CN116059370B

CN116059370B - Medical use of substances for knocking down or inhibiting long non-coding RNA JPX

Info

Publication number: CN116059370B
Application number: CN202211191187.9A
Authority: CN
Inventors: 陈宏山; 顾嘉铭; 李雪松; 董梦蝶; 陈佳婧; 陈明红; 张韵佳; 陈祥; 印全文; 郑龙彬
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2022-09-28
Filing date: 2022-09-28
Publication date: 2025-03-28
Anticipated expiration: 2042-09-28
Also published as: CN116059370A

Abstract

The present application provides the medical use of substances that knock down or inhibit long non-coding RNA JPX, specifically, the use of substances that knock down or inhibit lncRNA JPX in the preparation of drugs for preventing and treating cardiovascular diseases caused by aging; in patients with atherosclerosis caused by aging and in cells and animal models related to atherosclerosis, it was found that inhibiting lncRNA JPX can inhibit aging and delay the progression of atherosclerosis; the present application provides a new target for the diagnosis and treatment of vascular diseases related to atherosclerosis caused by aging, and opens up a new direction for the preparation of preventive and therapeutic drugs.

Description

Medical application of substances for knocking down or inhibiting long-chain non-coding RNA JPX

Technical Field

The invention belongs to the technical field of biological medicines, relates to medical application of substances for knocking down or inhibiting long-chain non-coding RNA JPX, and in particular relates to application of substances for knocking down or inhibiting lncRNA JPX in preparation of medicines for preventing and treating atherosclerosis related vascular diseases caused by aging.

Background

With the development of society, cardiovascular diseases (Cardiovascular diseases, CVDs) are becoming a worldwide problem threatening human health due to their high prevalence, high disability rate and high mortality rate, and by 2030, CVDs are expected to continue to be the leading cause of death worldwide. Among them, atherosclerosis (AS) is a common pathophysiological basis for stroke, ischemic heart disease and peripheral vascular disease, and is caused by various risk factors such AS hypertension, hyperlipidemia and genetics, and bad lifestyle aggravates the occurrence and development of AS, such AS high-fat and high-cholesterol diet, obesity, smoking, lack of exercise, sedentary sitting, etc. Lesion development in AS is a typical age-dependent process. AS is a chronic vascular inflammatory disease, whose main pathological feature is vascular intimal lipid deposition, accompanied by smooth muscle cell and fibrous matrix proliferation, gradually developing into AS plaques. AS research is advanced, researchers find that a variety of senescent cells in blood vessels are closely related to AS pathophysiological changes, while SASP secreted by senescent cells also causes AS plaque to progress and become unbalanced. Under the stimulation of pathogenic factors, aging vascular smooth muscle cells (Human vascular smooth muscle cells, HVSMCs) can form multiple SASPs, promote monocyte chemotaxis, can stimulate and influence the steady state of adjacent non-aging HVSMCs cells and tissues, aging HVSMCs can degrade protease through secretion matrix, so that cell proliferation is weakened, fibrous cap thinning is promoted, and plaque stability is influenced, and collagen generated by aging HVSMCs is less than normal HVSMCs, so that plaque stability is further influenced. Thus, cellular aging is increasingly being one of the major risk factors for AS cardiovascular disease.

With intensive research in epigenetic science, it is increasingly recognized that long non-coding RNAs (lncRNAs) exhibit a high degree of cell and tissue specificity and their role as modulators in cells. Many studies show that lncRNAs are closely related to embryo development, apoptosis, tissue growth and the like, and are a very wide-range regulatory factor expressed in human tissues. In recent years, the modification of lncRNA in the pathogenesis of AS has also attracted considerable attention. Studies prove that lncRNA H19, LNCRNA MALAT, lncRNA P21, LNCRNA ANRIL and the like can participate in processes of influencing cell proliferation, apoptosis and the like through two main regulation pathways of P53/P21 and P16/Rb, thereby participating in cell aging and the occurrence and development of AS. Many lncRNAs have been reported to modulate activation of nuclear factor κB (NF-. Kappa.B) and its downstream target genes, and these findings suggest that lncRNAs may play a key role in SASP-induced AS during aging. The lncRNA JPX gene is located at position 12 on the long arm of the X chromosome, and is an activator of the XIST gene and a molecular switch for the inactivation of the X chromosome. It can upregulate XIST expression and thus be involved in X chromosome inactivation. JPX as a lncRNA has been shown to target hundreds of autosomal genes, which prefer binding promoters, proximal regions of transcription initiation or transcription termination sites, exons and introns over their expression in the genome. JPX can regulate gene expression by expelling transcription factors or in conjunction with a number of important transcription factors. The related literature reports that lncRNA JPX can promote proliferation, migration and invasion of tumor cells and participate in the occurrence and development processes of various cancers such AS lung cancer, gastric cancer, cervical cancer and the like, but whether lncRNA JPX can participate in cell aging by regulating and controlling SASP genes has not been reported yet, so that the relation of influencing AS is not reported.

