Traditional Chinese medicine decoction functional miRNAs detection method

Abstract

The invention discloses a method for detecting functional miRNAs in a traditional Chinese medicine decoction. The method comprises the following steps: irradiating target cells with ultraviolet rays; splitting the to-be-detected cells, digesting DNA (Deoxyribose Nucleic Acid) and cutting target mRNA enzyme into a preset size; extracting the miRNA-AGO <-> target mRNA (messenger Ribonucleic Acid) silencing complex; connecting the 5'end of the target mRNA with the 3 'end of the target miRNA, and connecting a 3' end joint to the 3 'end of the miRNA-target mRNA hybrid chain; the invention discloses a method for detecting functional miRNAs in a traditional Chinese medicine decoction, which comprises the following steps of: carrying out phosphorylation on a 5'end of a target miRNA in a miRNA-target mRNA-3'joint nucleic acid chain, connecting the 5 'end of the target miRNA in a phosphorylation product with a 5' end joint, verifying a target mRNA sequence, and outputting a detection result. The technical detection result is real and reliable, and the method can be used for detecting functional miRNAs in the traditional Chinese medicine decoction.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a method for detecting functional miRNAs in traditional Chinese medicine decoctions. Background Art

[0002] miRNAs are a class of single-stranded non-coding small RNAs of about 22 nucleotides in length in cells. They can be loaded onto Argonaute proteins, bind to target mRNAs through the principle of base complementary pairing to form a silencing complex, regulate the translation of target mRNA proteins, and play an important role in post-transcriptional regulation of genes.

[0003] Cross-border gene regulation refers to plant miRNAs entering the animal body and regulating its protein translation by acting on animal mRNAs. In the prior art, the cross-border gene regulation of plant miRNAs has been discovered. Rice MIR-168a can withstand cooking and gastrointestinal digestion, be absorbed into the blood and into the liver tissue of mice, and target the inhibition of low-density lipoprotein receptor adapter protein 1 mRNA translation. Since then, the cross-border gene regulation of plant miRNAs has become a new research hotspot.

[0004] Plant miRNAs in Chinese herbal decoctions can be absorbed into the blood by gastrointestinal cells. The absorption mechanism includes two types: "endocytosis" and "systemic RNA interference defective protein 1 receptor-mediated". MiRNA monomers are mainly absorbed through receptor-mediated absorption, while miRNA encapsulated in extracellular microvesicles is mainly absorbed through endocytosis; after being absorbed, both forms of plant miRNAs are mainly present in the circulating blood in the form of extracellular microvesicles. Plant miRNAs in Chinese herbal decoctions have strong acid and temperature stability and can withstand the metabolic effects of RNA enzymes in the liver and blood, which is related to the following factors: ① The protective effect of extracellular microvesicles (such as exosomes); during the decoction process of Chinese herbal decoctions, plant fat can be promoted to form stable exosome-like microvesicles, which account for 0.5%~2.5% of the Chinese herbal decoctions, and a large number of plant miRNAs are enriched in these microvesicles; ② The protective effect of RNA binding proteins; ③ Methylation of the 3' end of plant miRNAs; ④ Some specific structures, such as GC-rich sequences.

[0005] Following the discovery of the cross-border gene regulation function of plant miRNAs in daily diet, in the existing technology, it was also found that honeysuckle MIR-2911 can withstand high-temperature boiling and stably exist in honeysuckle decoction, can be absorbed into the blood through the digestive tract, and reach the lung tissue of mice, inhibiting viral replication by acting on the mRNA of influenza A viruses such as H1N1, H5N1, and H7N9. By designing two miRNAs (MIR-471 and MIR-519) targeting hepatitis B virus surface antigen mRNA (HBsAg), they were transferred into the lettuce genome through transgenic technology, so that lettuce highly expressed MIR-471 and MIR-519 during growth. Subsequently, lettuce leaves were boiled into decoction and fed to hepatitis B model mice. The results showed that MIR-471 and MIR-519 were highly expressed in lettuce, stably exist in lettuce decoction, and absorbed by the digestive tract into the mouse liver cells to be tested, inhibiting HBsAg expression. A total of 20,758,257 small RNAs (sRNAs) were detected from 245 traditional Chinese medicine decoctions, of which 21,757 were consistent with known plant miRNA sequences, and a herbal sRNA library was constructed. Further in vitro experiments revealed that among the 213 sRNAs, 201 could regulate the expression of cytokines.

[0006] In addition to entering the body through the plant miRNAs contained in it to exert cross-border gene regulation, Chinese herbal decoctions can also exert gene regulation by differentially regulating the body's miRNAs. For example, it was found that Ma Xing Huo Qiao decoction can differentially regulate 15 miRNAs in mice, and then regulate 40 target mRNA translation proteins, achieving the effect of improving mouse pulmonary fibrosis.

[0007] Chinese herbal decoctions contain a large number of plant miRNAs, some of which can enter the animal body and regulate animal gene translation by acting on mRNAs, which may be one of the medicinal ingredients of Chinese herbal decoctions. In addition, Chinese herbal decoctions can cause animals to differentially express miRNAs, thereby playing a gene regulatory role. Here, these two types of miRNAs differentially regulated by Chinese herbal decoctions are collectively referred to as functional miRNAs in Chinese herbal decoctions.

[0008] At the same time, there may be multiple functional miRNAs in Chinese herbal decoctions, and not all plant miRNAs are functional miRNAs. For plant miRNAs or miRNAs differentially regulated by Chinese herbal decoctions to become functional miRNAs, they need to overcome multiple influencing factors, including gastrointestinal absorption selectivity, miRNA expression, AGO protein selectivity, base complementarity, target accessibility, double-strand stability, etc. The factors are described as follows: ① Absorption selectivity refers to the selective absorption of plant miRNAs by the gastrointestinal tract of the body. Plant miRNAs must be absorbed by the gastrointestinal tract to become functional miRNAs. ② miRNA expression level Usually, only plant miRNAs with higher expression levels in Chinese herbal decoctions are likely to become functional miRNAs. The reason is that the absorption rate of plant miRNAs in the gastrointestinal tract is low, only 0.04~4.5%, and in the target cells, miRNAs must reach more than 1000 copies / cell to effectively regulate the translation of target mRNA. ③AGO protein selectivity AGO protein is selective for loaded miRNAs, and only miRNAs that can be loaded onto AGO protein can become functional miRNAs. ④Base complementarity refers to the fact that miRNAs loaded onto AGO protein must be complementary paired with their target mRNA to play a cross-border gene regulatory role. ⑤Target site accessibility A large number of secondary structures can be formed within the mRNA itself through base complementary pairing. If the secondary structure is formed at the target site of the miRNA on the mRNA, it can block the binding of the miRNA to the mRNA and prevent it from playing a cross-border gene regulatory role, which is the so-called target site accessibility. ⑥Double-strand stability refers to the fact that the double-stranded structure formed by the miRNA and mRNA through base complementary pairing must reach a certain stability before it can finally play a cross-border gene regulatory role; In summary, the existing functional miRNAs detection technology can only detect base complementarity and double-strand stability, but cannot detect other factors, and its detection technology has a high false positive rate. Summary of the invention

[0009] In view of the problems of existing methods for detecting functional miRNAs in traditional Chinese medicine decoctions, the present invention provides a method for detecting functional miRNAs in traditional Chinese medicine decoctions to solve the problems raised in the above background technology.

