application in preparing a drug for resisting myocardial cell hypoxia injury. The drug of the present invention can reduce myocardial cell apoptosis caused by hypoxia.

[0006] Based on the above technical problems to be solved by the present invention:

[0007] One of the purposes of the present invention is to provide a use of siRNA for inhibiting the expression of BNIP3L protein in the preparation of a drug for resisting myocardial cell hypoxia injury.

[0008] Furthermore, the siRNA for inhibiting the expression of BNIP3L protein includes a sense strand and an antisense strand;

[0009] The sense strand is shown in SEQ ID No: 1; the antisense strand is shown in SEQ ID No: 2.

[0010] The second purpose of the present invention is to provide a siRNA that inhibits the expression of BNIP3L protein;

[0011] The siRNA that inhibits the expression of BNIP3L protein includes a sense strand and an antisense strand;

[0012] The sense strand is shown in SEQ ID No: 1; the antisense strand is shown in SEQ ID No: 2.

[0013] The third object of the present invention is to provide a pharmaceutical composition for preventing myocardial cell hypoxia injury, wherein the pharmaceutical composition comprises siRNA as an active ingredient;

[0014] Furthermore, the pharmaceutical composition also includes a carrier.

[0015] Furthermore, the vector is an acceptable vector for siRNA that pharmaceutically inhibits the expression of BNIP3L protein.

[0016] Furthermore, the carrier is one of cholesterol, virus, nanoparticle or liposome.

[0017] The beneficial effects of the present invention are as follows: Compared with the prior art, the present invention provides siRNA for inhibiting the expression of BNIP3L protein and its application in the preparation of a drug for preventing myocardial cell hypoxia injury. The BNIP3L-siRNA proposed in the present invention can inhibit the increase of myocardial cell autophagy level caused by hypoxia, reduce the level of oxidative stress, increase the level of ATP, improve mitochondrial function, and reduce myocardial cell damage caused by hypoxia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other embodiments can be obtained based on these drawings without paying creative work.

[0019] Figure 1Effects of si-BNIP3L on autophagy and mitophagy in hypoxic H9c2 cells; (A) Western Blot detection of changes in related protein expression after BNIP3L knockdown; (B) TOMM20 protein quantification results; (C) BNIP3L protein quantification results; compared with the Ctrl group, *P<0.05; compared with the HC group, #P<0.05;

[0020] Figure 2 Effects of si-BNIP3L on the viability of hypoxic H9c2 cells; compared with the Ctrl group, *P<0.05; compared with the HC group, #P<0.05;

[0021] Figure 3 The effect of si-BNIP3L on the ROS level of hypoxic H9c2 cells; (A) The effect of knocking down BNIP3L on the ROS level of hypoxic H9c2 cells (200×); (B) The quantitative results of ROS level; Compared with the Ctrl group, *P<0.05; Compared with the HC group, #P<0.05;

[0022] Figure 4 Effects of si-BNIP3L on mitochondrial function of hypoxic H9c2 cells; (A) Effect of knocking down BNIP3L on ATP content in hypoxic H9c2 cells; (B) Effect of knocking down BNIP3L on mROS level in hypoxic H9c2 cells (400×); (C) Quantification of mROS level; (D) Effect of knocking down BNIP3L on mitochondrial membrane potential level in hypoxic H9c2 cells (400×); (E) Quantification of mitochondrial membrane potential level; Compared with the Ctrl group, *P<0.05; Compared with the HC group, #P<0.05. DETAILED DESCRIPTION

[0023] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] The siRNA that inhibits the expression of BNIP3L protein was prepared by artificial synthesis.

[0027] The siRNA that inhibits the expression of BNIP3L protein is referred to as si-BNIP3L.

[0028] Example 1 :

[0029] The present invention provides the source of siRNA for inhibiting the expression of BNIP3L protein.

[0030] (1) si-BNIP3L design

[0031] Starting from the AUG start codon of the BNIP3L protein transcript, search for the downstream AA sequence and record the 19 nucleotides adjacent to the 3' end of each AA as candidate siRNA target sites. The sense strand has C or G at position 1 and U or A at position 19, and there are at least 3 U or A at positions 15 to 19 of the sense strand, or at least 5 U or A at positions 13 to 19.

