System and detection method for monitoring mRNA m6A methylation in hepatic stellate cells based on hybridization chain reaction

Abstract

The invention relates to a system for monitoring mRNA m6A methylation in hepatic stellate cells based on a hybridization chain reaction and a detection method, and belongs to the technical field of analysis and detection. In order to solve the problem of poor sensitivity and detection precision in the prior art, the invention provides a system for monitoring mRNA m6A methylation in hepatic stellate cells based on hybridization chain reaction and a detection method, the method comprises the following steps: contacting a target mRNA m6A methylation sample to be detected with a group of probes comprising a probe a and a probe b; the probe b is connected to an anti-rabbit IgG antibody to form a probe b attached with the anti-rabbit IgG antibody; combining with a rabbit monoclonal anti-m6A antibody; the rabbit monoclonal anti-m6A antibody can be combined with a target mRNA m6A methylated sample to be detected; a hairpin probe H1 monomer and a hairpin probe H2 monomer are subjected to a hybridization chain reaction to form a polymer, and imaging detection is carried out. The method has the advantages of high detection sensitivity, high detection selectivity and strong intuition.

Description

Technical Field

[0001] The present invention relates to a system and a detection method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction, and belongs to the technical field of analysis and detection. Background Art

[0002] N6-methyladenosine (m6A) methylation is one of the most common post-transcriptional modifications in eukaryotic RNA, occurring extensively in mRNA and non-coding RNAs such as lncRNA and tRNA. It plays a key role in regulating gene expression and is considered a core component of "RNA epigenetics."

[0003] m6A modification is dynamically regulated by three key protein types: writers: methyltransferase complexes (e.g., METTL3 / METTL14 / WTAP) catalyze m6A formation, with METTL3 being the core catalytic subunit. Erasers: demethylases (e.g., FTO and ALKBH5) remove methyl groups, maintaining the modification's dynamic equilibrium. Readers: proteins that recognize m6A (e.g., YTHDF1 / 2 / 3, YTHDC1, etc.) regulate gene expression by affecting RNA splicing, transport, translation, or degradation.

[0004] Liver fibrosis is a pathological change caused by excessive deposition of extracellular matrix (ECM) during liver tissue repair following chronic liver injury. The main cells involved include hepatic stellate cells (HSCs) and Kupffer cells (liver macrophages). Its core characteristic is the activation and transformation of hepatic stellate cells (HSCs) into myofibroblasts, which secrete large amounts of collagen (e.g., type I and type III), disrupting normal liver tissue architecture. Transforming growth factor β1 (TGF-β1), secreted by activated Kupffer cells, promotes the progression of non-alcoholic steatohepatitis (NASH) to liver fibrosis. During the progression from NASH to liver fibrosis, the posttranscriptional regulation of TGF-β1 is determined by m6A modification. The NF-κB pathway promotes m6A methylation of TGF-β1 mRNA by activating the methyltransferases METTL3 / METTL14, thereby exacerbating TGF-β1-mediated HSC activation. m6A participates in various life processes by regulating RNA metabolism, including posttranscriptional regulation and RNA splicing. Therefore, how to diagnose liver fibrosis and determine the condition by analyzing m6A methylation of mRNA is also crucial.

[0005] Currently, the main techniques used to detect m6A include antibody enrichment, chemical conversion, enzymatic methods, nanopore direct sequencing, and mass spectrometry. Antibody enrichment methods, including MeRIP-seq (m6A RNA immunoprecipitation sequencing) and miCLIP-seq (cross-linking immunoprecipitation sequencing), are based on m6A antibodies. However, these methods are unable to precisely locate methylation sites and are limited to a low base resolution of approximately 200 bases. However, these methods suffer from complex procedures, cumbersome methods, and high detection limits. Crucially, these methods rely on extracting RNA from cell populations, making them unable to effectively capture spatial localization and resulting in poor detection sensitivity and accuracy. Summary of the Invention

[0006] In response to the above problems in the prior art, the present invention provides a system and detection method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction, which solves the problem of how to avoid poor sensitivity and low detection accuracy.

