Traditional Chinese medicine gecko miRNA and application thereof

Abstract

The application discloses a traditional Chinese medicine gecko miRNA and application thereof. The application screens a traditional Chinese medicine gecko miRNA with potential anti-tumor effect by mining the miRNA from the traditional Chinese medicine gecko, and names the miRNA as gek-miR-2862, and the nucleotide sequence of the miRNA is shown as SEQ ID NO. 1. Research results show that the gek-miR-2862 can regulate multiple tumor-related signal pathways and inhibit liver cancer cell proliferation. Therefore, the traditional Chinese medicine gecko miRNA provided by the application can become a biological preparation for treating tumors and has potential drug development value, and provides a new solution for tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to a traditional Chinese medicine gecko miRNA and its application. Background Technology

[0002] Geckos, also known as scorpions, dragons, and palace lizards, are traditional insect-based medicinal materials used in traditional Chinese medicine for treating tumors. They are believed to promote blood circulation, remove blood stasis, nourish yin, reduce phlegm, and penetrate the meridians. Their clinical anti-tumor effects are significant. In recent years, numerous studies have demonstrated that geckos possess good broad-spectrum anti-tumor activity. Clinically, geckos have a clear anti-liver cancer effect, which has been confirmed by many studies. However, the effective active ingredients of geckos in anti-tumor treatment are not yet fully understood, limiting their further development for medicinal value. Previous research on gecko active ingredients has focused on gecko proteins and polypeptides. However, natural proteins and polypeptides are easily modified due to the ease with which amino acid residues can be modified, affecting their structural stability and pharmacological activity. Furthermore, proteins have large molecular weights and complex structures, thus posing many challenges to their practical clinical application. Compared to polypeptide drugs, nucleic acid drugs are easier to synthesize, have high specificity, and low drug toxicity, and are expected to make breakthrough progress in treating tumors for which traditional drugs are ineffective.

[0003] MicroRNAs (miRNAs) are a class of highly conserved endogenous non-coding small RNAs, consisting of 18-22 nucleotides, found in eukaryotes. They participate in regulating various complex pathophysiological processes in the human body by binding complementary to the 3' untranslated region (3-UTR) of target gene mRNAs. Currently, miRNAs have been found to affect cell cycle, proliferation, apoptosis, angiogenesis, epithelial-mesenchymal transition, and invasion in tumor cells, making them important factors in tumor development and progression. Related studies have shown that miRNAs combined with chemotherapy can improve the therapeutic effect of chemotherapeutic drugs, and combined with radiotherapy can enhance radiosensitivity. In tumor immunotherapy, they can enhance the recognition and killing function of adoptive T cells. Recent studies have found that miRNAs extracted from traditional Chinese medicine can enter mammals with good bioavailability and biological activity, and can regulate corresponding animal target genes across species to exert anti-tumor effects. However, domestic and international research on the cross-species regulation of miRNAs from traditional Chinese medicine mainly focuses on plant-based drugs, while there are currently no research reports on miRNAs from the insect-based traditional Chinese medicine gecko, and research on their anti-tumor effects remains lacking. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of this invention is to provide a traditional Chinese medicine gecko miRNA and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of this invention provides a traditional Chinese medicine gecko miRNA, named gek-miR-2862 according to the miRNA naming principle, wherein gek in gek-miR-2862 is an abbreviation of the Latin word Gekko for gecko, and miR represents miRNA. The nucleotide sequence of gek-miR-2862 (SEQ ID NO.1) is 5'-CGGGGAGGUGGAGCCUGGG-3', wherein 5'-GGGGAGG-3' is the seed region sequence of gek-miR-2862.

[0007] Preferably, the miRNA from the Chinese herbal gecko is obtained by small RNA high-throughput sequencing mining and analysis of the Chinese herbal gecko, followed by screening.

[0008] In a second aspect of the invention, the use of the above-mentioned traditional Chinese medicine gecko miRNA in the preparation of a medicament for treating tumors is provided.

[0009] Preferably, in the above-mentioned uses, the traditional Chinese medicine gecko miRNA can regulate tumor-related signaling pathways.

[0010] A third aspect of the present invention provides the use of the above-mentioned traditional Chinese medicine gecko miRNA in the preparation of a drug for inhibiting tumor proliferation.

