TWI451088B - A method for screening high risk of liver cancer - Google Patents

Description

Method for screening high risk of liver cancer

The present invention relates to a method of screening for cancer, and more particularly to a method of screening for cancer using methylated DNA as a biomarker.

Cervical cancer is one of the leading causes of death among women worldwide and in Taiwan. According to the 2002 World Health Organization (WHO), cervical cancer is the second leading cause of cancer death among women worldwide, second only to breast cancer; regular cervical cancer screening Detection is the best way to prevent cervical cancer. There are two main methods for screening for cervical cancer. One is the most common Pap smear, and the other is HPV testing. Pap smear is to remove the secretions from the uterine neck, to observe the presence of cancerous lesions in the epithelial cells, to detect cervical cancer at an early stage; and the HPV test is to reverse transcriptase The reverse transcription polymerase chain reaction (RT-PCR) examines the presence of human papilloma virus (HPV) virus genes in the samples.

However, because Pap smear requires a doctor's sampling and an examiner/pathologist to interpret the smear, in addition to prone to high false negative rate and delay the diagnosis and treatment of precancerous lesions. Moreover, the labor cost required is too high, which has difficulty in promotion for many developing countries; on the other hand, HPV testing is highly sensitive but easy to use. The high false positive rate not only makes the patient worry, but also wastes many medical resources on the follow-up examination of false positive patients; therefore, how to improve the accuracy and convenience of cervical cancer detection methods, It is one of the important topics to promote cervical cancer testing.

In the etiology of cervical cancer, infection with carcinogenic human papillomavirus (HPV) is the most significant risk factor; Zur Hausen's 2002 report shows that "high risk" human papillomavirus (HPV) produces E6/ E7 oncoprotein acts on the tumor suppressor gene p53/pRB , causing abnormal cell cycle regulation; in fact, human papillomavirus (HPV) DNA can be detected in all cervical cancer cases. However, human papillomavirus (HPV) is a necessary condition for cervical cancer, but it is not enough to cause cervical cancer; about 60% of low-grade squamous intraepithelial lesions , LSIL) will regress, 30% will persist, 5-10% will develop high-grade squamous intraepithelial lesions (HSIL), only less than 1% will become Cervical cancer. The persistent infection of HPV and the viral load may be the determinants of the development of highly squamous cell intraepithelial lesions (HSIL) and cancer; however, the molecular mechanisms of cervical cancer development remain to be confirmed.

Other factors, such as environmental and genetic changes, may also play an important role in the deterioration of cervical keratinocytes; and whether or not initiated by HPV, genetic changes that cause genomic instability have long been considered cervical An important mechanism of carcinogenesis, cytogenetic studies have shown that there are non-random chromosomal changes in cervical cancer cells; in addition, several molecular genetic studies have identified some The location of loss of heterozygosity (LOH), which may be associated with tumor suppressor genes (TSGs) when cervical cancer occurs.

Genomic deletions are considered to be important factors in tumor formation. For a long time, we have been accustomed to the idea that the coding in the genome depends on the four bases of ATCG. Knudson proposed the double-invasive theory as early as 1975. Two-hit theory), indicating that mutations or deletions accompanying some homologous tumor suppressor genes may cause or cause cancer; however, other information that affects phenotype may exist in the modified base 5-A In 5-methylcytosine, 5-methylcytosine was found in the palindromic sequence 5'-CpG-3' in mammalian cells, except for some called "CpG islands" in mammalian cells. Outside the region of (CpG islands, CGIs), most CpG dinucleotide pairs are methylated, and CpG islands refer to a large number of GC- and CpG in a region of approximately 1000 base pairs (1 Kb). -, usually located near the gene, and found near the promoter of a widely expressed gene. Methylation of cytosine occurs after DNA synthesis, from a-methyl donor nucleoside gland s- methionine (S -adenosylmethionine, SAM) cytosine methyltransferase the fifth carbon position by enzymes The enzyme reaction is carried out by DNA methyltransferase (DNMTs), which is the main methyltransferase of mammals, responsible for post-replicative restoration of the hemimethylation site. Methylation, known as maintenance methylation; conversely, DNMT3A and DNMT3B are thought to be primarily responsible for the new position of methylation, a step called de novo methylation.