[0007] One of the purposes of the present invention is to achieve the following technical solution: a system for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction, characterized in that the system comprises

[0008] Probe a, which is used to specifically hybridize with the target mRNA m6A methylation sample to be detected; the sequence of probe a is as follows:

[0009] 5'-GACGCTAATAGTTAGCTCGGCTTCGTCGTGGATCATGTATGACATGTC ACTAACATCATCCGACTGCTGCTTTTCTCCTCAA-3';

[0010] Rabbit monoclonal anti-m6A antibody, used to bind to the target mRNA m6A methylation sequence;

[0011] Probe b, the probe b is connected to the anti-rabbit IgG antibody via a biotin-streptavidin-biotin bridge; the sequence of the probe b is as follows:

[0012] 5'-AAAAAAAAAATACGAAGCGGAGCTGACACTCTT-3', the 5'-end of the probe b is modified with Biotin;

[0013] Hairpin probe H1:

[0014] 5'-ATAGTTGACACTCTTACAGCTCAACTAAAGAGTGTCAACTATTAGCGT C-3';

[0015] Hairpin probe H2:

[0016] 5'-TAGTTGAGCTGTAAGAGTGTCAACTATGACGCTAATAGTTGACACTCT TACAG-3', the 5'-end of the hairpin probe H2 is modified with FAM, and the 3'-end is modified with BHQ-1.

[0017] The present invention uses two proximity probes, namely probe a and probe b. One of the two proximity probes, probe a, can specifically hybridize and recognize the target mRNAm6A methylation sample to be tested (target mRNAm6A methylation sequence, etc.); the other probe b can be connected to the anti-rabbit IgG antibody through a biotin-streptavidin-biotin bridge, and the rabbit monoclonal anti-m6A antibody used to bind to the target mRNAm6A methylation sequence can be recognized by the anti-rabbit IgG antibody; in this way, during detection, the above-mentioned recognition process can bring probe a and probe b very close to each other. When close, the two probes (probe a and probe b) selected with specific sequences expose the overhanging ends, which can act as templates to complement the loop of the first hairpin probe (H1), open its stem-loop structure, and expose the originally closed sequence (called "sticky end"). The exposed H1 sticky end complements the loop of the second hairpin probe (H2), opens the stem-loop structure of H2, and simultaneously exposes the sticky end of H2. The sticky end of H2 can be combined with new H1 probe again, opens its structure, forms alternating H1-H2-H1-H2 ... linear long-chain polymer.It is equivalent to being able to carry out signal amplification by HCR when detecting, for the detection of target analyte to be measured, it is possible to more intuitively provide the expression and distribution of target substance in target analyte such as cell, actual operation is simple, does not require any pre-treatment process, greatly improves the intuitiveness of detection, reduces the construction time and cost of material, is more conducive to practical application.Meanwhile, the above-mentioned detection system of the present invention is used for the detection of mRNA m6A methylation in hepatic stellate cells, which is equivalent to a specific proximity probe (probe a and probe b) by design, and a group of hairpin probes (H1 and H2) used in combination, so that in the process of identification, it is possible to more accurately detect the material and distribution of mRNA m6A methylation in hepatic stellate cells, and there is the advantage that detection sensitivity is high.

[0018] The second object of the present invention is achieved by the following technical solution: a detection method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction, characterized in that the method comprises the following steps:

[0019] A. Contact the target mRNA m6A methylation sample with a set of probes including probe a and probe b; the sequence of probe a is as follows:

[0020] 5'-GACGCTAATAGTTAGCTCGGCTTCGTCGTGGATCATGTATGACATGTC ACTAACATCATCCGACTGCTGCTTTCTCCCTCAA-3'; the probe a is used to specifically hybridize with the target mRNAm6A methylation sample to be tested;

[0021] The probe b is connected to the anti-rabbit IgG antibody via a biotin-streptavidin-biotin bridge to form a probe b attached to the anti-rabbit IgG antibody; the sequence of the probe b is as follows:

[0022] 5'-AAAAAAAAAATACGAAGCGGAGCTGACACTCTT-3', the 5'-end of the probe b is modified with Biotin;

[0023] The probe b attached with anti-rabbit IgG antibody binds to the rabbit monoclonal anti-m6A antibody; the rabbit monoclonal anti-m6A antibody can bind to the target mRNAm6A methylation sample to be tested;

[0024] A hybridization chain reaction is performed using a hairpin probe H1 monomer and a hairpin probe H2 monomer to form a polymer; the sequence of the hairpin probe H1 is shown below:

[0025] 5'-ATAGTTGACACTCTTACAGCTCAACTAAAGAGTGTCAACTATTAGCGT C-3';

[0026] The sequence of the hairpin probe H2 is shown below:

[0027] 5'-TAGTTGAGCTGTAAGAGTGTCAACTATGACGCTAATAGTTGACACTCT TACAG-3', the 5'-end of the hairpin probe H2 is modified with FAM, and the 3'-end is modified with BHQ-1.

[0028] B. Perform imaging detection on the polymer obtained in step A.

