WO2019086041A1 - Method for assessing risk of liver diseases or cancer by means of micro-ribonucleic acid - Google Patents

Abstract

In the present disclosure, a new micro-ribonucleic acid has been developed by means of mice in which a glycine N-methyltransferase has been knocked out and high-throughput sequencing. The micro-ribonucleic acid can be used to assess whether an individual is at risk of suffering from liver diseases. Accordingly, also disclosed are primers and kits which can amplify the micro-ribonucleic acid by means of a polymerase chain reaction. Further disclosed is an inhibitor which can complement micro-ribonucleic acid and which acts as a pharmaceutical composition for treating liver diseases. Further disclosed is the in situ hybridization staining of a tissue section of an organ or tissue with the micro-ribonucleic acid to assess whether an individual is at risk of suffering from other cancers.

Description

Method for assessing the risk of liver disease or cancer using tiny RNA [Technical Field]

The present invention relates to an evaluation method, kit and pharmaceutical composition. In particular, the present invention relates to a method and kit for assessing whether an individual has a risk of developing liver disease, and a pharmaceutical composition for treating liver disease.

【current technology】

The liver of vertebrates is used to remove the toxicity of various metabolites, to synthesize proteins, and to make biochemical substances needed for digestion. Other roles of the liver in metabolism include regulating glycogen storage, breaking down red blood cells, and making hormones. The liver is also used as an auxiliary digestive gland for the production of bile salts, which are then stored by the gallbladder. The highly specialized tissues of the liver include hepatocytes, which regulate many biochemical reactions, including the synthesis and decomposition of small molecules and complex molecules necessary for normal viability. Since the liver is responsible for many of the above functions in the organism, when these functions are abnormal, it usually indicates that the organism may have liver disease.

Common symptoms of liver inflammation are hepatitis A, B, C, D, and E, which are caused by hepatitis virus infection or by sexual behavior. Inflammation of the liver may also be caused by other viruses of the Herpesviridae family, such as herpes simplex virus. Chronic infections caused by hepatitis B or C viruses are the main cause of liver cancer.

In addition, fatty liver is called when liver cells accumulate more than 5% of liver wet weight or more than 1/3 of liver cells per unit area of liver produce fat accumulation. Fatty liver occurs because triglycerides in the liver are not metabolized by the liver or transported to other parts, causing fat droplets to spread in liver cells, causing excessive infiltration of liver fat. Fatty liver disease can be divided into alcoholic fatty liver disease (AFLD) and nonalcoholic fatty liver disease (NAFLD). AFLD includes alcoholic hepatitis, fatty liver and cirrhosis, and severe liver cancer; NAFLD covers a wide range of liver diseases, including simple liver steatosis (NAFL), nonalcoholic steatosis (NASH), cirrhosis and liver cancer. . In addition, patients with NAFLD may have liver fibrosis.

Liver cancer is a malignant tumor that occurs in the liver or from the liver, and may also be transferred from other parts to the liver, called a liver metastasis. The main cause of liver cancer is hepatitis B caused by hepatitis B, hepatitis C or alcohol. Other causes include aflatoxin, NAFLD, and liver flukes.

Liver disease can be diagnosed or assessed through blood tests and medical imaging, and confirmed by tissue tests, such as the detection of various enzymes in the blood (such as alanine aminotransferase (GPT), aspartate aminotransferase (GOT)), detection Antibody or virus in serum. Methods for treating liver cancer include surgery, targeted therapy, and radiation therapy.

Due to persistent liver inflammation and damage caused by chronic liver disease, the follow-up usually leads to the onset of liver fibrosis, and then gradually fibrosis becomes more and more serious, leading to the development of cirrhosis and even liver cancer. In the distribution of fibrous tissue in liver histopathological sections, from normal to cirrhosis, it is divided into five stages: F0, F1, F2, F3, F4, etc.: F0 is normal liver tissue, F1 is mild fibrosis, and F2 is moderate fibrosis. F3 is severe fibrosis, F4 is a fibrous tissue that has been circled once, and pathologically diagnosed as cirrhosis.

At present, tens of millions of people with cirrhosis or death from liver cancer in the world do not have an effective and universal method to develop early diagnosis or evaluation of biomarker patterns to simulate clinical chronic hepatitis, cirrhosis or liver cancer pathological symptoms. Therefore, it is necessary to develop a convenient, extensive and accurate assessment of biomarkers.

In view of the shortcomings in the prior art, the applicant of this case, after careful experimentation and research, and a perseverance spirit, finally conceived the case and can overcome the shortcomings of the prior art. The following is a brief description of the case.

[Summary of the Invention]

In order to overcome the current failure to have a universal and effective technique for early evaluation of biomarkers, and thus to analyze and simulate various liver diseases (such as liver damage, clinical chronic hepatitis, liver cirrhosis, liver tumor, liver cancer, etc.), the present invention utilizes Generational sequencing technology has developed a potential novel microRNA as a biomarker for the microRNA expression common to both male and female liver cancers. Further, the present invention develops a technique for evaluating liver cirrhosis and early liver cancer biomarkers by serum, in the case where liver microscopic diseases are caused by the change of the microenvironment to release the specific microRNA of the liver tissue to the circulation of the blood system. In addition, the novel microribonucleic acid of the present invention can also be used to accurately detect and confirm the recurrence rate of liver cancer after surgery. Accordingly, the present invention provides an excellent test development platform and a rapid and convenient novel method, reagent, kit and pharmaceutical composition for evaluating biomarkers.

The present invention discloses a method for assessing whether an individual has a risk of developing liver disease, comprising: (a) providing a sample of the individual; and (b) performing quantitative reverse transcription polymerase chain reaction (qRT-PCR) on the sample to obtain a product. (c) confirming whether the product comprises a first picoruclease, the first picoruclease selected from the group consisting of hsa-miR-4791 (SEQ ID NO: 1), hsa-miR-3960 (SEQ ID NO: 2), and hsa-miR At least one of the group consisting of -4532 (SEQ ID NO: 3); and (d) when the product comprises the first picorucleic acid, the individual is identified as having a risk of developing liver disease.

In certain embodiments, the sample is a serum sample or a tissue sample, and step (d) further comprises: (d1) when the first microRNA is hsa-miR-4791, hsa-miR-3960, and hsa-miR-4532 When one of the groups is composed, the individual is assessed as having a risk of developing early liver cancer.

In certain embodiments, step (c) further comprises: (c1) confirming whether the product comprises a second picoruclease, the second picorucleic acid is hsa-miR-4508 (SEQ ID NO: 4) and hsa-miR- At least one of 4492 (SEQ ID NO: 5).