[0010] A method for detecting functional miRNAs in a traditional Chinese medicine decoction, the method comprising the following steps: S1, obtaining target cells of target receptors after drinking the Chinese herbal medicine decoction to be detected for a preset period of time, and irradiating the target cells with ultraviolet rays to obtain cells to be detected; S2, using a cell lysis solution to lyse the cells to be detected, using DNase to digest the DNA in the cells to be detected, and using RNase to enzymatically cut the target mRNA in the cells to be detected to a preset size, so as to obtain a pre-treated sample; S3, using an AGO protein antibody to adsorb the AGO protein in the pre-treated sample to extract the miRNA-AGO-target mRNA silencing complex from the pre-treated sample; S4, connecting the 5' end of the target mRNA of the miRNA-AGO-target mRNA silencing complex with the 3' end of the target miRNA to obtain a head-to-tail miRNA-target mRNA hybrid chain, and connecting the 3' end adapter to the 3' end of the miRNA-target mRNA hybrid chain to obtain a miRNA-target mRNA-3' adapter nucleic acid chain; S5, phosphorylating the 5' end of the target miRNA in the miRNA-target mRNA-3' linker nucleic acid chain to obtain a phosphorylated product, and connecting the 5' end of the target miRNA in the phosphorylated product to the 5' end linker to obtain a 5' linker-miRNA-target mRNA-3' linker nucleic acid chain; S6. Extract the 5' linker-miRNA-target mRNA-3' linker nucleic acid chain and sequence it to obtain the target miRNA sequence and the target mRNA sequence. Verify the target mRNA sequence based on the verification tool and the target miRNA sequence. If the verification is successful, compare the target mRNA sequence with the gene in the cell source based on the comparison tool to output the detection result.

[0011] According to the method for detecting functional miRNAs in traditional Chinese medicine decoctions proposed by the present invention, the beneficial effects of the present invention are: Using the "bound miRNA-target mRNA capture sequencing technology", functional miRNAs in traditional Chinese medicine decoctions can be detected. The detection results of this technology are true and reliable, reflecting the actual situation that the functional miRNAs in traditional Chinese medicine decoctions overcome the influencing factors such as gastrointestinal absorption selectivity (for plant miRNAs contained in traditional Chinese medicine decoctions), miRNA expression requirements, AGO protein selectivity, base complementarity, target site accessibility, double-strand stability, etc., enter the target cells to be detected, and finally act on the target mRNAs. Therefore, the detection results of the "bound miRNA-target mRNA capture sequencing technology" are very accurate and can be used to detect functional miRNAs in traditional Chinese medicine decoctions.

[0012] In addition, the method for detecting functional miRNAs in traditional Chinese medicine decoctions provided by the present invention may also have the following additional technical features: Preferably, step S1 is specifically as follows: The target cells were suspended, counted, and a certain number of target cells were selected. The cells were centrifuged at 900 rpm for 5 min and the supernatant was removed. Cold 1× Dulbecco's phosphate buffer was added to resuspend the cells and placed in a cross-linking instrument for UV irradiation cross-linking. Liquid nitrogen was added every 2 min to maintain the low temperature and the cells were mixed with a pipette every 4 min. The cross-linked products were transferred to a centrifuge tube and centrifuged at 4°C and 1400 rpm for 20 min. The supernatant was removed to obtain the cells to be tested.

[0013] Preferably, step S2 is specifically as follows: Take a complete lysis buffer and add dithiothreitol and a protease inhibitor to mix to obtain a cell lysis solution, take the cells to be tested and use the cell lysis solution to lyse and resuspend them and incubate them on ice, then add RQ1 DNA enzyme to remove DNA contamination, incubate at 37°C and 1000 rpm for 5 minutes to obtain an incubation product; T1 shearing enzyme with a final concentration of 0.0002 U / μL was added to the incubation product, incubated at 22°C for 15 min, and then RNase inhibitor was added for cutting, centrifuged at 4°C and 28000×g for 15 min, and then the supernatant was transferred and the precipitate was discarded to obtain a pretreated sample.

[0014] Preferably, step S3 is specifically as follows: Take a 1.5 mL LoBind tube and add protein G magnetic beads, centrifuge at 4000rpm for 2min, remove the supernatant, add cold 1×PBS-T buffer to repeatedly wash the magnetic beads and blow the magnetic beads, then add 1×PBS-T and rabbit anti-mouse IgG to resuspend the magnetic beads, rotate at room temperature with a flip mixer for 50min, centrifuge at 775×g for 1min and remove the supernatant, then add cold 1×PBS-T buffer to repeatedly wash the magnetic beads and add AGO protein antibody to resuspend the magnetic beads, rotate at 4°C for 8h, then wash repeatedly with cold 1×PXL and resuspend in 1×PXL to obtain a magnetic bead sample; The pre-treated sample was added to the magnetic bead sample and rotated overnight at 4°C to allow antigen-antibody specific binding, after which the supernatant was transferred to an enzyme-free sterile 1.5 mL EP tube and repeatedly washed with several pre-cooled buffers to obtain the miRNA-AGO-target mRNA silencing complex.

[0015] Preferably, step S4 is specifically as follows: The washed miRNA-AGO-target mRNA silencing complex was centrifuged at 775×g for 1 min, the supernatant was removed, the phosphorylation mixture was added, and the target mRNA 5' end phosphorylated miRNA-AGO-target mRNA silencing complex was obtained; the target mRNA 5' end phosphorylated miRNA-AGO-target mRNA silencing complex was repeatedly washed with 1×PNK, and then the ligation mixture was added, and then the first T4 RNA ligase was added to resuspend, and the mixture was rotated at 16°C for 8h, and then adenosine triphosphate and the first T4 RNA ligase were added again for re-ligation, and the mixture was rotated at 16°C for 8h to obtain a head-to-tail miRNA-target mRNA hybrid chain; The head-to-tail connected miRNA-target mRNA hybrid chains were repeatedly washed with 1×PNK, and then a dephosphorylation mixture was added and the mixture was placed in a metal bath at 37°C for 30 minutes to obtain a dephosphorylated product. A 3'-adapter ligation mixture was added to the dephosphorylated product, and the mixture was placed in a metal bath at 16°C for 8 hours and repeatedly washed with 1×PNK; Then add elution buffer and incubate at 22℃ and 1400rpm for 15min; centrifuge at 775×g for 1min, and transfer the supernatant to a new tube; repeat once, and add the supernatant obtained from the previous step to the supernatant of the previous step to obtain the miRNA-target mRNA-3' adapter nucleic acid chain in the silencing complex; add 1×PK buffer to the miRNA-target mRNA-3' adapter nucleic acid chain in the silencing complex, place in a metal bath at 37℃ for 20min, add DEPC water and RNA extraction reagent, incubate at room temperature at 1400rpm in a constant temperature mixer for 8min, then centrifuge at 18000×g and 4℃ for 10min, remove the water layer and transfer it to a LoBind tube, then add sodium acetate, glycerol blue and a 1:1 anhydrous ethanol / isopropanol mixture, and incubate at -20℃ overnight to obtain the miRNA-target mRNA-3' adapter nucleic acid chain.

[0016] Preferably, the phosphorylation mixture includes DEPC water, 10×PNK buffer, adenosine triphosphate, RNase inhibitor, and T4 PNK enzyme; the ligation mixture includes DEPC water, a first T4RNA ligase buffer, PEG 8000, potassium chloride, adenosine triphosphate, and RNase inhibitor; the dephosphorylation mixture includes DEPC water, 10×alkaline phosphatase buffer, alkaline phosphatase, and RNase inhibitor; the 3' linker ligation mixture includes DEPC water, 10×second T4RNA ligase buffer, PEG 8000, RNase inhibitor, 3' linker, and second T4RNA ligase; the elution buffer includes sodium bicarbonate, sodium dodecyl sulfate, and DEPC water; the 1×PK buffer includes 5xPK buffer, proteinase K, and DEPC water.

[0017] Preferably, step S5 is specifically as follows: Take the miRNA-target mRNA-3' adapter nucleic acid chain, centrifuge at 21000×g, 4°C for 30 min and remove part of the supernatant, wash with cold 80% ethanol, then centrifuge at 18000×g, 4°C for 10 min and remove part of the supernatant, add DEPC water after air drying and incubate on ice for 15 min, then add miRNA 5' end phosphorylation mixture and place in metal bath at 37°C for 30 min to phosphorylate the 5' end of the target miRNA in the miRNA-target mRNA-3' adapter nucleic acid chain; preparing a miRNA 5' end ligation mixture, adding the miRNA 5' end ligation mixture to the miRNA-target mRNA-3' adapter nucleic acid chain after the 5' end phosphorylation of the target miRNA, and placing in a metal bath at 16° C. for 8 hours to obtain a ligation product; DEPC water and RNA extraction reagent were added to the ligation product, incubated at 1400 rpm for 8 min, and then centrifuged at 18000×g and 4°C for 10 min. The aqueous layer was removed and transferred to a LoBind tube, and then sodium acetate, glycerol blue and a 1:1 mixture of anhydrous ethanol / isopropanol were added and incubated at -20°C overnight to obtain a 5' adapter-miRNA-target mRNA-3' adapter nucleic acid chain.

