Lncrna sequence and application thereof in preparation of medicine for treating pulmonary arterial hypertension

Abstract

The application discloses a kind of lncRNA sequences and its application in preparation treatment pulmonary arterial hypertension drug, it is related to biological medicine technical field, to solve the existing pulmonary arterial hypertension treatment drug There is poor targeting, cannot reverse pulmonary vascular remodeling, obvious side effect defects.This application technical point is: provide a kind of lncRNA sequence, the lncRNA sequence is LincRik-L09 full-length sequence, the LincRik-L09 full-length sequence is as shown in SEQ ID NO.1, the effective sequence of the LincRik-L09 full-length sequence is LincRik-L09 (91-155 nt) sequence, the LincRik-L09 (91-155 nt) sequence is as shown in SEQ ID NO.2.The application also discloses the application of the LincRik-L09 full-length sequence in preparation treatment pulmonary arterial hypertension drug, and the drug for treating pulmonary arterial hypertension comprising the LincRik-L09 full-length sequence;The drug is administered by AAV carrier-mediated gene delivery, liposome wrapping mode, targets pulmonary vascular tissue.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a lncRNA sequence and its application in the preparation of drugs for treating pulmonary arterial hypertension. Background Technology

[0002] Pulmonary hypertension (PAH) is a highly fatal cardiovascular disease with no radical cure, resulting in a low 5-year survival rate and a significant unmet medical need. The core pathological feature of PAH is pulmonary vascular remodeling, primarily driven by abnormal proliferation and metabolic reprogramming (the shift from oxidative phosphorylation to glycolysis) of pulmonary artery smooth muscle cells (PASMCs). Enhanced glycolysis leads to lactate accumulation, which regulates cellular function through protein lactylation modification; however, the role of non-histone lactylation in PAH remains unclear.

[0003] lncRNAs are key regulators of pulmonary vascular remodeling, but their known mechanisms of action are mostly concentrated in the ceRNA network, and research on their cross-regulation with metabolic reprogramming and lactylation modification remains lacking. Identifying novel lncRNAs that regulate pulmonary vascular remodeling and their core functional fragments, and clarifying their regulatory mechanisms with metabolism and epigenetics, is a crucial breakthrough for developing disease-related applications.

[0004] Current clinical drugs can only relieve symptoms and cannot reverse pulmonary vascular remodeling, and they also have significant side effects and limited efficacy. Therefore, identifying novel lncRNAs that regulate pulmonary vascular remodeling and their core functional fragments, and clarifying their regulatory mechanisms with metabolism and epigenetics, is crucial for developing disease-modifying therapies. Summary of the Invention

[0005] The technical problem to be solved by this invention is:

[0006] To address the shortcomings of existing pulmonary hypertension treatment drugs, such as poor targeting, inability to reverse pulmonary vascular remodeling, and significant side effects.

[0007] Therefore, providing a novel treatment strategy based on LincRik-L09 and clarifying its molecular mechanism for regulating PASMC proliferation is key to developing disease-modifying therapies.

[0008] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0009] This invention provides a lncRNA sequence, lncRNA sequence-1 being the full-length sequence of LincRik-L09, as shown in SEQ ID NO. 1; lncRNA sequence-2 being the effective sequence LincRik-L09 (91-155 nt) as shown in SEQ ID NO. 2.

[0010]

[0011] The effective sequence of LincRik-L09 (91-155 nt) is 5'-CCTGGCGGCCGCGATCCGTCTGGGGAGGCTGCTGCGGAGTGTGGAGACTCGCGTGAGCATGCCTT-3'.

[0012] This invention provides the application of the LincRik-L09 in the preparation of drugs for treating pulmonary hypertension.

[0013] The present invention provides a medicament for treating pulmonary hypertension comprising the LincRik-L09 sequence.

[0014] Furthermore, the drug is delivered via AAV vector-mediated gene delivery and liposome encapsulation, targeting pulmonary vascular tissue.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] The inventors discovered through research that LincRik-L09, through its effective sequence LincRik-L09 (91-155 nt), binds to SUZ12, inducing H3K27me3 modification in the LDHA promoter region, inhibiting LDHA transcription and lactate production, thereby reducing lactylation modification of TK1 at the K192 site, reducing TK1 activity, and blocking the cell cycle progression of PASMCs; under hypoxic conditions, the LincRik-L09 gene silencer inhibits its expression by recruiting ARNTs.

