Application of miR-486-5p marker in exosome in preparation of hepatocellular carcinoma diagnostic kit

Abstract

The invention discloses a miRNA marker for auxiliary diagnosis of hepatocellular carcinoma, and provides the possibility that miRNA derived from plasma exosome can be used as a biomarker to improve the prediction accuracy of hepatocellular carcinoma, and in addition, hsa-miR-486-5p in the plasma-derived exosome can be used as a predictive factor of hepatocellular carcinoma, so that selection of treatment modes is facilitated. In addition, the mechanism that the hsa-miR-486-5p plays a role in hepatocellular carcinoma cells is also researched through experiments.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a miR-486-5p marker in exosomes in the preparation of a hepatocellular carcinoma diagnostic kit. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Despite great advances in various prevention, screening, diagnosis, and treatment technologies, its morbidity and mortality rates remain high. In 2022, primary hepatocellular carcinoma became the sixth most common cancer and the third most common cause of cancer death worldwide. Although it takes decades for liver disease to develop into hepatocellular carcinoma, the prognosis of hepatocellular carcinoma patients remains poor due to late diagnosis of HCC patients, tumor metastasis, and limitations of treatment options. Therefore, there is an urgent need to gain a more comprehensive understanding of the pathogenesis of HCC at the molecular level in order to find indicators for early diagnosis of HCC and develop effective treatments.

[0003] Autophagy is a process in which a part of the material in the cell is encapsulated by autophagosomes and transported to lysosomes for degradation. Dysfunction in the autophagy process is associated with many diseases, including cancer. Studies have found that the expression of autophagy-related genes in HCC is decreased, and autophagy defects can lead to more aggressive cancer cell phenotypes and poor prognosis of HCC. Studies have shown that AFP can inhibit autophagy and promote malignant behaviors such as proliferation and invasion of hepatocellular carcinoma. Autophagy can degrade cell components to maintain cell function and survival, but when autophagy destroys most of the cytoplasm and organelles, exceeding a certain threshold, it will lead to irreversible cell death. A large number of studies have also shown the role of autophagy in causing cell death. Autophagy is regulated by multiple factors such as signaling pathways and metabolites. PTEN is an effective tumor suppressor gene, and its expression is reduced or absent in about half of HCC. The loss of PTEN causes the activation of PI3K, which triggers the increased activation of the downstream effector AKT. As the downstream of AKT, mTOR has a negative regulatory effect on autophagy.

[0004] As the main mode of intercellular communication, extracellular vesicles have been shown to be involved in various processes of tumor occurrence and development. Exosomes are the main type of extracellular vesicles, with a diameter of about 30-150nm, and are secreted by the fusion of multivesicular bodies with the plasma membrane. Exosomes participate in intercellular communication and regulate the microenvironment and immune function between cells by transporting various biomolecules such as proteins and RNA. Exosomes can be absorbed by receptor cells, thereby changing their functions. In recent years, a large number of studies have shown that exosomes play a role in the occurrence and development of various tumors, including metastasis, angiogenesis, immune escape, etc. And studies have shown that tumor exosomes are involved in regulating epithelial to mesenchymal transition (EMT), which is a key step in cancer metastasis. MicroRNAs (miRNAs) are the most important RNA species in exosomes. MicroRNAs in exosomes are mediators of intercellular signal transduction and are also important biomarkers for many diseases and drug resistance. MiRNAs are small non-coding RNAs (usually 18-24 nucleotides in length) that regulate gene expression by binding to the 3′ untranslated region (3′UTR) of target mRNAs, directly targeting mRNAs to cause degradation or inhibiting protein translation. Many miRNAs are abnormally expressed in tumors and participate in the biological process of tumor development as oncogenes or tumor suppressors according to the cellular functions of their target genes. Given that miRNAs are important regulators of biological processes, abnormal expression of miRNAs is involved in the development of a variety of diseases, including cancer. Tumor-derived miRNAs exist in the blood in a relatively stable form and can therefore serve as potential markers for the diagnosis or prognosis of malignant tumors, including HCC. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the miR-486-5p marker in exosomes in the preparation of a hepatocellular carcinoma diagnostic kit.