[0032] According to the following principles: siRNAs with GC content of about 30%-50% are more effective than those with higher GC content; compare potential sequences with the corresponding genome database, and exclude those sequences that are homologous to other coding sequences and ESTs. Finally, the target sites are located at 131, 200, 260, 408, 413, and 516 for chemical synthesis of siRNA. A negative control siRNA is also synthesized.

[0033] Negative control siRNA: positive strand: 5' to 3' UUCUCCGAACGUGUCACGUTT; (SEQ ID No. 3)

[0034] Antisense strand: 5' to 3' ACGUGACACGUUCGGAGAATT; (SEQ ID No. 4)

[0035] siRNA at site 131: positive strand: 5' to 3'GCAGCAAUGGCAACGGUAATT; (SEQ ID No.5)

[0036] Antisense strand: 5' to 3' UUACCGUUGCCAUUGCUGCTT; (SEQ ID No.6)

[0037] 200-site siRNA: positive strand: 5' to 3'CCAGCUUCCGUCUCUAUUUTT; (SEQ ID No7)

[0038] Antisense strand: 5' to 3'AAAUAGAGACGGAAGCUGGTT; (SEQ ID No.8)

[0039] siRNA at site 260: positive strand: 5' to 3' GCUCAAGAGGCAGUUCUCATT; (SEQ ID No.9)

[0040] Antisense strand: 5' to 3' UGAGAACUGCCUCUUGAGCTT; (SEQ ID No.10)

[0041] siRNA at site 408: positive strand: 5' to 3' GAAGAGAAGUUGAAAGUAUTT; (SEQ ID No.1)

[0042] Antisense strand: 5' to 3'AUACUUUCAACUUCUCUUCTT; (SEQ ID No. 2)

[0043] siRNA at site 413: positive strand: 5' to 3' GUGCAGACUGGGUAUCAGATT; (SEQ ID No.11)

[0044] Antisense strand: 5' to 3' UCUGAUACCCAGUCUGCACTT; (SEQ ID No.12)

[0045] siRNA at site 516: positive strand: 5' to 3' GCACAGCUACUCUCAGCAUTT; (SEQ ID No.13)

[0046] Antisense strand: 5' to 3'AUGCUGAGAGUAGCUGUGCTT. (SEQ ID No.14)

[0047] The above siRNA was directly synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0048] (2) Determination of the inhibitory activity of si-BNIP3L on BNIP3L protein expression

[0049] Rat-derived H9c2 cardiomyocytes were purchased from Wuhan Pronocell Life Science Co., Ltd. The cells were cultured at 5×10 14The cells were inoculated at a concentration of 100 μg / mL in a six-well plate, and 2 mL was added to each well. The cells were placed in a cell culture incubator (37°C, 5% CO2) for culture. When the cells in the well plate grew to 70%-80%, the cells were divided into three groups: (1) normoxic control group (NC), the cells were placed in a normoxic incubator (37°C, 5% CO2) for 48 h; (2) hypoxic group (HC), hypoxia intervention (37°C, 0.5% O2 hypoxia intervention) for 48 h; (3) hypoxic cell BNIP3L-siRNA (131 site siRNA, 200 site siRNA, 260 site siRNA, 408 site siRNA, 413 site siRNA or 131 site siRNA) intervention group, and the transfection working solution was prepared. 200 μL Opti-MEM, 15 μL transmate transfection reagent, and 6 μL BNIP3L-siRNA were added to a 1.5 mL sterile EP tube in sequence, and the mixture was evenly mixed and allowed to stand for 20 min. Then, the transfection working solution was added to each transfection well, and the cells were subsequently transferred to a hypoxia workstation for hypoxia intervention (37° C., 0.5% O 2 hypoxia intervention) for 48 h.