Loss of methylation of CpG dinucleotide, meaning general low methylation, is the first epigenetic abnormality in cancer cells; however, in the past few years Studies have shown that site-specific hypermethylation at specific locations (eg, some tumor suppressor genes) is associated with loss of function, which may provide selective advantages in cancer production; in promoter regions High methylation of the upper CpG island can cause chromatin remodeling by histone modification followed by successive gene silencing; in addition to chromosomal deletions and gene mutations In addition, epigenetic silencing of tumor suppressor genes caused by hypermethylation of promoters is also common in human cancers.

Recent epidemiological studies have shown that the concentration of serum folate (a major source of methyl) is associated with HPV infection and clearance; in the metabolism of the methyl cycle, enzymes Gene polymorphisms have also been reported to be involved in the development of cervical intraepithelial lesions; as with the concept of supergene evolution, studies of DNA methylation and cervical cancer are also prevalent, cervical cancer The DNA methylation study days and proliferation showed the possibility of using methylation as a screening for cervical cancer; due to the nature of genetic and environmental interactions, the degree of methylation of tumor suppressor genes is due to different genes and different ethnic groups. Different, different diseases may have different methylator phenotypes; however, the methylation phenotype of cervical cancer and its association with HPV genotype are still unknown, and what is specific in cervical cancer The genes are methylated and how many genes are needed to meet the needs of clinical applications. These issues remain issues that need to be identified in the future.

It can be seen that there are still many defects in the above-mentioned conventional cervical cancer screening methods, which is not a good designer, and needs to be improved.

In view of the shortcomings derived from the above-mentioned conventional cervical cancer screening methods, the inventors of the present invention have improved and innovated, and after years of painstaking research, finally succeeded in research and development of the method for screening cancer.

It is an object of the present invention to provide a method of screening for cervical cancer as a cancer screen for first line cervical cancer.

A second object of the present invention is to provide a method for screening for cervical cancer, which can be used as a screening for second-line cervical cancer in addition to screening for cervical cancer of the first line, and assists humans. HPV testing to achieve a more accurate screening for cervical cancer.

Another object of the present invention is to provide a method for diagnosing cancer, which can be applied to the detection of cervical cancer, and can also be applied to the detection of other cancers (such as ovarian cancer and liver cancer) to assist abnormal specimens. Diagnosis.

A method for screening cancers which achieves the above object of the invention is to detect the state of methylation of a target gene in a test subject cell as a screening indicator for the presence or absence of cancer, and the method comprises the following steps: Step 1 provides a test a sample; step 2 detecting a methylation status of a CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene consisting of SOX1, PAX1, LMX1A, NKX6-1, WT1, and ONECUT1; 3 According to the presence or absence of the methylation status of the target gene, it is determined whether the sample has cancer or precancerous lesions.

The test body is Pap smear, ascites, blood, urine, feces, sputum, oral mucosal cells, gastric juice, bile, cervical epithelial cells and the like.

The methylation status of the CpG sequence of the target gene is methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP) ), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC).

Wherein the target gene SOX1 has the nucleotide sequence as shown in SEQ ID No: 1.

Wherein the target gene PAX1 has the nucleotide sequence as shown in SEQ ID No: 2.

Wherein the target gene LMX1A has a nucleotide sequence as shown in SEQ ID No: 3.

Wherein the target gene NKX6-1 has the nucleotide sequence as shown in SEQ ID No: 4.

Wherein the target gene WT1 has the nucleotide sequence as shown in SEQ ID No: 5.

Wherein the target gene ONECUT1 has the nucleotide sequence as shown in SEQ ID No: 6.

The present invention further provides a method for screening for cervical cancer, which detects the state of methylation of a target gene in a test subject cell as a screening index for the presence or absence of cervical cancer, and the method comprises the following steps: Step 1 provides a The test subject; step 2 detects the methylation status of the CpG sequence of at least one target gene in the genomic DNA of the test subject, and the target gene is composed of SOX1, PAX1, LMX1A, NKX6-1, WT1 and ONECUT1; And step 3 determines whether the specimen has cervical cancer and precancerous lesions according to the presence or absence of the methylation status of the target gene.

The test subject is a Pap smear, blood, urine, cervical epithelial cells, and the like.

