CN103374630B - Methods to detect the risk of liver cancer - Google Patents

Abstract

The present invention relates to a method for detecting the probability of an individual suffering from liver cancer, comprising detecting the methylation level or expression level of microRNA miR-129-2 in a biological sample from the individual. When the methylation level of miR-129-2 in the biological sample is higher than the methylation level of miR-129-2 in a control sample, or the expression level of miR-129-2 in the biological sample is lower than the expression level of miR-129-2 in the control sample, it indicates that the individual is prone to or has suffered from liver cancer.

Description

Methods to detect the risk of liver cancer

FIELD OF THE INVENTION The present invention relates to biomarkers for human liver cancer.

Background technique

MicroRNAs (miRNAs) are short endogenous RNAs of approximately 22 nucleotides. By base-pairing with target mRNAs, miRNAs act as post-transcriptional regulators of gene expression, leading to disintegration of target mRNAs or inhibition of target mRNA expression.

Several studies have shown that miRNAs are abnormally expressed in cancer cells. Suppression of miRNA expression was observed in several cancer cell lines. It is generally speculated that miRNAs inhibit tumor formation by inhibiting oncogenes and regulating the expression of genes related to apoptosis or differentiation. This miRNA acts like a tumor suppressor gene (tumor suppressor gene), so it is also called "tumor suppressor microRNA" (tumorsuppressor miRNA). It is known that 68% of patients with chronic lymphocytic leukemia (CLL) have deletion or inhibition of miR-15 and miR-16. . On the other hand, compared with normal tissues of rectum and lung, miR-143 and miR-145 showed lower expression levels in rectal cancer and lung cancer tissues. In addition, miRNA let-7 is also inhibited in lung cancer cells, and the downregulation of miRNA let-7 can detect the poor prognosis of lung cancer.

Studies have also shown that DNA methylation plays an important role in the regulation of tumor suppressor microRNAs. DNA methylation refers to the phenomenon that cytosine of CpG dinucleotide is converted into 5-methylcytosine by DNA methyltransferase (DNA methyltransferase). CpG dinucleotides (also known as "CpG sites") are mostly located at the 5' end of the gene. Studies have shown that CpG sites can be found in the promoter region of about 70% of human genes. When the CpG site in the promoter region is hypermethylated, it is presumed that the transcription of the coding gene located downstream may be repressed and thus cannot be expressed. It is generally speculated that certain methyl-binding proteins, histamine deacetylase (HDAC) and co-repressors can recognize hypermethylated CpG sites, thereby changing the chromosome structure and forming inactive heterochromatin (heterochromatin) ). Several studies have shown that aberrant DNA methylation occurs in the early stages of cancer and may result in the failure of tumor suppressor genes to be expressed. Recent studies have shown that DNA methylation not only suppresses the expression of tumor suppressor genes, but also reduces the expression of tumor suppressor microRNAs.

Contents of the invention

An embodiment of the present case provides a method for detecting the probability of an individual suffering from liver cancer, including detecting the degree of methylation of microRNA miR-129-2 in a biological sample from the individual, wherein the degree of methylation is determined by the following Detected by the method described: combined bisulfite restriction analysis (combined bisulfite restriction analysis; COBRA), quantitative methylation-specific polymerase chain reaction (quantitativemethylation-specific polymerase chain reaction; q-MSP), bisulfite sequencing method (bisulfite sequencing), pyrosequencing (pyrosequencing), next generation sequencing (nextgeneration sequencing; NGS), and DNA methylation array chip analysis (DNAmethylation array analysis), etc., wherein, when miR- When the methylation degree of 129-2 is higher than that of miR-129-2 in the control sample, it means that the individual tends to suffer from liver cancer or has suffered from liver cancer.