[0050] Results: After digestion and collection of cells in each group, cell proteins were extracted and Western blot was performed to detect the expression of BNIP3L and mitochondrial outer membrane protein TOMM20. It was found that siRNA at site 408 could significantly inhibit the expression of BNIP3L and increase the level of TOMM20, with the best activity. Figure 1 As shown in the figure, compared with the hypoxia intervention group, the BNIP3L protein expression in the si-BNIP3L group at site 408 decreased by about 70%, and the protein knockdown effect was good. The expression of mitochondrial outer membrane protein TOMM20 increased significantly compared with the hypoxia group, and mitochondrial autophagy was weakened.

[0051] Example 2

[0052] The present invention proposes an experiment on the protective effect of si-BNIP3L at site 408 on cardiomyocytes under hypoxia.

[0053] After the hypoxia intervention of each group of cells in Example 1, the cell viability was determined by CCK8 method (Biosharp, BS350B). Alternatively, the collected cells were used to measure the ROS content using ROS kit (Solarbio, CA1410), and the ATP level was determined using ATP detection kit (Beyotime, S0026). Alternatively, the collected cells were used to detect the mitochondrial mROS level using MitoSOX Red kit (Thermo Fisher, M36008), and the mitochondrial membrane potential was determined using JC-1 detection kit (Beyotime, C2006).

[0054] The specific results are as follows:

[0055] 1) Effect of si-BNIP3L on the viability of hypoxic H9c2 cells. In order to explore the role of BNIP3L in hypoxic injury of H9c2 cardiomyocytes, the present invention used BNIP3L-siRNA to knock down BNIP3L before hypoxia intervention, and detected the biological effects of the knockdown group and the non-knockdown group. The results are as follows Figure 2 As shown, compared with the hypoxia intervention group, the cell viability of the BNIP3L knockdown group was significantly increased (P<0.05), indicating that si-BNIP3L has a protective effect on hypoxic H9c2 cells.

[0056] 2) Effect of si-BNIP3L on ROS levels in hypoxic H9c2 cells. Figure 3 As shown in the figure, compared with the hypoxia intervention group, the ROS level in the BNIP3L knockdown group was significantly decreased (P<0.05), indicating that si-BNIP3L can significantly reduce the oxidative stress level of H9c2 cells under hypoxia.

[0057] 3) Effects of si-BNIP3L on mitochondrial function in hypoxic H9c2 cells. The results of mitochondrial function related indicators are as follows Figure 4 Compared with the hypoxia intervention group, the ATP content of H9c2 cells in the BNIP3L knockdown group ( Figure 4 A) and mitochondrial membrane potential level ( Figure 4 D, E) significantly increased (P<0.05), and mROS levels ( Figure 4 B, C) showed a downward trend (P<0.05), indicating that si-BNIP3L can significantly improve the mitochondrial function of hypoxic H9c2 cells.

Claims

1. Application of siRNA that inhibits BNIP3L protein expression in the preparation of drugs to prevent myocardial cell hypoxia injury.

2. The use according to claim 1, characterized in that: The siRNA that inhibits the expression of BNIP3L protein includes a sense strand and an antisense strand; The sense strand is shown in SEQ ID No: 1; the antisense strand is shown in SEQ ID No:

2.

3. A siRNA for inhibiting the expression of BNIP3L protein, characterized in that: The siRNA for inhibiting the expression of BNIP3L protein is the siRNA used in any one of claims 1-2; The siRNA that inhibits the expression of BNIP3L protein includes a sense strand and an antisense strand; The sense strand is shown in SEQ ID No: 1; the antisense strand is shown in SEQ ID No:

2.

4. A pharmaceutical composition for preventing myocardial cell hypoxia injury, characterized in that: The pharmaceutical composition comprises the siRNA used in any one of claims 1-2 or claim 3 as an active ingredient.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition further comprises a carrier.

6. The pharmaceutical composition according to claim 4, characterized in that The vector is an acceptable vector for siRNA that pharmaceutically inhibits the expression of BNIP3L protein.

7. The pharmaceutical composition according to claim 4, characterized in that The carrier is one of cholesterol, virus, nanoparticle or liposome.

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