[0029] By contacting the target mRNAm6A methylation sample to be tested with a set of probes including probe a and probe b, and designing the corresponding probes for specific sequences, one of the two proximity probes, probe a, can specifically hybridize and recognize the target mRNAm6A methylation sequence (target mRNAm6A methylation sample to be tested); the other probe b can be indirectly connected to the anti-rabbit IgG antibody through a biotin-streptavidin-biotin bridge to form a probe b attached to the anti-rabbit IgG antibody, which can bind to the rabbit monoclonal anti-m6A antibody (or simply referred to as anti-m6A antibody). , and then identify the target mRNAm6A methylation sequence in the target mRNAm6A methylation sample to be tested; during the identification process, the corresponding hairpin probe H1 monomer and hairpin probe H2 monomer can undergo hybridization chain reaction (HCR) to form a polymer; this is equivalent to exposing the overhanging end when the two probes (probe a and probe b) of the above-selected specific sequence are close to each other through hybridization chain reaction. As a template, it can complementarily bind to the loop of the first hairpin probe (H1), open its stem-loop structure, and expose the originally closed sequence (called "sticky end"). The exposed H1 sticky end complementarily binds to the loop of the second hairpin probe (H2), opening the stem-loop structure of H2 and exposing the sticky end of H2 at the same time. The sticky end of H2 can then bind to the new H1 probe, opening its structure to form a linear long-chain polymer of alternating H1-H2-H1-H2...; then, the fluorescent groups contained in the designed probes, such as the modified groups in probe b and the modified groups in the hairpin probe H2, are used to amplify the signal in the HCR reaction. This allows for more sensitive and accurate signal detection in the subsequent imaging detection process, and the distribution can also be more intuitively observed through the imaging image.

[0030] In the above-mentioned detection method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction, preferably, the target mRNAm6A methylation sample to be tested in step A is selected from mRNAm6A methylation sequences or mouse hepatic stellate cells. The above-mentioned detection method can be directly used for the detection of mRNAm6A methylation sequences or for detection of corresponding effector cells, such as mouse hepatic stellate cells, for direct intracellular detection, which has the advantage of simpler operation while still ensuring the sensitivity and accuracy of the detection.

[0031] In the above-mentioned detection method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction, preferably, when the target mRNAm6A methylation sample to be detected is selected from the mRNAm6A methylation sequence, the step A specifically adopts the following method:

[0032] a. Incubate the biotin-labeled probe b with streptavidin in PBS buffer at a temperature of 35°C to 38°C.

[0033] b. Add biotin-labeled anti-rabbit IgG antibody and control the temperature to incubate at 35° C. to 38° C. to obtain probe b attached with anti-rabbit IgG antibody;

[0034] c. Incubating the obtained anti-rabbit IgG antibody-attached probe b with rabbit monoclonal anti-m6A antibody at a temperature of 35° C. to 38° C. to obtain the anti-rabbit IgG antibody-attached probe b after binding to the rabbit monoclonal anti-m6A antibody;

[0035] d. Mix the mRNA m6A methylation sequence, probe b attached with anti-rabbit IgG antibody after binding to rabbit monoclonal anti-m6A antibody, probe a, hairpin probe H1, and hairpin probe H2, and incubate at a controlled temperature of 35°C to 38°C for four days.

[0036] In the above-mentioned detection method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction, preferably, when the target mRNAm6A methylation sample to be detected is selected from mouse hepatic stellate cells, the step A specifically adopts the following method:

[0037] Aa. Incubate activated mouse hepatic stellate cells with rabbit monoclonal anti-m6A antibody at 35°C to 38°C for five minutes;

[0038] Ab, then add anti-rabbit IgG antibody labeled with streptavidin and biotin and incubate at a temperature of 35°C to 38°C;

[0039] Ac, then add a mixture containing probe a, probe b, hairpin probe H1 and hairpin probe H2 and control the temperature to be incubated at 35°C to 38°C to obtain the corresponding cells after the incubation is completed.

[0040] Through the above operation, the activated mouse hepatic stellate cells are first recognized and bound by the rabbit monoclonal anti-m6A antibody, and then the anti-rabbit IgG antibody system is attached indirectly through streptavidin and biotin, and probe a, probe b, hairpin probe H1 and hairpin probe H2 are subsequently added. During the HCR reaction process, it can be directly carried out in the cell, which has the advantages of simple operation and efficient detection, and can also ensure the sensitivity and accuracy of the detection, and can also ensure the intuitiveness of the subsequent imaging detection.