[0017] The full-length LincRik-L09 sequence or its effective sequence (91-155 nt) is delivered via AAV vector-mediated gene delivery, liposome encapsulation, or other methods, targeting pulmonary vascular tissue, and has the following beneficial effects:

[0018] Highly targeted: It specifically acts on PASMCs, regulates the core signal axis of pulmonary vascular remodeling, and has low off-target effects;

[0019] Novel Mechanism: The first time the "silencer-ARNT-LincRik-L09-SUZ12-LDHA-TK1 lactylation" regulatory axis has been revealed, providing a novel intervention target for pulmonary hypertension;

[0020] Functionally defined: The effective sequence LincRik-L09 (91-155 nt) can replace the full-length LincRik-L09 sequence and is easy to carry for in vivo delivery by delivery systems;

[0021] Significant therapeutic effects: It can effectively inhibit the proliferation of PASMCs, reverse pulmonary vascular remodeling, and improve hemodynamic parameters in both cell and animal models. Attached Figure Description

[0022] Figure 1 This is a diagram showing the expression characteristics of LincRik-L09 in the lung tissues of hypoxic PASMCs and SuHx mice in this embodiment of the invention.

[0023] Figure 2 This diagram illustrates the inhibitory effect of LincRik-L09 on hypoxia-induced PASMC proliferation in this embodiment of the invention.

[0024] Figure 3 This is a diagram illustrating the mechanism by which LincRik-L09 regulates LDHA-TK1 lactylation in pulmonary artery smooth muscle, as described in this invention.

[0025] Figure 4 This is a diagram illustrating how LincRik-L09, in this embodiment of the invention, recruits SUZ12 to inhibit LDHA transcription in pulmonary artery smooth muscle cells via its effective sequence LincRik-L09 (91-155 nt).

[0026] Figure 5 Overexpression of LincRik-L09 or its effective sequence LincRik-L09 (91-155nt) in the embodiments of the present invention can improve SuHx-induced pulmonary hypertension and pulmonary vascular remodeling.

[0027] Figure 6 This is an in vivo safety evaluation diagram of AAV6 overexpression of LincRik-L09 or its effective sequence LincRik-L09 (91-155 nt) in the embodiments of the present invention;

[0028] Figure 7 This is a diagram showing how the silencer in this embodiment of the invention regulates the expression of LincRik-L09 via the transcription factor ARNT. Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, exemplary embodiments or examples of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described embodiments or examples are merely some, not all, of the embodiments or examples of the present invention. All other embodiments or examples obtained by those skilled in the art based on the embodiments or examples of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0030] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0031] Example 1

[0032] 1. Preparation of LincRik-L09 and its effective sequence LincRik-L09 (91-155 nt): An overexpression plasmid was constructed carrying the LincRik-L09 gene (full-length sequence) or the gene encoding the effective sequence LincRik-L09 (91-155 nt). The recombinant vector was transfected into HEK293T cells, and after culturing for 48-72 hours, the full-length LincRik-L09 sequence or its effective sequence was obtained through RNA extraction and purification.

[0033] 2. In vitro activity verification

[0034] Cell culture: Mouse pulmonary artery smooth muscle cells (PASMCs) were cultured in smooth muscle cell culture medium and induced to proliferate under hypoxic conditions (3% O2);

[0035] Transfection treatment: Transfect the full-length overexpression LincRik-L09 vector, the effective LincRik-L09 (91-155nt) vector, or the empty vector into hypoxic PASMCs and culture for 48 hours.

[0036] Detection indicators: Cell counting kit-8 (CCK-8) to detect cell viability, EdU / Ki67 staining to detect proliferation capacity, flow cytometry to analyze cell cycle, Western blot to detect protein levels of LDHA, PCNA, Cyclin E, etc., lactate assay kit to determine lactate content, and enzyme-linked immunosorbent assay (ELISA) to detect TK1 activity.