Another embodiment of the present case provides a method for detecting the probability of an individual suffering from liver cancer, including detecting the expression level of microRNA miR-129-2 in a biological sample from the individual, wherein the expression level is determined by quantitative reverse transcription polymerization Detected by enzyme chain reaction (quantitative real time PCR; qRT-PCR) or microRNA array chip analysis (miRNA array analysis), wherein, when the expression level of miR-129-2 in the biological sample is lower than that in the control sample When the expression level of miR-129-2 is low, it means that the individual tends to suffer from liver cancer or has suffered from liver cancer.

Description of drawings

Figure 1 shows the detection of miR-129-2 A in normal liver tissue, normal liver cells, and six kinds of HCC cells (HepG2, HuH7, J7, Hep3B, Mahlavu, SK-Hep1) by COBRA analysis in an example of this case Kylated electropherogram.

Figures 2A-2F show the detection of normal liver tissue, normal liver cells, and six types of HCC cells (HepG2, HuH7, J7, Hep3B, Mahlavu, SK- The methylation status and ratio of miR-129-2 in Hep1).

FIG. 3A is a bar graph showing the expression levels of miR-129-2 in normal hepatocytes and HCC cells in an embodiment of the present case.

Fig. 3B is a histogram showing the expression degree of miR-129-2 in normal liver tissue and HCC tissue in one embodiment of the present case.

FIG. 3C is a histogram showing the degree of expression of miR-129-2 in HCC cells treated with or without 5-aza-cytidine (5-AzaC) in an embodiment of the present case.

4A and 4B are bar graphs showing the degree of expression of miR-129-2 in HuH7 and J7 cells with or without lenti-miR-129-2 infection in an embodiment of the present case.

Figure 4C is a histogram showing the colony formation of J7 cells infected with lenti-miR-129-2 or not in an embodiment of the present case.

Figure 4D is a histogram showing the colony formation of HuH7 cells infected with or without lenti-miR-129-2 in an embodiment of the present case.

5A is an electropherogram showing the methylation of miR-129-2 in tissue samples (42 pairs of HCC tissues) detected by COBRA analysis in an embodiment of the present case.

5B is a histogram showing the degree of methylation of miR-129-2 in tissue samples (42 pairs of HCC tissues) detected by COBRA analysis in an embodiment of the present case.

5C is a bar graph showing the degree of methylation of miR-129-2 in tissue samples detected by q-MSP reaction in an embodiment of the present invention.

FIG. 5D is a box plot showing the degree of methylation in HCC tissues and adjacent normal tissues in one embodiment of the present case.

6A is a histogram showing the degree of expression of miR-129-2 in HCC tissues and adjacent normal tissues detected by qRT-PCR in an embodiment of the present case.

Fig. 6B is a ratio diagram showing the expression ratio of miR-129-2 in HCC tissues and adjacent normal tissues in one embodiment of the present case.

7A is a bar graph showing the degree of methylation of miR-129-2 in HCC plasma, liver cirrhosis plasma and normal plasma detected by q-MSP reaction in an embodiment of the present invention.

FIG. 7B is a box plot showing the degree of methylation of miR-129-2 in HCC plasma, liver cirrhosis plasma and normal plasma in one embodiment of the present case.

7C is a ROC curve showing the sensitivity and specificity of calculating the degree of methylation of miR-129-2 between HCC plasma sample tissue and sample tissue composed of healthy plasma and liver cirrhosis plasma in an embodiment of the present case.

Detailed ways

The detailed implementation of the present invention is as follows. However, the following examples are only used to further disclose the technical content of the present invention, and should not be used to limit the scope of the present invention.

An embodiment of the present invention provides a novel microRNA (miRNA) as a biomarker for detecting human liver cancer. According to the results of the high-throughput CpG microarray platform (high-throughput CpGmicroarray platform), the inventors of this case screened out a potential miRNA, namely miR-129, from the methylated miRNA in hepatocellular carcinoma (HCC) cells. -2, as a detection marker for liver cancer.