[0018] Preferably, the miRNA 5' end phosphorylation mixture includes T4 PNK buffer, RNase inhibitor, adenosine triphosphate, and T4 PNK enzyme; the miRNA 5' end ligation mixture includes 10x first T4 RNA ligase, bovine serum albumin, adenosine triphosphate, 5' linker, and first T4 RNA ligase.

[0019] Preferably, the step of extracting the 5' linker-miRNA-target mRNA-3' linker nucleic acid chain and sequencing it to obtain the target miRNA sequence and the target mRNA sequence comprises: The 5' adapter-miRNA-target mRNA-3' adapter nucleic acid chain was centrifuged at 21000×g and 4°C for 30 min and washed repeatedly with cold 80% ethanol. After each washing, the nucleic acid chain was centrifuged at 18000×g and 4°C for 10 min, and then the supernatant was removed and air-dried. After air-drying, the nucleic acid chain was resuspended in DEPC water and RT primers and deoxynucleotide mixture were added. The nucleic acid chain was incubated at 65°C for 5 min in a PCR instrument, and then cooled on ice and centrifuged briefly. Add the RT mixture and perform reverse transcription according to the preset program. After completion, a reverse transcription product is obtained. Add the PCR mixture and the corresponding index primer to the reverse transcription product. Preheat the PCR thermal cycler to 98°C and place it in the PCR thermal cycler for thermal cycling according to the preset thermal cycling program. After completion, perform agarose gel electrophoresis, use an ultraviolet transilluminator to perform gel excision, and use the Qiaquick Gel Extraction Kit to perform gel recovery to purify the DNA obtained by reverse transcription and PCR. The purified DNA is sent for sequencing to obtain the target miRNA sequence and the target mRNA sequence.

[0020] Preferably, the RT mixture includes 5× SuperScript reverse transcriptase buffer, dithiothreitol, RNase inhibitor, and SuperScript reverse transcriptase; and the PCR mixture includes 2× PCR premix, RP1 primer, and DEPC water.

[0021] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 A pairing diagram of miRNA and target mRNA provided in an embodiment of the present invention; Figure 2 Results of human experiment, rat experiment and cell experiment provided by the embodiments of the present invention; Figure 3 A graph showing the experimental results of embryonic lung fibroblasts infected with 229E coronavirus and intervention with Qingfei Paidu Decoction provided in an embodiment of the present invention; Figure 4 A complementary pairing diagram of miR-214 and target mRNA sequences provided in an embodiment of the present invention.

[0024] The embodiments of the present invention will be further described below with reference to the accompanying drawings. DETAILED DESCRIPTION

[0025] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the examples. Several embodiments of the present invention are given in the examples. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0026] Example 1 In Example 1 of the present invention, a method for detecting functional miRNAs in a traditional Chinese medicine decoction is provided, the method comprising the following steps: S1, obtaining target cells of target receptors after drinking the Chinese herbal medicine decoction to be detected for a preset period of time, and irradiating the target cells with ultraviolet rays to obtain cells to be detected; Wherein, step S1 is specifically as follows: The target cells were suspended and counted, and a certain number of target cells were selected, centrifuged at 900 rpm for 5 min, and the supernatant was removed. Cold 1× Dulbecco's phosphate buffer was added to resuspend the cells, and the cells were placed in a UV crosslinker for UV irradiation crosslinking. Liquid nitrogen was added every 2 min to maintain the low temperature and the cells were mixed with a pipette every 4 min. The crosslinked products were transferred to a centrifuge tube and centrifuged at 1400 rpm at 4°C for 20 min. The supernatant was removed to obtain the cells to be tested; Specifically, first select the target cells of each tissue, count the cell suspension of each group, and take 10 5 The cells were centrifuged at 900 rpm for 5 min, the supernatant was removed, 3 mL of cold 1× Dulbecco's phosphate buffer was added to resuspend the cells, the cells were transferred to a culture dish, and the cells were crosslinked in a UV instrument at 400 mJ / cm 2 UV irradiation cross-linking was performed for 16 minutes, and liquid nitrogen was added once every 2 minutes to maintain low-temperature cross-linking. Every 4 minutes, the sample was mixed with a pipette and the culture dish was rotated 180 degrees and then put back to continue cross-linking. After the cross-linking was completed, the sample was transferred to a 15 mL centrifuge tube, and the culture dish was rinsed with 1 mL of cold 1× Dulbecco's phosphate buffer. This was repeated 3 times, and the sample was transferred to the above 15 mL centrifuge tube. The sample was centrifuged at 1400 rpm at 4°C for 20 minutes. After the supernatant was removed, it was stored at -80°C to obtain the cells to be tested. The purpose of using ultraviolet irradiation is to firmly bind AGO protein, miRNA, and target mRNA.

[0027] S2, using a cell lysis solution to lyse the cells to be detected, using DNase to digest the DNA in the cells to be detected, and using RNase to enzymatically cut the target mRNA in the cells to be detected to a preset size, so as to obtain a pre-treated sample; Wherein, step S2 is specifically as follows: Take a complete lysis buffer and add dithiothreitol and a protease inhibitor to mix to obtain a cell lysis solution, take the cells to be tested and resuspend them with the cell lysis solution and incubate them on ice, then add RQ1 DNA enzyme to remove DNA contamination, incubate at 37°C and 1000 rpm for 5 minutes to obtain an incubation product; T1 shearing enzyme with a final concentration of 0.0002 U / μL was added to the incubation product, incubated at 22°C for 15 min, and then RNase inhibitor was added for cutting, centrifuged at 4°C and 28000×g for 15 min, and then the supernatant was transferred and the precipitate was discarded to obtain a pretreated sample.

[0028] Specifically, first take 3 mL of complete lysis buffer, add 15 μL of dithiothreitol (Invitrogen, 18090010) and 120 μL of protease inhibitor (Roche, 11836170001) and mix well, take out the cells to be tested from the -80°C refrigerator and thaw on ice, resuspend each group with 500 μL of complete lysis buffer (containing dithiothreitol and protease inhibitors), and incubate on ice for 15 minutes; add 30 μL of RQ1 DNA enzyme (Promega, M610A) to each tube to remove DNA contamination, incubate at 37°C, 1000 rpm, and incubate for 5 minutes to obtain the incubation product, then add 1 μL of T1 shear enzyme (Thermo Fisher, EN0541) with a final concentration of 0.0002U / μL to each group of incubation products, and incubate at 22°C for 15 minutes; then, add 7 μL The enzyme digestion was terminated with RNase inhibitor (Promega, N2615), and the tube was centrifuged at 28,000 × g for 15 min at 4°C. The supernatant was transferred to a new tube and the precipitate was discarded to obtain the pre-treated sample.

[0029] S3, using an AGO protein antibody to adsorb the AGO protein in the pre-treated sample to extract the miRNA-AGO-target mRNA silencing complex from the pre-treated sample; Wherein, step S3 is specifically as follows: Take a 1.5 mL LoBind tube and add protein G magnetic beads, centrifuge at 4000rpm for 2min, remove the supernatant, add cold 1×PBS-T buffer to wash the magnetic beads repeatedly and blow the magnetic beads, then add 1×PBS-T and rabbit anti-mouse IgG to resuspend the magnetic beads, rotate 360° for 50min at room temperature with a flip mixer, centrifuge at 775×g for 1min and remove the supernatant, then add cold 1×PBS-T buffer to wash the magnetic beads repeatedly and add AGO protein antibody to resuspend the magnetic beads, rotate 360° at 4°C for 8h, then wash repeatedly with cold 1×PXL and resuspend in 1×PXL to obtain a magnetic bead sample; The pre-treated sample was added to the magnetic bead sample and rotated overnight at 4°C to allow antigen-antibody specific binding, after which the supernatant was transferred to an enzyme-free sterile 1.5 mL EP tube and repeatedly washed with several pre-cooled buffers to obtain the miRNA-AGO-target mRNA silencing complex.