[0041] In the above-mentioned detection method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction, preferably, the imaging detection in step B includes the following steps:

[0042] Dual-channel imaging was performed using a confocal microscope with excitation wavelengths of FAM 488 nm and DAPI 405 nm.

[0043] In the detection method for monitoring mRNA m6A methylation in hepatic stellate cells based on hybridization chain reaction described above, as preferred, the time of incubation one, incubation two, incubation three and incubation four is independently selected from 1h-2h; further preferred, the time of incubation five, incubation six and incubation seven is independently selected from 1h-2h. By controlling the incubation time, the recognition and binding can be more effectively guaranteed, and the effectiveness and sufficiency of recognition can be improved. For the incubation one, incubation two, incubation three, incubation four, incubation five, incubation six and incubation seven described above, it is only for better description and identification, and is not limited thereto, which is the meaning of incubation.

[0044] In summary, compared with the prior art, the present application has the following advantages:

[0045] 1. By designing the sequences of adjacent probe a and probe b, and combining the use of hairpin probes (H1 and H2) and indirect connection, the high-methylated mRNA in hepatic stellate cells can be more effectively recognized and detected more accurately, which has the advantages of high detection sensitivity, high detection selectivity and strong intuitiveness.

[0046] 2. By modifying the end of probe b and hairpin probe H2 in the system with a fluorescent group, after the HCR hybridization chain reaction in the detection, the signal can be effectively amplified, and the observation in the imaging detection is more intuitive. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 is the construction mechanism and detection principle diagram of the m6A-PHPEA system of the present application.

[0048] Figure 2 is the structural integrity and nanoparticle morphology characterization analysis of the m6A-PHPEA system by 10% non-denaturing polyacrylamide gel combined with transmission electron microscopy (TEM).

[0049] Figure 3 is the fluorescence detection verification analysis diagram of the feasibility of the m6A-PHPEA system of the present application.

[0050] Figure 4 is the fluorescence spectrum diagram and linear fitting equation of the m6A-PHPEA system of the present application for different target concentrations.

[0051] Figure 5 is the laser confocal microscopic imaging diagram of the corresponding cells after incubation in Example 4 of the present application. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further specifically described below through specific embodiments, but the present invention is not limited to these embodiments.

[0053] The system for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction of the present invention includes a probe a, which is used to specifically hybridize with a target mRNAm6A methylation sequence or a target mRNAm6A methylation sample (or analyte to be tested); the sequence of the above-mentioned probe a is as follows:

[0054] 5'-GACGCTAATAGTTAGCTCGGCTTCGTCGTGGATCATGTATGACATGTC ACTAACATCATCCGACTGCTGCTTTTCTCCTCAA-3';

[0055] The above-mentioned target mRNAm6A methylation sequence is equivalent to the target analyte to be analyzed by this detection method; the target analyte can be the detection and analysis of the whole nucleic acid sequence or the intracellular analysis, both of which can achieve the purpose of in situ analysis and have the advantage of simple operation.

[0056] Rabbit monoclonal anti-m6A antibody, which can bind to the target mRNA m6A methylation sequence;

[0057] Probe b, where probe b is linked to anti-rabbit IgG antibody via a biotin-streptavidin-biotin bridge; the sequence of probe b is as follows:

[0058] 5'-AAAAAAAAAATACGAAGCGGAGCTGACACTCTT-3', the 5'-end of the probe b is modified with Biotin;

[0059] Hairpin probe H1:

[0060] 5'-ATAGTTGACACTCTTACAGCTCAACTAAAGAGTGTCAACTATTAGCGT C-3';

[0061] Hairpin probe H2:

[0062] 5′-TAGTTGAGCTGTAAGAGTGTCAACTATGACGCTAATAGTTGACACTCT TACAG-3′, the 5′-end of the hairpin probe H2 is modified with FAM, and the 3′-end is modified with BHQ-1.

[0063] This detection method systematically combines rabbit monoclonal anti-m6A antibody recognition with in situ hybridization. Utilizing the recognition of probes a and b, rabbit monoclonal anti-m6A antibodies, and anti-rabbit IgG antibodies, it effectively identifies m6A methylation sites and targets target sequences, achieving bispecific recognition with high accuracy and sensitivity. When the proximity probes (probes a and b) come into close proximity, the two proximity probes (probes a and b) expose overhanging ends, which serve as templates for complementary binding to the loop of the first hairpin probe (H1), opening its stem-loop structure and exposing previously blocked sequences (referred to as "sticky ends"). The exposed H1 sticky ends then bind complementary to the loop of the second hairpin probe (H2), opening the H2 stem-loop structure and exposing the H2 sticky ends. The H2 sticky ends can then bind to new H1 probes, opening their structure and forming a long, linear polymer chain alternating between H1-H2-H1-H2...

