ShRNA (short hairpin Ribonucleic Acid) for inhibiting METTL7B gene expression and application thereof

Abstract

The invention relates to the technical field of gene drugs, the METTL7B provides a new target spot for treating cerebral arterial thrombosis, the specific effect of the m6A methylase METTL7B in clinical treatment of cerebral arterial thrombosis is found through research, the METTL7B can have a treatment effect on acute stage injury of focal cerebral ischemia reperfusion SD rats, and the METTL7B can be applied to clinical treatment of cerebral arterial thrombosis. It is preliminarily clear that METTL7B plays a role in treating acute ischemic stroke by inhibiting intracerebral apoptosis injury. A development direction and a technical basis are provided for searching, researching and developing exact and effective new treatment medicines for treating cerebral arterial thrombosis and the like and researching and developing accurate treatment action targets, and patients with cerebral arterial thrombosis and the like can be helped to get rid of illness and pain as soon as possible, so that more lives can be rescued.

Description

[Technical field]

[0001] The present invention relates to the technical field of gene medicine, and in particular to shRNA for inhibiting the expression of METTL7B gene and application thereof. [Background technology]

[0002] With the deepening of genetic engineering research, scientists have shown great interest in the use of genetic engineering to develop drugs. Recent studies have revealed the important involvement of RNA-related epigenetic mechanisms in gene expression regulation, including the various activities of non-coding RNA and RNA methylation. As the most common post-transcriptional modification of eukaryotic mRNA, m6A (N-6 methyladenosine) methylation modification is widely present in various eukaryotic organisms, such as yeast, plants, fruit flies and mammals, accounting for 80% of RNA methylation modification. It can functionally regulate the transcriptome of eukaryotic organisms, thereby affecting the splicing, nuclear export, localization, translation and stability of mRNA. More and more studies have shown that m6A methylation-related enzymes are often abnormally expressed in a variety of human brain tissues, inducing the expression of disease transcripts, cell proliferation, apoptosis and differentiation through m6A methylation and demethylation mechanisms. A large number of studies have confirmed that m6A modification is involved in the pathological process of cerebral ischemia-reperfusion injury. For example, Zheng et al. found that miR-421-3p inhibited the translation of p65 mRNA by targeting the m6A reader protein YTHDF1, preventing inflammatory response in cerebral ischemia / reperfusion injury; Diao et al. revealed that hypothermia activated PTEN and PI3K / Akt / GSK-3 signaling pathways through m6A modification to protect neurons from ischemia / reperfusion-induced cell pyroptosis; Si et al. confirmed that METTL3-mediated m6A methylation promoted the formation of early stress granules in acute ischemic stroke. Therefore, the role of abnormal m6A modification in a variety of diseases indicates that regulators or targeted genes of m6A methylation can serve as new candidates for clinical treatment. In addition, m6A can play its functional role by modifying substrate mRNA through interactions with other molecules (DNA, RNA, proteins, etc.). Scientists have confirmed that m6A-related genes regulate protein expression profiles at the post-transcriptional level to determine the progress and diversity of a series of important life activities such as cell differentiation and embryonic development, providing a theoretical basis for us to reveal the mechanism of disease occurrence from the perspective of RNA regulation, and providing new gene targets and molecular markers for disease diagnosis and treatment. [Summary of the invention]

[0003] In view of the above, it is necessary to study new target sites related to stroke for m6A methyltransferase, find new targets suitable for the preparation of stroke drugs, and verify the model of the target sites.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] Application of METTL7B gene as a therapeutic target for stroke.

[0006] The present invention also includes the use of an inhibitor for inhibiting the functional expression of the METTL7B gene in the preparation of a drug for treating cerebral stroke.

[0007] Furthermore, the nucleic acid sequence of the METTL7B gene is shown in SEQ ID NO.1.

[0008] Furthermore, the inhibitor is shRNA, and the nucleic acid sequence of shRNA is 5'-GGGAAAGGCTGTCAAATAA-3' (SEQ ID NO. 2).