[0037] 3. In vivo efficacy verification

[0038] Animal model: 6-8 week old male C57BL / 6J mice were used to induce a SuHx pulmonary hypertension model by using Sugen5416 (20 mg / kg, intraperitoneal injection) combined with hypoxia (FiO2=10%, 21 days), and then they were transferred to a normoxic environment for 21 days.

[0039] Dosage regimen: Adeno-associated virus type 6 empty vector (AAV6-NC) and viral vector carrying the full-length LincRik-L09 sequence (AAV6-LincRik-L09), and viral vector AAV6-LincRik-L09 (91-155 nt) carrying the effective LincRik-L09 sequence (91-155 nt) (10¹) were administered via intranasal instillation. 0 genome equivalents / only);

[0040] Detection indicators: PVSP was detected by right heart catheterization, hemodynamic parameters such as PAVTI and PAAT were detected by echocardiography, the RV / (LV+S) ratio was calculated, pulmonary vascular remodeling was observed by HE / Masson staining, and LincRik-L09 expression in lung tissue was detected by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR).

[0041] 4. Mechanism verification experiment

[0042] Interaction verification: RNA immunoprecipitation-real-time quantitative PCR (RIP-qPCR) was used to detect the binding of LincRik-L09 and SUZ12, and RNA pull-down assay was used to identify the binding region;

[0043] Transcriptional regulation validation: Chromatin immunoprecipitation-high-throughput sequencing (ChIP-seq) was used to detect the enrichment levels of SUZ12 and H3K27me3 in the LDHA promoter;

[0044] Validation of lactylation modification: The lactylation sites of TK1 were identified by co-immunoprecipitation (Co-IP) combined with liquid chromatography-mass spectrometry (LC-MS), and the K192 mutant was constructed to verify its function.

[0045] like Figure 1As shown, (A) GEO data analysis showed that hypoxia significantly downregulated LincRik-L09 expression; (B) RT-qPCR was used to detect LincRik-L09 expression in the lung tissue of SuHx-induced pulmonary hypertension mice (6 mice per group), and LincRik-L09 expression was specifically downregulated in the mouse lung tissue; (C) RT-qPCR was used to detect the expression of LincRik-L09 in pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs), and LincRik-L09 expression was downregulated in PASMCs, but there was no significant change in PAECs; (D) FISH-immunofluorescence was used to detect the expression of LincRik-L09 in lung tissue (red: LincRik-L09, green: α-SMA, blue: DAPI, scale bar: 50 micrometers). Cellular data were obtained from 3–6 independent experiments and are expressed as mean ± standard error (mean ± SEM); *p<0.05, **p<0.01, ***p<0.001. Nor: normoxic; Hyp: hypoxic; OE: LincRik-L09 overexpression plasmid; SuHx: sugen5416 combined with hypoxia-induced pulmonary hypertension mice. The results showed that LincRik-L09 was significantly downregulated in both the hypoxic PASMCs model and the pulmonary hypertension mouse model.

[0046] like Figure 2 As shown, (A) flow cytometry analysis of cell cycle; (B) Western blot detection of the expression of proliferation-related protein PCNA and cyclin E; (C) EdU / Ki67 staining detection of cell proliferation (red: EdU, green: Ki67, blue: DAPI, scale bar: 100 μm); (D) CCK-8 cell viability detection. Cell-level data were obtained from 3–6 independent experiments and are expressed as mean ± standard error (mean ± SEM); *p<0.05, **p<0.01, ***p<0.001. Nor: normoxic; Hyp: hypoxic; OE: LincRik-L09 overexpression plasmid; NC: negative control. The results showed that overexpression of LincRik-L09 significantly inhibited hypoxia-induced pulmonary artery smooth muscle cell proliferation.