Disclosure of Invention

The application provides a medical application of a knock-down lncRNA JPX substance aiming at the problems.

The aim of the invention can be achieved by the following technical scheme:

The application of substances knocked down or inhibited lncRNA JPX in preparing medicaments for preventing and treating cardiovascular diseases caused by aging. As a preferred aspect of the present invention, the knockdown or suppression lncRNA JPX substance includes a small interfering RNA knockdown lncRNA JPX or a gene editing system knockdown lncRNA JPX, such as a CRISP/Cas9 gene editing system or the like, which is currently known to be capable of knocking out or suppressing a gene of interest.

As a further preferred aspect of the present invention, the knockdown lncRNA JPX siRNA sequences are shown as CCAGUUAAUAGUAUUGUGUTT (SEQ ID NO. 1) and ACACAAUACUAUUAACUGGTT (SEQ ID NO. 2). Preferably, the medicine for preventing and treating cardiovascular diseases caused by aging comprises medicines for preventing and treating atherosclerosis.

LncRNA JPX as a target point is applied to screening medicaments for preventing and treating cardiovascular diseases caused by aging.

A method for screening the medicines for preventing and treating cardiovascular diseases caused by senility includes such steps as providing the medicines candidate to Ras-induced senility HVSMCs, detecting lncRNA JPX expression, if lncRNA JPX is inhibited, indicating that said medicines candidate have the in-vitro activity for preventing and treating cardiovascular diseases caused by senility, or providing the medicines candidate to animal model of cardiovascular diseases caused by senility, detecting lncRNA JPX expression, if lncRNA JPX is inhibited, indicating that said medicines candidate have in-vivo activity for preventing and treating cardiovascular diseases caused by senility.

LncRNA JPX is used as a detection target in the preparation of auxiliary detection reagents for atherosclerosis caused by aging.

Use of a substance for detecting lncRNA JPX in the preparation of a reagent for the assisted detection of atherosclerosis caused by aging.

Preferably, the substance detected lncRNA JPX is a substance detected lncRNA JPX quantitatively.

As a further preferred aspect of the present invention, the substance for quantitative detection lncRNA JPX is a lncRNA JPX specific primer pair.

The beneficial effects are that:

According to the application, expression of lncRNA JPX in aortic tissue of an Apoe ^-/- mouse and human vascular smooth muscle caused by aging is studied by Western Blot, cell immunofluorescence and other methods. The method comprises the steps of constructing and synthesizing a knock-out mouse with specific interference lncRNA JPX, selecting an Apoe ^-/- mouse with the age of 8 weeks, randomly dividing the Apoe ^-/- mouse into a control group and a lncRNA JPX knock-out group, and carrying out intravenous injection on the control group and the lncRNA JPX knock-out group twice weekly to obtain a nonspecific control or lncRNA JPX gapmeRs (10 mg/kg of each mouse), and continuously injecting and feeding the mice with a high-fat diet for 16 weeks to construct an AS mouse model. The application defines the regulating mechanism of lncRNA JPX on HVSMCs for the first time, effectively prevents the aging of HVSMCs and AS caused by the aging, provides a new prevention and treatment drug development path and drug action target point for AS diagnosis and treatment, and has very important medicinal value.

Drawings

FIG. 1 is a schematic representation of the expression levels of lncRNA JPX in normal HVSMCs (represented by Con in the figure) and Ras-induced aging HVSMCs (represented by Ras in the figure);

The detection method was to detect their lncRNA JPX expression levels in HVSMCs of normal and Ras-induced senescence by real-time quantitative PCR (RT-qPCR).