[0009] The present invention has the following beneficial effects:

[0010] 1. The present invention has discovered the specific role of m6A methylase METTL7B in the clinical treatment of ischemic stroke through research. The inventor team has conducted further experimental related research and found that silencing METTL7B can reduce the expression levels of MDM2, p300, Bax, and Puma, which are closely related to the proliferation and apoptosis of p53 downstream in the brain, thereby playing a therapeutic role in ischemic stroke. The best time to use METTL7B for treatment is after the clinical treatment subject has symptoms of ischemic stroke. METTL7B can be used as a target site for stroke treatment, and has a therapeutic effect on the acute phase injury of focal cerebral ischemia-reperfusion SD rats. It has also been preliminarily clarified that METTL7B plays a therapeutic role in acute ischemic stroke by inhibiting cell apoptosis damage in the brain. It provides a development direction and technical basis for finding and developing new and effective therapeutic drugs for the treatment of cerebrovascular diseases such as ischemic stroke and developing precise therapeutic targets, which is conducive to patients with cerebrovascular diseases such as ischemic stroke to get rid of pain and torture as soon as possible and save more lives.

[0011] 2. The present invention down-regulates and silences METTL7B, which is beneficial to avoiding the occurrence of cerebral ischemia / reperfusion injury, and has a protective effect on the neural damage in the acute phase of focal cerebral ischemia-reperfusion SD rats. It can be promoted and applied in the preparation of drugs for treating ischemic stroke, and has a good application prospect. It also provides a direction and technical basis for finding and developing new and effective therapeutic drugs for treating cerebrovascular diseases such as ischemic stroke, which is beneficial for patients with cerebrovascular diseases such as ischemic stroke to get rid of the pain as soon as possible.

Brief Description of the Drawings

[0012] Figure 1 Figure 3 shows the neurological score after MCAO / R using mNSS.

[0013] Figure 2This is a picture of TTC-stained brain tissue sections.

[0014] Figure 3 This is a quantitative analysis of the volume of cerebral infarction in mice.

[0015] Figure 4 Representative images of HE staining to detect cytopathology in the ischemic penumbra.

[0016] Figure 5 Representative images of NeuN immunohistochemical staining to detect cytopathology in the ischemic penumbra.

[0017] Figure 6 Representative images of TUNEL staining analysis of ischemic penumbra cytopathology.

[0018] Figure 7 This is a quantitative statistical graph of NeuN immunohistochemical staining to detect cytopathology in the ischemic penumbra.

[0019] Figure 8 This is a quantitative statistical graph of TUNEL staining to detect cytopathology in the ischemic penumbra.

[0020] Fig. 9 This is a graph showing the detection of mRNA levels of genes related to the downstream signaling pathway of P53-mediated apoptosis.

[0021] Fig.10 To detect the protein levels of genes related to the downstream signaling pathway of P53-mediated cell apoptosis. [Specific implementation method]

[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0023] Embodiment 1:

[0024] An embodiment of the present invention provides a METTL7B, whose cDNA sequence is SEQ ID NO.1.

[0025] The expression of METTL7B was detected and its sequence was obtained as follows:

[0026] 1. Preparation of focal cerebral ischemia-reperfusion animal model:

[0027] The rat middle cerebral artery occlusion (MCAO) model was prepared by referring to the modified Longa suture embolism method. Male SD rats aged 7-8 weeks and weighing 18-21g were raised in an SPF environment. First, the skin of the rat's neck was cleaned with a shaver to expose the complete skin tissue. Then, the skin was carefully cut with surgical scissors, and a 1.5cm longitudinal incision was made in the middle of the neck. At the same time, the surgical microscope was turned on. Under the microscope, thicker forceps were used to carefully open the tissue around the neck of the SD rat in the field of vision to avoid mechanical damage. At this time, the subtracheal muscles were separated to expose the common carotid artery (CCA). The muscles and fascia adhered to the common carotid artery were further peeled off with microtweezers. The bifurcation of CCA was visible upward, and the internal carotid artery (ICA) and external carotid artery (ECA) were visible forward. The ECA near the bifurcation of the proximal CCA was ligated. Since the suture could be artificially avoided from entering the pterygopalatine artery (PPA), the PPA was not ligated. Afterwards, a small incision was made on the blood vessel near the CCA ligation with microscissors, and a 0.18mm nylon thread plug with a head coated with silicone was inserted into the CCA through the small incision. To avoid massive bleeding in the artery when inserting the thread plug, a slipknot was tied at the middle end of the blood vessel before the thread plug was inserted. The slipknot was then loosened and gently pushed, causing the thread plug to advance along the direction of the internal carotid artery. After passing the intersection of ECA and ICA, it continued to be inserted along the direction of the ICA blood vessel until it reached the middle cerebral artery (MCA) of the SD rat. It was stopped when slight resistance was felt and it could not go deeper. Calculated from the bifurcation, the thread plug entered the MCA for about 12 to 14 mm, basically blocking the blood flow to the brain provided by the middle cerebral artery of the SD rat. After 60 minutes of cerebral ischemia, the suture was slowly withdrawn, and the external carotid artery was ligated again. Then, the silk thread at the common carotid artery was loosened to restore the blood flow of the right carotid artery, the incision was sutured, and then the rats were placed in a cage after disinfection with iodine and kept at room temperature of 25°C to end the MCAO and complete the focal cerebral ischemia-reperfusion model. The whole process was monitored by laser Doppler blood flow meter to ensure the successful preparation of the model. From the start of the operation to the awakening of the animal, a thermometer was used to ensure that the body temperature of the SD rats was maintained at 36.5°C ± 1°C. After anesthesia of the sham group animals, only the bifurcation of the internal and external carotid arteries was exposed, and no suture was inserted.

[0028] 2. Experimental groups and medication:

[0029] C57BL / 6 male mice were weighed and randomly divided into four groups: sham operation group (Sham), cerebral ischemia model group (Vehicle), silent negative control group (sh-NC), METTL7B silent treatment group (sh-METTL7B), overexpression negative control group (oe-NC), and METTL7B overexpression intervention group (oe-METTL7B); among them, the nucleic acid sequence of shRNA was 5'-GGGAAAGGCTGTCAAATAA-3' (as shown in SEQ ID NO.2); the nucleic acid sequence of shRNA in the overexpression negative control group was: 5'-TTCTCCGAACGTGTCACGT-3' (as shown in SEQ ID NO.3) as the control.

[0030] Administration method: According to the rat's weight, 10% chloral hydrate (4.0 ml / kg) was injected intraperitoneally for anesthesia, and the rat was fixed in a prone position to keep the skull horizontal and in the middle. The hair was trimmed, iodine was used for disinfection, and the top of the skull was exposed. 2 O 2 Wipe the top of the skull and expose the bregma. According to the rat brain atlas, the coordinates of the left ventricle are: anterior-posterior direction = 0.8 mm, mediolateral direction = ±1.4 mm, dorsoventral direction = 3.5 mm relative to bregma. Plasmids containing sh-METTL7B or sh-NC, oe-METTL7B or oe-NC were slowly injected into each lateral ventricle (3 μL per hemisphere, injection rate of 0.15 μL / min, injection time more than 15 minutes), and the needle was left in place for 5 minutes after the injection was complete. After slowly lifting and removing the microinjection needle, the drill hole was sealed with bone wax. The rat was removed from the stereotaxic instrument, the wound was sutured, and the rat was returned to the cage after iodine disinfection.

[0031] 3. Neuromotor function score:

[0032] mNSS was used to score the neurological function of SD rats after 1h of ischemia and 24h of reperfusion. The modified neurobehavioral score (mNSS) includes five aspects of research: sensory testing, motor experiments, balance beam tests, loss of reflexes and abnormal movements, epilepsy, myoclonus, and dystonia. The total score of this behavioral test is 18 points. Normal rats score 0 points, and the highest behavioral defect is 18 points. The specific scoring labels for each item are as follows. Mild injuries are 1-6 points; moderate injuries are 7-12 points; and severe injuries are 13-18 points. The experimental results are as follows Figure 1As shown, the neuromotor score of the sh-METTL7B silencing treatment group was significantly lower than that of the model group, and the neuromotor score of the sh-METTL7B silencing treatment group was significantly lower than that of the shRNA-NC group. The results of the overexpression intervention group were just the opposite, indicating that silencing METTL7B can significantly improve the neuromotor dysfunction of SD rats after cerebral ischemia-reperfusion.