In certain embodiments, when the first picorucleic acid is hsa-miR-3960 and the second picorucleic acid is hsa-miR-4508, the individual is assessed as having a risk of developing liver fibrosis or early cirrhosis. In certain embodiments, when the first picorucleic acid is hsa-miR-4791, hsa-miR-3960, and hsa-miR-4532 and the second picorucleic acid is hsa-miR-4508 and hsa-miR-4492 Individuals are assessed as having a risk of developing liver cancer or a high-risk group for postoperative recurrence of liver cancer. In certain embodiments, when the first picorucleic acid is hsa-miR-4791 and hsa-miR-4532 and the second picorucleic acid is hsa-miR-4508 and hsa-miR-4492, the individual is evaluated as having The risk of developing liver cancer or a high risk group for postoperative recurrence of liver cancer. In certain embodiments, when any of hsa-miR-4791, hsa-miR-3960, hsa-miR-4532, hsa-miR-4508, and hsa-miR-4492 is present in the product, the individual is It was assessed as having a risk of developing liver cancer. In certain embodiments, when the first picorucleic acid is hsa-miR-4532 and the second picorucleic acid is hsa-miR-4508 and hsa-miR-4492, the individual liver cancer cells are assessed to have metastatic potential. In certain embodiments, step (b) further comprises genetically sequencing the product.

The invention further discloses a kit for assessing whether an individual has a risk of suffering from liver diseases, comprising: a plurality of primer sets for quantitative polymerase chain reaction of the individual samples to obtain a product, each pair of primer sets Paired with the first picoruclease, the first picoruclease is selected from the group consisting of hsa-miR-4791 (SEQ ID NO: 1), hsa-miR-3960 (SEQ ID NO: 2), and hsa-miR-4532 (SEQ ID NO: 3) at least one of the group consisting of; and a gene sequencing reagent set for sequencing the product, wherein when the product comprises the first microRNA, the individual is identified as having a risk of developing liver disease.

The invention further discloses a pharmaceutical composition for treating liver disease in an individual, comprising: a first gene fragment, wherein the first gene fragment is complementary to the first picorucleic acid, and the first microRNA is selected from the group consisting of hsa-miR At least one of the group consisting of -4791 (SEQ ID NO: 1), hsa-miR-3960 (SEQ ID NO: 2), and hsa-miR-4532 (SEQ ID NO: 3).

In certain embodiments, the pharmaceutical composition further comprises a second gene fragment complementary to the second picorucleic acid, and the second picorucleic acid is hsa-miR-4508 (SEQ ID NO: 4) and hsa-miR-4492 At least one of (SEQ ID NO: 5).

The invention further discloses a method for assessing whether an individual has a risk of developing cancer comprising: (a) providing a tissue section of the individual from the organ or tissue of the individual; (b) administering the first tiny Ribonucleic acid and a second picoruclease to tissue section and in situ hybridization staining, wherein the first microRNA is selected from hsa-miR-4791 (SEQ ID NO: 1), hsa-miR-3960 (SEQ ID NO: 2) and at least one of the group consisting of hsa-miR-4532 (SEQ ID NO: 3), and the second picorucleic acid is hsa-miR-4508 (SEQ ID NO: 4) and hsa-miR- At least one of 4492 (SEQ ID NO: 5); and (c) when the tissue section is stained, indicating that the organ or tissue of the individual is at risk of developing cancer.

The invention further discloses a method of assessing whether an individual has a risk of developing cancer, comprising: (a) providing a sample of the individual derived from the serum or tissue of the individual; (b) quantifying the reverse transcription polymerase chain of the sample Reaction (qRT-PCR) to obtain a product; (c) confirming whether the product contains a first picoruclease, and the first picoruclease is selected from hsa-miR-4791 (SEQ ID NO: 1), hsa-miR-3960 ( At least one of the group consisting of SEQ ID NO: 2) and hsa-miR-4532 (SEQ ID NO: 3); and (d) when the product comprises the first picorucleic acid, the individual is confirmed to have the cancer Risk of cancer selected from liver cancer, gastric cancer, colorectal cancer, prostate cancer, lung cancer, kidney cancer, breast cancer, cervical cancer, esophageal cancer, ovarian cancer, bladder cancer, lymphoma, skin cancer, pancreatic cancer, and cancer At least one of the group consisting of tongue cancer.

In certain embodiments, step (c) further comprises: (c1) confirming whether the product comprises a second picoruclease, and the second picorucleic acid is hsa-miR-4508 (SEQ ID NO: 4) and hsa-miR- At least one of 4492 (SEQ ID NO: 5).

The term "small RNA (sRNA)" or "small non-coding RNA" refers to a molecule within 200 nucleotides in length, the main function of which is RNA silencing, or For RNA interference. Small ribonucleic acids can be subdivided into double-stranded small interfering RNA (siRNA) and single-stranded microRNAs (miRNAs or miRs).

The term "microRNA (miRNA or miR)" as used herein refers to a non-coding RNA of about 15 to 25 nucleotides in eukaryotes that regulates the expression of other genes. miRNAs are derived from some 300-1000 base primary mini-RNAs (pri-miRNAs) that are transcribed from the DNA but cannot be translated into proteins. They are processed to 70-90 bases and stems (stem). -loop) The pre-mature microRNA (pre-miRNA) of the structure is digested by Dicer to become a functional mature mini-RNA (miRNA). miRNAs regulate post-transcriptional gene expression by binding to messenger RNA (mRNA).

The term "individual" is used to refer to mammals. In certain embodiments, mammals include, but are not limited to, rodents, primates. Rodents include, but are not limited to, rats, mice. Primates include but are not limited to people.

The terms "male mouse" and "mother" in the examples herein refer to "male mouse" and "mother mouse", respectively. In the examples of the present invention, mice are used as examples, but those having ordinary knowledge in the art can be deduced from experimental results of mice to other rodents (e.g., rats) or other mammals.

The term "liver cancer cells" as used herein refers to low metastatic Huh6 and high metastatic Mahlavu cell lines in liver cancer cells, but those of ordinary skill in the art understand any mammalian or rodent liver cancer cell or hepatocellular carcinoma. All are covered by the scope of the invention.

The term "assessment" as used herein refers to an indirect assessment of the disease's intermediate assessment by means of testing or experimentation, and the medical staff assesses whether the individual is at risk of developing the disease based on the assessment.

The term "cirrhosis" in this article refers to the result of pathologically being judged by mild fibrosis and gradually progressing to moderate and severe fibrosis, eventually forming a circle of hepatic fibrous tissue. Cirrhosis also indicates more severe liver fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and advantages of the present invention will become more apparent to those skilled in the <

Figure 1 is a graphical representation of the number of common tumor-associated miRNAs in GNMT knockout mice versus wild type male and female mice.