[0006] In one aspect, the present invention provides the use of the miR-486-5p marker in exosomes in the preparation of a diagnostic kit for hepatocellular carcinoma.

[0007] Furthermore, the miR-486-5p marker in the exosomes inhibits autophagy of hepatocellular carcinoma cells.

[0008] Furthermore, the miR-486-5p marker in the exosomes targets and inhibits the tumor suppressor gene PTEN in hepatocellular carcinoma cells.

[0009] Furthermore, the miR-486-5p marker in the exosomes activates the AKT / mTOR signaling pathway in hepatocellular carcinoma cells, thereby inhibiting the autophagy process.

[0010] Beneficial effects:

[0011] In this invention, the miRNA expression data of plasma exosomes of patients with hepatocellular carcinoma before and after surgery were analyzed. The miR-486-5p in plasma exosomes was significantly enriched, so exosome miR-486-5p can be used as a potential biomarker for diagnosing hepatocellular carcinoma. We then explored the mechanism of action of miR-486-5p, and found that it can target the tumor suppressor gene PTEN and activate the downstream AKT / mTOR signaling pathway, thereby inhibiting the autophagy process. Therefore, miR-486-5p in exosomes regulates the function of autophagy through the PTEN-AKT signaling pathway, and suggests a potential diagnostic value in HCC. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results show that miR-486-5p in plasma exosomes of patients with hepatocellular carcinoma is significantly increased; 1A shows the expression analysis of miRNA in plasma exosomes of patients with hepatocellular carcinoma before and after tumor resection; 1B is the expression level of miR-486-5p in plasma exosomes of normal people and patients with hepatocellular carcinoma; 1C is the ROC curve of plasma exosome miR-486-5p predicting hepatocellular carcinoma; 1D is the expression level of miR-486-5p in primary tumors and portal vein tumor thrombi matched with hepatocellular carcinoma patients in GEO database GSE76903. The results are mean ± standard error (SEM); **p<0.01;

[0013] Figure 2 Figure 2 is the characterization of exosomes derived from hepatocellular carcinoma cells; 2A is the size and morphology of isolated exosomes observed by transmission electron microscopy, scale bar: 100 μm; 2B is the size distribution of exosomes measured by nanoflow cytometry, with a peak diameter of approximately 100 nm; 2C is a protein immunoblot analysis of exosomes and whole cell lysates using antibodies against exosome protein markers (including CD9, CD63, CD81, TSG101 and Alix) and exosome negative protein markers (GM130); 2D is a confocal imaging showing Dio-labeled exosomes (green) delivered to DAPI-labeled Huh7 cells (blue), with red arrows indicating delivered exosomes, scale bar: 10 μm.

[0014] Figure 3Figure 3 shows the regulation of autophagy by miR-486-5p in hepatocellular carcinoma cells; 3A is the detection of miR-486-5p content in different hepatocellular carcinoma cell lines by RT-qPCR; 3B is the analysis of the expression of autophagy-related proteins in Hep3B and Huh-7 cells after overexpression of miR-486-5p by western blotting; 3C is the analysis of the expression of autophagy-related proteins in MHCC97H and HCCLM3 cells after inhibition of miR-486-5p by western blotting; 3D is the infection of Hep3 with adenovirus expressing mCherry-EGFP-LC3. B and Huh-7 cells, and then observed using a confocal microscope, scale bar: 10 μm, randomly selected fields of view to quantify the number of yellow and red spots per cell under each condition; 3E MHCC97H and HCCLM3 cells were infected with adenovirus expressing mCherry-EGFP-LC3, and then observed using a confocal microscope, scale bar: 10 μm, randomly selected fields of view to quantify the number of yellow and red spots per cell under each condition; the results are mean ± standard deviation (SD); *p<0.05, **p<0.01, ***p<0.001;