The test subject is an abnormal Pap smear.

The test subject is a cervical cell sample that is positive for HPV testing.

The methylation status of the CpG sequence of the target gene is methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP) ), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC).

Wherein the target gene SOX1 has the nucleotide sequence as shown in SEQ ID No: 1.

Wherein the target gene PAX1 has the nucleotide sequence as shown in SEQ ID No: 2.

Wherein the target gene LMX1A has a nucleotide sequence as shown in SEQ ID No: 3.

Wherein the target gene NKX6-1 has the nucleotide sequence as shown in SEQ ID No: 4.

Wherein the target gene WT1 has the nucleotide sequence as shown in SEQ ID No: 5.

Wherein the target gene ONECUT1 has the nucleotide sequence as shown in SEQ ID No: 6.

The invention further provides a method for screening ovarian cancer, which is to detect the state of methylation of a target gene in a test subject cell, and to serve as a screening index for the presence or absence of ovarian cancer, the method comprising the following steps: Step 1 provides a test a sample; step 2 detecting a methylation status of a CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene consisting of SOX1, PAX1, LMX1A; and step 3 according to the methylation status of the target gene Whether or not the specimen has ovarian cancer and precancerous lesions.

The test subject is ascites, blood, urine, and the like.

The methylation status of the CpG sequence of the target gene is methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP) ), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing (pyrosequencing) ).

Wherein the target gene SOX1 has the nucleotide sequence as shown in SEQ ID No: 1.

Wherein the target gene PAX1 has the nucleotide sequence as shown in SEQ ID No: 2.

Wherein the target gene LMX1A has a nucleotide sequence as shown in SEQ ID No: 3.

The present invention further provides a method for screening for liver cancer, which is a method for detecting the methylation status of a target gene in a test subject cell as a screening index for the presence or absence of liver cancer, and the method comprises the following steps: Step 1 provides a test sample Step 2: detecting the methylation status of CpG sequence of at least one target gene in the genomic DNA of the test subject, the target gene is composed of SOX1 and NKX6-1; and step 3 according to the presence or absence of methylation status of the target gene Determine whether the specimen has liver cancer and precancerous lesions.

The test subject is ascites, blood, urine, feces, gastric juice, bile, and the like.

The methylation status of the CpG sequence of the target gene is methylation-specific PCR (MSP), quantitative methylation-specific PCR (QMSP) ), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing (pyrosequencing) ).

Wherein the target gene SOX1 has the nucleotide sequence as shown in SEQ ID No: 1.

Wherein the target gene NKX6-1 has the nucleotide sequence as shown in SEQ ID No: 4.

Embodiment 1 Materials and methods

First, the test materials

The test material contained a complete range of cervical lesions including normal samples (n=45), low-grade squamous cell intraepithelial lesions (LSIL, n=45), and highly squamous intraepithelial lesions (HSIL, n=58). ), squamous cell carcinoma (SCC, n=109); the test material also contains a series of complete ovarian tumor samples, including ovarian benign tumor samples (n=36), ovarian marginal tumor samples (n=6) ), ovarian malignant tumor samples (n=122); all cervical samples and ovarian samples were taken from the gynecological tumor tissue bank of the Taipei Military Academy. The genomic DNA of each sample was extracted with the Qiagen DNA kit and illuminated with PicoGreen. The DNA was quantified by absorption and the quality of the DNA was detected by gel electrophoresis.

In addition, hepatocyte samples contained normal liver cell samples (n=13), chronic hepatitis (n=15), cirrhosis (n=40), hepatocellular carcinoma (HCC, n=54); all livers The cell samples were taken from the general surgical tumor tissue bank of the Taipei Three Military General Hospital. The genomic DNA of each liver cell sample was also extracted with the Qiagen DNA kit, and the DNA was quantified by PicoGreen fluorescence absorption method, and the DNA quality was detected by gel electrophoresis.