Figures 2(A), 2(B) and 2(C) are the types of (A) standard pathways for predicting miRNA target genes by IPA, (B) types of diseases and functions, and (C) functions of liver diseases and functions, respectively. Schematic diagram of sexual analysis.

Figure 3 (A) is a schematic diagram showing the ROC analysis of liver cirrhosis evaluated by serum miR-4791, -4532 and -4492.

Figures 3(B), 3(C), and 3(D) assess cirrhosis with (B) miR-4508, (C) miR-3960, and (D) LC miRs panel (miR-3960+miR-4508), respectively. Schematic diagram of ROC analysis.

Figures 4(A), 4(B), 4(C), 4(D), 4(E), 4(F), and 4(G) are respectively (A)miR-4791, (B)miR- 3960, (C) miR-4532, (D) miR-4492, (E) miR-4508, (F) HCC-4miRs panel (miR-4791, -4532, -4492, and -4508) and (G) HCC- The 5miRs panel evaluates the ROC analysis of HCC.

Figure 5 (A) and 5 (B) are the miRNA inhibitors and miRNA mimics for the hepatoma cell line Huh6 for canceration ability test for 8 to 72 hours (A) percentage of viable cells and (B) percentage of dead cells schematic diagram. **P<0.01, ns is not statistically significant.

Figure 5 (C) and 5 (D) are the miRNA inhibitors and miRNA mimics for the hepatocellular carcinoma cell line Mahlavu for canceration ability test for 8 to 72 hours (C) percentage of viable cells and (D) percentage of dead cells schematic diagram. **P<0.01, ns is not statistically significant.

6(A) and 6(B) are schematic diagrams showing (A) percentage of viable cells and (B) percentage of dead cells of a miRNA inhibitor and a miRNA mimetic for the canceration ability of hepatoma cell line Huh6, respectively. *P<0.05, **P<0.01, ***P<0.001, ns was not statistically significant.

Figures 6(C) and 6(D) are schematic diagrams of (A) percentage of viable cells and (B) percentage of dead cells in a hepatocellular carcinoma cell line Mahlavu tested for miRNA inhibitors and miRNA mimics, respectively. **P<0.01, ***P<0.001, ns is not statistically significant.

Figures 7 (A) and 7 (B) are schematic representations of (A) viable cell percentage and (B) percentage of dead cells for miRNA inhibitor treated Huh6 cells for 72 hours, respectively. *P<0.05, **P<0.01, ***P<0.001.

Figures 7(C) and 7(D) are schematic representations of (C) viable cell percentage and (D) percentage of dead cells for miRNA inhibitor treated Mahlavu cells for 72 hours, respectively. *P<0.05, **P<0.01, ***P<0.001.

Figures 8(A) and 8(B) are schematic diagrams showing the (A) migration ability and (B) invasive ability of miRNA inhibitors to inhibit Huh6 cells for 48 hours, respectively. **P<0.01, ***P<0.001.

Figures 8(C) and 8(D) are graphical representations of miRNA inhibitors inhibiting (12) migration ability of Mahlavu cells for 12 hours and (D) invasive ability, respectively. *P<0.05, ***P<0.001.

Figures 8(E) and 8(F) are schematic representations of miRNA inhibition of (E) migration ability and (F) invasive ability of Mahlavu cells, respectively. **P<0.01, ***P<0.001.

[Embodiment]

The inventions set forth in the present application will be fully understood by the following examples, so that those skilled in the art can do so. However, the implementation of the present invention may not be limited to the embodiments by the following embodiments. Other embodiments can be devised by those skilled in the art in light of the spirit of the disclosed embodiments, which are within the scope of the invention.

1. Animal experiment:

Wild-type mice without specific pathogens (SPF) were purchased from the National Laboratory Animal Center. The production, breeding and animal experiments of the mice were reviewed and approved by the Experimental Animal Care and Use Committee of Kaohsiung Medical University. The experimental mice used included: 12-week-old wild-type C57BL/6JNarl (B6) male rats (WTM) and mother rats (WTF), 12-week-old B6 GNMT ^-/- reject male mice (KOM) and mother. Mouse (KOF), B6 GNMT ^-/- male (KMA) and female (KFA) with tumor-adjacent liver tissue (also known as paracancerous tissue), and hepatocellular carcinoma (HCC) B6 GNMT ^-/- male mouse (KMT) and mother mouse (KFT).

2. RNA preparation:

Mice were sacrificed by carbon dioxide for dissection, tissue was placed in liquid nitrogen for rapid freezing or RNA isolation was performed immediately. use

Total RNA was isolated by reagent (Invitrogen, Carlsbad, CA, USA). The isolated RNA was treated with DNase I to remove contamination of residual genomic DNA. The purity and concentration of the RNA samples were determined by UV spectroscopy (NanoDrop, Wilmington, DE, USA) and RNA integrity was further determined in an Agilent Bioanalyzer (Santa Clara, CA, USA). When performing RNA sequencing, all samples have 8 or better RNA Integrity Number (RIN).

3. Small RNA (small RNA) sequencing:

According to the manufacturer's instructions, to a small RNA samples TruSeq ^TM preparation kit (Illumina Inc., San Diego, USA ) to build a library of small RNA from the total RNA. Briefly, 1 to 4 μg of total RNA was sequenced with RNA 3' linker and RNA 5' using cut-off T4 RNA-binding enzyme 2 (New England Biolabs, Ipswich, MA, USA) and T4 RNA-binding enzyme, respectively. The joint is glued. RNA ligated to the 5' and 3' ends was converted to the first cRNA using SuperScript II (Invitrogen, Carlsbad, CA, USA) and Illumina miniRNA reverse transcription-primers. The obtained first strand of cDNA was subjected to 11 cycles of PCR amplification using a small RNA primer set (Illumina Inc., San Diego, CA, USA). The PCR amplified library was purified using a 6% Novex TBE polyacrylamide gel electrophoresis (PAGE) gel (Invitrogen, Carlsbad, CA, USA). The amplified small RNgilent 2100 Bioanalyzer uses a highly sensitive DNA chip (Agilent) and is double-ended sequenced in HiSeq2000 (Illumina Inc., San Diego, CA, USA) with a 100 nucleotide read length (Illumina paired) -end sequencing 100bp). Each library of amplifications is prepared with unique index primers such that the library of amplifications can be assembled into first-class cell lanes.