[0064] The detection method of the present invention can be specifically analyzed in the following manner, which specifically comprises the following steps:

[0065] The target mRNA m6A methylation sample to be detected (or the target analyte to be detected) is contacted with a set of probes including probe a and probe b; the sequence of probe a is as follows:

[0066] 5'-GACGCTAATAGTTAGCTCGGCTTCGTCGTGGATCATGTATGACATGTC ACTAACATCATCCGACTGCTGCTTTCTCCCTCAA-3'; Probe a is used to specifically hybridize with the target mRNA m6A methylation sample to be tested;

[0067] Probe b is linked to the anti-rabbit IgG antibody via a biotin-streptavidin-biotin bridge to form a probe b attached to the anti-rabbit IgG antibody; the sequence of the probe b is shown below:

[0068] 5'-AAAAAAAAAATACGAAGCGGAGCTGACACTCTT-3', the 5'-end of the probe b is modified with Biotin;

[0069] Probe b attached with anti-rabbit IgG antibody binds to rabbit monoclonal anti-m6A antibody; rabbit monoclonal anti-m6A antibody can bind to the target mRNA m6A methylation sample;

[0070] A hybridization chain reaction is performed using a hairpin probe H1 monomer and a hairpin probe H2 monomer to form a polymer; the sequence of the hairpin probe H1 is shown below:

[0071] 5'-ATAGTTGACACTCTTACAGCTCAACTAAAGAGTGTCAACTATTAGCGT C-3';

[0072] The sequence of the hairpin probe H2 is shown below:

[0073] 5'-TAGTTGAGCTGTAAGAGTGTCAACTATGACGCTAATAGTTGACACTCT TACAG-3', the 5'-end of the hairpin probe H2 is modified with FAM, and the 3'-end is modified with BHQ-1.

[0074] The polymer obtained above is subjected to imaging detection. The polymer formed by the modified fluorescent group after HCR hybridization chain reaction can effectively amplify the signal, thereby achieving better detection and analysis, and has the advantages of high sensitivity and high accuracy. Figure 1 As shown, the construction mechanism and detection principle display diagram of the m6A-HCPHA system of the present invention can clearly show the recognition process and the process of HCR hybridization chain reaction when the proximity probe approaches.

[0075] In order to further illustrate the above detection method of the present invention, the following is an analysis through a specific implementation process.

[0076] Example 1

[0077] Construction and characterization of a hybridization chain reaction method (named m6A-HCPHA) for monitoring mRNA m6A methylation in hepatic stellate cells

[0078] To verify the assembly of the m6A-HCPHA system, this study characterized the system by non-denaturing polyacrylamide gel (10% nPAGE) and transmission electron microscopy (TEM).

[0079] The sequence of probe a in this embodiment is as follows:

[0080] 5'-GACGCTAATAGTTAGCTCGGCTTCGTCGTGGATCATGTATGACATGTC ACTAACATCATCCGACTGCTGCTTTTCTCCTCAA-3'

[0081] The sequence of probe b is as follows:

[0082] 5'-AAAAAAAAAATACGAAGCGGAGCTGACACTCTT-3', the 5'-end of the probe b is modified with Biotin;

[0083] Hairpin probe H1: Its sequence is as follows:

[0084] 5'-ATAGTTGACACTCTTACAGCTCAACTAAAGAGTGTCAACTATTAGCGT C-3';

[0085] Hairpin probe H2: Its sequence is as follows:

[0086] 5'-TAGTTGAGCTGTAAGAGTGTCAACTATGACGCTAATAGTTGACACTCT TACAG-3', the 5'-end of the hairpin probe H2 is modified with FAM, and the 3'-end is modified with BHQ-1.

[0087] The specific experimental methods are as follows:

[0088] 1. Preparation of m6A-HCPHA System

[0089] (11) Biotin-labeled probe b (50 μM, 2 μL) was incubated with streptavidin (50 μM, 2 μL) in 1× PBS buffer at 37°C for 1 h;

[0090] (12) adding biotin-labeled anti-rabbit IgG antibody (1 μL) to the above solution and incubating at 37°C for 1 h to obtain probe b attached with anti-rabbit IgG antibody;

[0091] (13) The prepared anti-rabbit IgG antibody-attached probe b (5 μL) was incubated with rabbit monoclonal anti-m6A antibody (5 μL) at 37°C for 1 h;

[0092] (14) Take 0.5 μL of the target mRNA mA methylation sample, 0.5 μL of the probe b prepared in step (13) above and attached with anti-rabbit IgG antibody after binding to rabbit monoclonal anti-m6A antibody, 0.5 μL of probe a, 1 μL of H1 and 1 μL of H2, mix them thoroughly, and control the temperature to incubate at 37°C for 1 hour to obtain mA-HCPHA solution.

