Application of long-chain non-coding RNA (Ribonucleic Acid) in preparation of gastric cancer diagnosis and treatment product

Abstract

The invention discloses an application of long-chain non-coding RNA (Ribonucleic Acid) in preparation of a gastric cancer diagnosis and treatment product. The long-chain non-coding RNA is HIF1A-AS3. The expression level difference of the lncRNA HIF1A-AS3 in human peripheral blood and tissues shows that the lncRNA HIF1A-AS3 can be used as a gastric cancer diagnosis marker for early screening and diagnosis of gastric cancer; meanwhile, a novel RNA drug is constructed, can selectively target a tumor microenvironment, is used for precise treatment of the gastric cancer, provides a brand new treatment target for targeted treatment of the gastric cancer, and has good transformation and application values.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of long-chain non-coding RNA in the preparation of gastric cancer diagnosis and treatment products. Background Art

[0002] Gastric cancer (GC) is one of the most common malignant tumors with high morbidity and mortality worldwide, with a particularly high incidence in East Asia (such as China, Japan, and South Korea). Despite recent advances in diagnosis and treatment, the prognosis for GC remains suboptimal, particularly for patients in the advanced stage, with a low 5-year survival rate. The treatment of GC is highly dependent on the stage of the disease. Surgical resection of early-stage GC can offer a better prognosis, but early-stage GC often presents with subtle symptoms, a low detection rate, and a lack of specificity. Most patients are already in the locally advanced or advanced stages at the time of diagnosis, making treatment more challenging and associated with a higher risk of recurrence and metastasis.

[0003] Currently, treatment strategies for gastric cancer primarily include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Surgical resection is the primary method for curing early-stage gastric cancer, but for patients with locally advanced and advanced disease, single-agent treatment is limited in effectiveness, and multidisciplinary therapy (MDT) is often required to develop individualized treatment plans. In recent years, with the advancement of molecular biology and immunology research, the application of targeted therapies (such as anti-HER2 therapy and anti-angiogenic therapy) and immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors) has brought new hope to some patients with advanced gastric cancer. However, gastric cancer is highly heterogeneous, and responses to treatment vary significantly among patients. Therefore, optimizing treatment regimens, improving treatment efficacy, and reducing side effects remain key research priorities.

[0004] Furthermore, early diagnosis and screening of gastric cancer are crucial for improving prognosis. Because early symptoms are often subtle, many patients miss the optimal treatment window by the time they are diagnosed. Therefore, exploring more effective early diagnostic markers and screening methods, as well as developing novel therapeutic drugs targeting specific molecular targets for gastric cancer, are crucial areas for future research.

[0005] Long non-coding RNA (lncRNA) is a class of non-coding RNA molecules exceeding 200 nucleotides in length. Although they do not encode proteins, they play an important role in regulating gene expression. In recent years, lncRNAs have attracted considerable attention in cancer research due to their high tissue specificity, stability, sensitivity, and specificity, holding great potential as diagnostic biomarkers. Furthermore, lncRNAs participate in tumorigenesis, progression, metastasis, and drug resistance through regulating gene expression, signaling pathways, and epigenetic modifications, thus holding great promise as therapeutic targets. Summary of the Invention

[0006] One of the purposes of the present invention is to provide the use of lncRNA HIF1A-AS3 as a marker in the preparation of gastric cancer diagnostic products. The nucleotide sequence of the lncRNA HIF1A-AS3 is as follows:

[0007] >NR_144368.1Homo sapiens HIF1A antisense RNA 3(HIF1A-AS3),long non-coding RNA

[0008] (SE Q ID NO.1).

[0009] A second object of the present invention is to provide a reagent for detecting the expression level of lncRNA HIF1A-AS3 for use in preparing a gastric cancer diagnosis / screening product. The nucleotide sequence of the lncRNA HIF1A-AS3 is as follows:

[0010] >NR_144368.1Homo sapiens HIF1A antisense RNA 3(HIF1A-AS3),long non-coding RNA

[0011] (SE Q ID NO.1).