FIG. 2 is a schematic representation of the Western Blot detection of P16, P21, P53 protein expression levels following the knock-out HVSMCs of lncRNA JPX, ras stimulus;

The detection method comprises the steps of knocking down lncRNA JPX after HVSMCS SILNCRNA JPX stimulation for 24 hours, and then, after Ras stimulation for 24 hours, detecting the expression level of the P16, P21 and P53 proteins by Western Blot.

FIG. 3 is a schematic representation of the aging of the β -galactosidase staining assay HVSMCs with Ras stimulation by knocking out lncRNA JPX from HVSMCs;

The detection method comprises the steps of knocking down lncRNA JPX after HVSMCS SILNCRNA JPX stimulation for 24 hours, then, after 24 hours of Ras stimulation, detecting HVSMCs aging by using a beta-galactosyltransferase staining experiment.

FIG. 4 shows the detection of SASP gene expression levels of IL-6, IL-8, IL-1β, CCL2, ICAM-1, TNF- α, etc. by knocking out lncRNA JPX in HVSMCs, applying Ras stimulus, and real-time quantitative PCR (RT-qPCR), wherein the detection method comprises the steps of knocking down lncRNA JPX by applying HVSMCS SILNCRNA JPX stimulus for 24 hours, applying Ras stimulus for 24 hours, extracting cellular RNA, and detecting SASP gene expression levels of IL-6, IL-8, IL-1β, CCL2, ICAM-1, TNF- α, etc. by qRT-PCR.

Fig. 5 is a schematic representation of the expression levels of lncRNA JPX in vascular tissues of Normal (NC) and High Fat (HFD) fed Apoe ^-/- mice.

The detection method comprises selecting 8-week-old Apoe ^-/- mice, randomly dividing into control group and high-fat feeding group, feeding for 16 weeks with high-fat diet, and constructing AS mouse model. The mice vascular tissue was then harvested and RNA extracted for qRT-PCR experiments to detect expression levels of lncRNA JPX in normal diet and high fat feeding vessels.

FIG. 6 is a graph of the detection result of the oil red O staining of the mouse model;

The detection method comprises selecting 8-week-old Apoe ^-/- mice, randomly dividing into a control group and a lncRNA JPX knockout group, performing intravenous injection of nonspecific control or lncRNA JPX gapmeRs (10 mg/kg of each mouse) twice a week on the control group and the lncRNA JPX knockout group mice, and continuously injecting and feeding with high-fat diet for 16 weeks to construct an AS mouse model. The aortic vessels were then harvested and plaque size was detected by oil red O staining.

FIG. 7 is a schematic diagram showing the detection of P16, P21 and P53 protein expression levels of mouse vascular tissues by Western Blot;

The detection method comprises selecting 8-week-old Apoe ^-/- male mice, randomly dividing into a control group and a lncRNA JPX knockout group, performing intravenous injection of nonspecific control or lncRNA JPX gapmeRs (10 mg/kg of each mouse) twice a week on the control group and the lncRNA JPX knockout group mice, and continuously injecting and feeding with high-fat diet for 16 weeks to construct an AS mouse model. The mouse vascular tissue is then harvested, mouse vascular tissue protein is extracted, and P-STING, STING, P-TBK1, P-IRF3, P16, P21, P53 protein expression levels are detected by Western Blot.

FIG. 8 is a graph showing the result of immunofluorescence detection of a mouse model;

The detection method comprises selecting 8-week-old Apoe ^-/- male mice, randomly dividing into a control group and a lncRNA JPX knockout group, performing intravenous injection of nonspecific control or lncRNA JPX gapmeRs (10 mg/kg of each mouse) twice a week on the control group and the lncRNA JPX knockout group mice, and continuously injecting and feeding with high-fat diet for 16 weeks to construct an AS mouse model. Expression levels of P21 and alpha-SMA in sections of rat aortic tissue were measured by OCT embedding after extraction of rat aortic tissue, and immunofluorescence detection of P21 and alpha-SMA expression after frozen sections, red (A) for P21, green (C) for alpha-SMA, blue (B) for nucleus, and white arrows for red and green co-localized yellow regions.