[0015] Figure 4 miR-486-5p targets PTEN; 4A left figure shows the target genes of miR-486-5p predicted by the database, and the right figure shows a list of 11 common target genes; 4B uses protein immunoblotting to detect the expression of PTEN in Hep3B and Huh-7 cells after overexpression of miR-486-5p; 4C left figure is a schematic diagram of the dual luciferase reporter experiment of miR-486-5p targeting PTEN 3'UTR, and the right figure is transfected with miR-486-5p (or negative control, NC) and luciferase reporter plasmid in HEK293T cells and detect luciferase activity, and the luciferase reporter plasmid is PTEN 3'UTR wild type (WT) or PTEN 3'UTR mutant (MUT); the results are mean ± standard deviation (SD); *p<0.05, **p<0.01, ***p<0.001;

[0016] Figure 5Figure 5 shows that miR-486-5p affects autophagy by activating the AKT / mTOR signaling pathway; 5A shows the KEGG enrichment analysis using differentially expressed genes after overexpression of miR-486-5p in WRL-68 cells; 5B uses protein immunoblotting to detect the expression levels of p-AKT and PTEN in different cell lines; 5C uses protein immunoblotting to detect the expression of p-AKT, p-mTOR, p-ULK1 (Ser-757), AKT, mTOR and ULK1 proteins in Hep3B and Huh-7 cells after overexpression of miR-486-5p; 5D uses protein immunoblotting to detect the inhibition of p-AKT protein in Hep3B and Huh-7 cells using different doses of MK2206; 5E uses protein immunoblotting to detect the expression of p-AKT in H Inhibition of p-mTOR protein by using different doses of Rapamycin in Hep3B and Huh-7 cells; 5F is a protein immunoblot assay to detect the expression level of LC3B protein when MK2206 or Rapamycin was added to Hep3B and Huh-7 cells with or without overexpression of miR-486-5p; 5G is an adenovirus expressing mCherry-EGFP-LC3 infected Hep3B and Huh-7 cells, overexpressing miR-486-5P and then adding MK2206 or Rapamycin, and then observing using a confocal microscope, scale bar: 10 μm, randomly selected fields of view to quantify the number of yellow and red spots per cell under each condition; the results are mean ± standard deviation (SD); *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] Example 1

[0019] 1.1 Isolation of plasma exosomes

[0020] (1) 2 mL of plasma was extracted from the patient before and after surgery and centrifuged at 3,000 g for 15 min at 4°C to remove cells and debris.

[0021] (2) The plasma supernatant was centrifuged at 4°C, 3,000 g for 15 min to ensure that no platelets remained.

[0022] (3) The plasma supernatant after centrifugation was immediately diluted 20-fold in 1× PBS buffer and centrifuged at 15,000 g at 4°C for 40 min.

[0023] (4) Filter the plasma supernatant using a 0.22 μm filter membrane and place the filtered supernatant in an ultracentrifuge tube.

[0024] (5) Centrifuge at 4°C, 120,000 g for 4 h. Add 1 mL of Trizol (Invitrogen) for lysis for 5 min, and then extract RNA.

[0025] 1.2 Extraction of total RNA from plasma exosomes by Trizol method

[0026] (1) Add 200 μL of chloroform to the Trizol lysate containing exosomes, shake vigorously for 15 seconds, let stand at room temperature for 2 minutes, and then centrifuge at 4°C and 12,000g for 15 minutes.

[0027] (2) Pipette 400 μL of supernatant, add an equal volume of isopropanol, shake slowly 15 times, and let stand at room temperature for 10 min; then centrifuge at 4°C, 12,000 g for 10 min.

[0028] (3) Discard the supernatant, add 1 mL of 75% ethanol, invert five times, and then centrifuge at 4°C, 12,000 g for 5 min.

[0029] (4) Discard the ethanol, centrifuge briefly, and absorb the residual ethanol.

[0030] (5) Air-dry for 1 min and add 20–30 μL of nuclease-free water to dissolve the RNA.

[0031] (6) The extracted RNA concentration and purity were measured using a NanoDrop One micro-spectrophotometer (Thermo Scientific) (), and the extracted RNA was then stored in a -80 °C refrigerator or used for the next experiment.