[0030] Specifically, prepare the magnetic beads first, pipette 800 μL protein G magnetic beads (Invitrogen, 10004D) into a 1.5 mL LoBind tube, centrifuge at 4000 rpm for 2 min, and discard the supernatant; wash the magnetic beads 3 times with 1200 μL cold 1× PBS-T buffer, pH 7.2, and gently pipette to resuspend the magnetic beads (each wash requires centrifugation at 4000 rpm for 2 min, and then discard the supernatant, and all washing steps are performed in this way throughout the experiment); add 611 μL 1× PBS-T buffer, pH 7.2, and 56 μL rabbit anti-mouse IgG (Jackson, 315-005-008) to resuspend the magnetic beads; rotate 360° for 50 min at room temperature using a flip mixer, centrifuge at 775 × g for 1 min, discard the supernatant, and wash 3 times with 1200 μL cold 1× PBS-T, pH 7.2; add 653 μL 1× PBS-T buffer, pH 7.2 and 13.3 μL AGO protein antibody (Millipore, MABE56), resuspend the magnetic beads; rotate 360° at 4°C for 8 hours, wash 4 times with 1200 μL cold 1×PXL; resuspend in 820 μL 1×PXL to make 4 200 μL magnetic bead samples, take out the pre-treated samples, aspirate them into the magnetic beads to resuspend the magnetic beads, rotate at 4°C overnight, that is, antigen-antibody specific binding, to obtain miRNA-AGO-target mRNA silencing complex; At the same time, after obtaining the miRNA-AGO-target mRNA silencing complex, it needs to be washed. The washing process is as follows: first take out the sample, centrifuge it at 775×g for 1min, transfer the supernatant to an enzyme-free sterile 1.5mL EP tube, and store it at -80°C. Wash 3 times with each of the following pre-cooled buffers (950 μL each time): 1×PXL, 5×PXL, high-stringency buffer, high-salt buffer, and 1×PNK; among them, the second and third washes with high-stringency buffer and high-salt buffer need to be incubated on ice for 1min, and then centrifuged at 775×g for 1min to remove the supernatant.

[0031] S4, connecting the 5' end of the target mRNA of the miRNA-AGO-target mRNA silencing complex with the 3' end of the target miRNA to obtain a head-to-tail miRNA-target mRNA hybrid chain, and connecting the 3' end adapter to the 3' end of the miRNA-target mRNA hybrid chain to obtain a miRNA-target mRNA-3' adapter nucleic acid chain; Wherein, step S4 is specifically as follows: The washed miRNA-AGO-target mRNA silencing complex was centrifuged at 775×g for 1 min, the supernatant was removed, the phosphorylation mixture was added, and the target mRNA 5' end phosphorylated miRNA-AGO-target mRNA silencing complex was obtained; the target mRNA 5' end phosphorylated miRNA-AGO-target mRNA silencing complex was repeatedly washed with 1×PNK, and then the ligation mixture was added, and then the first T4 RNA ligase was added to resuspend, and the mixture was rotated at 16°C for 8h, and then adenosine triphosphate and the first T4 RNA ligase were added again for re-ligation, and the mixture was rotated at 16°C for 8h to obtain a head-to-tail miRNA-target mRNA hybrid chain; The head-to-tail connected miRNA-target mRNA hybrid chain was repeatedly washed with 1×PNK, and then a dephosphorylation mixture was added and the mixture was placed in a metal bath at 37°C for 30 minutes to obtain a dephosphorylated product. A 3'-adapter ligation mixture was added to the dephosphorylated product, and the mixture was placed in a metal bath at 16°C for 8 hours, and the mixture was repeatedly washed with 1×PNK; Then add elution buffer and incubate at 1400rpm at room temperature for 15min; centrifuge at 775×g for 1min, and transfer the supernatant to a new tube; repeat once, and add the supernatant obtained from the previous step to the supernatant of the previous step to obtain the miRNA-target mRNA-3' adapter nucleic acid chain in the silencing complex; add 1×PK buffer to the miRNA-target mRNA-3' adapter nucleic acid chain in the silencing complex, place in a metal bath at 37°C for 20min, add DEPC water and RNA extraction reagent, incubate at 1400rpm at room temperature in a constant temperature mixer for 8min, then centrifuge at 18000×g and 4°C for 10min, remove the water layer and transfer it to a LoBind tube, then add sodium acetate, glycerol blue and a 1:1 anhydrous ethanol / isopropanol mixture, and incubate at -20°C overnight to obtain the miRNA-target mRNA-3' adapter nucleic acid chain.

[0032] Specifically, the washed miRNA-AGO-target mRNA silencing complex was centrifuged at 775×g for 1 min, the supernatant was removed, 80 μL of phosphorylation mixture was added, and the mixture was placed in a metal bath at 37°C for 30 min, and rotated at 800 rpm for 5 seconds every 6 min to obtain a miRNA-AGO-target mRNA silencing complex with phosphorylation at the 5' end of the target mRNA; the silencing complex was washed 3 times with 950 μL 1×PNK, left in the final washing buffer, and incubated on ice until the ligation mixture was ready; the mixture was centrifuged at 775×g for 1 min, the supernatant was removed, 450 μL of ligation mixture was added for resuspending, and 50 μL of the first T4 RNA ligase (New England, M0204L) was added, and after rotating at 16°C for 8 h, 0.25 μL of adenine nucleoside triphosphate and 25 μL of the above silencing complex were added again. First, T4 RNA ligase (New England, M0204L) was used for ligation and rotation was performed at 16°C for 8 h to obtain the end-to-end connected miRNA-target mRNA hybrid chain; Specifically, the end-to-end linked miRNA-target mRNA hybrid chain was centrifuged at 775×g for 1 min, the supernatant was removed, and the mixture was washed three times with 950 μL 1×PNK, and then 80 μL of dephosphorylation mixture was added and placed in a metal bath at 37°C for 30 min to obtain a dephosphorylated product; the dephosphorylated product was washed twice with 950 μL 1×PNK-EGTA, incubated on ice for 1 min during the second wash, and then centrifuged at 775×g for 1 min, the supernatant was removed, and the mixture was washed three times with 950 μL 1×PNK, and 80 μL of 3'-adapter connection mixture was added, and placed in a metal bath at 16°C for 8 h, and the thermostatic mixer program was set to "16°C, 0 rpm, 8 min and 16°C, 1000 rpm, 1 min" for continuous circulation, and after 4 h, it was gently rotated at 800 rpm for 5 seconds to mix once, and the sample was taken out after the 8 h incubation, and 950 μL Wash 3 times with 1× PNK; place samples in the last wash buffer and keep on ice until the elution buffer is prepared; Specifically, 100 μL of elution buffer was then added to the silencing complex, and the mixture was incubated at 22°C and 1400 rpm for 15 min; the mixture was centrifuged at 775 × g for 1 min, and the supernatant was transferred to a new tube; the mixture was repeated once, and the supernatant was added to the supernatant of the previous step to obtain 200 μL of miRNA-target mRNA-3' linker nucleic acid chain in the silencing complex, and 10 μL was transferred to a new enzyme-free sterile tube, marked, and stored at -80°C; 50 μL of 1×PK buffer was added to the miRNA-target mRNA-3' linker nucleic acid chain in the silencing complex, and the mixture was placed in a metal bath at 37°C for 20 min, and 250 μL of RNA extraction reagent was added, and the mixture was incubated at 22°C and 1400 rpm for 8 min; the mixture was centrifuged at 18000 × g, 4°C for 10 min; about 200 μL of the upper aqueous layer was taken out to a new test tube, and 20 μL of sodium acetate was added to each tube; 2.5 μL of glycerol blue and 500 μL of a 1:1 mixture of anhydrous ethanol / isopropanol at -20°C overnight to precipitate the miRNA-target mRNA-3' adapter nucleic acid chain, thereby obtaining the miRNA-target mRNA-3' adapter nucleic acid chain; Wherein, the phosphorylation mixture includes DEPC water, 10×PNK buffer, adenosine triphosphate, RNase inhibitor, and T4 PNK enzyme; the ligation mixture includes DEPC water, a first T4RNA ligase buffer, PEG 8000, potassium chloride, adenosine triphosphate, and RNase inhibitor; the dephosphorylation mixture includes DEPC water, 10×alkaline phosphatase buffer, alkaline phosphatase, and RNase inhibitor; the 3' linker ligation mixture includes DEPC water, 10×second T4RNA ligase buffer, PEG 8000, RNase inhibitor, 3'linker, and second T4RNA ligase; the elution buffer includes sodium bicarbonate, sodium dodecyl sulfate, and DEPC water; the 1×PK buffer includes 5x PK buffer, proteinase K, and DEPC water; Specifically, for the 360 ​​μL phosphorylation mixture, it includes 293.4 μL of DEPC water, 36 μL of 10× PNK buffer (New England, M0201S), 3.6 μL of 100 mM adenosine triphosphate (Thermo Fisher, R0041), 9 μL of RNase inhibitor, and 18 μL of T4 PNK enzyme (New England, M0201S); for the 2025 μL ligation mixture, it includes 1445.6 μL of DEPC water, 225 μL of the first T4 RNA ligase (New England, M0204L) buffer, 270 μL of PEG 8000 (50% w / v) (New England, M0204L), 5.6 μL of potassium chloride (4 M) (Sigma, P9541), 22.5 μL of adenosine triphosphate, and 56.3 μL of RNase inhibitor. 1 μL; for 360 μL of dephosphorylation mixture, it included 301.5 μL of DEPC water, 36 μL of 10× alkaline phosphatase buffer, 13.5 μL of alkaline phosphatase, and 9 μL of RNase inhibitor; for 360 μL of 3' adapter ligation mixture, it included 189 μL of DEPC water, 36 μL of 10× second T4 RNA ligase (New England, M0351L) buffer, 72 μL of PEG 8000 (50% w / v) (New England, M0351L), 9 μL of RNase inhibitor, 3' adapter (10 μM) 36 μL, and second T4 RNA ligase (New England, M0351L) 180 μL; for 929 μL of elution buffer, it included 186 μL of 500 mM sodium bicarbonate (Sigma, S6014), sodium dodecyl sulfate (10%, w / v) (Invitrogen, 24730020) 93μL, DEPC water 650μL; for 225μL of 1×PK buffer, it includes 5x PK buffer 45μL, proteinase K (Roche, 03115887001) 45μL, DEPC water 135μL.