4. Small RNA sequencing data analysis:

The analysis process consists of six steps: (1) trimming the adapter sequence and quality control with the FASTX tool kit (http://hannonlab.cshl.edu/fastx_toolkit/); (2) comparing the Bowtie software with the miRDeep2 software For the reference genome; (3) calculate the expression level by RNA-Seq by Expectation Maximization (RSEM); (4) normalize the miRNA expression level per million readings (RPM); 5) using a log2 fold change to rise to 1.8 fold and a decrease of 0.6 fold to confirm miRNAs with differential expression; and (6) using the Encyclopedia of DNA Elements (ENCODE) and the Cancer Genome Atlas (The Cancer Genome) Altas, TCGA) database analysis of miRNA expression profiles with log2 fold change P < 0.05.

5. Analysis of functional categories:

use

Ingenuity Pathway Analysis (IPA) and the Database of Annotation (Visualization and Integrated Discovery, DAVID) web tools (http://david.abcc.ncifcrf.gov/) and Kyoto Gene and The functional type is analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) Path Database (http://www.genome.jp/kegg/pathway.htm).

6.microRNA real-time polymerase chain reaction (microRNA RT-qPCR):

Cell and tissue RNA was extracted with 100 μl of serum using Trizol (Invitrogen, Carlsbad, CA, USA), and 10 fmol (5.6 x 10 ⁸ copies) of cel-miRNA-39 mimic (Qiagen) was added as a calibration quantitative control to a final concentration of 1 fmol. Direct-zol and to trace RNA preparation kit (Zymo Research) extracting RNA, to ^{All-in-One TM miRNA qRT} -PCR primers and detection kit (Genecopoeia) for RT-qPCR.

7. Cell transfection with RNA oligonucleotides:

Using hsa-miR-4791, -3960, -4532, -4492, and -4508 inhibitors (hairpin RNA complementary to the microRNA sequence) / mock (double-stranded RNA identical to the microRNA sequence) and negative control cel -miR-67 inhibitor/mimetic (Dharmacon, USA) or scrambled siRNA sequence: 5'-UUC UCC GAA CGU GUC ACG UTT-3' (Justice) and 5'-ACG UGA CAC GUU CGG AGA ATT-3' (antisense stocks) served as a negative control. Oligonucleotide transfection was performed with TransIT TKO Reagent (Mirus, USA) or lipofectamine RNAiMAX (Invitrogen), and samples were collected 36 hours after transfection, followed by analysis.

8. Results:

Samples of 8 groups of mice include 12-week-old wild-type male and female liver tissues (WTM and WTF), GNMT ^-/- eliminated male and female liver tissues (KOM and KOF), GNMT ^-/- HCC tissues (KMT, and KFT) and adjacent liver tissues (KMA and KFA) of mice and mothers. Table 1 shows the small RNA sequencing reading alignments and distributions of the 8 groups of mice, where the original reading is the number of sequences detected by RNA sequencing, and the query reading is the number of sequences that have been clipped by RNA, but can be matched and read. Values are the number of sequences mapped to known mouse RNA or genome. miRNAs greater than or equal to 18 nucleotides have preferred enantiomeric matching reads (results not shown), while sequence reads are between 21 and 25 nucleotides in length ranging from about 36.8% to 64.9%. Next, miRNA profiling was performed to analyze new miRNAs in HCC during the period from early (12 weeks old) to late (HCC formation) in GNMT ^−/− male and female rats. In addition, the following research has been conducted to identify miRNAs and new miRNAs with significant changes in tissues and serum, which can be used to detect liver fibrosis and early HCC as potential biomarkers. .

Table 1. Statistics of reading and distribution of raw data of small RNA sequencing

In addition, mini-RNA deep-sequence analysis confirmed the gene annotation and distribution of small RNAs. As shown in Table 2, most of the fragments were miRNAs.

Table 2. Genomic annotation and distribution of small non-coding RNAs

*piRNA:piwi-interacting RNA

**snRNA: small nuclear RNA

Please refer to Figure 1, which is a schematic diagram showing the number of common tumor-associated miRNAs in GNMT knockout mice and wild type male and female mice. In FIG. 1, a new miRNA is predicted from an unnotated region of sequencing data using miRdeep 2.0 software, using a fold change equal to or greater than 1.8 times and equal to or less than 0.6 times as a critical value. When mice with hepatocellular carcinoma (HCC) tissue were compared with wild-type mice (control group), there were statistically significant differences in the differential expression of 36 miRNAs, of which 24 were known (miRbase). The miRNA and 10 new miRNAs are up-regulated (in the wild-type control group, there is a fold change equal to or greater than 1.8 times, see the number surrounded by two circles in the upper left and lower left panels of Figure 1), and 12 The known miRNA and the four new miRNAs are down-regulated (equal changes of 0.6 times or less, see the number enclosed by the intersection of two circles in the upper right and lower right panels of Figure 1). That is, 24 known up-regulated miRNAs and 12 known down-regulated miRNAs were found in the HCC tissues of GNMT ^-/- male and female rats, and 10 up-regulated novel miRNAs and 4 down-regulations were found. Novel miRNAs. Small RNA sequence analysis revealed that known miRNA profiles have differential expression in all samples.

Next, compare and analyze tumor tissue with non-tumor tissue using the miRbase 20th Edition database and the University of California Santa Cruz (UCSC) genomic database, from known/new mice. The BLAST corresponding genes of the miRNA and the human miRNA are aligned to find the homologous position of the corresponding miRNA with high similarity to the human gene. Thus, as shown in Table 3, from 3 new mouse miRNAs (ie KMU-"miR"-337 (5'-ggg gcg gcg gcg gcg gcg gcg acu c-3'), KMU-"miR"-422 (5'-ccc ggg gag ccc ggc ggg-3') and KMU-"miR"-708 (5'-cgg ggc ugg gcg cgc gcg-3')) confirmed 4 human miRNAs (hsa-miR-3960, respectively) (5'-ggc ggc ggc gga ggc ggg gg-3'; SEQ ID NO: 2; abbreviated as miR-3960), hsa-miR-4532 (5'-ccc cgg gga gcc cgg cg-3'; SEQ ID NO :3; abbreviated as miR-4532), hsa-miR-4492 (5'-ggg gcu ggg cgc gcg cc; SEQ ID NO: 5; abbreviated as miR-4492) and hsa-miR-4508 (5'-gcg ggg Cug ggc gcg cg-3'; SEQ ID NO: 4; abbreviated as miR-4508)).