[0032] 1.3 High-throughput sequencing of exosomal RNA

[0033] High-throughput RNA sequencing analysis of isolated exosomes was performed by RiboBio. Total RNA was extracted using Trizol (Invitrogen). RNA quantity and integrity were assessed using the Agilent 2200 TapeStation (Life Technologies) and Agilent Technologies for Illumina according to the manufacturer’s instructions. Small RNA libraries were prepared from 1 μg of total RNA from each sample using the Multiplex Small RNA Library Preparation Kit (NEB). The libraries were then sequenced on the HiSeq 2500 platform (Illumina) of RiboBio using a single-end 50 bp protocol.

[0034] 1.4 Screening of differentially expressed genes

[0035] The p-value and fold change value of the test were used to screen differentially expressed genes, and the screening criteria were up-regulation fold change value ≥ 1.0 and P value < 0.05.

[0036] 1.5. RT-qPCR experiment of miR-486-5p in plasma exosomes

[0037] The blood samples of the participants were used to perform RT-qPCR experiments on miR-486-5p. Upstream and downstream primers were designed based on the sequence of miR-486-5p, and U6 was used as an internal reference. The specific primer sequences of miR-486-5p are shown in the following table.

[0038]

[0039] The separation of plasma exosomes and the extraction of exosomal RNA were performed according to 1.1 and 1.2 of Example 1.

[0040] Removal of genomic DNA: Prepare the following reaction system in an RNase-free centrifuge tube: 1 μL of 5×g DNA wiper Mix, 1 μg of total RNA, add nuclease-free water to make the total volume 5 μL, and use a pipette to gently pipette to mix, and react in a PCR instrument at 42°C for 2 min.

[0041] Synthesize cDNA: 1 μL 10×RT Mix, 1 μL HiScriptIII Enzyme Mix, 0.5 μL reverse transcription primer (4 μM), 2.5 μL nuclease-free water, plus one-step mixture, the total reaction system is 10 μL. React at 37°C for 15 min and 85°C for 5 s in a PCR instrument.

[0042] The qPCR reaction system was configured to include 5 μL of 2×AceQ qPCR SYBR Green Master Mix (Novozyme), 0.1 μL of upstream primer (20 μM), 0.1 μL of downstream primer (20 μM), 1 μL of cDNA, and 3.8 μL of dd H2O. The following procedures were performed on the qPCR instrument:

[0043] Stage 1 (pre-denaturation): 95°C, 5 min;

[0044] Stage 2 (cyclic reaction, 40 cycles): 95°C, 10 s, 60°C, 30 s;

[0045] Stage 3 (melting curve): 95°C, 15 s, 60°C, 60 s, 95°C, 15 s;

[0046] Data processing was performed using the ΔΔCT method. First, the CT value difference (ΔCT) between miR-486-5p and the internal reference gene U6 was calculated, and then the ΔCT difference (ΔΔCT) between the hepatocellular carcinoma group and the normal control group was calculated. Finally, the relative expression of the target gene was calculated using the formula 2^(-ΔΔCT). A relative expression greater than 1 indicates gene upregulation, and less than 1 indicates downregulation.

[0047] The results showed that miRNAs were the main RNA species in exosomes. The miRNAs in plasma exosomes of patients with hepatocellular carcinoma before and after tumor resection were sequenced, and a volcano map of differentially expressed genes was made. The results showed that miR-486-5p was significantly increased in plasma exosomes of patients with hepatocellular carcinoma before surgery ( Figure 1 A).

[0048] Then, plasma from 6 healthy subjects and 19 patients with hepatocellular carcinoma was collected for verification, and the content of miR-486-5p in plasma exosomes was detected. The results showed that the expression level of miR-486-5p in plasma exosomes of patients with hepatocellular carcinoma was significantly increased ( Figure 1 B).

[0049] The ROC curve was drawn using the content of miR-486-5p in plasma exosomes from 6 healthy subjects and 19 patients with hepatocellular carcinoma. The area under the ROC curve was 0.917 ( Figure 1 C), indicating its potential as an aid in the diagnosis of hepatocellular carcinoma.

