LncRNA marker for cerebral arterial thrombosis and application of LncRNA marker

Abstract

The invention relates to the technical field of biology, and particularly discloses a cerebral arterial thrombosis LncRNA marker and application thereof.The cerebral arterial thrombosis LncRNA marker comprises lncRNA SETD5-AS1, the difference of the lncRNA SETD5-AS1 in a cerebral arterial thrombosis group and a cerebral thrombosis high-risk group has statistical significance, differential expression of the lncRNA SETD5-AS1 in the cerebral arterial thrombosis group and a healthy group also has statistical significance, and the lncRNA SETD5-AS1 can be used for detecting cerebral arterial thrombosis. Differential expression of the lncRNA SETD5-AS1 in a cerebral apoplexy high-risk group and a healthy group also has statistical significance, and it is indicated that the lncRNA SETD5-AS1 has a medium-level prediction value for screening of cerebral arterial thrombosis high-risk groups and early diagnosis of diseases of cerebral arterial thrombosis high-risk groups.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a LncRNA marker for ischemic stroke and its application. Background Art

[0002] Ischemic stroke is a vascular disease caused by thrombosis of a cerebral artery, resulting in localized brain ischemia and hypoxia. It has become the second leading cause of disability and mortality worldwide. The fundamental pathological factor in ischemic stroke is intravascular thrombosis, which leads to focal neuronal loss and brain tissue necrosis. Fifty percent of ischemic strokes are caused by atherosclerosis and plaque rupture, 20% are cardioembolic, 25% are lacunar infarctions caused by small vessel disease, and 5% are caused by specific causes such as extracranial arterial dissection. After a stroke, the body undergoes multiple pathological processes, including microglial activation, mitochondrial dysfunction, and endoplasmic reticulum stress. After a stroke, neuronal morphology changes, and programmed cell death pathways are activated. Damaged brain cells release substances such as sphingosine 1-phosphate and chemokines, prompting microglia to rapidly migrate to the lesion site, undergoing dramatic changes in their morphology and function, and phagocytizing dying cells and cell debris. After brain tissue is deprived of oxygen, glycolysis is activated to compensate for energy loss. The resulting lactic acid lowers pH, causing acidosis and a decrease in mitochondrial membrane potential. Simultaneously, impaired energy metabolism inactivates the ion pumps upon which ATPase depends, leading to intracellular calcium overload and mitochondrial damage. After blood flow is restored to the brain, ischemia-reperfusion injury affects the homeostasis of the endoplasmic reticulum (ER), causing the accumulation of numerous unfolded, misfolded proteins in the ER, activating the unfolded protein response, further exacerbating brain damage, and even triggering cell apoptosis.

[0003] With the rapid development of microarray technology and nucleic acid sequencing technology, scientists have discovered that only 2% of the sequences in the human genome encode proteins, and the number of protein-coding genes is less than 30,000. The remaining nearly 98% of the genome sequences transcribe to produce a large number of non-coding RNAs with a wide variety. These ncRNAs are important components of the complex regulatory network in the body.

[0004] Long noncoding RNA (lncRNA) is a class of large, noncoding RNAs (ncRNAs) that regulate diverse biological processes. They are widely distributed, typically exceeding 200 bases in length, and lack or have little protein-coding capacity due to the lack of a valid open reading frame (ORF). As a new field in molecular biology, lncRNAs, acting as RNAs, regulate gene expression at multiple levels, primarily through epigenetic, transcriptional, and post-transcriptional regulation. As a crucial component of the mammalian transcriptome, the functions of lncRNAs remain under investigation. Initially, lncRNAs were considered byproducts of RNA polymerase II transcription, representing "noise" and "dark matter" of genomic transcription, devoid of biological function. However, recent research has revealed the crucial role of lncRNAs in normal cellular activity and their involvement in the development and progression of various tumors and other diseases. lncRNAs are not only involved in cellular physiological processes such as nuclear trafficking, transcriptional regulation, protein degradation, genomic imprinting, and X-chromosome silencing, but have also been implicated in the pathogenesis of diseases such as breast cancer, prostate cancer, non-small cell lung cancer, and lymphocytic leukemia. lncRNAs can implement their functions through mechanisms such as gene imprinting, chromatin remodeling, splicing regulation, mRNA degradation, and translational regulation. Although recent progress has been made in lncRNA research, their functions and mechanisms of action remain to be further explored. Key methods and tools for studying lncRNAs include microarrays, RNA sequencing, real-time quantitative PCR, Northern blotting, in situ hybridization, RNAi, RIP, and bioinformatics prediction. The development and utilization of research technologies are crucial for understanding biological mechanisms.

