Serum exosome miRNA marker related to liver cancer diagnosis, and application thereof

A marker and exosome technology, applied in the field of serum exosomal miRNA markers, can solve the problems of normal cell transformation, high transfer rate and high recurrence rate restricting the long-term curative effect of HCC patients, and insufficient sensitivity of diagnostic indicators. The effect of less serum volume, delaying and stopping disease progression, and avoiding invasive diagnosis

Inactive Publication Date: 2020-05-29
NANJING DRUM TOWER HOSPITAL
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, surgical resection is still the most effective treatment for HCC, but the high metastasis rate and high recurrence rate of HCC seriously restrict the long-term efficacy of HCC patients
At the same time, the commonly used HCC-specific diagnostic indicators in clinical practice are not sensitive enough, and because the liver has a strong compensatory ability, when patients present with symptoms, most of them are already in the middle or late stage of cancer, and less than 10% of them can get radical surgery
Therefore, abnormal expression of individual miRNAs may also transform normal cells into tumor cells

Method used

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  • Serum exosome miRNA marker related to liver cancer diagnosis, and application thereof
  • Serum exosome miRNA marker related to liver cancer diagnosis, and application thereof
  • Serum exosome miRNA marker related to liver cancer diagnosis, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: has-miRNA-21-5p in exosomes promotes the proliferation of liver cancer cells

[0031] 1.1) Cell source: Human liver cancer cell line HCCLM3 (human highly metastatic liver cancer cells) was purchased from the Cell Bank of the Chinese Academy of Sciences (catalogue number: TCHu 94), MHCC97H (human high metastatic liver cancer cells) cells were purchased from Fudan University Liver Cancer Institute (Shanghai, China), human normal liver cells LO2, and human hepatic stellate cells (HSCs) were purchased from Fudan University Liver Cancer Institute (Shanghai, China);

[0032] 1.2) Extraction and identification of exosomes from liver cancer cell lines

[0033]1.2.1) Cultivate human hepatoma cell lines HCCLM3, MHCC97H and human hepatocyte LO2: human hepatoma cell lines HCCLM3 and MHCC97H were used in high-sugar DMEM medium containing 10% fetal bovine serum (containing penicillin 100 U / ml, streptomycin 100 μg / ml) ml) at 37°C and 5% CO2 in a cell incubator for culture ...

Embodiment 2

[0084] Example 2: has-miRNA-21-5p in exosomes promotes the migration of liver cancer cells

[0085] 2.1) Referring to the transfection step of step 1.4.2), HSCs cells were transfected with has-miRNA-21-5p mimic and negative control miRNA-mimic-NC;

[0086] 2.2) Preparation of cell suspension: collect transfected HSCs cells, and make 1.0x10 with serum-free DMEM high-glucose medium 6 cell suspension per ml;

[0087] 2.3) Scratch: After the cells adhere to the wall, scratch along the pre-marked line. During the scratching process, use a 200μL pipette tip to scratch straight along the marked line to create multiple parallel scratches. The strength is to scrape off the cells. It shall prevail without leaving traces on the culture plate; then rinse with PBS twice to remove floating cells; then culture with DMEM high glucose without FBS and continue to incubate for 24 hours in a 37°C, 5% CO2 incubator;

[0088] 2.4) Observation of results: Observe the scratch repair process of cell...

Embodiment 3

[0090] Example 3: Liver cancer cell exosomes has-miRNA-21-5p promotes the growth of subcutaneous xenografts

[0091] 3.1) Material source: has-miRNA-21-5p inhibitor (has-miRNA-21-5p inhibitor) was purchased from Guangzhou Ruibo Biotechnology Company;

[0092] The sequence of has-miRNA-21-5p inhibitor is: upstream sequence has-miRNA-21-5p inhibitor-F: 5'-UAGCUUAUCAGACUGAUGUUGA-3' (SEQ ID NO.2), downstream sequence has-miRNA-21-5pinhibitor- R: 5'-AUCGAAUAGUCUGACUACAACU-3' (SEQ ID NO.3);

[0093] 3.2) Preparation of HCCLM3 and MHCC97H cells that inhibit the expression of has-miRNA-21-5p:

[0094] 3.2.1) HCCLM3 and MHCC97H cells were cultured and passaged in a 37°C, 5% CO2 cell incubator with high-glucose DMEM medium containing 10% fetal bovine serum (containing 100 U / ml penicillin and 100 μg / ml streptomycin);

[0095] 3.2.2) Prepare 2 mL of HCCLM3 and MHCC97H cell suspensions with a density of 4*10^4 cells / mL respectively, add 100 μL of cell suspension / well into a 96-well plate...

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Abstract

The invention belongs to the field of biological diagnosis and medicine, and particularly relates to a serum exosome miRNA marker related to liver cancer diagnosis and application thereof. The markeris has-miRNA-21-5p which is used as a biomarker of liver cancer, whether a detected person has liver cancer or liver cancer risk or not is diagnosed by detecting the expression level of the marker, the marker has the characteristics of convenience in detection, small damage and the like, and meanwhile, the quantification is accurate, the sensitivity and the specificity of detection can be improved, and the has-miRNA-21-5p and a specific amplification primer thereof can be used for preparing a liver cancer diagnosis kit, so that the rapid and auxiliary diagnosis can be conveniently carried outat the early stage, and a basis is provided for further deeply examining or rapidly and accurately mastering the disease state of a patient by a clinician.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a serum exosome miRNA marker related to the diagnosis of liver cancer and an application thereof. Background technique [0002] Hepatocellular carcinoma (HCC) is the sixth most common malignant tumor worldwide and the third leading cause of cancer-related death. At present, surgical resection is still the most effective treatment for HCC, but the high metastasis rate and high recurrence rate of HCC seriously restrict the long-term efficacy of HCC patients. At the same time, the commonly used HCC-specific diagnostic indicators in clinical practice are not sensitive enough, and because the liver has a strong compensatory ability, most patients are already in the middle or late stage of cancer when they present symptoms and seek treatment, and less than 10% of them can receive radical surgery. Therefore, elucidating the specific mechanism of HCC occurrence and development, ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11A61K31/7088A61P35/00A61P1/16
CPCA61K31/7088A61P1/16A61P35/00C12Q1/6886C12Q2600/158C12Q2600/178
Inventor 施晓雷任昊桢王经琳丁义涛
Owner NANJING DRUM TOWER HOSPITAL
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