[0050] In addition, the GEO database (GSE76903) showed the expression levels of miR-486-5p in the primary tumors and portal vein tumor thrombi of 20 HCC patients. The results showed that the expression level of miR-486-5p in the portal vein tumor thrombi of 14 HCC patients (70%) was increased compared with that in the primary tumors ( Figure 1 D). Hepatocellular carcinoma combined with portal vein tumor thrombus is a more serious condition in patients with hepatocellular carcinoma and is closely related to poor prognosis. We believe that miR-486-5p in hepatocellular carcinoma cells may be associated with poor prognosis in patients with hepatocellular carcinoma. In addition, exosomes can carry miR-486-5p and be absorbed by tumor cells, playing an important role in the progression of hepatocellular carcinoma.

[0051] Therefore, miR-486-5p in exosomes can assist in the diagnosis of hepatocellular carcinoma, and miR-486-5p may be associated with the poor prognosis of hepatocellular carcinoma.

[0052] Example 2

[0053] 2.1 Isolation of exosomes from tumor cells

[0054] 8 h before collecting the tumor cell supernatant, the culture medium was replaced with serum-free medium to exclude the influence of exosomes contained in the serum on the experiment. The supernatant of tumor cells was collected, first centrifuged at 300g for 10 minutes to remove cell debris, the supernatant was collected and centrifuged at 300g for 10 minutes to remove cell debris again, then centrifuged at 4°C, 12,000g for 90 minutes to remove larger extracellular vesicles, and finally the supernatant was centrifuged at 4°C, 120,000g for 240 minutes, and the obtained precipitate, i.e., exosomes, was lysed or resuspended in PBS for further analysis.

[0055] 2.2 Transmission electron microscopy observation of exosomes

[0056] 30 μL of the exosome suspension was placed on a carbon-coated formvar grid for 10 min and then stained with phosphotungstic acid (pH 6.8) for 5 min. The samples were observed under a transmission electron microscope and photographed.

[0057] 2.3 Nanoflow cytometry

[0058] The standard microspheres contain particles of different sizes: PS110, Si180, Si240, Si300, Si590, Si880, Si1300, PS500. A standard reference coordinate system is set in the machine according to the distribution of the standard microspheres. 200 μL of exosome suspension is compared with the size of the nanoflow standard microspheres through the nanoflow analyzer to determine the size distribution range of the exosomes.

[0059] 2.4 Protein immunoblotting

[0060] The exosomes obtained by centrifugation were lysed in NP-40 lysis buffer (Beyotime) to prepare exosome lysate, and the protein concentration of exosomes was determined using the BCA assay (Thermo Scientific). The whole cell lysate was used as a control, and the proteins were separated by 5-10% SDS-PAGE gel electrophoresis and transferred to NC membrane for detection. The following antibodies were used in this example: CD9 (Invitrogen), CD63 (Invitrogen), CD81 (Invitrogen), TSG101 (Abcam), Alix (Abcam), GM130 (Cell Signaling Technology), anti-mouse antibody (Proteintech) and anti-rabbit antibody (Proteintech).

[0061] 2.5 Exosome tracing experiment

[0062] First, resuspend the exosomes obtained by centrifugation with 1mL of PBS. Add 1μL of Dio staining solution (5mM) and incubate at room temperature for 20 minutes. Then centrifuge at 4℃, 120,000g for 2 hours, discard the supernatant, and resuspend the exosome precipitate with culture medium. Aspirate the cell culture medium that was plated in the confocal dish one day in advance, wash it 3 times with PBS, add culture medium, and then add the stained exosomes at a final protein concentration of 40μg / mL, and place it in a cell culture incubator for 24 hours. Discard the cell supernatant, wash it 2 times with PBS, and then add 500μL of 4% paraformaldehyde solution to fix it for 20 minutes. Discard the fixative, wash it 2 times with PBS, slowly add 100μL of DAPI staining solution to the middle part of the confocal dish, incubate it for 1 minute, and observe and take pictures with a confocal microscope.

