Application of a miRNA combined marker in the preparation of a kit for diagnosing and detecting early liver cancer

Abstract

The present invention discloses an application of a miRNA combined marker in the preparation of a kit for diagnosing and detecting early liver cancer. The miRNA combined markers are has-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p and hsa-miR-382-5p derived from plasma exosomes. Early liver cancer is primary HCC. The miRNA combined marker proposed in the present invention is not limited to whether the patient has hepatitis B HBV positive and liver cirrhosis LC symptoms, and has high sensitivity and strong specificity.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and medical diagnosis, and to the application of a miRNA combined marker, and in particular to the application of a miRNA combined marker in the preparation of a kit for diagnosing and detecting early liver cancer. Background Art

[0002] Primary liver cancer is one of the malignant tumors with the highest morbidity and mortality rates worldwide. Hepatocellular carcinoma (HCC) is the main form of primary liver cancer, which usually evolves from chronic liver diseases such as cirrhosis and HBV infection. The five-year survival rate of early HCC is 50%-70%, while that of patients with advanced liver cancer is only 10%-19%. Therefore, early diagnosis and screening are the key to liver cancer treatment. Due to the lack of early diagnostic methods that can effectively distinguish between patients with early liver cancer and patients with chronic liver disease, the current diagnosis of HCC still mainly relies on imaging, serum AFP and tissue biopsy, which has a certain lag, and most patients are already in the late stage at the time of initial diagnosis. Therefore, there is an urgent need to develop new biomarkers for the early diagnosis of liver cancer patients in clinical practice.

[0003] Exosomes are a type of extracellular vesicles with a diameter of 40-160nm (average 100nm), which can be secreted by most cells and enter the blood circulation system. Exosomes carry biological information such as membrane proteins, nuclear proteins, DNA, mRNA, non-coding RNA, etc., participate in the important regulation of cell activities, and play an important role in tumor metastasis, immune regulation mechanism, and disease occurrence and development. MicroRNA (miRNA) is a type of non-coding single-stranded RNA with a length of 19-24nt. Generally, it plays a role in post-transcriptional regulation by partially or completely complementary pairing with the target messenger RNA (mRNA), resulting in the degradation of mRNA or the inhibition of translation. It plays a role in various links of biological development and the occurrence and development of various diseases including tumors. At the same time, compared with other types of biological information in exosomes, miRNA fragments are smaller and more stable. Therefore, miRNA derived from exosomes can be used as a potential tumor molecular marker.

[0004] Although some miRNAs have been proposed in the art for dynamic monitoring and early diagnosis of liver cancer, the effects of hepatitis B and cirrhosis on the diagnosis results are not considered. The present invention is based on abnormally expressed miRNAs in peripheral blood exosomes to avoid the effects of hepatitis B and cirrhosis on the diagnosis results, and proposes targeted miRNA combined markers for early diagnosis of liver cancer. Summary of the invention

[0005] The first object of the present invention is to address the deficiencies in the prior art and to provide an application of a peripheral blood exosome miRNA combined marker in the preparation of a kit for diagnosing or detecting early liver cancer, wherein the miRNA combined marker is has-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p and hsa-miR-382-5p derived from plasma exosomes.

[0006] The second object of the present invention is to provide a kit for preparing a diagnosis or detection of early liver cancer, the kit comprising: a detection reagent for specifically detecting human has-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p and hsa-miR-382-5p.

[0007] Preferably, the early liver cancer is primary HCC.

[0008] The third object of the present invention is to provide the application of peripheral blood exosome miRNA combined markers as auxiliary diagnostic indicators for early liver cancer, wherein the miRNA combined markers are has-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p and hsa-miR-382-5p derived from plasma exosomes.

[0009] The fourth object of the present invention is to provide an early liver cancer serum exosome marker, including has-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p and hsa-miR-382-5p.

[0010] The fifth object of the present invention is to provide an early liver cancer diagnostic preparation, comprising miRNA combined markers has-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p and hsa-miR-382-5p.

[0011] The advantages and beneficial effects of the present invention are as follows:

[0012] 1. Exosomal miRNAs are a new type of liquid biopsy biomarker that is more sensitive, stable, non-invasive, requires less sample volume, and is more easily accepted by patients. Its successful development will help in the auxiliary diagnosis of liver cancer.

[0013] 2. Products designed based on multiple miRNAs are more sensitive and specific than products designed based on single miRNAs, and can reduce the errors caused by individual expression differences of a single indicator, making the results more accurate.

[0014] 3. The miRNA combined marker proposed in the present invention is not limited to whether the patient is HBV positive and has liver cirrhosis LC symptoms, and has high sensitivity and strong specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The detection results of miRNA panel were used to identify HCC patients and non-HCC patients for ROC curve analysis.

[0016] Figure 2 The ROC curve was used to analyze the test results of liver cancer serological marker alpha-fetoprotein (AFP) to identify HCC patients and non-HCC patients.

[0017] Figure 3 For ROC curve analysis, miRNA panel was used to identify the detection results of patients with liver cancer (HCC) and non-liver cancer, breast cancer (BRCA) and non-breast cancer, lung adenocarcinoma (LUAD) and non-lung adenocarcinoma, lung squamous cell carcinoma (LUSC) and non-lung squamous cell carcinoma, pancreatic cancer (PAAD) and non-pancreatic cancer in the TCGA database. DETAILED DESCRIPTION

[0018] The present invention is further analyzed below in conjunction with specific embodiments.

[0019] Exosome extraction kit Exo-Quick exosome precipitation solution (EXOQ5SA-1, SBI, United States), miRNeasy Serum / Plasma Kit (217184, QIAGEN, Germany), MGIEasy Small RNA Library Prep Kit (1071073, MGI, China) and NGS sequencer were used for miRNA sequencing.