2. Differential Methylation Hybridization (DMH) using CpG island microarrays

The DNA of 30 cervical cancer tissue samples were mixed together, and the DNA of 10 normal Pap smear samples were mixed together. The DNA of the sample was digested with restriction enzyme Mse I and ligated to the linker ( On the linkers), the methylation-sensitive restriction enzymes Hpa II and Bst UI were subsequently digested, and the DNA was used as a template for PCR for 20 cycles. Increased and labeled with fluorescent dye, the DNA of the normal Pap smear sample is labeled with the fluorescent dye Cy3, and the DNA of the cervical cancer tissue sample is labeled with the fluorescent dye Cy5; Needles, hybridization with CpG island microarrays containing 8,640 CpG island tags, CGI database (http://derlab.med.utoronto.ca/CpGIslands/) Identify the selected CpG islands. The microarray data was analyzed in the circular-features mode of the GenePix 6.0 software, marking the repeatedly selected clones, and filtering to remove unacceptable features; the ratio of Cy5 to Cy3 was greater than The locus of 2.0 is a gene with a high degree of methylation in a mixed cervical cancer tissue sample, and thus accepts a gene position with a ratio greater than 2.0.

3. Bisulfite modification, methylation-specific PCR (MSP) and bisulfite sequencing (BS)

Sulfite modification using Chemicon's DNA modification kit (Chemicon, Ternecula, CA): 1 μg of sample genomic DNA, chemical modification of genomic DNA with sodium sulfite, in single-stranded In DNA, all unmethylated cytosines undergo deamination and turn into uracil, while methylated cytosines are not modified and remain in the state of 5-methylcytosine. Finally, after the reaction The sample DNA was dissolved in 70 μl of 55 ° C TE buffer (TE buffer) for methylation specific PCR (MSP).

The normal DNA of human peripheral blood was subjected to sulfite modification as a control group with an unmethylated promoter sequence; and human normal DNA was treated with SssI methyltransferase (methyltransferase, New England). Biolabs, Beverly, MA) was treated to obtain a positive control group with a methylated dual gene.

1 μg of sample genomic DNA after sulfite modification, and control and positive control DNA were used for methylation-specific PCR amplification with MSP primers. The MSP primers were divided into two types, one of which was specifically identifiable. The MSP primer (U) of the methylation gene sequence, and the MSP primer (M) which can specifically recognize the methylation gene sequence, the MSP primer sequence of each target gene is shown in Table 1; methylation-specific PCR reaction The total volume of the material was 25 μl, containing 1 μl of modified template DNA, 1.5 pmol of each primer, 0.2 mmol/L dNTPs, and 1 unit Gold Taq DNA polymerase (Applied Biosystems, Foster City, CA); 5 minutes at 95 ° C, followed by 95 ° C dissociation (denature) 30 seconds, appropriate primer bonding (annealing temperature bonding 30 seconds, 72 ° C synthesis 30 seconds for the cycle, dissociation, bonding, synthesis steps a total of 35 cycles, after The reaction was further carried out at 72 ° C for 5 minutes. The amplified product was electrophoresed on a 2.5% agar colloid containing ethidium bromide (EtBr) and observed under ultraviolet light.

The primer type M represents an MSP primer that can specifically recognize the methylation gene sequence.

The primer type U represents an MSP primer that can specifically recognize the sequence of the unmethylated gene.

All samples were subjected to at least two independent sulfite modification and methylation-specific PCR. In the PCR reaction using MSP primer (M) which can specifically recognize the methylation gene sequence, if the same sample could not be used When the PCR product is synthesized more than twice, the sample is considered to be unmethylated; the PCR product amplified by the MSP primer (M) which can specifically recognize the methylation gene sequence is selected to the pCR4-TOPO vector (Invitrogen, In Carlsbad, CA), at least 5 independent strains (clones) were selected for sulfite sequencing (BS). The primers used for sulfite sequencing (BS) are shown in Table 2. The sulfite sequencing was performed on a sequencer (Applied Biosystems, Foster City, CA).

4. The methylation gene is expressed in cervical cancer cell lines by treatment with 5'-aza-2'-deoxycytidine.

First, in the HeLa cervical cancer cell line, methylation-specific PCR (MSP) was used to test the methylation status of a gene that may have a high degree of methylation, and a gene having methylation was selected. The HeLa cervical cancer cell line was treated with 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (Sigma Chemical Co.) for 4 days to render the cell line unexposed due to methylation. Genes can be re-expressed and analyzed for gene expression by RT-PCR; Total RNA was extracted using Qiagen RNeasy kit (Qiagen, Valencia, CA) and DNase I was added to remove DNA contamination; 1 μg per sample Total RNA was synthesized by Superscript II reverse transcriptase and random hexamer (Invitrogen); the synthesized cDNA was PCR amplified by PCR master mix reagents kit (Applied Biosystems) and placed at temperature. The reaction was carried out in a thermal cycler (GeneAmp 2400 PE, Applied Biosystems), and the amplified cDNA was analyzed by RT-PCR. The RT-PCR primers used for each target gene are shown in Table 3.