[0063] The results showed that in order to verify that exosomes can carry miR-486-5p to hepatocellular carcinoma cells, exosomes derived from hepatocellular carcinoma cells were separated by differential ultracentrifugation and their characteristics were identified.

[0064] First, transmission electron microscopy was used to identify the structural characteristics and size of exosomes. The isolated exosomes showed a typical cup-shaped structure and were nanometer-sized ( Figure 2 A). The size of the exosomes was then detected by nanoflow cytometry. Compared with the standard microspheres, the size of the isolated exosomes ranged from 80nm to 300nm, with a peak of about 100nm, which was consistent with the particle size of the exosomes ( Figure 2 B). Finally, protein immunoblotting was used to detect exosome markers. The results showed that CD9, CD63, CD81, TSG101, and Alix proteins in exosomes were positive, while GM130 protein was negative, which was consistent with the protein marker characteristics of exosomes ( Figure 2C). We then labeled HCCLM3 tumor cell-derived exosomes and Huh-7 tumor cells with Dio (green) and DAPI (blue). After incubation, confocal imaging showed the presence of Dio spots in recipient tumor cells, indicating that exosomes released by HCCLM3 tumor cells were delivered to Huh-7 tumor cells ( Figure 2 D) The results verified that exosomes released by tumor cells can be taken up by other tumor cells.

[0065] Example 3

[0066] 3.1 Cell transfection: 100,000 cells were plated in a 6-well plate one day before transfection. For each transfection sample, transfection was performed as follows: First, 40 pmol miR-486-5p or negative control was diluted in 200 μL of Opti-MEM medium and gently mixed. Then 2 μL Lipofectamine2000 was diluted in 200 μL Opti-MEM medium. Gently mix and incubate at room temperature for 5 minutes. After incubation for 5 minutes, the diluted miR-486-5p or negative control and the diluted Lipofectamine 2000 were mixed, gently mixed, and then incubated at room temperature for 20 minutes. After the incubation, the mixture was added to the wells containing cells and the culture plate was gently shaken to mix. The culture medium can be replaced after 4-6 hours.

[0067] 3.2 The treated cells were lysed using NP-40 lysis buffer (Biyuntian) and the protein concentration in the cells was determined using the BCA assay (Thermo Scientific). Proteins were separated by 5-10% or 5-13.5% SDS-PAGE gel electrophoresis and transferred to NC membrane for detection. The following antibodies were used in this example: Beclin-1 (CellSignaling Technology), LC3B (Novus), p62 (Abcam), β-Actin (Proteintech), anti-mouse antibody (Proteintech) and anti-rabbit antibody (Proteintech).

[0068] 3.3 Autophagy flow detection: 24 hours before viral infection, 50,000 cells were plated in confocal culture dishes. Then, cells were infected with mCherry-EGFP-LC3 adenovirus, cells were cultured with virus for 12 hours, culture medium was changed, and negative control, miR-486-5p, inhibitor negative control or miR-486-5p inhibitor were transfected. After 48 hours of transfection, fluorescence intensity was evaluated using confocal microscopy (Zeiss LSM 800); yellow dots (mCherry+EGFP+) represent autophagosomes, while red dots (mCherry+EGFP-) represent autolysosomes, and data are expressed as the number of autolysosomes or autophagosomes per cell.

[0069] The results showed that in vitro cell experiments were used to explore the biological functions of elevated miR-486-5p in plasma exosomes of hepatocellular carcinoma patients in tumor cells. The expression levels of miR-486-5p in different hepatocellular carcinoma cell lines were detected. The results showed that the expression levels of miR-486-5p in Hep3B and Huh-7 cells were low, while the expression levels of miR-486-5p in MHCC-97H and HCC-LM3 cells were high ( Figure 3 A).