[0033] 4. Determination of cerebral infarction volume:

[0034] The rats were decapitated and their brains were removed 24 hours after ischemia-reperfusion. The brain tissues were quickly removed, and the olfactory bulbs, cerebellum and lower brainstem were removed and frozen at -20°C for 20 minutes. Then, the SD rat brain tissue was sliced ​​continuously in the coronal plane from front to back starting from 3 mm away from the frontal pole using a brain mold, and then cut into 5 slices at equal distances. Placed in 1% TTC, incubated at a constant temperature of 37°C for 10 minutes, the brain slices were turned over from time to time to ensure uniform staining. After TTC staining, normal tissue appears red and infarcted tissue appears white. Use Image J software to scan the SD rat brain slices and calculate the percentage of the infarct volume to the contralateral brain volume. As shown in the attached figure. Figure 2 and Figure 3 As shown, compared with the model group and the negative control group, the cerebral infarction volume in the METTL7B silencing treatment group was significantly reduced, while the results in the overexpression intervention group were just the opposite.

[0035] 5. HE staining:

[0036] Dry the sections in an oven at 65°C. Dewax the paraffin sections, usually using xylene or limonene, and then hydrate them through an ethanol series. Stain with hematoxylin for 5-10 minutes. The dye can penetrate the cell nucleus and make it bluish-purple. Wash briefly, then stain with eosin for 1-3 minutes. The dye stains the cytoplasm and other tissue structures red. The stained sections need to be dehydrated again through an ethanol series. Use xylene or other transparent agents for transparency. Cover the sections with mounting media and cover with a coverslip. Observe and analyze the stained tissue sections under an optical microscope to assess the extent and characteristics of brain damage. As shown in the attached Figure 4 , Figure 5 and Figure 6 As shown in the figure, 24 hours after cerebral ischemia-reperfusion, compared with the sham operation group, the number of cells in the cerebral cortex of rats in the model group and the negative control group was significantly reduced, the cell nuclei were condensed and the degree of vacuolation was severe; after METTL7B silencing treatment, the number of cells in the cortex of SD rats increased significantly compared with the model group, and the cell morphology was also restored. The results of the overexpression intervention group were just the opposite.

[0037] 6. TUNEL staining:

[0038] Neuronal apoptosis was detected by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP end labeling) assay. Cells or brain tissue samples were fixed on slides with ice-cold 4% glutaraldehyde for 10 minutes and then permeabilized with 0.1% (vol / vol) Triton X-100 (Sigma) for 5 minutes. The slides were incubated with the TUNEL incubation mixture at 37°C for 1.5 hours in a humid and dark environment. The cell nuclei were then stained with 4,6-diphenylamine-2'-phenylhydrazine hydrochloride (DAPI). TUNEL-positive staining was observed and photographed using a fluorescence microscope. The apoptotic index refers to the percentage of TUNEL-positive cells (positive cells / 100% total cells). As shown in the attached Figure 7 and Figure 8 As shown in the figure, 24 hours after cerebral ischemia-reperfusion, compared with the sham operation group, the neuronal apoptosis in the cerebral cortex of rats in the model group and the negative control group was significantly increased; after METTL7B silencing treatment, the neuronal apoptosis induced by MCAO / R was significantly reduced; the results of the overexpression intervention group were just the opposite.

[0039] 7. Real-time fluorescence quantitative PCR (qRT-PCR) detection of MDM2, p300, Bax, and Puma levels

[0040] Total RNA from tissues was extracted by TRIzol method, and the concentration of total RNA was determined by UV absorbance. RNA was reverse transcribed into cDNA using SuperScript III First-Strand Synthesis kit and oligo(dT) primer according to GenBank guidelines and instruction manual. RT-PCR reaction was then performed using SYBR Green PCR kit on 96-well plates using ABIPRISM7500 real-time PCR system. RNA was reverse transcribed into cDNA and PCR amplified in 20 μL reaction system. Amplification conditions were: initial denaturation at 95°C for 30 seconds; denaturation at 95°C for 5 seconds; annealing at 60°C for 20 seconds; extension at 72°C for 20 seconds, 40 cycles. Then the Ct value of the sample was obtained. Compared with GAPDH, the relative mRNA expression of the target gene was calculated using the 2-ΔΔCT method. Compared with the sham operation group, the mRNA expression level of P53 signaling pathway in the cerebral cortex tissue of rats in the model group and the negative control group was increased; after METTL7B silencing treatment, the protein expression level of P53 and the mRNA expression level of P53 signaling pathway were significantly decreased; the results of the overexpression intervention group were just the opposite.