Table 3. Homologous miRNAs and homologous sequences associated with new mouse homologous miRNAs

Then, further compare the cancer Genome Atlas of Liver Cancer (TCGA-LC) database to find 11 positively regulated miRNAs (including hsa-miR-146b-5p, -132- 3p, -425-5p, -140-3p, -181a-5p, -182-5p, -183-5p, -96-5p, -21a-5p, -34a-5p and -10b-5p), 7 Down-regulated miRNAs (including hsa-miR-451a, -144-3p, -101-3p, -99a-5p, -125b-5p, -145a-3p, and -122-5p) and five data-free miRNAs (including hsa-miR-4791, -3960, -4532, -4508, and -4492) (see Table 4). To confirm whether the expression levels of these miRNAs in mouse liver cancer are consistent with the clinical findings of the TCGA-LC database, for 49 pairs of HCC tumors and adjacent tumor tissues and another group of HCC patients (371 HCC tumor tissues from the TCGA database) And 49 non-tumor tissue components) for analysis. The results confirmed that the amount of miRNA expression in mice was similar to that of clinical hepatocellular carcinoma in the TCGA-LC database (see Table 4). In addition, a novel miRNA (hsa-miR-4791; 5'-ugg aua uga uga cug aaa-3'; SEQ ID NO: 1; abbreviated as miR-4791) which was not presented in the TCGA-LC database was also confirmed.

Table 4. Expression profiles of tumor-associated miRNAs (known miRNAs) commonly found in TCGA-LC databases on miRNA sequences

a statistically significant results (bold)

b Multiple change - HCC_372T/49TA is greater than 1 in the direction of change with a p value of <0.05 and is adjusted upwards. Conversely, the direction of change with a fold change - HCC_372T/49TA less than 1 is a p value < 0.05 and is downward adjusted.

T: tumor tissue; TA: adjacent tumor tissue; NT: non-tumor tissue; TCGA-LIHC: cancer genome mapping program - hepatocellular carcinoma

In addition, 14 novel miRNAs (containing 10 up-regulated and 4 down-regulated novel miRNAs) of GNMT ^-/- mice indicated in Fig. 1 were also analyzed for expression levels, and the results are shown in Table 5.

Table 5. 14 new miRNA expression levels in GNMT ^-/- mice

Next,

Path analysis (IPA) and DAVID network tools predict functional analysis of miRNA target genes. Based on the predicted results of miRTarbase and TargetScan 7.0, these 11 up-regulated miRNAs and 7 down-regulated miRNAs were expected to target all 1594 genes and analyzed for their (A) standard pathway type, (B) disease. And the type of function and (C) functional analysis of liver disease and function.

Please refer to FIG. 2(A), which is a schematic diagram showing the types of standard pathways for predicting miRNA target genes by IPA. Figure 2 (A) The 1-34 items on the abscissa are in order: (1) the molecular mechanism of cancer, (2) p53 information transmission, (3) cell cycle: G1/S checkpoint regulation, (4) Fang Hydrocarbon receptor signaling, (5) STAT3 pathway, (6) PTEN signaling, (7) NF-κB signaling, (8) regulation of epithelial-mesion transition, (9) HGF signaling, (10) Glucocorticoid receptor signaling, (11) Wnt/β-catenin signaling, (12) IL-8 signaling, (13) IL-6 signaling, and (14) LPS-stimulated MAPK signaling , (15) ERK/MAPK signaling, (16) RAR activation, (17) EGF signaling, (18) PPARα/RXRα activation, (19) Myc-regulated apoptosis signaling, (20) by TSP1 Inhibition of angiogenesis, (21) liver fibrosis/hepatic stellate cell activation, (22) PPAR information transmission, (23) T cell receptor information transmission, (24) toll-like receptor Body information transmission, (25) IL-17 information transmission, (26) acute phase response information transmission, (27) HIF1α information transmission, (28) TGF-β information transmission, (29) insulin receptor information transmission, (30) ) mTOR information transmission, (31) IGF-1 information transmission, (32) ) Type II diabetes information transmission, (33) fat production pathway, (34) tight junction information transmission. As can be seen from Fig. 2(A), 11 up-regulated miRNAs and 7 down-regulated miRNAs mainly point to cancer molecular mechanisms, p53 signaling, and cell cycle associated with cancer signaling pathways.

Please refer to FIG. 2(B), which is a schematic diagram of the types of diseases and functions of miRNA target genes predicted by IPA. Figure 2 (B) The first to the 27th items on the abscissa are: (1) RNA expression, (2) cell viability, (3) cell migration, (4) cell invasion, and (5) cell colony formation. , (6) cell type, (7) metastasis, (8) DNA synthesis, (9) cell transformation, (10) protein metabolism, (11) cell cytotoxicity of tumor cell lines, (12) joint inflammation Role, (13) cytoskeletal organization, (14) DNA damage response of cells, (15) cell communication, (16) development of induced pluripotent stem cells, (17) cellular glycolysis, (18) glycogen Synthesis, (19) DNA fragmentation, (20) autophagy, (21) granulosa gland dysfunction, (22) cell constant, (23) drug resistance of tumor cell lines, (24) active oxygenates Production, (25) viral infection, (26) lipid formation, and (27) protein breakdown. As can be seen from Figure 2(B), 11 up-regulated miRNAs and 7 down-regulated miRNAs are mainly directed to the function of cancer cells (eg, RNA expression, cell viability, cell migration, cell invasion, cell colony formation). )related.

Please refer to FIG. 2(C), which is a functional analysis diagram of liver disease and function of predicting miRNA target genes by IPA. As can be seen from Fig. 2(C), 11 up-regulated miRNAs and 7 down-regulated miRNAs mainly point to hepatocellular carcinoma, increased alkaline phosphatase activity of hepatocyte apoptosis, and worsened liver metastasis. Therefore, by integrating the results of Figure 2(A)-2(C), the functional roles and information transmission pathways of five novel miRNAs and cell proliferation and metastasis were further evaluated. The function of a potential target is related to the negative regulation of the cell cycle, immune response, and inflammatory effects.

Next, 60 paired HCC samples from the Taiwan Liver Cancer Network (TLCN) cohort were used to validate five novel miRNAs (miR-4791, -3960, -4532, -4508, and -4492). The expression levels of these five miRNAs were compared between adjacent liver samples (TA) and liver tissues (T) of matched HCC samples, and the expression was quantified by reverse transcription polymerase chain reaction (qRT). - PCR) OK. The expression level of each sample was normalized to the sample mean of adjacent liver tissue of the tumor. A significant increase in the extent of the five novel miRNAs in the liver tissue was observed. Consistent with the results of the mouse model, the expression levels of these five miRNAs (miR-4791, -3960, -4532, -4508, and -4492) were found in the liver cancer tumor tissues of 60 matched patients. It is rising (results not shown).

Next, the expression of these 5 miRNAs in healthy persons (control group, n=36), cirrhosis patients (n=51), and HCC patients (n=55) was examined. The expression levels of the five miRNAs were confirmed by qRT-PCR of serum samples, and the expression amount was normalized to cel-miR-39 (control group). Five miRNAs were observed to have a significant increase in HCC patients, and that miR-3960 and miR-4508 were significantly higher in cirrhotic patients than in healthy controls (control group) (results not shown).