[0070] Autophagy is a process in which a part of the cell's material is encapsulated by autophagosomes and transported to lysosomes for degradation. Dysfunction in the autophagy process is associated with many diseases, including cancer. Studies have shown that the expression of autophagy-related genes is decreased in hepatocellular carcinoma, and autophagy defects can lead to a more aggressive cancer cell phenotype and a poor prognosis for hepatocellular carcinoma. Therefore, miR-486-5p was overexpressed in Hep3B and Huh-7 cells, and protein immunoblotting results showed that the expression levels of autophagy marker proteins Beclin-1 and Microtubule3 (LC3) were decreased, while the expression level of p62 was increased ( Figure 3 B). However, when miR-486-5p was knocked down in MHCC-97H and HCC-LM3 cells, western blotting results showed that the expression levels of autophagy marker proteins Beclin-1 and Microtubule 3 (LC3) increased, while the expression level of p62 decreased ( Figure 3 C). Western blotting results showed that miR-486-5p could inhibit autophagy in liver cancer cells.

[0071] Then, the effect of miR-486-5p on autophagic flux was studied, and liver cancer cells were infected with mCherry-EGFP-LC3 adenovirus. Immunofluorescence was evaluated by confocal microscopy. Red represents the mCherry fluorescence signal, and green represents the EGFP fluorescence signal, which co-localize to form a yellow fluorescence signal. When the autophagosome fuses with the lysosome, the acid-sensitive EGFP green fluorescence is quenched, showing a red signal. Therefore, the yellow and red spots represent autophagosomes and autophagic lysosomes, respectively. The results showed that after overexpression of miR-486-5p in Hep3B and Huh-7 cells, both yellow and red spots were reduced ( Figure 3 D), while after knockdown of miR-486-5p in MHCC-97H and HCC-LM3 cells, both yellow and red spots increased ( Figure 3 E), which indicates that miR-486-5p can inhibit autophagy flux. In general, the experimental results show that miR-486-5p can inhibit autophagy in liver cancer cells.

[0072] Example 4

[0073] 4.1 Prediction of miRNA target genes: The target genes of miR-486-5p were predicted using the miRTarbase, TargetScan and miRDB databases respectively, and the prediction results of the three databases were intersected to obtain the most likely target gene.

[0074] 4.2 Protein immunoblotting experiment: The treated cells were lysed using NP-40 lysis buffer (Biyuntian), and the protein concentration in the cells was determined by BCA assay (Thermo Scientific). Proteins were separated by 5-10% gel electrophoresis and transferred to NC membrane for detection. The following antibodies were used in this example: PTEN (ABclonal), GAPDH (Proteintech), anti-mouse antibody (Proteintech) and anti-rabbit antibody (Proteintech).

[0075] 4.3 Dual luciferase reporter experiment: HEK293T cells were seeded in 24-well plates one day in advance, and PTEN 3'UTRWT or MUT plasmids were co-transfected with miR-486-5p mimics or controls the next day. The culture medium was replaced 6 hours after transfection, and the cells were washed with PBS, trypsinized and collected 24 hours later. An appropriate amount of lysis buffer was added, and the cells were lysed on a shaker at 4°C for 30 minutes, and then centrifuged at 12,000g for 10 minutes (4°C). 20μL of lysis buffer was added to a 96-well plate, and 100μL of buffer containing luciferase reaction substrate and luciferase reaction substrate II were added respectively, and the activities of Firefly and Renilla luciferase were detected in turn. The binding ability of miR-486-5p to PTEN 3'UTR was evaluated by calculating the ratio of Firefly / Renilla enzyme activity.

[0076] The results showed that the miRTarbase, TargetScan and miRDB databases were used to predict potential target genes to explore the biological function of miR-486-5p in hepatocellular carcinoma, and 11 target genes were obtained ( Figure 4 A). Among them, the tumor suppressor gene PTEN is one of the 11 potential targets. The protein immunoblotting method was used to verify the expression of miR-486-5p in Hep3B and Huh-7 cells, and the results showed that the protein level of PTEN was significantly decreased after expression of miR-486-5p ( Figure 4 B). A dual luciferase reporter assay was then performed. When miR-486-5p was overexpressed in HEK293T cells overexpressing the PTEN 3'UTR WT plasmid, the luciferase activity was significantly reduced. However, when HEK293T cells overexpressed the PTEN 3'UTR MUT plasmid, overexpression of miR-486-5p did not reduce the luciferase activity compared with the control group. The results of the dual luciferase reporter assay confirmed the direct interaction between miR-486-5p and PTEN's 3′-UTR ( Figure 4 C). Taken together, these results suggest that miR-486-5p directly targets the tumor suppressor gene PTEN.