[0033] S5, phosphorylating the 5' end of the target miRNA in the miRNA-target mRNA-3' linker nucleic acid chain to obtain a phosphorylated product, and connecting the 5' end of the target miRNA in the phosphorylated product to the 5' end linker to obtain a 5' linker-miRNA-target mRNA-3' linker nucleic acid chain; Wherein, step S5 is specifically as follows: Take the miRNA-target mRNA-3' adapter nucleic acid chain, centrifuge at 21000×g, 4°C for 30 min and remove part of the supernatant, wash with cold 80% ethanol, then centrifuge at 18000×g, 4°C for 10 min and remove part of the supernatant, add DEPC water after air drying and incubate on ice for 15 min, then add miRNA 5' end phosphorylation mixture and place in metal bath at 37°C for 30 min to phosphorylate the 5' end of the target miRNA in the miRNA-target mRNA-3' adapter nucleic acid chain; preparing a miRNA 5' end ligation mixture, adding the miRNA 5' end ligation mixture to the miRNA-target mRNA-3' adapter nucleic acid chain after the 5' end phosphorylation of the target miRNA, and placing in a metal bath at 16° C. for 8 hours to obtain a ligation product; DEPC water and RNA extraction reagent were added to the ligation product, incubated at 1400 rpm for 8 min, then centrifuged at 18000×g and 4°C for 10 min, the water layer was removed and transferred to a LoBind tube, and then sodium acetate, glycerol blue and a 1:1 mixture of anhydrous ethanol / isopropanol were added, and incubated at -20°C overnight to obtain a 5' adapter-miRNA-target mRNA-3' adapter nucleic acid chain; Specifically, the sample was taken out from the -20°C refrigerator, centrifuged at 21,000×g, 4°C for 30 min; part of the supernatant was removed, leaving an appropriate amount of supernatant (based on not sucking out the blue precipitate solid), washed once with 900 μL of cold 80% ethanol, and centrifuged at 18,000×g, 4°C for 10 min; part of the supernatant was removed, leaving an appropriate amount of supernatant (based on not sucking out the blue precipitate solid), washed once with 200 μL of cold 80% ethanol, and centrifuged at 18,000×g, 4°C for 10 min; the supernatant was removed, based on not sucking out the blue precipitate solid, and air-dried for about 15 min; 10.5 μL of DEPC water was added, incubated on ice for 15 min, and then 4.5 μL of miRNA 5' end phosphorylation mixture was added and placed in a metal bath at 37°C for 30 min; then 5 μL of miRNA was added The 5' end connection mixture was placed in a metal bath at 16°C for 8h, and the 5' end of the miRNA in the phosphorylated product was connected to the 5' end adapter; 200 μL DEPC water and 250 μL RNA extraction reagent were added; incubated at 1400rpm for 8 min at room temperature in a constant temperature mixer; centrifuged at 4°C, 18,000×g, 4°C for 10 min; about 200 μL of the water layer was taken out and transferred to a new 1.5 mL LoBind tube, 20 μL sodium acetate; 2.5 μL glycerol blue and 500 μL of a 1:1 mixture of anhydrous ethanol / isopropanol were placed at -20°C overnight to precipitate the 5' adapter-miRNA-target mRNA-3' adapter nucleic acid chain, thereby obtaining the 5' adapter-miRNA-target mRNA-3' adapter nucleic acid chain.

[0034] Specifically, the miRNA 5' end phosphorylation mixture includes T4 PNK buffer, RNase inhibitor, adenosine triphosphate, and T4 PNK enzyme; the miRNA 5' end ligation mixture includes 10x first T4 RNA ligase, bovine serum albumin, adenosine triphosphate, 5' linker, and first T4 RNA ligase; Among them, for 20.25 μL of phosphorylation mixture, it includes 6.75 μL of T4 PNK buffer, 2.25 μL of RNase inhibitor, 6.75 μL of 10 mM ATP (New England, M0204L), and 4.5 μL of T4 PNK enzyme; for 22.5 μL of ligation mixture, it includes 4.5 μL of 10x first T4 RNA ligase (New England, M0204L), 9 μL of bovine serum albumin (1 mg / mL) (New England, B9000S), 2.25 μL of 10 mM ATP, 4.5 μL of 5' linker (50 μM), and 2.25 μL of first T4 RNA ligase (New England, M0204L) (10 U / μL).

[0035] S6, extracting the 5' linker-miRNA-target mRNA-3' linker nucleic acid chain and sequencing it to obtain a target miRNA sequence and a target mRNA sequence, verifying the target mRNA sequence based on a verification tool and the target miRNA sequence, and if the verification is successful, comparing the target mRNA sequence with the gene in the cell source based on a comparison tool to output a detection result; The step of extracting the 5' linker-miRNA-target mRNA-3' linker nucleic acid chain and sequencing it to obtain the target miRNA sequence and the target mRNA sequence includes: The 5' linker-miRNA-target mRNA-3' linker nucleic acid chain was centrifuged at 21000×g and 4°C for 30 min and washed repeatedly with cold 80% ethanol. After each washing, the solution was centrifuged at 18000×g and 4°C for 10 min, and then the supernatant was removed and air-dried. After air-drying, the solution was resuspended in DEPC water and RT primer and deoxynucleotide mixture were added. The solution was incubated at 65°C for 5 min in a PCR instrument, and then cooled on ice and centrifuged briefly. Add the RT mixture and perform reverse transcription according to the preset program. After completion, a reverse transcription product is obtained. A PCR mixture and a corresponding index primer are added to the reverse transcription product. The PCR thermal cycler is preheated to 98° C. and placed in the PCR thermal cycler for thermal cycling according to the preset thermal cycle program. After completion, agarose gel electrophoresis is performed, and gel excision is performed using an ultraviolet transilluminator and gel recovery is performed using a Qiaquick Gel Extraction Kit to purify the DNA obtained by reverse transcription. The purified DNA is sent for detection sequencing to obtain the target miRNA sequence and the target mRNA sequence; Among them, the preset program is "incubation at 50℃ for 45min, incubation at 55℃ for 15min, incubation at 95℃ for 5min, and 4℃ ∞", and the preset thermal cycle program is "98℃ for 30 seconds once; 98℃ for 10 seconds, 52℃ for 30 seconds, and 72℃ for 30 seconds for 34 cycles; 72℃ for 5min once; 4℃ ∞", and the corresponding index primers include RPI1, RPI2, RPI3, and RPI4. After agarose gel electrophoresis, the sample bands are observed, and a gel of about 130bp to 350bp in size is cut out under a UV transilluminator. The Qiaquick GelExtraction Kit is used for gel recovery to purify the DNA, and then it is sent for detection sequencing. After sequencing, verification is first required. The target miRNA sequence is input into the BiBiServ2 online analysis platform for the target mRNA sequence to analyze whether the two exist. Figure 1If the complementary pairing pattern shown is consistent, it can be verified that the target mRNA in the above hybrid chain is the target mRNA of the functional miRNA of the traditional Chinese medicine decoction, which can be used for subsequent annotation analysis; after that, the target mRNA sequence is input into NCBI, compared with the gene of the cell source species, and the biological information such as the target mRNA name, function, gene ID, etc. is annotated to output the detection result.