[0012] Furthermore, the reagent includes a PCR reagent for detecting the expression level of lncRNA HIF1A-AS3.

[0013] Furthermore, the PCR reagent for detecting the expression level of lncRNA HIF1A-AS3 is a primer pair for detecting lncRNA HIF1A-AS3, and the primer pair includes a forward primer (5'-GTCTTTCCTCTCCGTCCCAG-3' (SEQ ID NO. 2)) and a reverse primer (5'-CAGACACCCGATCTCCGTG-3' (SEQ ID NO. 3)).

[0014] A third object of the present invention is to provide the use of lncRNA HIF1A-AS3 inhibitors in the preparation of gastric cancer treatment products.

[0015] Furthermore, the lncRNA HIF1A-AS3 inhibitor is siRNA.

[0016] Furthermore, the nucleotide sequence of the siRNA is: siRNA-S: GCAUAAGAGACGGAAUCUATT (SEQ ID NO. 4), siRNA-A: UAGAUUCCGUCUCUUAUGCTT (SEQ ID NO. 5).

[0017] The novel gastric cancer diagnostic marker lncRNA HIF1A-AS3 provided by the present invention is innovative, highly specific, highly sensitive, widely applicable, and easy to detect. Its AUC value reaches 80%, which meets clinical monitoring standards and can be used for early screening and diagnosis of gastric cancer.

[0018] The novel RNA drug provided by this invention can selectively target the tumor microenvironment, reducing damage to normal tissues and significantly reducing toxic side effects. Experimental studies have confirmed its remarkable tumor growth inhibition effect. It boasts high biocompatibility, strong targeting, good stability, low immunogenicity, simple preparation, and easy dissemination. It can be used for the precision treatment of gastric cancer, providing a new therapeutic target for targeted gastric cancer treatment and possessing promising translational and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results show the expression level of HIF1A-AS3 in the peripheral blood of gastric cancer patients.

[0020] Figure 2 The results show the expression level of HIF1A-AS3 in gastric cancer patient tissues.

[0021] Figure 3 This is the ROC curve of HIF1A-AS3 in human serum.

[0022] Figure 4 This is the ROC curve of HIF1A-AS3 in human tissues.

[0023] Figure 5 The results of in vivo imaging of targeted exosomes.

[0024] Figure 6 and Figure 7 These are the results of animal experiments targeting exosomes.

[0025] Figure 8 The results of tissue staining targeting exosomes. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] The materials involved in the following examples include: serum total RNA extraction kit (miRNeasy Serum / Plasma Advanced Kit), total RNA extraction reagents (Trizol, isopropanol, chloroform, anhydrous ethanol), reverse transcription reagents, HIF1A-AS3-specific qRT-PCR primers, qRT-PCR reagents, 293T cells, HIF1A-AS3-specific siRNA, cell transfection reagent Lipo2000, tumor-associated fibroblast targeting peptide (CRYYRITY), Balb / C nude, and immunohistochemistry kit.

[0030] in:

[0031] (1) RNA reverse transcription reagents: 10× RT Mix; HiScript Enzyme Mix; Random hexamers; Oligo(dT); RNA-free ddH2O.

[0032] (2) qRT-PCR reaction reagents: 2×AceQ qPCR Sybr green mix; ddH2O; HIF1A-AS3 specific qRT-PCR upstream primer and downstream primer.

[0033] Example 1

[0034] Methods: Serum samples from 46 gastric cancer patients and 32 healthy controls were collected from Zhenjiang First People's Hospital between January 2021 and January 2024. Twenty-two pairs of gastric cancer tumor tissue and adjacent adjacent tissues were collected from clinical gastric cancer patients at Nantong Cancer Hospital between January 2022 and January 2024.