[0011] Preferably, in the above-mentioned uses, the traditional Chinese medicine gecko miRNA can inhibit the proliferation of liver cancer cells HepG2 and Huh7.

[0012] In this invention, a tumor refers to a new growth formed when local tissue cells lose normal regulation of their growth at the gene level under the influence of various carcinogenic factors, leading to their clonal abnormal proliferation. Here, the tumor type primarily refers to liver cancer.

[0013] Preferably, the miRNA from the Chinese herbal medicine gecko is a modified miRNA from the Chinese herbal medicine gecko. The modification methods include, but are not limited to, glycosylation modification, methylation modification, hydrocarbon modification, and nucleic acid modification. Among them, the glycosylation modification can be 2-methoxy-glycosylation modification, hydrocarbon-glycosylation modification, glycan ring modification, etc.

[0014] The present invention has the following beneficial effects:

[0015] This invention identifies and sequences miRNAs from the traditional Chinese medicine gecko, and then screens them for potential anti-tumor activity. The resulting miRNA is named gek-miR-2862, and its nucleotide sequence is shown in SEQ ID NO. 1. The results indicate that gek-miR-2862 can regulate multiple tumor-related signaling pathways and inhibit the proliferation of liver cancer cells. Therefore, the gecko miRNA provided by this invention may serve as a biological agent for the treatment of liver cancer, possessing potential drug development value and offering a new solution for liver cancer treatment. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A flowchart illustrating the process of constructing a cDNA library;

[0018] Figure 2 A graph showing the statistical results of the length distribution of sRNA fragments from gecko slices;

[0019] Figure 3 The diagram shows the secondary structure of the precursor and the mature sequence of gek-miR-2862;

[0020] Figure 4 This is a comparison chart showing the differential expression of gek-miR-2862 in different samples;

[0021] Figure 5 Figure showing the results of KEGG enrichment pathway analysis for the target gene gek-miR-2862;

[0022] Figure 6A Figure showing the effect of gek-miR-2862 on the proliferation of HepG2 liver cancer cells;

[0023] Figure 6B The figure shows the effect of gek-miR-2862 on the proliferation of Huh7 liver cancer cells. Detailed Implementation

[0024] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention may be implemented in other embodiments without these specific details.

[0025] Example 1

[0026] This embodiment provides a method for mining miRNAs in the traditional Chinese medicine gecko using small RNA high-throughput sequencing.

[0027] 1. Experimental Materials and Methods

[0028] 1.1 Reagents and Materials

[0029] The geckos were purchased from the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine (manufacturer: Hunan Renshang Zhengyuan Chinese Herbal Pieces Co., Ltd., batch number: 2020070104, specification: 5g). Trizol reagent is a single-phase mixture of guanidine thiocyanate and phenol, which can effectively dissolve RNA.

[0030] 1.2 Experimental Methods

[0031] 1.2.1 Extraction of total RNA from geckos:

[0032] (1) Sample preparation: Gecko slices were ground with liquid nitrogen. After being completely ground, 100mg was weighed with an electronic balance, 1ml of Trizol lysis buffer was added, and homogenized with a homogenizer. The mixture was placed at 15-30℃ for 5min to completely separate the nucleic acid-protein complex.

[0033] (2) Phase separation: Add 200 μl of chloroform to the mixture, cap the tube, shake vigorously for 15 seconds to obtain a mixture, place at room temperature for 3 minutes, and centrifuge. At this time, the mixture is separated into a red lower layer (i.e., phenol-chloroform phase), an intermediate phase, and an upper colorless aqueous phase (RNA is present in this colorless aqueous phase).

[0034] (3) RNA precipitation: Transfer the aqueous phase containing RNA to a new tube and add isopropanol (0.5 ml isopropanol per 1 ml of Trizol reagent used for initial homogenization). Incubate the sample at room temperature (15-25°C) for 10 min, then centrifuge at 12,000 g (≈13,000 rpm) for 10 min at 4°C. RNA precipitation, which is usually not visible before centrifugation, forms a gel-like precipitate at the bottom or sides of the tube.