[0036] Specifically, the RT mixture includes 5× SuperScript reverse transcriptase buffer, dithiothreitol, RNase inhibitor, and SuperScript reverse transcriptase; the PCR mixture includes 2× PCR premix, RP1 primer, and DEPC water; For 31.5 μL of RT mixture, it includes 18 μL of 5× SuperScript reverse transcriptase buffer (Invitrogen, 18090010), 4.5 μL of dithiothreitol (Invitrogen, 18090010), 4.5 μL of RNase inhibitor (40 U / μL), 4.5 μL of SuperScript reverse transcriptase (200 U / μL) (Invitrogen, 18090010); for 81 μL of PCR mixture, it includes 45 μL of 2× PCR premix (New England, M0531S), 4.5 μL of RP1 (10 μM), and 27 μL of DEPC water; It should be noted that the purified DNA is specifically reverse transcribed from the target miRNA and the target mRNA corresponding to the target miRNA, so the target miRNA sequence and the target mRNA sequence can be obtained by detecting the DNA sequence and based on the complementary relationship.

[0037] In order to further verify the effect of the present invention, the present invention uses a recommended prescription for preventing and treating coronavirus infection - Qingfei Paidu Decoction as an example for illustration, wherein Qingfei Paidu Decoction includes the following components: ephedra 9g, licorice root 6g, apricot kernel 9g, gypsum 30g, cinnamon twig 9g, oriental rhizome 9g, umbellate 9g, atractylodes 9g, poria 15g, bupleurum 16g, scutellaria 6g, ginger pinellia 9g, ginger 9g, aster 9g, winter flower 9g, beam dry 9g, asarum 6g, yam 12g, immature bitter orange 6g, tangerine peel 6g, patchouli 9g; First, through high-throughput sequencing, a total of 586,176 small RNAs containing 16 to 30 nucleotides were detected from Qingfei Paidu Decoction, of which only 701 could be identified as known miRNAs, accounting for only 0.12% of the total number of detections (701 / 586,176*100%). Most of them could not be identified due to incomplete information in the plant gene library. This result verifies the rationality of the present invention to use small RNAs containing 16 to 30 nucleotides as the miRNAs expression profile in traditional Chinese medicine decoctions; Among the many functional miRNAs in Qingfei Paidu Decoction, there is one miRNA, named miR-214, which can act on the coronavirus replication-related gene, namely the phosphoglycerate kinase 1 gene (PGK1). Therefore, in this example, a series of experiments were conducted on miR-214: Afterwards, the present invention conducted a corresponding rat experiment. In the rat experiment, 20 female SD rats were randomly divided into two groups, the Qingfei Paidu Decoction group (n=9) and the control group (n=11). The experimental group mice were gavaged with warm Qingfei Paidu Decoction at a dose of 10.5 mg / g, once in the morning and once in the evening, and the control group was simultaneously gavaged with warm boiled water; rat plasma was collected on day 12, and the content of Qingfei Paidu Decoction miRNA in the plasma of the two groups was detected and compared by high-throughput sequencing. The results showed that the content of miR-214 in the plasma of the Qingfei Paidu Decoction group was significantly higher than that of the control group ( P <0.05), such as Figure 2 B in In order to verify the above results, the present invention conducted a population experiment. The population study used healthy adult women as the research subjects, and adopted a repeated measurement design research program. After obtaining ethical approval, 3 female college students were recruited as volunteers. After signing the informed consent form, 180 ml of Qingfei Paidu Decoction was taken at warm temperature. 5 minutes before oral administration, 0.5 and 1 hour after oral administration, EDTA anticoagulant tubes were used to collect peripheral blood of volunteers, and the supernatant was centrifuged to obtain plasma samples at different time points before and after oral administration. High-throughput sequencing technology was used to detect the changes in the content of Qingfei Paidu Decoction miRNA in the plasma of volunteers 5 minutes before oral administration, 0.5 and 1 hour after oral administration, and the miRNAs of Qingfei Paidu Decoction were analyzed according to the following standards: The first category meets all of the following conditions: (1) contained in the miRNA expression profile of Qingfei Paidu Decoction; (2) not present in the peripheral venous blood of volunteers before oral administration (i.e., 0 h); and (3) contained in the peripheral blood of volunteers after oral administration.

[0038] The second category meets all of the following conditions: (1) contained in the miRNA expression profile of Qingfei Paidu Decoction; (2) present in the peripheral venous blood of volunteers before and after oral administration, but the content after oral administration was significantly higher than that before oral administration.

[0039] The third category meets all of the following conditions: (1) not present in Qingfei Paidu Decoction; (2) with significantly increased or decreased levels in the peripheral venous blood of volunteers before and after oral administration; and (3) known or predicted human miRNAs.

[0040] In the miRNA expression profile of Qingfei Paidu Decoction (i.e., the 586,176 small RNAs found above), 462 were classified as Qingfei Paidu Decoction functional miRNAs, of which 210 were in the first category and 252 were in the second category. In addition, 198 human miRNAs were found to be classified as the third category of Qingfei Paidu Decoction functional miRNAs. Therefore, a total of 660 small RNAs were found to be possible functional miRNAs of Qingfei Paidu Decoction; The distribution of miR-214 in the plasma of the volunteers was then analyzed. The results showed that after oral administration, the content of miR-214 in the plasma of the volunteers showed a trend of gradual increase over time, and at 0.5 hours after oral administration, the content in plasma was statistically different from that before oral administration (P<0.05). Figure 2 A in Furthermore, an experiment of human embryonic lung fibroblasts infected with 229E coronavirus and Qingfei Paidu Decoction intervention was implemented. The experiment set up a normal control group, an infection model group and a decoction group, with 3 replicates in each group; before virus infection, Qingfei Paidu Decoction was added to the cell culture fluid of the decoction group to make its final concentration of 24 mg / ml, and DMEM was used instead of Qingfei Paidu Decoction, and the normal cell group and the infection model group were operated simultaneously; after 24 hours of culture, the cells in the infection model group and the decoction group were transfected with 229E coronavirus at MOI=0.1, and the cell culture flask was shaken every 15 minutes to make the virus and cells evenly contact, and the transfection was carried out for 2 hours; then, the virus solution was aspirated and discarded, and the cell maintenance solution was added, and the virus solution was replaced by DMEM, and the normal control group was operated simultaneously; it was placed at 5% The cells were cultured continuously under CO2 and 37°C for 48 hours. During this period, the virus culture fluid was collected at 0, 6, 12, 24, and 48 hours after infection. The concentration of viral nucleic acid in the virus culture fluid of each group was detected and compared by reverse transcription-fluorescence quantitative PCR (RT-qPCR) to analyze the antiviral effect of Qingfei Paidu Decoction in vitro. At the same time, RT-qPCR was used to detect and compare the expression levels of miR-214 in each group to analyze the effect of Qingfei Paidu Decoction on the expression level of miR-214. The results showed that Qingfei Paidu Decoction could significantly inhibit the proliferation of 229E coronavirus in cells (such as Figure 2 C in the figure verified the antiviral effect of Qingfei Paidu Decoction; and the expression level of miR-214 in the decoction group was significantly higher than that in the infection model group (such as Figure 2 D in the figure verified that Qingfei Paidu Decoction can significantly increase the expression level of miR-214 in the body.