[0035] Total RNA was isolated from peripheral blood and tissue specimens of gastric cancer patients and healthy controls using a serum total RNA extraction kit and RNA extraction reagent. HIF1A-AS3 cDNA was prepared using a universal reverse transcription kit. HIF1A-AS3 cDNA was reacted with the cDNA using qRT-PCR primers and reagents, respectively, to measure HIF1A-AS3 expression in peripheral blood and tissues of gastric cancer patients and healthy controls. Unpaired t-tests were used to compare HIF1A-AS3 expression levels between gastric cancer patients and healthy controls. Receiver operating characteristic (ROC) curves were drawn using serum from healthy controls and serum from gastric cancer patients as the disease group, while adjacent tissues from gastric cancer patients served as the control group and gastric cancer tissue as the disease group. The area under the curve (AUC) was calculated to evaluate the sensitivity and specificity of HIF1A-AS3 for gastric cancer diagnosis.

[0036] The specific process is as follows:

[0037] (1) Extraction of total RNA from peripheral serum: Prepare serum or plasma or thaw frozen samples. Transfer 200 μL of serum or plasma to a 2 mL microcentrifuge tube. Add 60 μL of Buffer RPL. Close the tube cap and vortex for 50 seconds. Add 20 μL of Buffer RPP. Close the tube cap and mix vigorously by vortexing for about 20 seconds. Centrifuge at 12,000 g for 3 minutes at room temperature to allow the precipitate to aggregate. Transfer the supernatant to a new microcentrifuge tube, add an equal volume of isopropanol, and mix. Transfer the entire sample to an RNeasy UCP MinElute column. Close the cap and centrifuge at ≥8,000 g for 15 seconds. Add 700 μL of Buffer RWT to the RNeasy UCP MinElute spin column. Close the cap and centrifuge at ≥8,000 g for 15 seconds. Add 500 μL of 80% ethanol to the RNeasy UCP MinElute spin column. Close the cap gently and centrifuge at ≥8,000 g (≥10,000 rpm) for 2 minutes to wash the spin column membrane. Place the RNeasy UCPMinElute spin column in a new 2 mL collection tube. Open the spin column cap and centrifuge at full speed for 5 minutes to dry the membrane. Add 20 μL of RNase-free water directly to the center of the membrane, close the cap, and centrifuge at full speed for 1 minute to elute the RNA to obtain the serum RNA sample.

[0038] (2) Extract RNA from tissue samples: Take an appropriate volume of tumor tissue and paired adjacent cancer tissue, cut them into pieces and place them in an RNase-free EP tube, add 300 μL of lysis buffer, grind into a uniform solution, add 400 μL of Trizol lysis buffer, and let it stand at room temperature for 5 minutes; add 140 μL of chloroform (1 / 5 volume of lysis buffer), vortex for 15 seconds, let it stand on ice for 5 minutes, and centrifuge at 12,000 g, 4°C for 15 minutes; transfer the upper aqueous phase to a new EP tube, add an equal volume of isopropanol and mix; let it stand on ice for 10 minutes, centrifuge at 12,000 g, 4°C for 10 minutes, discard the isopropanol, add 1 ml of 75% ethanol, let it stand at room temperature for 5 minutes, centrifuge at 12,000 g, 4°C for 5 minutes, discard the supernatant, dry the bottom precipitate until it is transparent, and add an appropriate amount of RNase-free water to dissolve it to obtain a tissue RNA sample.

[0039] (3) Reverse transcription

[0040] According to the kit instructions, the reaction system is as follows:

[0041] Components Dosage 10×RT Mix 2μL HiScript Enzyme Mix 2μL HiScript Enzyme Mix 2μL Random hexamers 1 μL Oligo(dT) 1 μL RNA 1 μg

[0042] Add RNA-free ddH2O to 20 μL and set the program in the PCR instrument: 85℃ for 15 min, 37℃ for 5 s. Store cDNA at 4℃ until use and store it at -80℃ for long-term storage.