[0005] At present, there are no reports on the role of lncRNA SETD5-AS1 in ischemic stroke at home and abroad. Summary of the Invention

[0006] The purpose of this application is to overcome the deficiencies of the above-mentioned prior art and provide a LncRNA marker for ischemic stroke and its application.

[0007] To achieve the above objectives, the technical solutions adopted in this application are:

[0008] The present application provides an application of a lncRNA marker in the preparation or screening of ischemic stroke diagnostic products, wherein the lncRNA marker includes lncRNA SETD5-AS1.

[0009] In the technical solution of the present application, lncRNA SETD5-AS1 is significantly differentially expressed in patients with ischemic stroke and has the potential to serve as a biomarker for early warning and diagnosis of ischemic stroke.

[0010] Moreover, experiments have shown that the difference in lncRNA SETD5-AS1 between the ischemic stroke group and the high-risk stroke group is statistically significant, the differential expression of lncRNA SETD5-AS1 between the ischemic stroke group and the healthy group is also statistically significant, and the differential expression of lncRNA SETD5-AS1 between the high-risk stroke group and the healthy group is also statistically significant, indicating that lncRNA SETD5-AS1 has a moderate predictive value for screening high-risk populations for ischemic stroke and early diagnosis of its onset.

[0011] As a preferred embodiment of the application described in this application, the product includes reagents for detecting the expression level of lncRNA SETD5-AS1 by detecting RT-PCR, real-time quantitative PCR, in situ hybridization, chip or high-throughput sequencing platform.

[0012] As a preferred embodiment of the application described in this application, the product includes a chip, a preparation or a kit.

[0013] As a preferred embodiment of the application described in the present application, compared with the healthy control group, the expression level of lncRNA SETD5-AS1 is increased in patients with ischemic stroke.

[0014] Preferably, the source of the lncRNA SETD5-AS1 includes plasma.

[0015] The experiment showed that the difference in plasma lncRNA SETD5-AS1 between the ischemic stroke group and the high-risk stroke group was statistically significant (P<0.05), with an AUC of 0.860, and a moderate level of diagnostic accuracy; the differential expression of plasma lncRNA SETD5-AS1 between the ischemic stroke group and the healthy group was statistically significant (P<0.05), with an AUC of 0.675, and a low level of diagnostic accuracy; the differential expression of plasma lncRNA SETD5-AS1 between the high-risk stroke group and the healthy group was statistically significant (P<0.05), with an AUC of 0.744, and a moderate level of diagnostic accuracy; suggesting that lncRNA SETD5-AS1 has a moderate level of predictive value for screening high-risk populations for ischemic stroke and early diagnosis of its onset.

[0016] The present application also provides the use of a reagent for detecting the expression level of a lncRNA marker, the use comprising:

[0017] 1) Preparation of reagents for diagnosing ischemic stroke; or

[0018] 2) Prepare a diagnostic kit for ischemic stroke.

[0019] As a preferred embodiment of the application described in this application, the ischemic stroke includes acute ischemic stroke.

[0020] The present application also provides a kit for diagnosing ischemic stroke, which can measure the expression level of the lncRNA SETD5-AS1 in a sample.

[0021] The present application also provides the use of a reagent that specifically inhibits the expression level of lncRNA SETD5-AS1 in the preparation of a drug for treating ischemic stroke.

[0022] Compared with the prior art, this application has the following beneficial effects:

[0023] The present application provides a lncRNA marker for ischemic stroke and its application. The difference in lncRNA SETD5-AS1 between the ischemic stroke group and the high-risk stroke group is statistically significant. The differential expression of lncRNA SETD5-AS1 between the ischemic stroke group and the healthy group is also statistically significant. The differential expression of lncRNA SETD5-AS1 between the high-risk stroke group and the healthy group is also statistically significant, indicating that lncRNA SETD5-AS1 has a moderate predictive value for screening high-risk populations for ischemic stroke and early diagnosis of its onset. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the expression level cluster heat map in Example 1 (Note: the horizontal axis represents three groups of different clinical samples, and the vertical axis represents different differentially expressed genes. The color depth indicates the level of gene expression. Among them, (A1, A2), (B1, B2), and (C1, C2) represent peripheral venous blood samples of three patients with cerebral infarction before onset and within 24 hours of onset, respectively);