FIG. 9 is a schematic diagram showing the detection of SASP gene expression levels of IL-6, IL-8, IL-1β, CCL2, ICAM-1, TNF- α, etc. by real-time quantitative PCR (RT-qPCR) in vascular tissues of mice;

The detection method comprises selecting 8-week-old Apoe ^-/- male mice, randomly dividing into a control group and a lncRNA JPX knockout group, performing intravenous injection of nonspecific control or lncRNA JPX gapmeRs (10 mg/kg of each mouse) twice a week on the control group and the lncRNA JPX knockout group mice, and continuously injecting and feeding with high-fat diet for 16 weeks to construct an AS mouse model. Mouse aortic tissue RNA was extracted and the SASP gene expression levels of IL-6, IL-8, IL-1β, CCL2, ICAM-1, TNF- α, etc., were detected by qRT-PCR.

Detailed Description

The following examples will provide those skilled in the art with a thorough understanding of the present invention and are not intended to limit the present invention in any way.

The following examples relate to cell, reagent sources:

Human Vascular Smooth Muscle Cells (HVSMCs) were purchased from science.

Apoe ^-/- mice were purchased from Venlhua laboratory animal technologies Co., ltd, 8 week old male mice.

The cell experiments described in the following examples were all approved by the ethical committee of the university of south Beijing medical science.

SiJPX Synthesis by Shanghai Ji Ma Gene. The nucleotide sequence of the sense strand is 5'-CCAGUUAAUAGUAUUGUGUTT-3' (SEQ ID NO. 1), and the nucleotide sequence of the antisense strand is 5'-ACACAAUACUAUUAACUGGTT-3' (SEQ ID NO. 2).

EXAMPLE 1 lncRNA JPX test for detection of age-induced AS-related Properties

To explore the levels of lncRNA JPX in normal and Ras-induced senescence HVSMCs, it was verified whether lncRNA JPX was involved in AS, and this example used qRT-PCR to detect lncRNA JPX expression levels in normal and Ras-induced senescence HVSMCs.

Referring to Oncogenic ras Provokes Premature CELL SENESCENCE Associated with Accumulation of p and p16.sup.INK4a, the experimental procedure for constructing senescence HVSMCs was as follows:

(1) Culturing 5x10 ⁶ Phoenix cells in a10 cm dish;

(2) Changing the liquid the next day, adding 6mL of fresh culture medium, and continuing culturing;

(3) Mu.g of the plasmid of interest (pBabe-H-Ras ^V12) +0.4. Mu. gVSV-G was dissolved in 1mL of Opti-MEM and gently mixed;

(4) mu.L lipo2000 was dissolved in 1mL Opti-MEM and gently mixed;

(5) Standing at room temperature for 5min;

(6) Mixing plasmid and lipo2000, and standing for 20min;

(7) Dripping the mixed solution into a culture dish for culturing the seeded cells, and gently shaking and uniformly mixing;

(8) Culturing at 37 ℃ for 15 hours, changing the liquid, discarding the culture medium, and adding 6mL of fresh culture medium into each dish of cells;

(9) Culturing for 48h, collecting cell first supernatant, filtering with 0.45 μm filter membrane, and preserving at 4deg.C;

(10) After further culturing the cells for 8 hours with the addition of 6mL of fresh medium, the second supernatant was collected again, filtered with a 0.45 μm filter membrane, and stored at 4 ℃):

(11) HVSMCs was cultured in six well plates;

(12) At the time of infection, mixing a proper amount of the first supernatant with a culture medium, and culturing for 12 hours at 37 ℃;

(13) The infection process was repeated with the second supernatant and screening was performed with 2. Mu.g/mL puromycin for 3 days.