[0043] (4) HIF1A-AS3 qRT-PCR experiment

[0044] The experiment was carried out according to the kit instructions. The reaction system was as follows:

[0045] Components volume 2×AceQ qPCR Sybr green mix 10 μL 1 μM forward primer 0.4μL 1 μM backward primer 0.4μL cDNA template 2μL RNA-free ddH2O 7.2μL

[0046] The HIF1A-AS3 primer was synthesized by Shanghai Bioengineering, and the sequence is as follows:

[0047]

[0048] The PCR reaction program was set (pre-denaturation at 95°C for 10 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, for 40 cycles). Gene expression in each group was analyzed using 2^(-ΔΔCt). The Ct value represents the number of cycles at which the fluorescence intensity of each sample reached the threshold.

[0049] The calculation formula is as follows: ΔCT = target gene CT value - reference gene CT value, ΔΔCT = target gene ΔCT of experimental group - target gene ΔCT of control group, relative value = 2^(-ΔΔCt).

[0050] The experimental results were processed according to the above principles and formulas to analyze the differential expression of HIF1A-AS3 in peripheral blood and tissues between gastric cancer patients and healthy controls. Prism9 statistical software was used to analyze the differences in HIF1A-AS3 expression in peripheral blood and tissues between gastric cancer patients and healthy controls using an unpaired t-test. P < 0.05 was considered statistically significant. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value. ROC curves were constructed using Prism9, and the area under the curve (AUC) was calculated to evaluate the sensitivity and specificity of HIF1A-AS3 expression in peripheral blood and tissues for diagnosing gastric cancer.

[0051] like Figure 1 and Figure 2 As shown, HIF1A-AS3 is expressed in the peripheral blood of gastric cancer patients ( Figure 1 ) and organizations ( Figure 2 ) were significantly increased compared with the healthy controls.

[0052] like Figure 3 and Figure 4 As shown, the area under the curve of human serum HIF1A-AS3 was 0.8081, the sensitivity was 87.0%, and the specificity was 65.6 ( Figure 3 ); the area under the curve of HIF1A-AS3 in human tissue was 0.8209, the sensitivity was 76.2%, and the specificity was 81.0% ( Figure 4 ); It shows that HIF1A-AS3 has a good diagnostic value for gastric cancer, with the area under the AUC curve reaching more than 80%, making it a relatively reliable specific biomarker for early screening and diagnosis of gastric cancer.

[0053] Example 2

[0054] Previous research by the inventors' research group has found that hypoxia can induce high expression of HIF1A-AS3 in human mesenchymal stem cells (MSCs) and promote their transformation into tumor-associated fibroblasts (CAFs). Based on this, this example constructed an engineered exosome-based targeted delivery system for HIF1A-AS3 siRNA. The specific preparation method is as follows: 293T cells are seeded in a 10 cm cell culture dish. When the cell density reaches 70%, si-HIF1A-AS3 (0.6 nmol / dish) and lipo8000 transfection reagent (30 μL / dish) are mixed and added to the 293T cells. After 48 hours of culture in DMEM medium containing 10% FBS without exosomes, the supernatant is collected and centrifuged at 300g, 4°C for 10 minutes to remove cells and dead cells. After centrifugation, the supernatant is collected and centrifuged at 2000g, 4°C for 10 minutes to remove cell debris. After centrifugation, the supernatant is collected and centrifuged at 10,000g, 4°C for 30 minutes to remove large membrane vesicles. After centrifugation, the supernatant is collected and centrifuged at 100,000g, 4°C for 240 minutes. After centrifugation, the supernatant is removed as much as possible and the bottom pellet is resuspended in PBS to obtain 293T-EX-SI-HIF1A-AS3. 100 μg of 293T-SI-HIF1A-AS3 exosomes (293T-EX-SI-HIF1A-AS3) were incubated with 100 μg of CRYYRITY at 37°C for 30 minutes to form the CRYYRITY-293T-EX-SI-HIF1A-AS3 targeted delivery therapeutic system. The tumor-targeting effect of CRYYRITY was then verified using in vivo imaging experiments, and the in vivo therapeutic efficacy of the CRYYRITY-293T-EX-SI-HIF1A-AS3 system was validated using a Balb / c nude subcutaneous tumor model.

[0055] The nucleotide sequence of the si-HIF1A-AS3 is: siRNA-S: GCAUAAGAGACGGAAUCUATT, siRNA-A: UAGAUUCCGUCUCUUAUGCTT.