Next, the five miRNAs (miR-4791, -3960, -4532, -4508, and -4492) were evaluated by the operator operating characteristic (ROC) curve to assess whether the individual was at risk for HCC and/or cirrhosis. Non-invasive assessment markers. As shown in Figures 3(A), 3(B), 3(C), and 3(D), miR-4508 and miR-3960 showed an area under the curve of the ROC (Area under curve, AUC) of 0.73 and 0.79, respectively. (See Figures 3(B) and 3(C)), and the combined group of these two miRNAs (a mixture of miR-4508 and miR-3960, also known as the LC miRs panel) has a fairly high AUC in patients with cirrhosis. The value is 0.84 (see Figure 3(D)). Thus, when an individual's serum was tested to have miR-3960 and miR-4508, the individual was assessed as having a risk of developing liver fibrosis or early cirrhosis.

Please refer to Figures 4(A) to 4(G), which are (A)miR-4791, (B)miR-3960, (C)miR-4532, (D)miR-4492, (E)miR- 4508, (F) HCC-4miRs panel and (G) HCC-5miRs panel to evaluate the ROC analysis of HCC. The AUC of the five miRNAs alone were 0.79 (miR-4791, Figure 4(A)), 0.74 (miR-3960, Figure 4(B)), 0.81 (miR-4532, Figure 4(C)), 0.76 ( miR-4492, Figure 4(D)), 0.88 (miR-4508, Figure 4(E)), and a combined group of 4 miRNAs (HCC-4miRs panel containing miR-4791, miR-4532, miR-4492 and The AUC of miR-4508, excluding miR-3960, see Figure 4(F), was 0.95, and the combined set of 5 miRNAs (HCC-5miRs panel, see Figure 4(G)) was 0.96. Further, as shown in Table 6, the optimal threshold for the sum of sensitivity and specificity is the maximum for cirrhosis and HCC, and the Yoden index (the maximum potential efficacy of biomarkers) is the general ROC curve. Total measurement. The combined group of 4 miRNAs (HCC-4miRs panel) and the combined group of 5 miRNAs (HCC-5miRs panel) can distinguish HCC patients from healthy people (control group) as potential biomarkers for HCC, and 2 A pooled set of miRNAs (LC miRs panel) can be used as a biomarker for cirrhosis (see Table 7). Thus, when an individual's serum was tested to have miR-4791, miR-3960, miR-4532, miR-4508, and miR-4492, the individual was assessed as having a risk of developing cirrhosis and liver cancer.

Table 6. Evaluation of miR-4791, -3960, -4532, -4508, and -4492 in serum samples from patients with HCC and cirrhosis

*5 in 1: HCC-5miRs panel

Table 7. Expression levels of five serum miRNAs in cirrhosis and HCC patients

Pearson chi-square test, ***: <0.001, **: <0.01

Next, the monitoring values of the serum miRNA pooled group were determined by the cut-off value to compare the pre-operative and post-operative conditions of the patients in the non-recurrent group and the relapse group. Here, the combined components of 5 miRNAs, 4 miRNAs, and 5 miRNAs were low and high. As shown in Table 8, the combination of 5 miRNAs, 4 miRNAs, and 5 miRNAs was associated with recurrence and non-recurrence after surgery. Moreover, after surgical resection of tumor tissues in the non-recurrent group, the expression levels of the five individual miRNAs and the combined groups were significantly reduced.

Table 8. Comparison of the expression levels of five miRNAs between the pre-operative period and the post-operative period without recurrence and relapsed patients

Fisher's accuracy test, ***: <0.001, **: <0.01, *: <0.05, #: <0.1

The expression levels of five miRNAs in patients with no recurrence and recurrence were analyzed from preoperative to postoperative period:

This is to analyze the expression of 5 miRNAs in patients with non-recurrence (n=9) from 1.58 to 7.25 years before surgery and from 0.25 to 6.00 years after surgery (n=24) before surgery. The amount is determined by the Mann Whitney U test and the Wilcoxon test. In these 33 HCC patients, the amount of miRNA expression in the serum did not decrease after surgery (results not shown). Next, the expression levels of five miRNAs in the unrelapsed and relapsed groups of HCC patients before and after surgery were analyzed. The expression levels of the two miRNA pooled groups (mixed miR-4508 and miR-3960) were significantly reduced in the post-operative samples compared to the non-recurrent group of the pre-operative samples (results not shown). Furthermore, the expression levels of the three miRNAs (miR-3960, -4532, and -4508) were significantly lower in the non-relapsed samples than in the pre- and post-operative miRNA expression of the relapsed samples (results not shown) ).

Five miRNA inhibitors and miR-4791 mimics induce liver cancer cell death:

In this test, living cells were detected by living cell fluorescent dye Calcein AM (Invitrogen, Carlsbad, CA, U.S.A.), and dead cells were detected with the dye Ethidium homodimer (EthD-III). The liver cancer cell lines used were Huh6 and Mahlavu.

First, Huh6 and Mahlavu cells (3000well/96well disks) were transfected with miRNA inhibitors or mimics (50 nM total) for 8, 24, 48 to 72 hours. Live cells in Huh6 and Mahlavu cells were stained with Calcein AM, dead cells in Huh6 and Mahlavu cells were stained with EthD-III, and live cells and dead cells were each negatively controlled for 8 hours (NC, Caenorhabditis elegans) (Caenorhabditis elegans) miRNAs are standardized.

As shown in Figures 5(A) to 5(D) and 6(A) to 6(D), the five new miRNAs (miR-4791, -3960, -4532, -4508 and -4492) inhibitors each effectively reduced live cells in hepatoma cells Huh6 and Mahlavu and increased their dead cells, and these five new miRNAs (miR-4791, -3960, -4532, -4508, and -4492) Compared to the inhibitor, the mimetic did not significantly increase the viable cells in the liver cancer cells Huh6 and Mahlavu. Therefore, the inhibitors of these five novel miRNAs (miR-4791, -3960, -4532, -4508, and -4492) are effective for inhibiting the growth of liver cancer cells, and can be used as a pharmaceutical composition for inhibiting the treatment of liver cancer.