The total RNA extraction experiment steps are as follows:

(1) The treated samples were collected and total RNA was extracted as described in Trizol kit. Using RNase gun head, DEPC water and wearing mask (preventing RNase and RNA degradation);

(2) Cells were washed with PBS and 1mL Trizol was added to each well;

(3) After 10s, transferring the cell lysate to EP, and standing on ice for 10min;

(4) Adding 200 μl of chloroform, shaking, mixing, and placing on ice for cracking for 10min;

(5) Centrifuging at 12000rpm at 4 ℃ for 15min;

(6) Carefully sucking the supernatant to a new EP pipe, adding isopropanol, mixing the mixture upside down, and standing the mixture on ice for 10min;

(7) Centrifuging at 12000rpm and 4 ℃ for 15min, and discarding supernatant;

(8) Pouring the residual liquid on paper to suck, adding 75% ethanol diluted by DEPC water, and slightly blowing off by a gun to obtain white feather-like precipitate;

(9) Centrifuging at 4deg.C, 12000rpm,15min;

(10) Removing supernatant ethanol, sucking to dry, and air drying at vent hole of cell room super clean bench (5-10 min)

(11) Adding DEPC water to dissolve RNA;

(12) The NanoDrop measures the concentration of RNA, and the RNA is stored in a refrigerator at-80 ℃ for standby.

The reverse transcription experiment steps are as follows:

(1) Using II 1st Strand cDNA Synthesis Kit reverse transcription was carried out in a total reaction volume of 20. Mu.L, and the specific composition was as follows:

RNase free ddH₂O To 20μL

Total RNA 1μg

II Buffer plus 4μL

(2) After mixing evenly, reverse transcription is carried out by a PCR instrument:

25°C 5min

42°C 30min

85°C 5min

(3) After the reverse transcription, the cDNA was diluted with DEPC water at a ratio of 1:3 or 1:4 and stored at-80℃for further use.

The Real-time PCR experiment steps are as follows:

(1) This experiment is carried out The qPCR SYBR GREEN MASTER Mix carries out relative quantitative detection on the target gene, and the PCR reaction system is as follows:

cDNA template 1. Mu.L

qPCR SYBR Green Master Mix 5μL

Upstream primer TGCAGTCAGAAGGGAGCAAT, 1. Mu.M 1. Mu.L

Downstream primer CACCGTCATCAGGCTGTCTT, 1. Mu.M 1. Mu.L

2 Mu L of ultrapure water

(2) Grouping, calculating a system (finally adding cDNA);

(3) Sealing the membrane, centrifuging the 384-hole plate, and then placing the membrane into a fluorescent quantitative PCR instrument;

(4) The reaction was performed on a Bio-Rad 480 type I quantitative PCR instrument.

The results of the assay are shown in FIG. 1, where lncRNA JPX expression levels were significantly elevated in Ras-induced senescence HVSMCs compared to the normal group.

To further determine whether lncRNA JPX is involved in HVSMCs senescence, leading to AS, we constructed small interfering RNAs (sirnas) of lncRNA JPX. After the siRNA knockdown lncRNA JPX is transfected in HVSMCs, ras is stimulated, cellular proteins are extracted, and expression of senescence markers such as P16, P21, P53 and the like is detected through Western Blot.

The Western Blot detection assay procedure was as follows:

(1) Preparing SDS polyacrylamide gel, namely preparing separating gel according to the molecular weight of protein, adding various reagents into a 50mL centrifuge tube according to different proportions to prepare the separating gel, vortex shaking for 30s, uniformly mixing, pouring into a vertical laminated glass plate to the middle upper part of the glass plate, slowly adding ddH ₂ O for liquid sealing, and solidifying at room temperature for 45min. Concentrating the gel, namely uniformly mixing the reagent according to the formula, pouring the mixture above the separating gel to the top end of the glass plate, inserting a comb (avoiding mixing bubbles), and adding the rest liquid from two sides of the comb by using a liquid dispenser for standby after the gel is solidified.

(2) And loading the sample, carrying out electrophoresis separation, namely loading electrophoresis, carrying out instantaneous centrifugation on the protein sample before loading, and then slightly shaking up. The sample is generally applied in terms of the volume of the solution having a total mass of 30. Mu.g of protein. The gel plates are fixed in an electrophoresis tank of an electrophoresis device, after 1x electrophoresis buffer solution is filled between the two gel plates, the comb is pulled out for sample loading, and after sample loading is finished, the electrophoresis is started after the liquid is filled. The 80V constant pressure electrophoresis was run until bromophenol blue reached the junction of the concentrated gel and the separation gel and the protein samples of each lane were on substantially the same horizontal line, 120V was set, and the electrophoresis was terminated until bromophenol blue was near the bottom.