[0025] Figure 2 To screen out the differential expression changes of lncRNAs before and after ischemic stroke (where A, B, and C represent 3 patients, HR: high-risk stroke group, CI: 24h ischemic stroke group);

[0026] Figure 3 Bubble chart of KEGG signaling pathway enrichment analysis results (Note: the horizontal axis represents the enrichment ratio, and the vertical axis represents the signaling pathway. The redder the color, the stronger the enrichment, and the bluer the color, the weaker the enrichment. The size of the bubble represents the number of genes enriched in the pathway);

[0027] Figure 4Figure 2 is the expression difference of plasma SETD5-AS1 among three groups of samples (HC is the healthy control group, HR is the high-risk group for stroke, and CI is the ischemic stroke group. Note: * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001);

[0028] Figure 5 is the ROC curve diagram (ac are the ROC curves corresponding to plasma SETD5-AS1 between the ischemic stroke group and the high-risk stroke group, the ischemic stroke group and the healthy control group, and the high-risk stroke group and the healthy control group, respectively);

[0029] Figure 6 The figure shows the difference in expression of lncRNA SETD5-AS1 between ischemic stroke patients and healthy controls (AIS is the ischemic stroke group; Control is the healthy control group, note: ** indicates P < 0.01). DETAILED DESCRIPTION

[0030] In order to better illustrate the purpose, technical solutions and advantages of this application, this application will be further described below with reference to the accompanying drawings and specific embodiments.

[0031] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are all the same.

[0032] Example 1: Screening for differential lncRNAs in ischemic stroke

[0033] Three patients participating in the Stroke High-Risk Screening and Intervention Program at Chenzhou Hospital Affiliated to the University of South China were selected. They were assessed by our medical staff as high-risk for stroke and had peripheral venous blood samples stored in our biobank. Within 24 hours of onset, they presented to our hospital and were diagnosed with ischemic stroke. Four milliliters of peripheral venous blood were collected, and basic and clinical data were collected. Plasma total RNA was extracted using the TRIZOL method, and appropriate amounts of RNA were sent to the BGI Genomics Laboratory in Wuhan, Hubei Province, for high-throughput sequencing and bioinformatics analysis. High-throughput transcriptomic sequencing of plasma from these three high-risk stroke patients and within 24 hours of ischemic stroke onset identified 96 differentially expressed lncRNAs, of which 80 were upregulated and 16 were downregulated. The top 6 differentially expressed lncRNAs with potential research value were screened out, namely the upregulated SETD5-AS1, LOC100288069, LOC105377989, MIR133A1HG and the downregulated XIST and RNFT1-DT genes, among which SETD5-AS1 showed the most significant difference. (As shown below Figure 1-2). KEGG PATHWAY analysis of differentially expressed genes was performed, and it was found that lncRNA SETD5-AS1 may participate in the regulation of ischemic stroke through autophagy ( Figure 3 ).

[0034] Example 2: Differential expression of lncRNA SETD5-AS1 in ischemic stroke

[0035] Statistical analysis of the general clinical data of 47 healthy subjects, 54 subjects at high risk of stroke, and 57 patients with ischemic stroke (Table 1) revealed no statistically significant differences in gender or age among the three groups (P>0.05). There was also no statistically significant difference in the relative expression level of SETD5-AS1 associated with the presence of high-risk factors for stroke (P>0.05).

[0036] Table 1 General clinical data of the subjects

[0037]

[0038] Plasma SETD5-AS1 and β-Actin were detected by qPCR in the healthy control group, the high-risk stroke group, and the ischemic stroke group. The relative expression levels of SETD5-AS1 in the plasma of the three groups of samples were calculated by combining the expression level of the internal reference gene β-Actin. The results were expressed as median (interquartile range) ( Figure 4 ).