Five miRNA inhibitors induce liver cancer cell death:

In this assay, miRNA inhibitors (10 nM) were mixed with C. elegans miRNA (40 nM), individual miRNA5 inhibitors (50 nM), or 5 miRNA inhibitors (10 nM each, 50 nM each) were mixed. Huh6 and Mahlavu cells were transfected for 72 hours. Live cells in Huh6 and Mahlavu cells were stained with Calcein AM, dead cells in Huh6 and Mahlavu cells were stained with EthD-III, and live cells and dead cells were each negatively controlled for 8 hours (NC, C. elegans miRNA). Standardize. As shown in Figures 7(A) to 7(D), the five miRNAs (miR-4791, -3960, -4532, -4508, and -4492) were combined together with low doses, respectively, compared to the negative control group. The enhanced mode inhibits the growth of liver cancer cells Huh6 and Mahlavu. Therefore, it has been confirmed again that the inhibitors of these five novel miRNAs (miR-4791, -3960, -4532, -4508, and -4492) can effectively inhibit the growth of liver cancer cells, and can be used as a pharmaceutical composition for inhibiting the treatment of liver cancer.

Inhibition effect of five miRNA inhibitors on migration and invasion of liver cancer cells:

In this study, the mitigation migration assay and invasion assay were used to study the inhibitory effects of five miRNA inhibitors or mimics on the migration and invasion of liver cancer cells Huh6 and Mahlavu at 12, 24 and 48 hours. All experiments were repeated 3 times independently and the data were calculated as the mean of the three replicates and presented as mean ± standard deviation.

In the perforation migration assays of Figures 8(A) and 8(C), miR-4532, miR-4492 and miR-4508 have a high effect on the metastasis of Huh6 and Mahlavu cells, and the inhibitors do not metastasize to low metastatic Huh6 cells. The effects of low metastatic Mahlavu cells were significantly inhibited. In the invasion ability tests of Figures 8(B) and 8(D), both miR-4352 and miR-4508 enhanced the invasive ability of Huh6 and Mahlavu cells, and the inhibitors were all attenuated. There was no statistically significant effect of miR-4492 on the invasive ability of liver cancer cells. In addition, the results of Figures 8(E) and 8(F) showed that the total inhibitors were 15 nM miR-4532, 15 nM miR-4492, 15 nM miR-4508, and 2 at a total transfection concentration of 45 nM. miRNA inhibitors mixed (miR-4352 and miR-4508 5nM plus 35nM C. elegans miRNA negative control group, 45nM total), 3 miRNA inhibitors mixed (5nM miRNA plus 30nM C. elegans miRNA negative control group) 45nM), inhibitor 45nM miR-4532, 45nM miR-4492, 45nM miR-4508, 2 miRNA inhibitors mixed (15nM miR-4352, 15nM miR-4508 and 15nM C. elegans miRNA negative control group) or 3 The miRNA inhibitors were mixed (15nM miR-449, 15nM miR-4532 and 15nM miR-4508), and the mixture of three miRNA inhibitors significantly inhibited the migration and invasion ability of liver cancer cells. Taken together, these results demonstrate that overexpression of miRNA-3960, miRNA-4532, miRNA-4492 and/or miRNA-4508 allows hepatoma cells to enter cell arrest and miR-4532, miR-4492 and/or miR-4508 Overexpression promotes the migration and invasion of liver cancer cells. Therefore, the miRNA inhibitor complementary to miR-4532, miR-4492 and/or miR-4508, and the medicinal composition prepared by the miRNA inhibitor can inhibit the migration and invasion ability of liver cancer cells, thereby allowing the growth of liver cancer cells. Stopped and entered apoptosis.

Assessment of multiple organ tumor differences by miRNA in situ hybridization:

This experiment is based on miRNA in situ hybridization to stain formalin-fixed paraffin embedded (FFPE) sections to evaluate multiple organ tumors (cancer tissue matrix, BCN962a) , Bioman). This experiment was performed by in situ hybridization (ISH) staining of paraffin-embedded sections to confirm the expression of miR-4791, miR-3960, mir-4532, miR-4492 and miR-4508 in different cancer tissues. First, the lock nucleic acid (LNA)-based probes labeled with ISH DIG are miR-4791 probes (5'-TTT CAG TCA TCA TAT CCA-3'), miR-3960 probes (5'-CCC CCG CCT). CCG CCG CCG CC-3'), mir-4532 (microRNA precursor) probe (5'-CAG GAT ACC AGG AGC TTC ACC GC-3'), miR-4492 probe (5'-GGC GCG CGC CCA GCC CC-3'), and miR-4508 probe (5'-CGC GCG CCC AGC CCC GC-3'). Control group probes include a negative control group probe (heterologous miRNA) and a positive control group probe (U6 small nuclear RNA (snRNA)). (H) The total integration of miRNA cell forward is determined by a randomly selected microscope field of view. The mean is normalized to the U6 snRNA score and the original magnification is 200X and 400X. LNA U6 snRNA (0.75 nM), LNA miRNA probe and hybrid miRNA probe (40 nM per probe) were hybridized for 16 hours at 45 °C. Multiple tissue tumors or adjacent normal tissues with adjacent tissues of cancer include 17 cancer organs (liver, stomach, large intestine, prostate, lung, kidney, breast, cervix, esophagus, with matching or mismatched adjacent normal tissues) Ovarian, bladder, lymph nodes, skin, pancreas, testicles, tongue, and placenta), including tumor-nodule-metastasis (TNM) classification, clinical stage, and pathological grade (60 cases/ 96 core). Each organ or tissue is taken from three normal human individuals, with a single core in each case. T is a tumor, including hepatoma, esophageal squamous cell carcinoma, gastric adenocarcinoma, colorectal adenocarcinoma, prostate adenocarcinoma, lung adenocarcinoma, renal bright cell carcinoma, breast invasive ductal carcinoma, cervical scale Cell carcinoma, ovary high grade severe papillary adenocarcinoma, low bladder urinary epithelial carcinoma, right lower jaw lymph node, large B-cell lymphoma, scalp squamous cell carcinoma, pancreatic ductal adenocarcinoma, testicular embryo Sexual cancer, and tongue embryonal rhabdomyosarcoma. TA is: normal adjacent tissue, adjacent tissue of liver cancer, normal adjacent tissues of esophagus, normal adjacent tissues of the stomach, normal adjacent tissues of the large intestine, benign prostatic hyperplasia, normal adjacent tissues of the lungs, normal adjacent tissues of the kidney, normal adjacent to the breast Tissue (breast adenosis with ductal dilatation), normal adjacent tissues of the cervix, normal adjacent tissues of the ovary, normal adjacent tissues of the bladder, reactive lymph node hyperplasia, normal adjacent tissues of the scalp, normal adjacent tissues of the pancreas, The normal adjacent tissue and placenta tissue of the testis.