[0056] (1) In vivo imaging experiments

[0057] A 1:1 mixture of gastric cancer cells HGC-27 and hypoxia-induced MSCs was subcutaneously injected into Balb / c nude mice. After the tumors grew to an appropriate size, 100 μg of Dir-stained 293T-EX and CRYYRITY-293T-EX were injected into the tail vein, respectively. In vivo imaging was performed at four time points: 4 h, 8 h, 12 h, and 24 h after injection.

[0058] The 293T-EX and CRYYRITY-293T-EX preparation methods are as follows: 293T cells are seeded in a 10 cm cell culture dish and cultured in DMEM medium containing 10% FBS without exosomes for 48 hours. The 293T cell supernatant is collected and centrifuged at 300g at 4°C for 10 minutes to remove cells and dead cells. The supernatant is then centrifuged at 2000g at 4°C for 10 minutes to remove cell debris. The supernatant is then centrifuged at 10,000g at 4°C for 30 minutes to remove large membrane vesicles. The supernatant is then centrifuged at 100,000g at 4°C for 240 minutes. After centrifugation, the supernatant is removed as much as possible and the pellet is resuspended in PBS to obtain the 293T-EX sample. The CRYYRITY-293T-EX sample is obtained by incubating 293T-EX and CRYYRITY at a 1:1 ratio at 37°C for 30 minutes.

[0059] The specific steps of the Dir staining are as follows: take 100 μg of 293T-EX and CRYYRITY-293T-EX respectively, add PBS to 1 ml, add 5 μl of Dir dye to each, incubate at 37 degrees Celsius for 30 minutes, and after incubation, add the mixed solution to a 100kD ultrafiltration tube at 2000g, 4°C, and centrifuge for 30 minutes to obtain a sample with complete staining in the ultrafiltration tube.

[0060] (2) Construction of nude mouse subcutaneous tumor model

[0061] Balb / c nude mice were subcutaneously injected with HGC-27 gastric cancer cells (HGC), a 1:1 mixture of HGC-27 and MSCs cultured in a normoxia environment (HGC+nMSC), and a 1:1 mixture of HGC-27 and MSCs cultured in a hypoxic environment (HGC+hMSC). Once tumors reached appropriate size, they were treated with oxaliplatin (OXA, 5 mg / kg, once every three days for six doses) and exosomes from each group (CRYYRITY-293T-EX-SI-NC and CRYYRITY-293T-EX-SI, 100 μg / mouse, once every three days for six doses). Once tumors reached appropriate size, tumors and major organs were harvested from the mice and analyzed by Tunel staining, H&E staining, and immunohistochemistry.

[0062] The CRYYRITY-293T-EX-SI-NC represents 293T cell exosomes that encapsulate HIF1A-AS3-siRNA-NC and are modified with CRYYRITY on the surface. The preparation process refers to the "CRYYRITY-293T-EX-SI-HIF1A-AS3 targeted delivery therapeutic system". HIF1A-AS3-siRNA-NC is provided by Shanghai Jima Pharmaceutical Technology Co., Ltd.; CRYYRITY-293T-EX-SI represents 293T cell exosomes that encapsulate HIF1A-AS3-siRNA and are modified with CRYYRITY on the surface.