[0039] Statistical results showed that the relative expression levels of SETD5-AS1 in the ischemic stroke group and the healthy control group were 369.281 (1617.35) and 87.730 (372.30), respectively, with a statistically significant difference (P < 0.05). Furthermore, the relative expression levels of both groups were significantly higher than that of the high-risk stroke group (19.776 (65.60)), with a statistically significant difference (P < 0.05). This suggests that SETD5-AS1 is significantly differentially expressed in ischemic stroke patients and has the potential to serve as a biomarker for early warning and diagnosis of ischemic stroke.

[0040] Example 3: Analysis of the value of plasma SETD5-AS1 in the early diagnosis of ischemic stroke

[0041] The ROC curve was used to analyze the diagnostic value of plasma SETD5-AS1 in the early stages of ischemic stroke. The horizontal axis of the ROC curve represents 1-specificity at different cutoff points, and the vertical axis represents sensitivity. The closer the area under the curve is to 1, the higher the diagnostic value.

[0042] The ROC curve analysis was performed to analyze the differential expression of plasma SETD5-AS1 in the three groups ( Figure 5), it was found that the difference in plasma SETD5-AS1 between the ischemic stroke group and the high-risk stroke group was statistically significant (P<0.05), with an AUC of 0.860, which had a moderate level of diagnostic accuracy (Table 2); the differential expression of plasma SETD5-AS1 between the ischemic stroke group and the healthy group was statistically significant (P<0.05), with an AUC of 0.675, which had a low level of diagnostic accuracy (Table 3); the differential expression of plasma SETD5-AS1 between the high-risk stroke group and the healthy group was statistically significant (P<0.05), with an AUC of 0.744, which had a moderate level of diagnostic accuracy (Table 4); suggesting that SETD5-AS1 has a moderate level of predictive value for the screening of high-risk ischemic stroke populations and the early diagnosis of their onset.

[0043] Table 2 ROC curve analysis of SETD5-AS1 between the high-risk stroke group and the ischemic stroke group

[0044]

[0045] Table 3 ROC curve analysis of SETD5-AS1 between ischemic stroke group and healthy group

[0046]

[0047]

[0048] Table 4 ROC curve analysis of SETD5-AS1 between high-risk stroke group and ischemic stroke group

[0049]

[0050] Example 4: lncRNA SETD5-AS1 expression is elevated in patients with acute ischemic stroke Peripheral venous blood specimen:

[0051] (1) Ischemic stroke patient group: 53 patients who visited the stroke green channel of the Department of Neurology of Chenzhou First People's Hospital within 24 hours of onset from March 2023 to November 2023 and were diagnosed with acute ischemic stroke were consecutively included.

[0052] (2) Healthy control group: 54 healthy subjects who underwent physical examinations at the Health Management Center of Chenzhou First People's Hospital during the same period and whose gender and age were matched were selected.

[0053] The diagnostic criteria for ischemic stroke patients refer to the eighth edition of the "Neurology" textbook and the "Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke in China 2018".

[0054] This application collected peripheral venous blood from 53 patients with acute ischemic stroke (AIS) within 24 hours of onset and 54 healthy controls, and compared relevant clinical information. There was no statistical difference between the two groups of subjects in terms of gender, age, smoking history, drinking history, and diabetes history (P>0.05). The blood pressure, triglycerides, and low-density lipoprotein in the ischemic stroke patient group were higher than those in the control group, and the differences were statistically significant (P<0.05) (as shown in Table 5).

[0055] Plasma RNA was extracted from peripheral venous blood, and the plasma lncRNA SETD5-AS1 level was detected by RT-qPCR and the relative expression was calculated. The results showed that the expression level of plasma lncRNA SETD5-AS1 in the ischemic stroke group was significantly higher than that in the healthy control group, and the difference was statistically significant (P < 0.01). Figure 6 ).

[0056] Table 5

[0057]

[0058]

[0059] Example 5. Effects and mechanisms of lncRNA SETD5-AS1 on neuronal autophagy during OGD / R and MCAO

[0060] This study intends to simulate ischemic stroke by constructing cell and animal models, and explore the role and mechanism of lncRNA SETD5-AS1 on neuronal autophagy during OGD / R and MCAO through in vitro and in vivo experiments. Plasma will be widely collected from clinical healthy people, people at high risk of ischemic stroke, and patients with ischemic stroke to detect the expression differences of lncRNA SETD5-AS1, explore its role in regulating ischemic stroke and its feasibility as a molecular marker, explore its molecular mechanism, and provide new molecular targets for early screening, diagnosis, and intervention of ischemic stroke.