[0047] like Figure 3As shown, (A) Left panel: Lactate content detected by the kit; Right panel: LDHA expression detected by Western blot; (B) PCNA expression detected by Western blot; (C) Left panel: TK1 lactylation modification site prediction; Middle panel: TK1 lactylation modification detected by Co-IP; Right panel: TK1 activity detected by ELISA; (D) Left panel: TK1-K192 lactylation modification site detected by LC-MS; Middle panel: Effect of K192 mutation on TK1 lactylation level detected by Co-IP; Right panel: Effect of K192 mutation on TK1 activity detected by ELISA. Cellular data were obtained from 3-6 independent experiments and are expressed as mean ± standard error (mean ± SEM); *p<0.05, **p<0.01, ***p<0.001. Nor: normoxic; Hyp: hypoxic; OE: LincRik-L09 overexpression plasmid; NC: negative control; La: lactate. The results showed that LincRik-L09 negatively regulates LDHA and lactate levels in pulmonary artery smooth muscle cells; lactylation modification of the lysine residue at position 192 of the TK1 protein increased the activity of the TK1 protein.

[0048] like Figure 4As shown, (A) FISH-immunofluorescence detection of co-localization of LincRik-L09 and SUZ12, scale bar: 50 μm; (B) RNA immunoprecipitation-real-time quantitative PCR (RIP-qPCR) experiment to detect the binding of LincRik-L09 and SUZ12; (C) RNA pull-down experiment to detect the binding of LincRik-L09 (91-155 nt) and SUZ12; (D) RIP-qPCR experiment to detect the abundance of SUZ12 and H3K27me3 in the LDHA gene promoter region; (E) Top: Distribution of H3K27me3 peak in the LDHA gene promoter region in the ChIP-seq database and design partitioning of subsequent study regions (G1: -1100~-900 nt, G2: +100~+600 nt, G3: +1500~+1800 nt relative to TSS); Middle and bottom: ChIP-qPCR. Experiments identified the G2 region of the LDHA gene promoter as an H3K27me3-rich region; (F) Western blot analysis detected the effect of SUZ12 knockdown on LDHA expression; (G) CCK-8 assay detected the effect of SUZ12 knockdown on the viability of hypoxic pulmonary artery smooth muscle cells. Cellular data were obtained from 3–6 independent experiments and are expressed as mean ± standard error (mean ± SEM); *p<0.05, ***p<0.001. Nor: normoxic; Hyp: hypoxic; OE: LincRik-L09 overexpression plasmid; NC: negative control; ns: no significant difference; si: siRNA. The results showed that LincRik-L09 recruits SUZ12 through its effective sequence LincRik-L09 (91–155 nt), influencing histone H3K27me3 modification in the LDHA gene promoter region to regulate LDHA transcription-mediated pulmonary artery smooth muscle cell proliferation.

[0049] like Figure 5As shown, (A) Schematic diagram of animal experimental design: Adeno-associated virus type 6 (AAV6) carrying different sequences dissolved in physiological saline was instilled into the nasal cavity of mice (nasogastric feeding method), and the sequences carried by the virus were delivered to the lungs of the mice through inhalation; (B) Pulmonary vasopressor pressure (PVSP) was measured by right heart catheterization; (C) Echocardiographic parameters: Pulmonary artery blood flow velocity time integral (PAVTI), pulmonary artery acceleration time (PAAT), and ejection fraction (EF%) were measured for each group of mice; (D) The weight ratio of the right ventricle to the left ventricle plus interventricular septum (RV / (LV+S)) was measured for each group of mice; (E) Representative image of α-SMA immunofluorescence staining, showing the myomodulation of pulmonary arterioles (α-SMA: green; 4',6-diamidino-2-phenylindole (DAPI): blue; scale bar: 50 μm); (F) Masson's trichrome staining of pulmonary arteries (top image, scale bar: 50 μm) and hematoxylin-eosin (HE) staining (bottom image, scale bar: 50 μm). (G) Representative images (50 μm) showing pulmonary vascular remodeling in each group; (H) Representative images of α-SMA and pan-lactylation co-stained in pulmonary arterioles (red: pan-lactylation; green: α-SMA; blue: DAPI; scale bar: 50 μm); (I) Thymidine kinase 1 (TK1) activity assay showing TK1 activity in lung tissue; (J) Representative images of α-SMA and Ki67 co-stained in pulmonary arterioles (green: α-SMA; red: Ki67; blue: DAPI; scale bar: 50 μm). Six mice were included in each group. Data are expressed as mean ± standard error (mean ± SEM); *p<0.05, **p<0.01, ***p<0.001. Nor: normoxic; SuHx: pulmonary hypertension mice induced by hypoxia combined with sugen5416; ns: no significant difference; NC: negative control; L: full-length LincRik-L09 sequence; S: effective LincRik-L09 sequence (91-155nt). The results showed that the SuHx mouse model was successfully established. Overexpression of the full-length LincRik-L09 sequence or its effective LincRik-L09 sequence (91-155nt) significantly inhibited pulmonary hypertension, pulmonary vascular remodeling, TK1 activity, and cell proliferation in the model mice.