[0035] (4) RNA washing: Discard the supernatant and wash the RNA precipitate with 75% ethanol (use at least 1 mL of 75% ethanol per mL of Trizol reagent for initial homogenization), vortex mix, and centrifuge at 12,000 g (≈13,000 rpm) for 5 min at 4 °C.

[0036] (5) RNA reconstitution: Discard the supernatant, air dry at room temperature for 10 min, add 40 μl of enzyme-free water to dissolve the RNA, and incubate in a metal bath at 60°C for 10 min;

[0037] (6) Use an enzyme-linked immunosorbent assay (ELISA) reader to determine the concentration and purity of the extracted RNA: Extract RNA (concentration greater than 100 ng / ul, 260 / 280 ratio between 1.8 and 2.0) for subsequent experiments and store at -80℃ to prevent degradation.

[0038] 1.2.2 Sequencing of total RNA from geckos:

[0039] (1) Document building: The document building process is as follows Figure 1 As shown.

[0040] The specific method is as follows: The TruSeq Small RNA Library Prep Kit is used to construct a library. Taking advantage of the special structure of the 3' and 5' ends of small RNA (the 5' end has a complete phosphate group and the 3' end has a hydroxyl group), 3 μg of total RNA is used as the starting sample. 3' adapters are added to both ends of the small RNA first, followed by 5' adapters. Then, cDNA is synthesized by reverse transcription. Subsequently, the target DNA fragments are separated by PCR amplification and PAGE gel electrophoresis, and the cDNA library is obtained by gel excision and recovery.

[0041] (2) Library testing: After the library is constructed, the library is first initially quantified using Qubit2.0 and diluted to 1 ng / L. Then, the insert size of the library is detected using a highly sensitive Agilent 2100. After the insert size meets the expectations, the effective concentration of the library is accurately quantified using the Q-PCR method (effective concentration of library > 2 nM) to ensure the quality of the library.

[0042] (3) Sequencing: After the libraries pass the library inspection, different libraries are pooled according to the effective concentration and the target amount of sequencing data before being sequenced using Illumina SE50. A sequencing-by-synthesis method is used, simultaneously adding DNA polymerase, adapter primers, and four types of dNTPs with base-specific fluorescent labels to the reaction system. The 3-OH groups of these four dNTPs are chemically protected, so only one dNTP can be added at a time. After the dNTPs are added to the synthetic strand, all unused free dNTPs and DNA polymerase are washed away. Next, the buffer required for fluorescence excitation is added, and the fluorescence signal is excited by a laser, with optical equipment recording the fluorescence signal. Finally, computer analysis is used to convert the optical signal into sequencing bases. After the fluorescence signal is recorded, chemical reagents are added to quench the fluorescence signal and remove the 3-OH protecting groups of the dNTPs, so that the next round of sequencing reaction can proceed. Illumina's sequencing technology, which adds only one dNTP at a time, effectively solves the problem of accurate measurement of homopolymer length, ultimately obtaining the total length of the gecko. The raw RNA sequencing data; steps (1)-(3) above were completed by Beijing Novogene Co., Ltd.

[0043] (4) Sequencing data quality assessment:

[0044] The raw image data files obtained from high-throughput sequencing are converted into sequenced reads through base calling analysis, referred to as Raw Data or Raw Reads. These reads contain sequence information and corresponding sequencing quality information. To ensure the quality of subsequent information analysis, Raw Data needs to be evaluated and processed, specifically including:

[0045] (i) Sequencing Error Rate Distribution Check: The sequencing error rate distribution check is used to detect whether there are base positions with abnormal sequencing error rates within the sequencing length range. Generally, the sequencing error rate for each base position should be below 0.5%. The sequencing error rate e of a base can be expressed by the formula: Qphred = -10 log10(e) The Qphred value is calculated using a model that predicts the probability of errors in base identification during the base recognition process and is known. A simplified correspondence between base recognition and Phred scores in Illumina Casava 1.8 is shown in Table 1 below. If more than 30% of the bases in a sequence have a sequencing error rate less than 99%, the sequence is considered low-quality and needs to be discarded.