[0020] The sequences of has-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p and hsa-miR-382-5p of the present invention can be obtained through the public website http: / / www.mirbase.org / and determined by the corresponding Accession number. They are existing known sequences and thus will not be explained in detail.

[0021] Example 1 Subjects and Sample Extraction

[0022] The samples were provided by Singapore General Hospital (approved by Singapore SingHealth CIRB B, approval number CIRB Ref: 2007 / 447 / B). Among them, 86 HCC patients, 154 non-HCC liver disease patients, and 12 healthy subjects. Among the non-HCC liver disease patients, 59 were HBV-positive cirrhotic patients, 89 were HBV-positive non-cirrhotic patients, and 6 were HBV-negative cirrhotic patients.

[0023] Example 2 Extraction of exosomes

[0024] Use a disposable syringe to collect 2 ml of peripheral blood from the subject, quickly transfer it into an EDTA anticoagulant tube, and mix it. Separate plasma within 2 hours of collection. The specific steps are as follows: 1) Transfer whole blood to a 2 ml centrifuge tube, centrifuge at room temperature, 500 g for 10 min; 2) Take the upper plasma, centrifuge at 4 ° C, 2000 g for 10 min; 3) Take the upper plasma, centrifuge at 4 ° C, 16000 g for 10 min; 4) The separated plasma is directly used for exosome extraction or frozen in a -80 ° C refrigerator.

[0025] Exosomes were extracted according to the instructions of the exosome extraction kit Exo-Quick exosome precipitation solution (EXOQ5SA-1, SBI, United States). 1) Plasma was added to a centrifuge tube and 63 μl of Exo-Quick was added to every 250 μl of plasma; 2) Centrifuge at 1500 g, 4°C for 30 minutes and discard the supernatant; 3) Centrifuge at 1500 g, 4°C for 5 minutes and discard the supernatant; 4) Resuspend in 200 μl PBS and store at -20°C.

[0026] Example 3 Extraction of exosomal miRNA

[0027] miRNA was extracted according to the instructions of the miRNeasy Serum / Plasma Kit (217184, QIAGEN, Germany). The specific steps are as follows: 1) add 5 times the volume of QIAzol Lysis Reagent to the plasma separated in Example 2, vortex mix or pipette mix; 2) incubate at room temperature for 5 minutes; 3) add an equal volume of chloroform and shake for 15 seconds; 4) incubate at room temperature for 2-3 minutes; 5) centrifuge at 4°C, 12000g for 15 minutes; 6) transfer the upper layer liquid to a new centrifuge tube, add 1.5 times the volume of anhydrous ethanol, pipette mix to obtain a sample; 7) take 700 μl of the sample to RNeasy MinElute spin column (placed in a 2 ml collection tube), cover the lid, centrifuge at room temperature>8000g for 15 seconds, and discard; 8) repeat step 7 once; 9) add 700 μl of RWTbuf to RNeasy MinElute spin column, centrifuge at room temperature>8000g for 15 seconds, and discard; 10) add 500 μl of RPE buf to RNeasy MinElute spin 11) Add 500μl 80% ethanol (freshly prepared) to the RNeasy MinElute spin column, centrifuge at room temperature>8000g for 2min, and discard; 12) Place the RNeasy MinElute spin column in a new collection tube, open the lid, and centrifuge at high speed for 5min; 13) Place the RNeasy MinElute spin column in a 1.5ml collection tube, add 14μl RNase-free water, and centrifuge at high speed for 1min.

[0028] Example 4 NGS sequencing analysis of miRNA

[0029] The extracted miRNA was detected by NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, USA), and the library was constructed using MGIEasy Small RNA Library Prep Kit (1071073, MGI, China), and sequenced using BGISEQ-500. The obtained data were analyzed for miRNA expression levels using three R language packages, namely DESeq2, limma, and edgeR. The differentially expressed miRNAs were considered to be has-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p, and hsa-miR-382-5p.

[0030] Example 5 Validation of miRNA combination for the prediction of primary HCC occurrence

[0031] For subjects such as HCC patients, non-HCC liver disease patients, and healthy people, has-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p, and hsa-miR-382-5p were used as detection objects, and the ROC curve was drawn using the "ROC curve graph" function of SPSS software. The analysis showed that if all four genes were significantly downregulated (|log2 Fold Change|>1.5, p-value<0.05), the AUC value was 0.86 ( Figure 1 ). The AUC value of the serum marker alpha-fetoprotein (AFP) for distinguishing HCC from non-HCC patients was 0.72 ( Figure 2 ).

[0032] Analysis based on data from the TCGA (The Cancer Genome Atlas) database showed that the AUC of the miRNA combination panel of the present invention in HCC was 0.85, the AUC in breast cancer was 0.60, the AUC in lung adenocarcinoma was 0.59, the AUC in lung squamous cell carcinoma was 0.59, and the AUC in pancreatic cancer was 0.52, indicating that the miRNA combination of the present invention has strong specificity in HCC ( Figure 3 ).

[0033] The above embodiments are not limitations of the present invention, and the present invention is not limited to the above embodiments. As long as the requirements of the present invention are met, they belong to the protection scope of the present invention.

Claims

1. Use of a reagent for detecting peripheral blood exosome miRNA combined markers in the preparation of a kit for diagnosing or detecting early liver cancer, characterized in that The miRNA co-markers were hsa-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p and hsa-miR-382-5p derived from plasma exosomes.

2. A kit for diagnosing or detecting early liver cancer, characterized in that The kit includes: a detection reagent for specifically detecting human peripheral blood exosome miRNA hsa-miR-1307-3p, hsa-miR-221-3p, hsa-miR-323b-3p and hsa-miR-382-5p.

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