[0050] like Figure 6As shown, mice were administered the drug continuously for 3 weeks, and pathological sections of the heart, liver, spleen, and kidneys were examined. Six mice were in each group. No significant morphological changes were observed in the heart, liver, spleen, and kidneys. Nor: normoxic; SuHx: mice with pulmonary hypertension induced by hypoxia combined with sugen5416; ns: no significant difference; NC: negative control; L: full-length sequence of LincRik-L09; S: effective sequence LincRik-L09 (91-155nt). The results indicate that nasogastric administration of AAV6 carrying LincRik-L09 (AAV6-LincRik-L09) or AAV6 carrying LincRik-L09 (91-155nt) (AAV6-LincRik-L09(91-155nt)) to mice had no significant effect on the tissue morphology of important organs other than the lungs.

[0051] like Figure 7As shown, (A) a schematic diagram of the LincRik-L09 genome loci was drawn based on the SilencerDB database and ChIP-seq data, showing the H3K27 trimethylation (H3K27me3) enriched region (silencer region); (B) the H3K27me3 level of the LincRik-L09 gene in PASMCs under normoxic and hypoxic conditions was detected by ChIP-qPCR in 3 independent experiments; (C) the effects of silencer decoy treatment, CRISPR-Cas9-mediated silencer knockout (KO), and corresponding control groups on LincRik-L09 expression in hypoxic PASMCs were analyzed by RT-qPCR in 6 independent experiments; (D) representative images of EdU staining (left, green) and Ki67 staining (right, red) show that the proliferation capacity of hypoxic PASMCs is enhanced after decoy or knockout of the inhibitory silencer (3 independent experiments, scale bar: 50). (e) Cell nuclei were stained with DAPI (blue); (f) CCK-8 assay confirmed that the cell viability of hypoxic PASMCs decreased after the silencer was inhibited or knocked out, 6 independent experiments; (g) ChIP-qPCR verified the binding of ARNT to the positive strand of the 5' proximal region of the LincRik-L09 silencer, 6 independent experiments; (h) ChIP-qPCR analyzed the enrichment of ARNT on the hypoxia response element (HRE) of the LincRik-L09 promoter in PASMCs under normoxic and hypoxic conditions, 6 independent experiments; (f) RT-qPCR analyzed the expression level of LincRik-L09 in hypoxic PASMCs after transfection with ARNT siRNA or scramble siRNA (negative control siRNA), 6 independent experiments; (c) CCK-8 assay showed that the cell viability of hypoxic PASMCs decreased after ARNT knockdown, 6 independent experiments. Data are expressed as mean ± standard error (mean ± SEM); *p<0.05, **p<0.01, ***p<0.001. Nor: normoxic; Hyp: hypoxic; NC: negative control; si: siRNA. The results indicate that the silencer mediates pulmonary artery smooth muscle cell proliferation by recruiting the transcriptional repressor ARNT to regulate LincRik-L09 expression.

[0052] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A lncRNA, characterized in that, The lncRNA is LincRik-L09 (91-155 nt), as shown in SEQ ID NO.

2.

2. The use of LincRik-L09 (91-155 nt) as described in claim 1 in the preparation of a drug for treating pulmonary hypertension.

3. A medicament for treating pulmonary hypertension comprising LincRik-L09 (91-155 nt) as described in claim 1.

4. The drug according to claim 3, characterized in that, The drug is delivered via AAV vector-mediated gene delivery and liposome encapsulation, targeting pulmonary vascular tissue.

Images