The microRNA (miRNA) mentioned here refers to the miRNA of human beings (Homo sapiens), which can be obtained from public databases, such as miRBases database (http://mirbase.org) or NCBI database (http://www.ncbi.nlm .nih.gov/), etc. MicroRNA (miRNA) forms a mature body (mature) after cleavage and processing from a precursor (precursor) with a stem-loop structure. If the mature body is the active fragment located at the 5' end of the original precursor, it is called "5p"; if the mature body is the active fragment located at the 3' end of the original precursor, it is called "3p". According to this case, miR-129-2 is the precursor of the 90bp linear sequence shown in sequence identification number 1. In the examples described later and in Figures 4A and 4B, "miR-129-2-5p" indicates the mature active fragment of miR-129-2 located at the 5' end, and "miR-129-2-3p" indicates the active fragment located at the miR- 129-23' mature active fragment.

An individual who is prone to liver cancer or has suffered from liver cancer can be judged according to that the degree of methylation of miR-129-2 in the biological sample from the individual is higher than that of the corresponding miR-129-2 in the control sample. Detectable biological samples can be obtained from fresh or frozen liver cancer tissues, cells, blood, serum, plasma, etc., but are not limited thereto. Control samples can be from normal tissues, cells, blood, serum, plasma, etc. of healthy individuals diagnosed as disease-free, or from normal tissues or cells of the same tissue type adjacent to the tumor being tested.

The methylation status of miRNA can be determined by combined bisulfite restriction analysis (combined bisulfite restriction analysis; COBRA), quantitative methylation-specific polymerase chain reaction (q-MSP), bisulfite sequencing (bisulfite sequencing), pyrosequencing (pyrosequencing), next generation sequencing (next generation sequencing; NGS), or DNA methylation array chip analysis (DNAmethylation array analysis) and other methods to confirm. Bisulfite Restriction Combinatorial Analysis (COBRA) means that the detected DNA is first treated with bisulfite, then amplified by PCR, and then cleaved and decomposed by restriction enzymes to obtain Know the DNA methylation degree or the analysis method of the methylation situation. In the present invention, the primer pair used for COBRA analysis of miR-129-2 includes the nucleotide sequence shown in sequence identification number 4 or 5, but is not limited thereto. For detailed COBRA operation procedures, please refer to the records in DNA Methylation Protocols, Methods in Molecular BiologyTM, HUMANA Press, Totowa, New Jersey, vol.200, p.71-86.

The methylation profile of miRNA can be analyzed by bisulfite sequencing and quantitative methylation-specific polymerase chain reaction (q-MSP). When the DNA sequence is treated with sodium bisulfite, the cytosine (cytosine; C) on the DNA sequence will be converted into uracil (U), but for 5-methylcytosine (5-methylcytosines; mC) It has no effect. Thus, sodium bisulfite treatment induces specific changes in DNA sequence revealing information about methylation status. In one embodiment of the present case, the primer pair shown in SEQ ID No. 8 or 9 can be used to analyze the methylation status of miR-129-2 by q-MSP, but is not limited thereto. The detailed q-MSP operation procedure can be found in DNA Methylation Protocols, Methods in Molecular BiologyTM, HUMANAPress, Totowa, New Jersey, vol.200, p.71-86.

Another embodiment of this case provides a method for detecting the probability of an individual suffering from liver cancer, including detecting the expression level of microRNA miR-129-2 in a biological sample from the individual, when the expression of miR-129-2 in the biological sample When the degree is lower than the expression degree of miR-129-2 in the control sample, it means that the individual tends to suffer from liver cancer or has suffered from liver cancer.

The expression level of miRNA can be detected by quantitative reverse transcription polymerase chain reaction (quantitative real time PCR; qRT-PCR) or microRNA array chip analysis (miRNAarray analysis). qRT-PCR is an improved PCR reaction, which is characterized in that amplified DNA can be detected in real-time during the PCR reaction. The detectable fluorescence cycle number in the exponential phase of qRT-PCR is called cycle threshold (Ct). The Ct value, or the difference in Ct value relative to the reference gene (ΔCt), can be used for the quantification of target DNA. In an embodiment of the present case, the pair of primers used to detect the expression level of miR-129-2 by qRT-PCR may include the nucleotide sequence shown in SEQ ID No. 6 or 7, but is not limited thereto. The expression degree of miRNA down-regulation in human liver cancer tissues is consistent with the methylation status of the miRNA, indicating that the miRNA is a reliable biomarker for liver cancer detection.