[0041] Then, in this example, human embryonic lung fibroblasts were infected with 229E coronavirus and Qingfei Paidu Decoction was used for intervention. The experiment set up a negative control group, an infection model group, a miR-214 group, and a miR-NC group, with 3 replicates in each group. The details are as follows: (1) Human embryonic lung fibroblasts were cultured in T25 culture flasks; (2) Prepare intervention solution of appropriate concentration and start transfection when the cells grow to 70-80%. The transfection treatment factors of each group are shown in Table 1: Table 1 Comparison of transfection treatments in each group

[0042] (3) 24 hours after transfection, the transfection solution in the culture bottle was discarded, and 229E coronavirus solution was added to the culture bottle at a multiplicity of infection of 0.1, i.e., virus inoculation. During the inoculation period, the culture bottle was shaken every 15 minutes to keep the virus solution evenly distributed in the culture bottle. The inoculation treatments of each group are shown in Table 2 below: Table 2 Comparison of the inoculation treatments in each group

[0043] (4) 2 hours after infection, discard the virus solution in the culture bottle, add cell maintenance medium, and proliferate the virus under 5% CO2 and 37°C conditions. After 48 hours, collect the virus culture medium and cells for subsequent testing; (5) RT-qPCR was used to detect the concentration of viral nucleic acid in the virus culture medium, and human embryonic lung fibroblasts were used to detect the virus titer in the culture medium, expressed as the half-pathogenic dose (TCID50), to analyze the antiviral effect of Qingfei Paidu Decoction on differentially regulating miR-214; Note: Both indicators were converted to log2 values ​​for statistical analysis.

[0044] (6) Western Blotting was used to detect the expression level of intracellular PGK1 protein and to evaluate the inhibitory effect of Qingfei Paidu Decoction on intracellular PGK1 by differentially regulating miR-214.

[0045] The results showed that compared with the infection model group, the virus titer and viral nucleic acid concentration in the miR-214 group were significantly reduced (P < 0.05), while the virus titer and viral nucleic acid concentration in the miR-NC group were not statistically different from those in the infection model group (P > 0.05). Figure 3 A in Figure 3 B in the figure). The results show that miR-214 can significantly inhibit the proliferation of 229E coronavirus, and the inhibitory effect excludes the influence of cell stress on nucleic acid chain. Note: The normal control group is a negative control group, and no viral nucleic acid and titer were detected, so it is not shown in the figure.

[0046] Western Blotting showed that Figure 3 As shown in Figure C, the expression level of PGK1 protein in the cells of the infection model group and the miR-NC group was significantly higher than that of the normal control group (P < 0.05), which indicates that viral infection can increase the expression level of PGK1 protein in cells, because the replication of coronavirus requires the assistance of PGK1 protein; Figure 3 As shown in D, the expression level of PGK1 protein in the miR-214 group was significantly lower than that in the other three groups (P < 0.05), indicating that miR-214 can target the PGK1 gene and inhibit its protein translation, verifying the detection results of the "bound miRNA-target mRNA capture sequencing technology".

[0047] At the same time, the present invention conducts specific target mRNA verification for miR-214, as follows: First, the miR-214 sequence and the target mRNA sequence differentially regulated by Qingfei Paidu Decoction in the above hybrid chain are input into the BiBiServ2 online analysis platform, and the complementary pairing is shown in FIG4 , and the complementary pairing binding energy is -29.3 kcal / mol, Figure 4 It can be seen that the differentially regulated miR-214 of Qingfei Paidu Decoction forms a good complementary pairing with the target mRNA sequence, which is consistent with the classical mode of action of miRNA (as shown in Figure 1). It verifies that the target mRNA in the above hybrid chain is the target mRNA of the differentially regulated miR-214 of Qingfei Paidu Decoction, and subsequent annotation analysis can be performed; Secondly, the target mRNA sequence of "Qingfei Paidu Decoction Differentially Regulated miR-214-Target mRNA Hybrid Chain" was entered into NCBI and compared with the human RNA library. The results showed that the target mRNA sequence was derived from the 937-964 sequence of human phosphoglycerate kinase 1 (PGK1), and the PGK1 gene number was NM_000291.4. Overexpression of this gene is conducive to the replication of coronavirus.

[0048] In summary, the present invention utilizes the "bound miRNA-target mRNA capture and sequencing technology" to detect functional miRNAs in traditional Chinese medicine decoctions. The detection results of this technology are true and reliable, reflecting the actual situation that the functional miRNAs in traditional Chinese medicine decoctions overcome the influencing factors such as gastrointestinal absorption selectivity (for plant miRNAs contained in traditional Chinese medicine decoctions), miRNA expression requirements, AGO protein selectivity, base complementarity, target site accessibility, double-strand stability, etc., enter the target cells to be detected, and finally act on the target mRNAs. Therefore, the detection results of the "bound miRNA-target mRNA capture and sequencing technology" are very accurate and can be used to detect functional miRNAs in traditional Chinese medicine decoctions.

[0049] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0050] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting functional miRNAs in traditional Chinese medicine decoctions, characterized in that: The method comprises the following steps: S1, obtaining target cells of target receptors after drinking the Chinese herbal medicine decoction to be detected for a preset period of time, and irradiating the target cells with ultraviolet rays to obtain cells to be detected; S2, using a cell lysis solution to lyse the cells to be detected, using DNase to digest the DNA in the cells to be detected, and using RNase to enzymatically cut the target mRNA in the cells to be detected to a preset size, so as to obtain a pre-treated sample; S3, using an AGO protein antibody to adsorb the AGO protein in the pre-treated sample to extract the miRNA-AGO-target mRNA silencing complex from the pre-treated sample; S4, connecting the 5' end of the target mRNA of the miRNA-AGO-target mRNA silencing complex with the 3' end of the target miRNA to obtain a head-to-tail miRNA-target mRNA hybrid chain, and connecting the 3' end adapter to the 3' end of the miRNA-target mRNA hybrid chain to obtain a miRNA-target mRNA-3' adapter nucleic acid chain; S5, phosphorylating the 5' end of the target miRNA in the miRNA-target mRNA-3' linker nucleic acid chain to obtain a phosphorylated product, and connecting the 5' end of the target miRNA in the phosphorylated product to the 5' end linker to obtain a 5' linker-miRNA-target mRNA-3' linker nucleic acid chain; S6. Extract the 5' linker-miRNA-target mRNA-3' linker nucleic acid chain and sequence it to obtain the target miRNA sequence and the target mRNA sequence. Verify the target mRNA sequence based on the verification tool and the target miRNA sequence. If the verification is successful, compare the target mRNA sequence with the gene in the cell source based on the comparison tool to output the detection result.

2. The method for detecting functional miRNAs in traditional Chinese medicine decoctions according to claim 1, characterized in that: Step S1 is specifically as follows: The target cells were suspended, counted, and a certain number of target cells were selected. The cells were centrifuged at 900 rpm for 5 min and the supernatant was removed. Cold 1× Dulbecco's phosphate buffer was added to resuspend the cells and placed in a cross-linking instrument for UV irradiation cross-linking. Liquid nitrogen was added every 2 min to maintain the low temperature and the cells were mixed with a pipette every 4 min. The cross-linked products were transferred to a centrifuge tube and centrifuged at 4°C and 1400 rpm for 20 min. The supernatant was removed to obtain the cells to be tested.

3. The method for detecting functional miRNAs in traditional Chinese medicine decoctions according to claim 1, characterized in that: Step S2 is specifically as follows: Take a complete lysis buffer and add dithiothreitol and a protease inhibitor to mix to obtain a cell lysis solution, take the cells to be tested and use the cell lysis solution to lyse and resuspend them and incubate them on ice, then add RQ1 DNA enzyme to remove DNA contamination, incubate at 37°C and 1000 rpm for 5 minutes to obtain an incubation product; T1 shearing enzyme with a final concentration of 0.0002 U / μL was added to the incubation product, incubated at 22°C for 15 min, and then RNase inhibitor was added for cutting, centrifuged at 4°C and 28000×g for 15 min, and then the supernatant was transferred and the precipitate was discarded to obtain a pretreated sample.