[0093] 2. Non-denaturing polyacrylamide gel analysis

[0094] (21) Take a 1.0 mm thin plate, a thick plate and a 1.0 mm comb, rinse them with running water, and then rinse them with ultrapure water and alcohol.

[0095] (22) Use a hair dryer to dry the electrophoresis plate, making sure the glue-filled surface of the electrophoresis plate is kept clean.

[0096] (23) Place a soft cushion on the glue rack, clamp the two plates with a clamp and place them on the glue rack. Use a 5mL pipette to draw the glue solution and pour it between the two glass plates. Be careful not to have bubbles. Then insert a comb. When there are mountain-like patterns in the glue holes, turn on the faucet, untie the clamp, rinse the glass plates with running water, pull out the comb, and shake off the water in the glue holes to prevent them from clogging.

[0097] (24) Take 8 μL of sample, add 2 μL of 6× loading buffer and 2 μL of 10× SYBR Green I, mix, and centrifuge gently. Before running the gel, use a pipette to add 10 μL of the sample mixture to the corresponding gel wells. Fill the electrophoresis tank with 0.5× TBE buffer and run at 90 V constant voltage mode for 50 min.

[0098] (25) After the electrophoresis is completed, carefully pry open the gel with a gel remover, remove the gel and place it in the center of the Bio-Red gel electrophoresis instrument, and use Image Lab analysis software for imaging analysis.

[0099] 3. Transmission Electron Microscopy (TEM) Characterization

[0100] 10 μL of the prepared m6A-HCPHA solution was dropped onto a carbon-supported copper grid (Holey Carbon Grid, 300 mesh) and allowed to stand at 37°C for 30 min. The residual liquid was removed by filter paper after adsorption. After secondary drying, the solution was placed in the sample chamber of a transmission electron microscope (200 kV, JEM-F200). Bright field images were collected at an accelerating voltage of 200 kV, and particle size statistical analysis was performed using Gatan Microscopy Sμite software.

[0101] The specific analysis results are as follows Figure 2 As shown in the figure, it can be seen that Figure 2 As shown in A, lane 2 is a mixture of probe a and probe b, and lane 4 is a mixture of probe a, probe b and TGF-β mRNA. The migration rate was significantly reduced, which effectively confirmed the construction of m6A-HCPHA. Further TEM characterization showed ( Figure 2 B) shows that m6A-HCPHA presents a well-monodispersed spherical nanostructure with an average particle size of 115±10 nm.

[0102] Example 2

[0103] Verification of the feasibility of the m6A-HCPHA system

[0104] To demonstrate the feasibility of the m6A-HCPHA system for detecting m6A RNA, we used hybridization chain reaction (HCR) for signal amplification to detect m6A sites in TGF-β mRNA in vitro. The specific experimental methods are as follows:

[0105] 1. Add 180 μL of pre-prepared 1× PBS to 20 μL of the solution described in Example 1 to make the final volume 200 μL. Vortex and mix thoroughly, then measure the fluorescence.

[0106] 2. Open the F-7000 software and set the parameters: set the excitation wavelength (EX WL) to 492 nm, the emission wavelength range (EM Start WL-EM Start WL) to 500-600 nm, the excitation and emission gratings (EX Slit, EM Slit) to 5 nm, the photomultiplier tube voltage (PMT Voltage) to 600 V, and the response time (Response) to auto.

[0107] 3. Gently add 200 μL of sample mixture into the quartz cuvette along the wall to avoid bubbles. Align the light side of the quartz cuvette with the light outlet, place it into the sample slot, close the cover, click Measure, and save the experimental results.

[0108] The results are as follows Figure 3 As shown in Figure 2, compared with the fluorescent signal generated by Probe-a of the targeting m6A sequence (Target), when Probe-a is not used, the fluorescent signal drops sharply. This shows that the m6A-HCPHA system of the present invention can target a specific sequence, so that specific m6A sites can be identified. And only when probe a and probe b are present at the same time, there will be a significantly rising fluorescent signal. On the contrary, the group lacking probe a, probe b or probe a replaced by a non-specific probe will not produce a strong fluorescent signal. In addition, the fluorescent signal generated by target m6A RNA is higher than unmodified m6A, indicating that the m6A-HCPHA system of the present invention is good for detecting the ability of m6A signals, and has the advantage of high sensitivity.