[0061] (1) Establish a cellular oxygen-glucose deprivation / re-glucose reoxygenation (OGD / R) model and study the relationship between lncRNA SETD5-AS1 and neuronal autophagy after OGD / R.

[0062] 1) Using hypoxia-inducible factor 1-1α (HIF-1α) as the hypoxia evaluation index, determine the optimal time and optimize the OGD / R modeling conditions.

[0063] Neuroblastoma cells in the logarithmic growth phase were cultured in serum-free, low-glucose DMEM medium. The culture dishes were placed in a hypoxia chamber to induce oxygen-glucose deprivation, creating a state of hypoxia. After 1, 2, and 3 hours, the cells were removed from the chamber, the low-glucose medium was discarded, and the cells were returned to normal culture medium. Total RNA was extracted and subsequently analyzed by RT-qPCR. A control group was treated identically, and the qPCR amplification products were subjected to agarose gel electrophoresis. The relative expression of HIF-1α was analyzed to determine the optimal hypoxia duration.

[0064] 2) After OGD / R, the expression changes of lncRNA SETD5-AS1 in neuroma blasts were detected.

[0065] Neuroma blast cells in the logarithmic growth phase were selected and treated with OGD / R. Total RNA was extracted from the cells and reverse transcribed into cDNA. The expression changes of lncRNA SETD5-AS1 in the cells were detected by qPCR.

[0066] 3) After OGD / R, the occurrence of autophagy in neuroma blasts and its effects on cell viability and proliferation were detected.

[0067] Neuroblastoma cells were cultured to the logarithmic growth phase and treated with OGD / R. The expression levels of autophagy-related proteins were detected by Western Blot. CCK8 and EDU were used to detect cell viability and proliferation after autophagy, respectively.

[0068] 4) lncRNA SETD5-AS1 was overexpressed and knocked down respectively, and after OGD / R, the autophagy, cell viability and proliferation of neuroma blasts were detected.

[0069] Neuroblastoma cells were cultured to the logarithmic growth phase to verify the transfection efficiency. They were transfected with overexpression plasmids pcDNA3.1SETD5-AS1 and si-SETD5-AS1 for 24 h, respectively. After OGD / R treatment, autophagy-related proteins, cell proliferation, and viability were detected by immunofluorescence, Western Blot, CCK8, and EDU, respectively.

[0070] 5) Based on the research results, this application will further use primary neural cells or iPS (induced pluripotent stem cells) to differentiate into neural cells, establish an OGD / R cell model, and study the role of lncRNA SETD5-AS1 in promoting autophagy in ischemic stroke neural cells.

[0071] Primary mouse neurons were isolated, culture conditions were established in vitro, and an OGD / R model was optimized for primary cells. LncRNA SETD5-AS1 expression, cell proliferation, and viability were measured. PCDNA3.1 SETD5-AS1 and si-SETD5-AS1 were transfected into primary neurons, and lncRNA SETD5-AS1 expression and autophagy were measured after OGD / R. Given the difficulty in isolating and culturing primary neurons, an alternative approach is to use induced pluripotent stem cells (iPS) (either mouse or human iPS cells can be purchased or prepared) to induce neuronal differentiation (established differentiation induction protocols are available). LncRNA SETD5-AS1 expression, cell viability, and autophagy were then measured. LncRNA SETD5-AS1 expression, cell viability, and autophagy were measured after transfection with pcDNA3.1 SETD5-AS1 and si-SETD5-AS1 and OGD / R.

[0072] The induction of iPS cells involves the following steps: ① Introducing exogenous genes (such as OSKM, Nanog, and line28) or their expression products into recipient cells, initiating transcriptional expression of the early embryonic genome within them; ② Placing the introduced recipient cells onto a feeder layer, the culture medium is changed to the appropriate ES cell culture medium on the second day, and then every 4-48 hours until ES-like clones emerge; ③ When the primary iPS clones reach 100-200 μm, they are passaged and plated onto a new feeder layer for proliferation. Early passages are primarily performed mechanically, with individual clones being picked, digested, and then plated onto a 96-well plate lined with feeder cells. After the second generation of clones reaches 5-6 generations, they can be subcultured using type IV collagenase digestion. Starting with the 96-well plate, the cells are gradually expanded and expanded until they can be plated onto a 60 mm dish. Once a sufficient cell mass is obtained, they are cryopreserved.