The biological function of miRNA in liver cancer tissue can be further confirmed by the formation of cell colony (colony formation). In one embodiment of the present case, miR-129-2 was inserted into viral nucleic acid and entered into liver cancer (HCC) cells through infection. The miRNA can inhibit the colony formation of the cell in HCC cells, showing that the miRNA acts as a tumor suppressor gene in human liver cancer tissue.

Example

Materials and Methods

Acquisition of patient samples, cell culture conditions and treatment of 5-aza-cytidine (5-AzaC)

Hepatocellular carcinoma (HCC) cells HepG2, HuH7, J7, Hep3B, Mahlavu and SK-Hep1 were cultured in DMEM (Dulbecco’s modified Eagle’s medium) medium at 37°C and 5% CO2. 24 hours before subsequent treatment, HuH7 cells (1×105 cells/well) and other liver cancer cells (3×105 cells/well) were seeded in 6-well plates, respectively. Cells were treated with 5-azaC (Sigma) at 5 μM for 3 days. Fresh medium containing 5-azaC was replaced every 24 h. 42 pairs of clinical samples of liver cancer (HCC), including tumor tissue and normal tissue adjacent to the tumor; 41 plasma samples of liver cancer, 8 plasma samples of liver cirrhosis, and 10 plasma samples of healthy people were obtained from Chi Mei Hospital, Tainan, Taiwan. Normal adult liver tissue chromosomal DNA (control) subjected to bisulfite sequencing and methylation-specific polymerase chain reaction (MSP) was purchased from US Biological. Methylated DNA (CpGenome Universal Methylated DNA) as a control group of methylation-specific polymerase chain reaction (MSP) was purchased from Chemicon.

Combinatorial Bisulfite Restriction Analysis (COBRA) and Bisulfite Sequencing

Use EZ DNA Methylation Kit (Zymo Research) to process chromosomal DNA (1 μg) from clinical samples of liver cancer (HCC), liver cancer (HCC) cells, and adult normal liver cells for bisulfite treatment and PCR amplification Reaction (amplification). In the combinatorial bisulfite restriction assay (COBRA), the above-mentioned PCR product was treated with endonuclease BstUI at 60oC overnight to decompose it. Resolved DNA fragments can be visualized on agarose gum stained with 1.5% (w/v) ethidium bromide (EtBr). On the other hand, the PCR products used for bisulfite sequencing were cloned in the pJET1.2/blunt clone vector with the CloneJET PCR clone kit (Thermo Fisher Scientific). After being transformed into Escherichia coli (E.coli), about 10 selected strains were selected and sequenced with the ABI sequencing system (Applied Biosystems).

Real-time quantitative methylation analysis

Amplification (amplification) of fluorescent SYBR green real-time methylation-specific PCR (qMSP) was performed on the DNA treated with bisulfite as described above. Add 1X PCR buffer, 0.25mM dNTP, 0.25μM forward primer and 0.25μM reverse primer, 1.5U FastStart Taq DNA polymerase (Roche) and 1μl Fluorescent SYBR green (Cambrex) was diluted 1000-fold in a total volume of 20 μl. The amplification reaction (amplification) was carried out in the ABI Prism7000 sequence detection system, according to the following thermocycling conditions: 94°C, 7 minutes; then 94°C, 30 seconds, 62°C, 30 seconds and 72°C, 30 seconds 40 cycles of seconds. According to the above-mentioned records, the Ct value difference between β-actin (β-actin) and miR-129-2 is calculated with the following formula to obtain the degree of methylation:

For tissue samples: 2 ^{[Ct(β-actin)-Ct(miR-129-2)]} × 100;

For plasma samples: 2 ^{[Ct(β-actin)-Ct(miR-129-2)]} × 1000.