[0077] Example 5

[0078] 5.1 Protein immunoblotting experiment: The treated cells were lysed using NP-40 lysis buffer (Biyuntian), and the protein concentration in the cells was determined by BCA assay (Thermo Scientific). Proteins were separated by 5-10% or 5-13.5% SDS-PAGE gel electrophoresis and transferred to NC membrane for detection. The following antibodies were used in this example: P-AKT (Cell Signaling Technology), AKT (Cell Signaling Technology), P-mTOR (Cell Signaling Technology), mTOR (Cell Signaling Technology), P-ULK1 (ser757) (Cell Signaling Technology), ULK1 (Cell Signaling Technology), LC3B (Novus), GAPDH (Proteintech), β-Actin (Proteintech), anti-mouse antibody (Proteintech) and anti-rabbit antibody (Proteintech).

[0079] 5.2 Autophagic flux detection

[0080] Same as 3.3 in Example 3

[0081] The results showed that RNA-Seq analysis was performed on WRL-68 cells transfected with miR-486-5p and negative control to understand the changes in the whole gene transcriptome caused by miR-486-5p. The analysis found many differentially expressed genes, and then KEGG enrichment analysis was performed on these differentially expressed genes. The results showed that the PI3K / Akt signaling pathway was significantly enriched ( Figure 5 A). PI3K / Akt and mTOR signaling pathways have been shown to be closely related to PTEN, which further suggests that PTEN may be a candidate target of miR-486-5p.

[0082] We then verified the protein expression of PTEN and AKT in liver cancer cell lines, and the results showed that PTEN was inversely proportional to p-AKT expression ( Figure 5 B). Overexpression of miR-486-5p in hepatocellular carcinoma cells can activate p-AKT and downstream p-mTOR, which subsequently leads to enhanced phosphorylation of mTOR's downstream protein ULK1 (ser757), resulting in decreased ULK1 protein activity ( Figure 5C). ULK1 is the initiator of autophagy, which also explains why miR-486-5p inhibits autophagy. These results indicate that miR-486-5p activates the AKT / mTOR pathway in HCC cells.

[0083] Based on the above-mentioned fact that overexpression of miR-486-5p in vitro can activate the AKT / mTOR signaling pathway and affect autophagy, we wanted to explore the effect of specific blocking of the AKT / mTOR pathway on miR-486-5p-induced autophagy inhibition in Hep3B and Huh-7 cells. First, we confirmed the effectiveness of different doses of AKT inhibitor (MK-2206) and mTOR inhibitor (rapamycin) ( Figure 5 D, E). Western blot results showed that the addition of MK2206 or Rapamycin to overexpress miR-486-5p increased the protein expression level of the autophagy marker protein LC3Ⅱ ( Figure 5 F). The confocal results also showed that the number of autophagosomes and autolysosomes represented by yellow and red spots increased after the addition of MK2206 and rapamycin, indicating that the autophagic flux increased ( Figure 5 G). In general, our experimental results showed that overexpression of miR-486-5p could activate the AKT / mTOR signaling pathway in liver cancer cells, and confirmed that miR-486-5p affects autophagy through this signaling pathway.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Application of miR-486-5p marker in exosomes in the preparation of diagnostic kit for hepatocellular carcinoma.

2. The use according to claim 1, characterized in that: The miR-486-5p marker in the exosomes inhibits autophagy of hepatocellular carcinoma cells.

3. The use according to claim 1, characterized in that: The miR-486-5p marker in the exosomes targets and inhibits the tumor suppressor gene PTEN in hepatocellular carcinoma cells.

4. The use according to claim 1, characterized in that: The miR-486-5p marker in the exosomes activates the AKT / mTOR signaling pathway in hepatocellular carcinoma cells, thereby inhibiting the autophagy process.

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