(3) Film transfer, namely, disassembling the glass plate and cutting glue. Cutting PVDF film according to gel size, activating with methanol for 1min in advance, soaking with sponge, filter paper and gel in 1x film transfer buffer solution for 5-10min, sequentially arranging, and discharging bubbles during the placing process. The film transfer tank is placed in a box, the constant pressure is 120V, the film transfer time is selected according to the molecular weight of protein (heat is generated in the film transfer process, and ice bags and water are placed around to simulate a low-temperature environment at 4 ℃).

(4) Sealing, namely taking out the membrane after turning, putting the membrane into 5% skimmed milk (prepared by TBS-T), and shaking and sealing on a room temperature decolorizing shaker for 1h.

(5) And (3) incubating the primary antibody, namely cleaning milk by using TBS-T after the sealing is finished, sealing the membrane in a plastic sealing membrane, adding the corresponding primary antibody, and shaking the membrane in a 4 ℃ refrigerator overnight.

(6) And combining the secondary antibodies, namely washing the membrane with TBS-T for four times respectively for 10min, 5min and 5min, incubating the membrane with the corresponding secondary antibodies at room temperature on a decolorizing shaker under shaking for 1h, and washing the membrane with TBS-T for 10min, 5min and 5min in sequence.

(7) ECL color development, namely uniformly mixing ECL color development liquid A, B liquid in equal volume (in dark place) before color development, developing in a fluorescent chemiluminescence gel imaging system, photographing and storing records.

As shown in FIG. 2, the expression level of P16, P21 and P53 proteins in HVSMCs induced by Ras was decreased after interfering with lncRNA JPX compared with normal HVSMCs. Meaning lncRNA JPX is involved in the regulation of HVSMCs senescence, interference lncRNA JPX can reduce Ras-induced senescence, thereby inhibiting the development of AS.

Meanwhile, a beta-galactosyltransferase staining experiment (beta-Gal staining experiment) is carried out, the detection result is shown in figure 3, and after lncRNA JPX is interfered, the aging HVSMCs (blue) is obviously reduced.

The beta-galactosyltransferase staining experiment steps are as follows:

(1) The medium was aspirated and washed 3 times with 5min each with PBS.

(2) Adding appropriate volume of beta-galactosidase staining fixative to cover cells, and fixing at room temperature for no less than 15min.

(3) The fixative was aspirated and washed 3 times with PBS for 5min each.

(4) Adding a proper amount of dyeing working solution (1 mL is taken as an example of the preparation of the dyeing working solution)

(5) The cells are preferably immersed in the staining solution after incubation at 37℃overnight. Note that incubation at 37℃cannot be performed in a carbon dioxide incubator.

(6) The staining working solution was aspirated and washed 3 times with PBS for 5min each.

(7) The detection solution was added and observed under a normal optical microscope. Cells expressing the enzyme galactosidase which turned blue were readily observed under light microscopy.

At the same time, cell senescence can be further demonstrated by verifying the level of the SASP gene. As a result, as shown in FIG. 4, the interference lncRNA JPX was able to reduce the increase in SASP gene levels of IL-6, IL-8, IL-1β, ICAM-1, CCL2, TNF- α, etc., induced by Ras. The IL-6 upstream primer sequence used in this example was 5'-CTCCAGAACAGATTTGAGAG-3', the downstream primer sequence was 5'-GGGTCAGGGGTGGTTATTGC-3', the IL-8 upstream primer sequence was 5'-CTGAGGTGCCAGTGCATTAG-3', the downstream primer sequence was 5'-AGCACACCTCTCTTCCATCC-3', the IL-1β upstream primer sequence was 5'-TTGCCAGCCAGTGACACAAT-3', the downstream primer sequence was 5'-GAGAAGGTGGTTGTCTGGGAAT-3', the ICAM-1 upstream primer sequence was 5'-AGGTTGAACCCCACAGTCAC-3', the downstream primer sequence was 5'-TCTGAGACCTCTGGCTTCGT-3', the CCL2 upstream primer sequence was 5'-GATCTCAGTGCAGAGGCTCG-3', the downstream primer sequence was 5'-TCTGGGGAAAGCTAGGGGAA-3', the TNF- α upstream primer sequence was 5'-TAACAAGCCGGTAGCCCACG-3', and the downstream primer sequence was 5'-TCTTGATGGCAGACAGGATG-3'.