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

The total RNA (totalRNA) of hepatocytes and liver cancer clinical samples was extracted using TRIzol reagent (Invitrogen). Synthesis of cDNA was performed using Superscript III reverse transcriptase (Invitrogen) according to the user manual. Real-time quantitative polymerase chain reaction was performed on ABI Prism7000 sequence detection system using fluorescent SYBR Green PCR master mix (Applied Biosystems). The performance analysis of mature microRNAs was performed using TaqMan miRNA Analysis System (Applied Biosystems) according to the operation manual. The degree of relative expression was analyzed by ΔCt method, and U6 and RNU44 were internal controls.

cell colony formation

HuH7 and J7 cells were seeded in 96-well plates at a density of 2×104 cells/well. Cells were infected with recombinant lentivirus carrying miR-129-2 precursor or control lentivirus according to the operation manual. After 24 hours of infection, cells were mixed with 0.7% top agar and transferred to a 6-well plate containing 1% bottom agar per well. Transfectants were selected for four weeks with 5 μg/ml puromycin. The formed cell colonies were washed with PBS, fixed with absolute methanol, and stained with crystal violet solution (0.5% crystal violet solution) for 1 hour.

[Example 1] Confirmation of potential microRNAs regulated by DNA methylation

The methylation status of miR-129-2 in normal liver tissue, normal liver cells, and six types of hepatocellular carcinoma (HCC) cells (HepG2, HuH7, J7, Hep3B, Mahlavu, and SK-Hep1) was confirmed according to the above COBRA analysis method. The results are shown in Figure 1, showing that miR-129-2 was methylated in six HCC cells, but not in normal liver tissues and cells.

The methylation of miR-129-2 in HCC cells and normal liver cells was further detected by bisulfite sequencing. Overall, 33 CpG sites in miR-129-2 were examined. Hypomethylation was observed in normal liver cells and tissues (Figure 2A~2B). In normal liver tissues NL-1663 and NL-4149 and normal liver cells, the methylation ratios of miR-129-2 were 2.1%, 3.3% and 0.6%, respectively. In contrast, in HepG2, HuH7, J7, Hep3B, Mahlavu and SK-Hep1 HCC cells, the methylation ratios of miR-129-2 were 64.5%, 58.8%, 71.5%, 79.7%, 72.7% and 94.5%, respectively. % (as shown in Figure 2C~2F). These results indicate that hypermethylation of miR-129-2 is a common phenomenon in HCC cells compared to normal hepatocytes.

[Example 2] Expression of miR-129-2 regulated by DNA methylation in HCC cells

In order to assess whether the expression of miR-129-2 is regulated by DNA methylation, quantitative reverse transcription PCR (quantitative reverse transcription PCR) was used to examine the expression degree of miR-129-2 in normal liver cells and HCC cells. Compared with normal hepatocytes, miR-129-2 expression was decreased in all HCC cells (Fig. 3A). Similarly, the expression of miR-129-2 in HCC tissue samples was also about 16 times lower than that in normal liver tissues (as shown in Figure 3B). Moreover, after 5-aza-cytidine (5-aza-cytidine; 5-AzaC) treatment, the expression levels of miR-129-2 in HuH7 cells and J7 cells were significantly increased, respectively. 2.5 times and 16 times of cells (as shown in Figure 3C). It was deduced from these results that DNA methylation regulates the expression of miR-129-2, resulting in a decreased expression of miR-129-2 in HCC cells.