[0046] Table 1. Relationship between base recognition and Phred score

[0047]

[0048] (ii) Sequencing data filtering: The following sequences must be removed from the raw reads: ① Reads with a proportion greater than 10% of N (N indicates undetermined base information); ② Reads with 5' adapter contamination; ③ Reads without 3' adapter sequences and insert fragments; ④ PolyA / T / G / C reads (mostly continuous polyA / T / G / C, which may originate from sequencing errors and have low information entropy, can be ignored). The sequences obtained after removing the above sequences are the clean reads.

[0049] (5) Alignment with reference sequence: The length range of animal small RNA (sRNA) is 18–35 nt. Therefore, clean reads within this range were selected first, and then the sRNA was located in the Gekko gecko using bowtie (Langmead et al., 2009) software. The reference genome of *japonicus* (https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCF / 001 / 447 / 785 / GCF_001447785.1_Gekko_japonicus_V1.1 / GCF_001447785.1_Gekko_japonicus_V1.1_genomic.fna.g) and the corresponding annotation file (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&PROG_DEF=blastn&BLAST_SPEC=Assembly&ASSEMBLY_NAME=GCA_001447785.1) were mapped.

[0050] (6) miRNA analysis:

[0051] (i) Analysis of known miRNAs: The reads mapped to the reference sequence are compared with the specified range of sequences in miRBase to obtain detailed information on the sRNAs that match each sample, including the secondary structure of the matched known miRNAs, the sequence, length, and frequency of occurrence of the miRNAs in each sample;

[0052] (ii) Novel miRNA Analysis: For miRNAs whose biological characteristics were not identified, the miREvo (Wen et al., 2012) and mirdeep2 (Friedlander et al., 2011) software was used to predict novel miRNAs. The basic principle of prediction is to extract a reference sequence from a certain length of sRNA, analyze its secondary structure, Dicer restriction site information, energy, and other characteristics to predict novel miRNAs in the sample, and statistically analyze the sequence, length, and frequency of matching sRNAs in each sample, as well as the primal base distribution of miRNAs of different lengths and the primal base distribution of all miRNAs.

[0053] 2. Experimental Results:

[0054] The raw RNA sequences of geckos were obtained using small RNA high-throughput sequencing technology. The specific results of the raw data are shown in Table 2, and the meanings of the terms used are as follows:

[0055] Sample: Sample ID;

[0056] Reads: Counts the number of sequencing sequences in each raw sequencing file, arranged in four-line units.

[0057] Bases: The number of sequencing sequences multiplied by the length of the sequencing sequences, converted to units in gigabytes (G).

[0058] Error rate: refers to the sequencing error rate, calculated using formula 1;

[0059] Q20: The percentage of bases with a Phred value greater than 20 out of the total number of bases;

[0060] Q30: The percentage of bases with a Phred value greater than 30 out of the total number of bases;

[0061] GC content: Calculates the total number of G and C bases as a percentage of the total number of bases.

[0062] Table 2. Quality Analysis of Small RNA Sequencing Data

[0063]

[0064] As shown in Table 2, a total of 10,885,415 raw RNA sequences of geckos were obtained in this embodiment. Within the sequencing length range, the sequencing error rate at each base position was less than 0.05%, the Q20 and Q30 values ​​were greater than 95%, and the number of G and C bases accounted for 55.43% of the total number of bases.

[0065] After filtering the raw data, 20 sequences had an N% > 10% threshold, 25,229 were low-quality sequences, 9,255 had 5' connector contamination, 262,758 had no 3' connector or insert fragments, and 21,375 contained polyA / T / G / C. After removing these sequences, 10,266,778 clean reads were obtained, representing 94.32% of the raw data (see Table 3).

[0066] Table 3: Overview of Raw Data Filtering

[0067]

[0068] Statistical analysis of the length distribution of sRNAs in geckos was performed to obtain the peaks of the length distribution of sRNAs of different lengths, in order to identify the types of small RNAs. The results are shown in [Figure number missing]. Figure 2 .

[0069] Depend on Figure 2 As shown in Table 3, 10,266,778 clean reads were obtained in this embodiment after quality control. After sRNA length screening, the total number of sRNA reads was 5,087,208. Statistical analysis of the length distribution of these sRNAs showed that the length of animal sRNAs is generally between 18-35 nt, while miRNAs are concentrated between 21-22 nt. The high distribution of experimental samples with lengths between 21-22 nt indicates that the sequencing quality of this embodiment is qualified.