Example 2 mouse model test

To further verify the role of lncRNA JPX in aging-induced AS, this example constructed knockout mice that specifically interfere with lncRNA JPX. ANTISENSE LNA GAPMERS, an antisense oligonucleotide capable of inhibiting functions of mRNA and lncRNA with high efficiency, is generally 14-16 nucleotides long, is completely phosphorothioated, can enter cells through mediation of cell membrane surface receptors to play a role, and specifically interferes with lncRNA. Apoe ^-/- male mice of 8 weeks of age were selected and randomly divided into control and lncRNA JPX knockout groups (construction method reference ：A Smooth Muscle Cell-Enriched Long Noncoding RNA Regulates Cell Plasticity and Atherosclerosis by Interacting With Serum Response Factor), mice of control and lncRNA JPX knockout groups were given intravenous controls (sequence 5'-CCTTCCCTGAAGGTTCCTCC-3') or lncRNA JPX gapmeRs (sequence 5'-TTCTTGTCATTGCTCCCTTC-3') twice weekly per mouse at 10mg/kg, and fed a high fat diet for 16 weeks to construct an AS mouse model.

Meanwhile, the vascular tissues of the mice were harvested and subjected to RT-qPCR assay, and the results of the assay are shown in FIG. 5, and it can be seen that lncRNA JPX was significantly activated after the mice were given a high fat diet (HIGH FAT DIET, HFD).

The experimental procedure of RNA extraction from mouse tissue:

(1) After removing the tissue from the liquid nitrogen, the tissue is quickly taken to an ultra clean bench, placed in a small dish, and Trizol reagent is added, (100 mg of tissue is placed in 1.2mL of Trizol reagent), and the tissue is ground by a syringe, taking care of proficiency and quick grinding.

(2) Centrifuge at 4 ℃ for 5min at 12000 rpm, discard pellet. (ultra clean bench ready EP tube, after centrifugation, the supernatant was aspirated into the prepared EP tube)

(3) Chloroform was added to 200. Mu.L chloroform/mL Trizol, vigorously shaken for 15s (shaking by hand) and incubated at 15-30℃for 2-3min. Note that the vortex is disabled to avoid fragmentation of genomic DNA. (chloroform, also known as chloroform chloroform, is typically stored in a 4 ℃ refrigerator).

(4) Centrifuge at 12000 rpm for 15min at 4 ℃. The upper aqueous phase was aspirated into another centrifuge tube. Note that the water phase is carefully sucked, tens of millions of people do not need to suck the middle interface, and a small gun head can be adopted for sucking a small amount of water for many times. If DNA and protein are extracted at the same time, the lower organic phase is preserved and stored in a refrigerator at 4 ℃ for standby, otherwise, the lower organic phase is discarded.

(5) Isopropanol was added in a ratio of 500. Mu.L isopropanol/mL Trizol, and incubated at 15-30℃for 10min with shaking by hand.

(6) Centrifuging at 2-8deg.C for 10min at 12000g, removing supernatant, and precipitating RNA at the bottom of the tube.

(7) 75% Ethanol was added to 1mL of 75% ethanol/mL Trizol, mixed with vortexes, and suspended for precipitation. (Note: 75% ethanol in DEPC Water)

(8) Centrifuging at 7500 r.t.for 5min at 4deg.C, discarding supernatant as much as possible (after using up power supply of centrifugal mechanism, opening the cover of the centrifugal machine for 30min, cooling, and covering), air drying at room temperature or vacuum drying for 5-10min. (Note: RNA cannot be dried by centrifugation, RNA samples are not too dry, otherwise difficult to dissolve.)

(9) Adding DEPC water to dissolve RNA;

(10) The NanoDrop measures the concentration of RNA, and the RNA is stored in a refrigerator at-80 ℃ for standby.