[0063] (3) Immunohistochemical staining

[0064] The tissue sections were dried at 65°C overnight, dewaxed in xylene I and II solutions for 15 minutes, and then dehydrated in 100% ethanol, 90% ethanol, 85% ethanol, and 75% ethanol for 2 minutes each. The sections were then transferred to ddH2O for immersion and washing three times, each for 10 minutes. The sections were placed in 0.3% hydrogen peroxide solution for 10 minutes to inactivate endogenous peroxidase, and washed with PBS three times, each for 5 minutes. The sections were heated in boiling 1× citric acid buffer for 30 minutes, cooled to room temperature, and then transferred to PBS for washing for 5 minutes, for a total of 3 times. The moisture on the slides was wiped dry, and a special immunohistochemistry pen was used to draw a circle around the tissue and drop 5% Block with BSA for 1 hour. Add an appropriate amount of diluted Ki67 primary antibody solution to the specimen and place in a humidified chamber for overnight reaction at 4°C. Wash with PBS for 5 minutes three times. Add biotin-conjugated secondary antibody solution to the slide and incubate in a humidified chamber for 1 hour at room temperature. Wash with PBS for 5 minutes three times. Add diluted SABC solution to the slide and react at 37°C for 30 minutes. Wash with PBS for 5 minutes three times. Add DAB working solution to the tissue. When the tissue turns yellow-brown under a microscope, immediately place it in a glass jar filled with double-distilled water to terminate staining (ensuring consistent staining time for each group). Counterstain: Add hematoxylin solution to the tissue for 3 seconds until the tissue structure is clear. Rinse with running water for 1 hour until the water does not change color. Dehydrate: Soak sections in 75%, 85%, 90%, and 100% ethanol solutions for 3 minutes each. Transfer the sections to a glass jar filled with xylene and soak for 30 minutes before removing. Drop an appropriate amount of neutral resin onto the glass slide for sealing, let it dry, and then take pictures with a microscope.

[0065] like Figure 5 As shown, in vivo imaging experiments showed that modifying the tumor-associated fibroblast targeting peptide (CRYYRITY) on the surface of 293T-EX can significantly improve the targeting effect of 293T-EX on the tumor site, resulting in obvious enrichment at the tumor site and improving the therapeutic effect of exosomes.

[0066] like Figure 6 and Figure 7 As shown, the Balb / c nude subcutaneous tumor model showed that CRYYRITY-293T-EX-SI-HIF1A-AS3 had a good therapeutic effect on gastric cancer in vivo.

[0067] like Figure 8 As shown, H&E, Tunel, and immunohistochemistry results showed that CRYYRITY-293T-EX-SI-HIF1A-AS3 can significantly inhibit the proliferation of gastric cancer cells and promote the apoptosis of gastric cancer cells in vivo, with good therapeutic effects.

[0068] In summary, HIF1A-AS3 expression is significantly elevated in both peripheral blood and tissues of gastric cancer patients, demonstrating its valuable diagnostic value for early screening and diagnosis of gastric cancer. Furthermore, the development of an exosome-based targeted delivery system for HIF1A-AS3 siRNA has demonstrated promising therapeutic effects in vivo. The diagnosis and treatment of gastric cancer require multidisciplinary collaboration and personalized treatment plans. Early diagnosis and effective treatment are key to improving survival rates.

Claims

1. Use of lncRNA HIF1A-AS3 as a marker in the preparation of a gastric cancer diagnostic product, wherein the nucleotide sequence of the lncRNA HIF1A-AS3 is shown in SEQ ID NO.

1.

2. Use of a reagent for detecting the expression level of lncRNA HIF1A-AS3 in the preparation of a gastric cancer diagnosis / screening product, wherein the nucleotide sequence of the lncRNA HIF1A-AS3 is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that The reagents include PCR reagents for detecting the expression level of lncRNA HIF1A-AS3.

4. The use according to claim 3, characterized in that The PCR reagent for detecting the expression level of lncRNA HIF1A-AS3 is a primer pair for detecting lncRNA HIF1A-AS3.

5. The use according to claim 4, characterized in that The primer pair includes a forward primer 5'-GTCTTTCCTCTCCGTCCCAG -3' and a reverse primer 5'- CAGACACCCGATCTCCGTG -3'.

6. Application of lncRNA HIF1A-AS3 inhibitors in the preparation of gastric cancer treatment products.

7. The use according to claim 6, characterized in that The lncRNA HIF1A-AS3 inhibitor is siRNA.

8. The use according to claim 8, characterized in that The nucleotide sequences of the siRNA are: siRNA-S: GCAUAAGAGACGGAAUCUATT, siRNA-A: UAGAUUCCGUCUCUUAUGCTT.

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