[0070] The obtained small RNAs were compared with reference sequences to analyze the distribution of sRNAs on the reference genome. The results are shown in Table 4. A total of 4,369,793 reads matched the reference sequence, accounting for 85.90% of the total small RNA sequences. Among them, the alignment rate with the reference sequence in the same direction was 48.89%, and the alignment rate with the reference sequence in the opposite direction was 37.00%.

[0071] Table 4. Statistics on the Alignment Information between sRNA Sequencing Data and Reference Genome

[0072]

[0073] Reads aligned to the reference sequence were compared with identified miRNA sequences in miRBase to obtain the secondary structure, sequence, and length of known mature miRNAs. For miRNAs not aligned to the database, prediction was performed using the characteristic hairpin structure of their precursors, based on the widely used miRNA prediction software miREvo and mirdeep2, resulting in a new miRNA named gek-miR-2862, whose structure is shown below. Figure 3 As shown.

[0074] Depend on Figure 3 The results showed that gek-miR-2862 can form a stable stem-loop structure similar to miRNA precursors. The mature sequence of gek-miR-2862 is: 5'-CGGGGAGGUGGAGCCUGGG-3' (SEQ ID NO.1), where 5'-GGGGAGG-3' is the seed region sequence of gek-miR-2862.

[0075] Example 2

[0076] Unlike Example 1, we further screened for miRNAs specific to geckos.

[0077] 1. Experimental Materials and Methods

[0078] 1.1 Reagents and Materials

[0079] Human hepatic stellate cell line LX2 and human hepatocellular carcinoma cell lines HepG2 and Huh7 were all purchased from Zhejiang Meisen Cell Technology Co., Ltd.

[0080] 1.2 Experimental Methods

[0081] 1.2.1 Obtaining miRNA expression levels in geckos

[0082] The expression levels of all miRNAs identified from geckos in Example 1 were statistically analyzed, and the expression levels were normalized using TPM (Transcripts Per Million). TPM = read count × 1,000,000 / sum of all miRNA read counts.

[0083] 1.2.2 Detection of the expression level of gek-miR-2862 in gecko and human cells

[0084] (1) Extraction of total RNA from geckos: The steps are the same as in Example 1, and will not be repeated here.

[0085] (2) Cell culture: LX2, HepG2, and Huh7 cells were cultured in a complete medium containing 10% fetal bovine serum, 5% CO2, and 37°C. After 2-3 passages, cells in the logarithmic growth phase were prepared to contain 3×10⁻⁶ cells per milliliter. 5 A cell suspension of 1,000 μl was seeded into a 24-well plate at a rate of 500 μl per well.

[0086] (3) Total RNA extraction from cells:

[0087] Sample preparation: Add 200 μl of Trizol reagent to each well of a 24-well plate and aspirate several times with a pipette to promote cell lysis. Then transfer the sample to a centrifuge tube and incubate at room temperature for 5 min to facilitate complete separation of ribosomes from the homogenized sample.

[0088] Phase separation: Add 40 μl of chloroform, cap the sample, shake vigorously by hand for 15 seconds, and incubate at room temperature for 3 minutes. Centrifuge at 12,000 g for 15 minutes at 4°C. After centrifugation, the mixture is separated into a red lower layer (phenol-chloroform phase), an intermediate phase, and a colorless aqueous upper phase (RNA is present in this aqueous phase).

[0089] RNA precipitation: Transfer the above aqueous phase to a new tube and add isopropanol (0.5 mL isopropanol per 1 mL of Trizol reagent used for initial homogenization) to precipitate RNA from the aqueous phase. Incubate the sample at room temperature for 10 min, then centrifuge at 12,000 g for 10 min at 4 °C. The RNA precipitate is usually not visible before centrifugation, but forms a gel-like precipitate on the sides and bottom of the tube after centrifugation.

[0090] RNA washing: Discard the supernatant and wash the RNA precipitate once with 75% ethanol. Use at least 1 mL of 75% ethanol per mL of Trizol reagent for initial homogenization. Vortex mix and centrifuge at 12,000 g for 5 min at 4 °C.