The reverse transcription experiment steps are as follows:

RNase free ddH₂O To 20μL

Total RNA 1μg

II Buffer plus 4μL

25°C 5min

42°C 30min

85°C 5min

The Real-time PCR experiment steps are as follows:

cDNA template 1. Mu.L

qPCR SYBR Green Master Mix 5μL

Upstream primer, 1. Mu.M 1. Mu.L

Downstream primer, 1. Mu.M 1. Mu.L

2 Mu L of ultrapure water

(2) Grouping, calculating a system (finally adding cDNA);

And the aortic blood vessels of the mice were harvested, and plaque size was detected by oil red O staining, and the detection picture is shown in fig. 6. Vascular red plaque was significantly increased following HFD administration by Apoe ^-/-, while knockout lncRNA JPX significantly reduced plaque area.

The aortic root vascular oil red O staining procedure is as follows:

(1) After aortic tree separation, the aortic tree was placed in a clean six-well plate and fixed with 4% paraformaldehyde.

(2) The aorta fixed by paraformaldehyde is taken by micro forceps and put into a new six-hole plate, and rinsed by three distilled water for about 10 min.

(3) Three distilled water in the six-hole plate was sucked off by a pipette, 60% isopropyl alcohol solution was added, and the mixture was treated for 2 minutes.

(4) The isopropanol in the six-hole plate is sucked by a pipetting gun, the oil red O dye liquor filtered in advance is added, and the mixture is placed on a horizontal shaking table for dyeing for 1h.

(5) The oil red O dye solution in the six-hole plate is sucked by a pipette, and is rinsed for 1min by adding 60% isopropanol solution, and the steps are repeated for 3 times until the vascular background is not red.

(6) Residual vessel outer wall fat was carefully removed under a microscope with micro-scissors.

(7) And finally, tiling the dyed aortic tree on a black dissecting wax plate, and photographing.

Meanwhile, collecting mouse vascular tissue proteins, and detecting senescence markers such as P16, P21, P53 and the like by using Western Blot. As shown in FIG. 7, it was found that the aging markers such as P16, P21 and P53 were significantly activated after HFD administration in the control mice, while P16, P21 and P53 protein expression levels were significantly reduced after HFD administration in JPX gapmeRs mice.

Further vascular immunofluorescence was performed, after 16 weeks of high fat feeding, different groups of mouse aortic tissues were extracted and frozen section embedding medium (optimal cutting temperature compound, OCT) was used to embed, and after frozen section, immunofluorescence was performed to detect P21 and α -SMA expression, as shown in fig. 8, where red represents P21, green represents α -SMA, blue represents nucleus, and white arrows indicate red and green co-localized yellow regions. It can be seen that vascular endothelial P21 expression was significantly increased following HFD administration by Apoe ^-/-, co-localization of P21 with alpha-SMA was yellow, whereas vascular smooth muscle P21 expression was decreased following HFD administration by JPX gapmeRs mice, while co-localization of P21 with alpha-SMA was significantly decreased.

Different groups of mouse aortic tissues were extracted and RT-qPCR experiments were performed to detect SASP gene levels to further demonstrate lncRNA JPX effects on aging and AS. FIG. 9 shows that the control mice had significantly elevated levels of SASP genes such as IL-6, IL-8, IL-1. Beta., ICAM-1, CCL2, TNF-. Alpha.after HFD administration, whereas JPX gapmeRs mice had significantly reversed elevated levels of SASP genes such as IL-6, IL-8, IL-1. Beta., ICAM-1, CCL2, TNF-. Alpha.after HFD administration.

From the above examples, it can be seen that the inhibition or knockout lncRNA JPX of the aging-induced atherosclerosis patients and atherosclerosis-associated cells and animal models can inhibit aging and delay the progression of atherosclerosis. Therefore, substances inhibiting or knocking lncRNA JPX out can be used for preparing medicaments for treating cardiovascular diseases caused by aging, especially medicaments for treating atherosclerosis caused by aging, and lncRNA JPX can also be used as a target spot for screening medicaments for treating cardiovascular diseases caused by aging, especially medicaments for screening medicaments for treating atherosclerosis caused by aging.

The above examples are provided for illustrating the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the contents of the present invention and to implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.

Claims

1. Use of small interfering RNA for knocking down lncRNA JPX shown in SEQ ID NO.1 and SEQ ID NO.2 in the preparation of drugs for preventing and treating atherosclerosis.