[0109] Example 3

[0110] Fluorescence detection of TGF-β mRNA m6A content

[0111] Different concentrations of TGF-β mRNA were added to 20 μL of the m6A-HCPHA solution obtained in Example 1, and the fluorescence intensity was measured using a molecular fluorescence instrument. Figure 4 As shown, Figure 4 This is a graph of TGF-βmRNAm6A detection by nanomaterials. In the figure, A is a linear graph for detecting TGF-βmRNAm6A, and B is a fluorescence kinetic curve. It can be seen from the figure that at 519nm, the fluorescence intensity increases with the increase of TGF-βmRNAm6A concentration. The detection limit is 1pM, and the linear equation is F=0.06824C TGF-βmRNA +78.03(R 2=0.9912), which proves that the linear determination effect is good.

[0112] Example 4

[0113] Cell imaging assay analysis

[0114] Hepatic stellate cell activation plays a crucial role in the progression of liver fibrosis. During the progression from NASH to fibrosis, posttranscriptional regulation of TGF-β1 is determined by m6A modification. The NF-κβ pathway promotes m6A methylation of TGF-β1 mRNA by activating the methyltransferases METTL3 / METTL14, thereby exacerbating TGF-β1-mediated HSC activation and promoting the transition from NASH to fibrosis. Based on this biological characteristic, murine hepatic stellate cells (JS-1) were selected as effector cells in this example.

[0115] The specific experimental methods are as follows:

[0116] 1. Take the mouse hepatic stellate cells JS-1 in the logarithmic growth phase and digest them with 0.25% trypsin. 5 cells / well were seeded in a 24-well plate with a pre-installed glass slide, mixed by cross-wise method, and cultured at 37°C, 5% CO2 for 24 h;

[0117] 2. Discard the original culture medium, wash along the side wall with DEPC-PBS three times, and fix with 4% paraformaldehyde at room temperature for 15 minutes;

[0118] 3. Add 0.5% TritonX-100 solution to each well and permeabilize for 15 minutes at room temperature. Wash with DEPC-PBS. Block with 3% BSA solution at room temperature for 1 hour.

[0119] 4. Incubate with rabbit monoclonal anti-m6A antibody at 37°C for 3 h, wash three times with PBST, and add streptavidin- and biotin-labeled anti-rabbit IgG antibody (1:50) and incubate at 37°C for 1 h.

[0120] 5. Add a mixture containing Probe a, Probe b, H1, and H2, incubate at 37°C for 1 hour, wash three times with PBST, and stain the nuclei with DAPI for 10 minutes. Then, after antifade mounting, obtain the corresponding cells after incubation and perform dual-channel imaging using a laser confocal microscope (CLSM, Nikon A1R MP, Japan) with excitation wavelengths: FAM 488nm / DAPI 405nm.

[0121] The specific analysis results are as follows Figure 5 As shown in Figure 2, laser confocal microscopy images confirm the ability of the m6A-PHPEA system to detect m6A sites in TGF-β mRNA in JS-1 cells. Figure 5 ( Figure 5A and Figure 5 As can be seen in Figure B), a clear fluorescence signal can only be observed when both probe a and probe b are present. When probe a or probe b is not used, almost no fluorescence signal is observed, confirming that signal generation depends on the proximity binding of the two probes. In addition, a weak fluorescence signal appears when a random sequence probe or probe a targeting an unmethylated site is used. Statistical analysis confirms that the fluorescence signal of the target m6A mRNA is significantly different from that of the negative control ( Figure 5 B), consistent with the previous fluorescence detection results, further highlighting the high specificity of m6A-PHPEA.

[0122] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.

[0123] Although the present invention has been described in detail and certain specific embodiments have been cited, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

Claims

1. A system for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction, characterized in that: The system includes Probe a, which is used to specifically hybridize with the target mRNAm6A methylation sample; the sequence of the probe a is as follows: 5'-GACGCTAATAGTTAGCTCGGCTTCGTCGTGGATCATGTATGACATGTC ACTAACATCATCCGACTGCTGCTTTTCTCCTCAA-3'; Rabbit monoclonal anti-m6A antibody, used to bind to the target mRNA m6A methylation sequence; Probe b, the probe b is connected to the anti-rabbit IgG antibody via a biotin-streptavidin-biotin bridge; the sequence of the probe b is as follows: 5'-AAAAAAAAAATACGAAGCGGAGCTGACACTCTT-3', the 5'-end of the probe b is modified with Biotin; Hairpin probe H1: 5'-ATAGTTGACACTCTTACAGCTCAACTAAAGAGTGTCAACTATTAGCGT C-3'; Hairpin probe H2: 5'-TAGTTGAGCTGTAAGAGTGTCAACTATGACGCTAATAGTTGACACTCT TACAG-3', the 5'-end of the hairpin probe H2 is modified with FAM, and the 3'-end is modified with BHQ-1.