[0073] (2) A mouse middle cerebral artery occlusion (MCAO) model was constructed to analyze and verify the role of lncRNA SETD5-AS1 in inducing autophagy in ischemic stroke neurons.

[0074] 1) A mouse MCAO model was established to induce focal cerebral ischemia. Neurological deficits were assessed, cerebral infarction volume was determined by triphenyltetrazolium (TTC) staining, and lncRNA SETD5-AS1 expression and autophagy in mouse brain cells were detected.

[0075] Male C57 / BL mice were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital (30 mg / kg). A central incision was made in the neck, and the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully isolated. A small incision was made at the free end of the ECA. Sterile 40-gauge monofilament nylon suture was inserted through the opening along the ECA and carefully advanced toward the ICA. The suture plug was marked and secured, and the wound was sutured. After 90 minutes, the suture plug was removed, completing the MCAO model. Neurological deficits were then assessed using a scoring system: no deficit (0 points); mild focal deficit (left forepaw not fully extended); moderate focal deficit (left circle); severe focal deficit (left descending); and 4 points (inability to walk independently and decreased level of consciousness). Mice were sacrificed, and brain tissue was removed for determination of infarct volume using triphenyltetrazolium (TTC) staining. Mouse brain tissue was ground, cells were lysed, and total RNA was extracted. cDNA was synthesized by reverse transcription. The expression changes of lncRNA SETD5-AS1 in brain tissue were detected by qPCR, and autophagy-related proteins were detected by Western Blot.

[0076] 2) pcDNA3.1SETD5-AS1 and si-SETD5-AS1 were injected into the lateral ventricle of mice, respectively, and a mouse MCAO model was established to induce focal cerebral ischemia. Brain tissue damage, cell autophagy, and lncRNA SETD5-AS1 expression were detected.

[0077] pcDNA 3.1SETD5-AS1 and si-SETD5-AS1 were injected into the lateral ventricle of mice (the lateral ventricle injection site was stereotactically located (AP: -0.5 mm, ML: ±1.0 mm, DV: -2.5 mm). The MCAO model was established in mice using the above method. The mouse brain tissue was ground, the cells were lysed, and total RNA was extracted and synthesized into cDNA by reverse transcription. The expression changes of lncRNA SETD5-AS1 in brain tissue were detected by qPCR, and the cell autophagy in brain tissue was detected by Western Blot.

[0078] The experiment was divided into five groups:

[0079] ①Sham operation group;

[0080] ②MCAO group;

[0081] ③pcDNA 3.1SETD5-AS1+MCAO group;

[0082] ④si-SETD5-AS1+MCAO group;

[0083] ⑤pcDNA 3.1SETD5-AS1+si-SETD5-AS1+MCAO group.

[0084] 3) A mouse MCAO model was established to induce focal cerebral ischemia. After injection of pcDNA3.1SETD5-AS1 overexpression plasmid and si-SETD5-AS1 to change the expression of lncRNA SETD5-AS1, the expression of autophagy-related proteins in cells was detected by Western blot.

[0085] Mice were injected intracerebroventricularly with pcDNA 3.1SETD5-AS1 overexpression plasmid and si-SETD5-AS1, respectively. The MCAO model was established in mice using the above method. The mouse brain tissue was ground, the cells were lysed, and total cellular RNA was extracted. cDNA was synthesized by reverse transcription, and the expression changes of lncRNA SETD5-AS1 in brain tissue were detected by qPCR. The brain tissue was ground, the cells were lysed, and total protein was extracted. Western blotting experiments were performed using autophagy-related antibodies (LC3, Beclin 1, P62, etc.) to detect the expression of autophagy-related proteins in cells of mouse brain tissue.

[0086] The experiment was divided into five groups:

[0087] ①Sham operation group;

[0088] ②MCAO group;

[0089] ③pcDNA 3.1SETD5-AS1+MCAO group;

[0090] ④si-SETD5-AS1+MCAO group;

[0091] ⑤pcDNA 3.1SETD5-AS1+si-SETD5-AS1+MCAO group.

[0092] (3) Expand the clinical sample size and detect the differential expression of lncRNA SETD5-AS1 in the plasma of healthy people, people at high risk of ischemic stroke, and patients.