[0091] RNA reconstitution: Discard the supernatant, air dry at room temperature for 10 min, add 40 μl of enzyme-free water to dissolve, and incubate in a metal bath at 60 °C for 10 min.

[0092] The concentration and purity of the extracted total RNA were determined using an enzyme-linked immunosorbent assay (ELISA) reader: Total RNA (concentration greater than 100 ng / μl, 260 / 280 ratio between 1.8 and 2.0) was extracted for subsequent experiments.

[0093] (4) Real-time quantitative PCR (RT-PCR) was used to detect the expression level of gek-miR-2862.

[0094] Reverse transcription reaction: Bulge-Loop™ miRNA RT Primer is a miRNA-specific reverse transcription primer (Guangzhou Ruibo Biotechnology Co., Ltd.). It must be used with the corresponding target miRNA. The usage method of the internal control primer U6 is the same as that of miRNA.

[0095] gek-miR-2862 primer sequence:

[0096] Forward Primer: 5'-ACACTCCAGCTGGGGGGTCCGAGGTGG-3'(SEQ ID NO.2)

[0097] Reverse Primer:5'-TGGTGTCGTGGAGTCG-3'(SEQ ID NO.3)

[0098] U6 primer sequence:

[0099] Forward Primer: 5'-CGCTTCGGCAGCACATATAC-3'(SEQ ID NO.4)

[0100] Reverse Primer:5'-TTCACGAATTTGGCGTGTCATC-3'(SEQ ID NO.5)

[0101] The experiment was conducted by preparing a 10 μl RT reaction system according to the composition in Table 5 below. After mixing all components, the mixture was instantly centrifuged. The RT reaction program was: 42 °C for 60 min; 70 °C for 10 min.

[0102] Table 5. Composition of the reverse transcription reaction system

[0103]

[0104] Prepare a 20 μl qPCR reaction system according to the composition in Table 6 below, and perform miRNA qPCR. Gently mix the above reaction system (avoid vigorous vortexing). Use the three-step method for detection. The PCR reaction program is set according to Table 7 below, following step 2. -△△ct The relative expression level of gek-miR-2862 was calculated using the formula, and the experiment was repeated three times.

[0105] Table 6. Composition of miRNA qPCR reaction system

[0106]

[0107] Table 7. PCR reaction procedure

[0108]

[0109] 2. Experimental Results:

[0110] Based on the miRNA expression abundance (read counts) and expression level after TPM normalization in geckos, the sequences, read counts, and TPMs of the top 5 miRNAs are listed in Table 8, where nt represents nucleic acid bases.

[0111] Table 8. General information on the top 5 miRNAs expressed in gecko decoction pieces.

[0112]

[0113] As shown in Table 8, gek-miR-2862 had the highest expression level among novel miRNAs and ranked third in expression level among all miRNAs.

[0114] The expression levels of gek-miR-2862 in gecko, human hepatic stellate cell line LX2, and human hepatocellular carcinoma lines HepG2 and Huh7 were detected by real-time quantitative PCR. Results are shown below. Figure 4 .

[0115] Depend on Figure 4The results showed that real-time quantitative PCR detection of gek-miR-2862 expression levels in gecko, human hepatic stellate cell line LX2, and human hepatocellular carcinoma lines HepG2 and Huh7 revealed high expression of gek-miR-2862 in gecko tissues, with a significant difference compared to LX2, HepG2, and Huh7 cells (P<0.01). Conversely, the expression levels of gek-miR-2862 in human LX2, HepG2, and Huh7 cells were extremely low, with no statistically significant difference. This indicates that gek-miR-2862 is a unique miRNA found in geckos.

[0116] Performance testing

[0117] 1. Research on the regulation of multiple tumor-related signaling pathways by gek-miR-2862.

[0118] Experimental Methods: Bioinformatics methods were used to predict the target genes of gek-miR-2862 using online databases miRDB (http: / / mirdb.org / ), RNAhybrid (https: / / bibiserv.cebitec.uni-bielefeld.de / rnahybrid), and miRanda (http: / / www.microrna.org / microrna / home.do). The intersection of the predicted target genes from these three databases was analyzed using R software packages ("org.Hs.eg.db", "clusterprofiler", "enrichplot", "ggplot2", "GOplot", "pathview", "DOSE") to perform GO (Gene Ontology) function and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses on the key intersection target genes. Only items with a p-value < 0.05 were considered statistically significant, and the top 20 KEGG enriched pathways by p-value were selected. Experimental results are shown below. Figure 5 Among them, the red box indicates cancer-related pathways.