[Example 3] miR-129-2 acts as a tumor suppressor miRNA in HCC cells

To evaluate the biological function of miR-129-2 in HCC cells, HCC cells expressing miR-129-2 were first infected with a lentivirus carrying miR-129-2 precursor (lenti-miR-129-2) (HuH7-miR, J7-miR). The expression of mature miR-129-2 in HCC cells infected with lenti-miR-129-2 was then confirmed using Taqman analysis. Infection of lenti-miR-129-2 in HuH7 cells (HuH7-miR) overexpressed mature miR-129-2-5p and miR-129-2-3p, and their expression levels were higher than those in control infected cells (HuH7-Ctrl) Respectively increased about 2000 times and 1000 times (as shown in Figure 4A~4B). Similarly, the expression of miR-129-2-5p and miR-129-2-3p in lenti-miR-129-2 infected J7 cells (J7-miR) was higher than that of control infected cells (J7-Ctrl). The amount is increased by about 500 times and 1000 times respectively. Cell colony experiments demonstrated that miR-129-2 has the ability to reduce the non-attachment growth of HCC cells (anchorage-independent growth). Compared with control infected cells (J7-Ctrl), only 1/3 of the cell colony formation was observed in J7 cells expressing miR-129-2 (J7-miR) (Fig. 4C). Similarly, in the HuH7 (HuH7-miR) cells infected with the control lentivirus, an average of 47 colonies were formed per well (as shown in FIG. 4D ). In contrast, HuH7 cells expressing the control miRNA (HuH7-Ctrl) had little colony formation. These results show that miR-129-2 functions as a tumor suppressor miRNA in HCC cells.

[Example 4] Hypermethylation and down regulation of miR-129-2 in HCC clinical samples (down regulation)

To assess the methylation profile of miR-129-2 in clinical samples, 42 pairs of HCC tumors and normal tissues adjacent to the tumors were analyzed using COBRA (Fig. 5A). HCC tissues of 30 out of 42 pairs (~71%) showed hypermethylation of miR-129-2 (Fig. 5B). To further quantify the degree of methylation, methylation-specific PCR was performed as described above. Except for 3 cases (patient numbers 1111, 2000, and 2756), almost all HCC clinical samples showed a higher degree of methylation of miR-129-2 compared with normal tissues adjacent to the tumor (Fig. 5C~ 5D). Furthermore, there was a significant correlation between the results analyzed using q-MSP and the results analyzed using COBRA. These results show that miR-129-2 is highly methylated in HCC clinical samples, which is consistent with the aforementioned results in HCC cells.

In order to confirm whether the expression of miR-129-2 is downregulated by DNA methylation (downregulation), qRT-PCR analysis was performed. The results showed that miR-129-2 expression was decreased in 29 out of 42 HCC clinical samples (~69%) compared with tumor-adjacent normal tissues (Fig. 6A~6B).

These results show that miR-129-2 is hypermethylated and downregulates its expression in HCC samples, but not in tumor-adjacent normal tissues. This means that the differential expression and/or methylation degree of miR-129-2 in liver cancer and normal tissues can be used as a marker for HCC diagnosis.

[Example 5] Methylation degree of miR-129-2 in HCC plasma samples

41 HCC plasma samples, 8 cirrhotic plasma samples and 10 healthy plasma samples obtained from Chimei Hospital were subjected to q-MSP to confirm the degree of methylation of miR-129-2. Among the 41 HCC plasma samples, 37 samples (90%) had the degree of methylation of miR-129-2 exceeding 0.1 (as shown in Figure 7A). In contrast, none of the plasma samples from healthy donors had a methylation level of miR-129-2 exceeding 0.05, and only 2 samples from cirrhotic plasma samples had miR-129-2 methylation levels exceeding 0.1 (eg Figure 7A). For further statistical analysis, the degree of methylation obtained above was converted into a log2 value. Statistical analysis showed that the degree of methylation of miR-129-2 in HCC plasma was significantly higher than that in plasma of healthy subjects or liver cirrhosis (P<0.01) (Figure 7B). Receiver operating characteristic curve (ROC) analysis was used to confirm the sensitivity, specificity, area under the curve (AUC) and cutoff value of miR-129-2 in the diagnosis of liver cancer. When the cut-off value (cut-off value) is -2.36, HCC can be distinguished from healthy people and liver cirrhosis, with a sensitivity of 88% and a specificity of 100% (AUC=0.92, SE=0.039, 95%CI=0.843- 0.997, P<0.001) (as shown in Figure 7C). ROC curve analysis showed high accuracy in HCC diagnosis using miR-129-2.

Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this art can make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, this The scope of protection of an invention shall be defined in the scope of the appended patent application.

Claims (15)

1. detect individuality and suffer from a test kit for liver cancer probability, comprise for detecting the oligonucleotide from this individual biological specimen Microrna miR-129-2 methylation, wherein this miR-129-2 is the nucleotide sequence shown in recognition sequence number 1.

2. the individuality of the detecting described in claim 1 is suffered from the test kit of liver cancer probability, the methylation of wherein working as miR-129-2 in detected biological specimen during higher than the methylation of miR-129-2 in check sample, represents that this individuality tends to suffer from liver cancer or suffered from liver cancer.

3. the individuality of the detecting described in claim 1 is suffered from the test kit of liver cancer probability, and wherein this methylates and occurs in one or the above CpG position of this miR-129-2.

4. the individuality of the detecting described in claim 1 is suffered from the test kit of liver cancer probability, and wherein this methylation is detected by the method being selected from the following group forming: heavy sulfurous acid restriction combined analytical method (COBRA), quantitative Methylation specific PCR (q-MSP), heavy sulfurous acid sequencing method, tetra-sodium sequencing method, inferior generation sequencing (NGS) and DNA methylation array chip analysis method.

5. the individual test kit of suffering from liver cancer probability of the detecting described in claim 1, wherein, this oligonucleotide is the introduction pair shown in recognition sequence numbers 4 or 5.

6. the individuality of the detecting described in claim 5 is suffered from the test kit of liver cancer probability, and wherein this oligonucleotide is for the methylation with heavy sulfurous acid restriction combined analytical method (COBRA) detecting miR-129-2.

7. the individual test kit of suffering from liver cancer probability of the detecting described in claim 1, wherein this oligonucleotide is the introduction pair shown in recognition sequence numbers 8 or 9.

8. the individuality of the detecting described in claim 7 is suffered from the test kit of liver cancer probability, and wherein this oligonucleotide is for the methylation with quantitative Methylation specific PCR (q-MSP) detecting miR-129-2.

9. the individuality of the detecting described in claim 1 is suffered from the test kit of liver cancer probability, and wherein this biological specimen comprises hepatic tissue, blood, serum or blood plasma.

10. detect individuality and suffer from a test kit for liver cancer probability, comprise for detecting the oligonucleotide from this individual biological specimen Microrna miR-129-2 performance degree, wherein this miR-129-2 is the nucleotide sequence shown in recognition sequence number 1.

The individual test kit of suffering from liver cancer probability of detecting described in 11. claims 10, the performance degree of wherein working as miR-129-2 in detected biological specimen during lower than the performance degree of check sample miR-129-2, represents that this individuality tends to suffer from liver cancer or suffered from liver cancer.

The individual test kit of suffering from liver cancer probability of detecting described in 12. claims 10, wherein, this performance degree is detected by quantitative reverse transcriptional PCR (qRT-PCR) or Microrna array chip analysis method.

The individual test kit of suffering from liver cancer probability of detecting described in 13. claims 10, wherein this oligonucleotide is the introduction pair shown in recognition sequence numbers 6 or 7.

The individual test kit of suffering from liver cancer probability of detecting described in 14. claims 13, wherein this oligonucleotide is for the performance degree with quantitative reverse transcriptional PCR (qRT-PCR) detecting miR-129-2.

The individual test kit of suffering from liver cancer probability of detecting described in 15. claims 10, wherein this biological specimen comprises hepatic tissue, blood, serum or blood plasma.