[0093] Peripheral venous blood samples were collected within 24 hours of onset from 100 healthy subjects, 100 subjects at high risk of stroke, and 100 patients with ischemic stroke. Total plasma RNA was extracted and reverse transcribed into cDNA. qPCR was used to detect the expression differences of lncRNA SETD5-AS1 in the three groups. The above experiment was repeated three times.

[0094] (4) To explore the molecular mechanism by which lncRNA SETD5-AS1 promotes autophagy in ischemic stroke neurons. 1) Using the OGD / R cell model, we analyzed whether lncRNA SETD5-AS1 regulates autophagy in ischemic stroke neurons through the AKT-related pathway and inducing the expression of autophagy-related proteins LC3, Beclin1, and P62.

[0095] In the neuroblastoma OGD / R cell model, we investigated whether overexpression and knockdown of lncRNA SETD5-AS1 and treatment with a pAKT activator altered AKT and autophagy-related proteins such as LC3, Beclin1, and P62. We also investigated whether significant changes in neuronal autophagy and proliferation occurred.

[0096] ①CCK-8 and Edu were used to detect cell viability and proliferation; ②Real-time fluorescence quantitative PCR was used to detect the expression of lncRNA SETD5-AS1; ③Western blot and immunofluorescence were used to detect the expression changes of AKT and induced autophagy-related proteins such as LC3, Beclin1, and P62.

[0097] Experimental groups:

[0098] ①Control group;

[0099] ②OGD / R group; ③Overexpression group;

[0100] ④ Overexpression group + pAKT activator;

[0101] ⑤ Knockdown group;

[0102] ⑥Overexpression treatment group + knockdown combined treatment group.

[0103] 2) Using the MCAO model, we investigated whether lncRNA SETD5-AS1 promotes autophagy in ischemic stroke neurons by regulating the expression of LC3, Beclin1, and P62 through mediating the AKT-related pathway.

[0104] In animal models, we investigated whether overexpression and knockdown of the lncRNA SETD5-AS1 (MCAO induced by intracerebroventricular injection of pcDNA3.1SETD5-AS1 and si-SETD5-AS1) altered the expression of AKT, p-AKT, and the autophagy-related proteins LC3, Beclin1, and P62. We also investigated whether treatment with an AKT activator could attenuate autophagy and alleviate or aggravate cerebral infarction in mice after MCAO. Neurological deficits and infarct volumes were assessed. RNA and protein were extracted from brain tissue, and changes in the expression of AKT, p-AKT, and the autophagy-related proteins LC3, Beclin1, and P62 were detected by real-time quantitative PCR, Western blot, and immunofluorescence.

[0105] 3) This application will also use databases such as PITA, miRanda, RNAhybrid, and TargetScanVert to predict and combine the transcriptome sequencing results of the OGD / R cell model to analyze and predict the relevant target genes that lncRNA SETD5-AS1 may regulate, and then use the above-mentioned cell and animal models to explore the molecular mechanism by which lncRNA SETD5-AS1 promotes autophagy in ischemic stroke neurons.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. Application of lncRNA markers in the preparation or screening of ischemic stroke diagnostic products, characterized in that: The lncRNA marker includes lncRNA SETD5-AS1.

2. The use according to claim 1, characterized in that The product includes reagents for detecting the expression level of lncRNA SETD5-AS1 through RT-PCR, real-time quantitative PCR, in situ hybridization, chip or high-throughput sequencing platform.

3. The use according to claim 1, characterized in that The product includes a chip, a preparation or a kit.

4. The use according to claim 1, wherein Compared with healthy controls, the expression level of lncRNA SETD5-AS1 was increased in patients with ischemic stroke.

5. Use of a reagent for detecting the expression level of a lncRNA marker, characterized in that: The applications include: 1) Preparation of reagents for diagnosing ischemic stroke; or 2) Prepare an ischemic stroke diagnostic kit.

6. The use according to claim 5, characterized in that The ischemic stroke includes acute ischemic stroke.

7. A kit for diagnosing ischemic stroke, characterized in that: The kit is capable of measuring the expression level of the lncRNA SETD5-AS1 according to claim 1 in a sample.

8. Use of a reagent that specifically inhibits the expression level of lncRNA SETD5-AS1 in the preparation of a drug for the treatment of ischemic stroke.

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