2. A detection method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction, characterized in that: The method comprises the following steps: A. Contact the target mRNA m6A methylation sample with a set of probes including probe a and probe b; the sequence of probe a is as follows: 5'-GACGCTAATAGTTAGCTCGGCTTCGTCGTGGATCATGTATGACATGTC ACTAACATCATCCGACTGCTGCTTTCTCCCTCAA-3'; the probe a is used to specifically hybridize with the target mRNAm6A methylation sample to be tested; The probe b is connected to the anti-rabbit IgG antibody via a biotin-streptavidin-biotin bridge to form a probe b attached to the anti-rabbit IgG antibody; the sequence of the probe b is as follows: 5'-AAAAAAAAAATACGAAGCGGAGCTGACACTCTT-3', the 5'-end of the probe b is modified with Biotin; The probe b attached with the anti-rabbit IgG antibody binds to the rabbit monoclonal anti-m6A antibody; the rabbit monoclonal anti-m6A antibody can bind to the target mRNAm6A methylation sample to be tested; A hybridization chain reaction is performed using a hairpin probe H1 monomer and a hairpin probe H2 monomer to form a polymer; the sequence of the hairpin probe H1 is shown below: 5'-ATAGTTGACACTCTTACAGCTCAACTAAAGAGTGTCAACTATTAGCGT C-3'; The sequence of the hairpin probe H2 is shown below: 5'-TAGTTGAGCTGTAAGAGTGTCAACTATGACGCTAATAGTTGACACTCT TACAG-3', the 5'-end of the hairpin probe H2 is modified with FAM, and the 3'-end is modified with BHQ-1. B. Perform imaging detection on the polymer obtained in step A.

3. The detection method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction according to claim 2, characterized in that: The target mRNAm6A methylation sample to be tested in step A is selected from mRNAm6A methylation sequences or mouse hepatic stellate cells.

4. The detection method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction according to claim 3, characterized in that: When the target mRNA m6A methylation sample to be tested is selected from an mRNA m6A methylation sequence, the step A specifically adopts the following method: a. Incubate the biotin-labeled probe b with streptavidin in PBS buffer at a temperature of 35°C to 38°C. b. Add biotin-labeled anti-rabbit IgG antibody and control the temperature to incubate at 35° C. to 38° C. to obtain probe b attached with anti-rabbit IgG antibody; c. Incubating the obtained anti-rabbit IgG antibody-attached probe b with rabbit monoclonal anti-m6A antibody at a temperature of 35° C. to 38° C. to obtain the anti-rabbit IgG antibody-attached probe b after binding to the rabbit monoclonal anti-m6A antibody; d. Mix the mRNA m6A methylation sequence, probe b attached with anti-rabbit IgG antibody after binding to rabbit monoclonal anti-m6A antibody, probe a, hairpin probe H1, and hairpin probe H2, and incubate at a controlled temperature of 35°C to 38°C for four days.

5. The method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction according to claim 3, characterized in that: When the target mRNA m6A methylation sample to be tested is selected from mouse hepatic stellate cells, step A specifically adopts the following method: Aa. Incubate activated mouse hepatic stellate cells with rabbit monoclonal anti-m6A antibody at 35°C to 38°C for five minutes; Ab, then add anti-rabbit IgG antibody labeled with streptavidin and biotin and incubate at a temperature of 35°C to 38°C; Ac, then add a mixture containing probe a, probe b, hairpin probe H1 and hairpin probe H2 and control the temperature to be incubated at 35°C to 38°C to obtain the corresponding cells after the incubation is completed.

6. The method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction according to claim 2, 3, 4 or 5, characterized in that: The imaging detection in step B comprises the following steps: Dual-channel imaging was performed using a confocal microscope with excitation wavelengths of FAM 488 nm and DAPI 405 nm.

7. The method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction according to claim 4, characterized in that: The time of the first incubation, the second incubation, the third incubation and the fourth incubation are each independently selected from 1 hour to 2 hours.

8. The method for monitoring mRNAm6A methylation in hepatic stellate cells based on hybridization chain reaction according to claim 5, characterized in that: The time of the fifth incubation, the sixth incubation and the seventh incubation are each independently selected from 1 hour to 2 hours.

Images