4. The method for detecting functional miRNAs in traditional Chinese medicine decoctions according to claim 1, characterized in that: Step S3 is specifically as follows: Take a 1.5 mL LoBind tube and add protein G magnetic beads, centrifuge at 4000rpm for 2min, remove the supernatant, add cold 1×PBS-T buffer to repeatedly wash the magnetic beads and blow the magnetic beads, then add 1×PBS-T and rabbit anti-mouse IgG to resuspend the magnetic beads, rotate at room temperature with a flip mixer for 50min, centrifuge at 775×g for 1min and remove the supernatant, then add cold 1×PBS-T buffer to repeatedly wash the magnetic beads and add AGO protein antibody to resuspend the magnetic beads, rotate at 4°C for 8h, then wash repeatedly with cold 1×PXL and resuspend in 1×PXL to obtain a magnetic bead sample; The pre-treated sample was added to the magnetic bead sample and rotated overnight at 4°C to allow antigen-antibody specific binding, after which the supernatant was transferred to an enzyme-free sterile 1.5 mL EP tube and repeatedly washed with several pre-cooled buffers to obtain the miRNA-AGO-target mRNA silencing complex.

5. The method for detecting functional miRNAs in traditional Chinese medicine decoction according to claim 1, characterized in that: Step S4 is specifically as follows: The washed miRNA-AGO-target mRNA silencing complex was centrifuged at 775×g for 1 min, the supernatant was removed, the phosphorylation mixture was added, and the target mRNA 5' end phosphorylated miRNA-AGO-target mRNA silencing complex was obtained; the target mRNA 5' end phosphorylated miRNA-AGO-target mRNA silencing complex was repeatedly washed with 1×PNK, and then the ligation mixture was added, and then the first T4 RNA ligase was added to resuspend, and the mixture was rotated at 16°C for 8h, and then adenosine triphosphate and the first T4 RNA ligase were added again for re-ligation, and the mixture was rotated at 16°C for 8h to obtain a head-to-tail miRNA-target mRNA hybrid chain; The head-to-tail connected miRNA-target mRNA hybrid chain was repeatedly washed with 1×PNK, and then a dephosphorylation mixture was added and the mixture was placed in a metal bath at 37°C for 30 minutes to obtain a dephosphorylated product. A 3'-adapter ligation mixture was added to the dephosphorylated product, and the mixture was placed in a metal bath at 16°C for 8 hours, and the mixture was repeatedly washed with 1×PNK; Then add elution buffer and incubate at 1400rpm at room temperature for 15min; centrifuge at 775×g for 1min, and transfer the supernatant to a new tube; repeat once, and add the supernatant obtained from the previous step to the supernatant of the previous step to obtain the miRNA-target mRNA-3' adapter nucleic acid chain in the silencing complex; add 1×PK buffer to the miRNA-target mRNA-3' adapter nucleic acid chain in the silencing complex, place in a metal bath at 37°C for 20min, add DEPC water and RNA extraction reagent, incubate at 1400rpm at room temperature in a constant temperature mixer for 8min, then centrifuge at 18000×g and 4°C for 10min, remove the water layer and transfer it to a LoBind tube, then add sodium acetate, glycerol blue and a 1:1 anhydrous ethanol / isopropanol mixture, and incubate at -20°C overnight to obtain the miRNA-target mRNA-3' adapter nucleic acid chain.

6. The method for detecting functional miRNAs in traditional Chinese medicine decoctions according to claim 5, characterized in that: The phosphorylation mixture includes DEPC water, 10×PNK buffer, adenosine triphosphate, RNase inhibitor, and T4 PNK enzyme; the ligation mixture includes DEPC water, a first T4RNA ligase buffer, PEG 8000, potassium chloride, adenosine triphosphate, and RNase inhibitor; the dephosphorylation mixture includes DEPC water, 10×alkaline phosphatase buffer, alkaline phosphatase, and RNase inhibitor; the 3' linker ligation mixture includes DEPC water, 10×second T4RNA ligase buffer, PEG 8000, RNase inhibitor, 3' linker, and second T4RNA ligase; the elution buffer includes sodium bicarbonate, sodium dodecyl sulfate, and DEPC water; the 1×PK buffer includes 5x PK buffer, proteinase K, and DEPC water.

7. The method for detecting functional miRNAs in traditional Chinese medicine decoctions according to claim 1, characterized in that: Step S5 is specifically as follows: Take the miRNA-target mRNA-3' adapter nucleic acid chain, centrifuge at 21000×g, 4°C for 30 min and remove part of the supernatant, wash with cold 80% ethanol, then centrifuge at 18000×g, 4°C for 10 min and remove part of the supernatant, add DEPC water after air drying and incubate on ice for 15 min, then add miRNA 5' end phosphorylation mixture and place in metal bath at 37°C for 30 min to phosphorylate the 5' end of the target miRNA in the miRNA-target mRNA-3' adapter nucleic acid chain; preparing a miRNA 5' end ligation mixture, adding the miRNA 5' end ligation mixture to the miRNA-target mRNA-3' adapter nucleic acid chain after the 5' end phosphorylation of the target miRNA, and placing in a metal bath at 16° C. for 8 hours to obtain a ligation product; DEPC water and RNA extraction reagent were added to the ligation product, incubated at 1400 rpm for 8 min, and then centrifuged at 18000×g and 4°C for 10 min. The aqueous layer was removed and transferred to a LoBind tube, and then sodium acetate, glycerol blue and a 1:1 mixture of anhydrous ethanol / isopropanol were added and incubated at -20°C overnight to obtain a 5' adapter-miRNA-target mRNA-3' adapter nucleic acid chain.

8. The method for detecting functional miRNAs in traditional Chinese medicine decoctions according to claim 7, characterized in that: The miRNA 5' end phosphorylation mixture includes T4 PNK buffer, RNase inhibitor, adenosine triphosphate, and T4 PNK enzyme; the miRNA 5' end ligation mixture includes 10x first T4 RNA ligase, bovine serum albumin, adenosine triphosphate, 5' linker, and first T4 RNA ligase.

9. The method for detecting functional miRNAs in traditional Chinese medicine decoctions according to claim 1, characterized in that: The step of extracting the 5' linker-miRNA-target mRNA-3' linker nucleic acid chain and sequencing it to obtain the target miRNA sequence and the target mRNA sequence comprises: The 5' adapter-miRNA-target mRNA-3' adapter nucleic acid chain was centrifuged at 21000×g and 4°C for 30 min and washed repeatedly with cold 80% ethanol. After each washing, the nucleic acid chain was centrifuged at 18000×g and 4°C for 10 min, and then the supernatant was removed and air-dried. After air-drying, the nucleic acid chain was resuspended in DEPC water and RT primers and deoxynucleotide mixture were added. The nucleic acid chain was incubated at 65°C for 5 min in a PCR instrument, and then cooled on ice and centrifuged briefly. Add the RT mixture and perform reverse transcription according to the preset program. After completion, a reverse transcription product is obtained. Add the PCR mixture and the corresponding index primer to the reverse transcription product. Preheat the PCR thermal cycler to 98°C and place it in the PCR thermal cycler for thermal cycling according to the preset thermal cycling program. After completion, perform agarose gel electrophoresis, use an ultraviolet transilluminator to perform gel excision, and use the Qiaquick Gel Extraction Kit to perform gel recovery to purify the DNA obtained by reverse transcription and PCR. The purified DNA is sent for sequencing to obtain the target miRNA sequence and the target mRNA sequence.

10. The method for detecting functional miRNAs in traditional Chinese medicine decoctions according to claim 9, characterized in that: The RT mixture includes 5× SuperScript reverse transcriptase buffer, dithiothreitol, RNase inhibitor, and SuperScript reverse transcriptase; the PCR mixture includes 2× PCR premix, RP1 primer, and DEPC water.

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