[0119] Depend on Figure 5 The results showed that the three databases predicted a total of 986 target genes associated with gek-miR-2862. KEGG enrichment analysis showed that the target genes of gek-miR-2862 were significantly enriched in a variety of cancer-related pathways, such as the MAPK signaling pathway, Ras signaling pathway, Rap1 signaling pathway, and Hedgehog signaling pathway.

[0120] 2. Study on the effect of gek-miR-2862 on the proliferation of liver cancer cells

[0121] 2.1 Experimental Materials and Methods:

[0122] Experimental materials: Human liver cancer cell lines were the same as in Example 2, and the following materials were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.: gek-miR-2862 mimic (mimic, sequence: 5'-CGGGGAGGUGGAGCCUGGG-3'(SEQ ID NO.6), 3'-GCCCCUCCACCUCGGACCC-5'(SEQ ID NO.7)) and gek-miR-2862 negative control (NC, sequence: 5'-UUUGUACUACACAAAAGUACUG-3'(SEQ ID NO.8), 3'-AAACAUGAUGUGUUUUCAUGAC-5'(SEQ ID NO.9)).

[0123] Experimental methods:

[0124] (1) HepG2 and Huh7 liver cancer cells were cultured using the same method as in Example 2. Cells were collected by digestion and centrifugation, and seeded with cell suspension (100 μl / well) in 96-well plates, approximately 8 × 10⁸ cells per well. 3 Cells were mixed using a cross-hatching method to ensure even distribution within the wells, and then incubated in a 5% CO2, 37°C incubator for 24 hours. Transfection was performed once the cell confluence rate in the 96-well plate reached over 60%.

[0125] (2) Negative controls of gek-miR-2862 at concentrations of 50 nM, 100 nM, and 200 nM (NC, sequence: 5'-UUUGUACUACACAAAAGUACUG-3' (SEQ ID NO.8) and 3'-AAACAUGAUGUGUUUUCAUGAC-5' (SEQ ID NO.9)).

[0126] ) and the gek-miR-2862 mimic (mimic, sequence:

[0127] 5'-CGGGGAGGUGGAGCCUGGG-3'(SEQ ID NO.6),

[0128] 3'-GCCCCUCCACCUCGGACCC-5' (SEQ ID NO.7) was transfected into HepG2 and Huh7 liver cancer cells, respectively. The mixture added to each well of a 96-well plate was: 20 μl Opti-MEM + 0.085 μl liposomes + 0.5 μl mimic (the same volume of culture medium should be aspirated before adding). The 96-well plate was gently shaken in a cross-shaped motion and placed in a 5% CO2 incubator at 37°C for incubation.

[0129] (3) Cell proliferation was observed and detected at 24, 48, 72, and 96 hours post-transfection using the CCK-8 assay after transfection. The old culture medium was discarded, and a 9:1 mixture of complete culture medium and CCK-8 (100 μl / well) was added. The 96-well plates were then incubated for 2 hours. The absorbance of each experimental group was measured at 450 nm using a microplate reader, and the cell viability of each group was calculated. Results are shown below. Figure 6A and Figure 6B .

[0130] Cell survival rate calculation formula: Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0131] Wherein, As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution and drug solution); Ac: absorbance of control wells (containing cells, culture medium and CCK-8 solution, but no drug); Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but no cells and drug).

[0132] Depend on Figure 6A and Figure 6B The results showed that at 24, 48, 72, and 96 hours post-transfection, the gek-miR-2862100nM group significantly inhibited the proliferation of Huh7 and HepG2 liver cancer cells (Note: * indicates P<0.05 compared to the NC group, ** indicates P<0.01 compared to the NC group).

[0133] This invention is not limited to the specific embodiments described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this invention.

Claims

1. Use of a nucleotide sequence of: 5'-CGGGGAGGUGGAGCCUGGG-3' (SEQ ID NO. 1) for preparing a medicament for treating liver cancer or inhibiting proliferation of liver cancer cells.

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