As shown in Table 9, miRNA-4791 is expressed in liver, prostate, lung, breast, cervix, ovary, bladder, lymph nodes, skin, pancreas, and testicular tumor tissue compared to adjacent tumor tissue (TA). ) Significantly higher, and significantly lower in the stomach, large intestine, and esophageal tumor tissue (T). Compared with adjacent tumor tissues (TA), miR-3960 expression levels were significantly higher in liver, breast, cervix, esophagus, bladder, lymph nodes and skin tumor tissues (T), but in the stomach, large intestine, kidney, and testicular pills. Tumor tissue (T) was significantly lower. Compared with adjacent tumor tissues (TA), the expression level of mir-4532 is significantly higher in liver, prostate, breast, bladder, lymph node, skin and pancreatic tumor tissues (T), but in the stomach, large intestine, kidney, and child. Cervical, esophageal, ovarian, and testicular tumor tissues (T) were significantly lower. Compared with adjacent tumor tissues (TA), miR-4492 expression levels were significantly higher in liver, lung, breast, cervix, skin, pancreas and testicular tumor tissues (T), but in the stomach, large intestine, prostate, Renal, esophageal, ovarian, bladder, and lymph node tumor tissues (T) were significantly lower. Compared with adjacent tumor tissues (TA), the expression level of miR-4508 was significantly higher in liver, prostate, kidney, breast, cervix, ovary, lymph node, skin, pancreas and testicular tumor tissue (T). The stomach, large intestine, lung, and esophageal tumor tissue (T) were significantly lower.

Table 9. Different miRNA expression levels in multiple organ tumors

Table 9. Different miRNA expression levels in multiple organ tumors (continued)

^A density analysis was performed using ImageJ software. The total score of positive for miRNA cells is determined by a randomly selected microscope field of view. The mean is the normalization of U6snRNA scores. Values are expressed as mean ± standard deviation.

^b All P values are from a two-tailed t-test.

The change direction of ^c T>TA is upward adjustment and the p value is <0.05. Conversely, the direction of change of T < TA is downward adjustment and the p value is <0.05. T: tumor tissue; TA: tumor adjacent tissue.

The invention is a difficult and innovative invention, and has profound industrial value, so the application is made according to law. In addition, the invention may be modified by those skilled in the art without departing from the scope of the appended claims.

Claims (12)

1. A method of assessing whether an individual has a risk of developing liver disease, including:

   (a) providing a sample of the individual;

   (b) performing quantitative reverse transcription polymerase chain reaction (qRT-PCR) on the sample to obtain a product;

   (c) confirming whether the product comprises a first picorucleic acid selected from the group consisting of hsa-miR-4791 (SEQ ID NO: 1), hsa-miR-3960 (SEQ ID NO: 2), and hsa- At least one of the group consisting of miR-4532 (SEQ ID NO: 3);

   (d) When the product contains the first picorucleic acid, the individual is confirmed to be at risk of developing the liver disease.
2. The method according to claim 1, wherein the sample is a serum sample or a tissue sample, and step (d) further comprises:

   (d1) When the first picorucleic acid is one of the groups consisting of hsa-miR-4791, hsa-miR-3960, and hsa-miR-4532, the individual is evaluated as having a risk of developing early liver cancer.
3. The method of claim 1 wherein step (c) further comprises:

   (c1) confirming whether the product comprises a second picoruclease, the second picorucleic acid nucleic acid being at least one of hsa-miR-4508 (SEQ ID NO: 4) and hsa-miR-4492 (SEQ ID NO: 5) By.
4. The method according to claim 3, wherein when the first picoRNA is hsa-miR-3960 and the second picoRNA is hsa-miR-4508, the individual is evaluated as having hepatic fibers The risk of cirrhosis or early cirrhosis.
5. The method according to claim 3, wherein when the first picorucleic acid is hsa-miR-4791, hsa-miR-3960 and hsa-miR-4532 and the second picorucleic acid is hsa-miR- At 4508 and hsa-miR-4492, the individual was assessed as having a risk of developing liver cancer or a high risk group for postoperative recurrence of liver cancer.
6. The method according to claim 3, wherein when the first picorucleic acid is hsa-miR-4791 and hsa-miR-4532 and the second picorucleic acid is hsa-miR-4508 and hsa-miR- At 4492, the individual was assessed as having a risk of developing liver cancer or a high-risk group for postoperative recurrence of liver cancer.
7. The method according to claim 3, wherein any one of hsa-miR-4791, hsa-miR-3960, hsa-miR-4532, hsa-miR-4508, and hsa-miR-4492 is present in the At the time of the product, the individual was assessed as having a risk of developing liver cancer or a high risk group for postoperative recurrence of liver cancer.
8. The method according to claim 3, wherein when the first picorucleic acid is hsa-miR-4532 and the second picorucleic acid is hsa-miR-4508 and hsa-miR-4492, the individual Liver cancer cells were evaluated as having metastatic ability.
9. The method of claim 1 wherein step (b) further comprises genetically sequencing the product.
10. A method for assessing whether an individual has a risk of developing cancer, including:

    (a) providing a tissue section of the individual from which the organ or tissue of the individual is;

    (b) administering a first microRNA and a second picoruclease to the tissue section and performing in situ hybridization staining, wherein the first microRNA is selected from the group consisting of hsa-miR-4791 (SEQ ID NO: 1), hsa- At least one of the group consisting of miR-3960 (SEQ ID NO: 2) and hsa-miR-4532 (SEQ ID NO: 3), and the second microRNA is hsa-miR-4508 (SEQ ID NO) At least one of: 4) and hsa-miR-4492 (SEQ ID NO: 5);

    (c) When the tissue section is stained, it indicates that the organ or tissue of the individual is at risk of developing cancer.
11. A method of assessing whether an individual has a risk of developing cancer, including:

    (a) providing a sample of the individual derived from the serum or tissue of the individual;

    (b) performing quantitative reverse transcription polymerase chain reaction (qRT-PCR) on the sample to obtain a product;

    (c) confirming whether the product comprises a first picorucleic acid selected from the group consisting of hsa-miR-4791 (SEQ ID NO: 1), hsa-miR-3960 (SEQ ID NO: 2), and hsa- At least one of the group consisting of miR-4532 (SEQ ID NO: 3);

    (d) when the product comprises the first picorucleic acid, the individual is confirmed to be at risk of developing the cancer, wherein the cancer is selected from the group consisting of liver cancer, gastric cancer, colon cancer, prostate cancer, lung cancer, kidney cancer, breast cancer, cervical cancer At least one of the group consisting of esophageal cancer, ovarian cancer, bladder cancer, lymphoma, skin cancer, pancreatic cancer, testicular cancer, and tongue cancer.
12. The method of claim 11 wherein step (c) further comprises:

    (c1) confirming whether the product comprises a second picoruclease, the second picorucleic acid nucleic acid being at least one of hsa-miR-4508 (SEQ ID NO: 4) and hsa-miR-4492 (SEQ ID NO: 5) By.

Images