[0041] 8. Western blotting

[0042] SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate solution, decapitated, and the brain was quickly removed on ice, and the cerebral hemisphere was divided into the injured side and the non-injured side. Fresh brain tissue was taken from the peri-infarct area of ​​the injured side, and the brain tissue and lysis solution were fully homogenized at a mass volume ratio of 1:10. The homogenate was placed in an ice box and placed on a shaker for full lysis for 30 minutes. After centrifugation at 12000rpm for 15 minutes at 4℃, the supernatant was aspirated, 6× protein loading buffer was added according to the volume ratio, denatured at 100℃ for 15 minutes, and stored at -80℃ after aliquoting. The protein sample at -80℃ was taken out and dissolved, centrifuged in a centrifuge, added to the lane with a microinjector, separated by SDS-polyacrylamide constant pressure gel electrophoresis, and transferred to PVDF membrane using an electrotransfer system. 10% skim milk powder-TBST was blocked at room temperature for 1-2 hours. Primary antibody was added and incubated overnight on a shaker at 4℃. The next day, discard the primary antibody and add TBST to wash the membrane for 10 min × 4 times, add the secondary antibody labeled with horseradish peroxidase, incubate on a shaker at room temperature for 1 hour, rinse with TBST, and then add ECL luminescent substrate for color development. Tanon5200 fully automatic chemiluminescence imaging analysis system was used for development and analysis. The ratio of the gray value of the target protein to the gray value of the internal reference GAPDH was semi-quantitatively analyzed. Studies have shown that the expression levels of MDM2, p300, Bax, and Puma related to the apoptosis-related P53 signaling pathway play an extremely important role in cerebral ischemia-reperfusion injury. In the apoptosis damage caused by cerebral ischemia-reperfusion injury, P53 expression is significantly increased, and reducing its expression can significantly improve apoptosis damage; and the P53 signaling pathway is stress-induced upregulation in apoptosis damage, and further activation of this signaling pathway can also significantly improve apoptosis damage. Fig.10 As shown in the figure, 24 hours after cerebral ischemia-reperfusion, compared with the sham operation group, the protein expression level of P53 signaling pathway in the cerebral cortex tissue of SD rats in the model group and the negative control group was increased; after METTL7B silencing treatment, the protein expression level of P53 was significantly decreased, and the protein expression level of P53 signaling pathway was significantly decreased; the results of the overexpression intervention group were just the opposite.

[0043] From the above, it can be seen that the present invention down-regulates and silences METTL7B to avoid the occurrence of cerebral ischemia / reperfusion injury, has a protective effect on the acute phase of focal cerebral ischemia-reperfusion SD rats with neural damage, can be promoted and applied in the preparation of drugs for the treatment of ischemic stroke, has a good application prospect, and also provides a direction and technical basis for finding and developing new and effective therapeutic drugs for the treatment of cerebrovascular diseases such as ischemic stroke, which is conducive to the early relief of patients with cerebrovascular diseases such as ischemic stroke from pain. The above is only used to illustrate the technical solution of the present invention and is not a limitation. The equal modifications and changes made to the technical solution of the present invention by ordinary personnel in this field, as long as they do not deviate from the overall concept of the invention, are still within the scope of the invention.

[0044] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. Application of METTL7B gene as a target for stroke treatment.

2. Application of inhibitors that inhibit the functional expression of the METTL7B gene in the preparation of drugs for the treatment of stroke.

3. The use according to claim 1 or 2, characterized in that: The nucleic acid sequence of the METTL7B gene is shown in SEQ ID NO.

1.

4. The use according to claim 2, characterized in that The inhibitor is shRNA, and the nucleic acid sequence of shRNA is shown as SEQ ID NO.2.

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