5. Detection of human papillomavirus (HPV)

The presence of human papillomavirus (HPV) DNA in squamous cell carcinoma (SCC) was detected by L1 consensus PCR and reverse line blot. If there is more than the analysis range of the hybridization technique, The sequence of the novel human papillomavirus (novel HPV type) was confirmed by DNA sequencing.

Sixth, statistical analysis

Data analysis using statistical software SAS version 9.1, the relationship between methylation of genes and various clinical parameters (including HPV status), using X ² test ( X ² test) and Fisher's exact test (Fisher's exact test) Calculate and calculate the Odds ratios (ORs) of age and HPV infection and 95% confidence intervals (CI) in the logistic regression model. The alpha level is statistically significant (the alpha level) Of statistical significance) was set at p = 0.05; and the sensitivity and specificity of using HPV and methylation markers to diagnose cervical lesions were calculated.

Example 2 Screening of methylation index genes for cervical cancer

Differential methylation hybridization (DMH) was performed by CpG island microarrays to screen for genes with high methylation in cervical squamous cell carcinoma (SCC); CpG island microarrays ( The results of CpG island microarrays showed that there were 216 points in the cervical cancer tissue sample and the normal Pap smear samples with differential methylation. After removing the sequence repeats, 26 gene promoter region CpG islands were obtained. Promoter CGIs).

These gene promoters were sequenced and analyzed, and six genes were selected, including: SOX1 (SEQ ID No: 1), PAX1 (SEQ ID No: 2), LMX1A (SEQ ID No: 3), NKX6. -1 (SEQ ID No: 4), WT1 (SEQ ID No: 5), and ONECUT1 (SEQ ID No: 6), the details of which are shown in Table 4; as shown in Table 4, these six genes are all developing. The important transcription factors in the process, SOX1, PAX1, LMX1A, NKX6-1, and WT1 are important for the development of the brain, the nerve plate, the limbs, the islets, and the genitourinary organs. The ONUCUT1 is for the liver and pancreas. The performance of genes is important, but few studies have shown that these genes are involved in cancer.

CpG sequence analysis was performed on about 500 bp nucleotides before and after the transcription start point (+1) of each gene. As shown in Fig. 1, those having CpG sequences in each gene were labeled with "∣", and their MSPs were designed for each gene. Primers (as shown in Table 1) and sulfite sequencing (BS) primers (as shown in Table 2), each target gene was synthesized by methylation-specific PCR (MSP) and sulfite sequencing (BS) The location of the fragment is also shown in Figure 1.

Methylation-specific PCR (MSP) was then performed on mixed cervical cancer tissue samples (mixed with 30 samples) and mixed normal Pap smear samples (10 samples) to confirm the methylation of the six genes. Whether the phenomenon is different in different tissue samples, the results shown in Figure 2, these six genes in the mixed cervical cancer tissue samples are methylation phenomenon (as shown in the second column of Figure 2), and There was no methylation in the mixed normal Pap smear samples (as shown in the first column of Figure 2); further tests were performed on individual cervical cancer tissue samples, and 4 cervical cancer tissue samples (T1, T2) were taken. , T3, T4) and 4 normal samples (N1, N2, N3, N4) for methylation-specific PCR (MSP), respectively, with MSP primer (U) that can specifically identify the unmethylated gene sequence, and Methylation-specific PCR (MSP), which specifically recognizes the methylation gene sequence, was subjected to methylation-specific PCR (MSP). The results are shown in Figure 3. These six genes are methylated in individual cervical cancer tissue samples. (as shown in columns 1, 3, 5, and 7 of Figure 3), the same gene is not detected in normal samples. Group phenomenon occurs (as shown in FIG three columns of 9,11,13,15); From the above results, this six genes as gene methylation index of cervical cancer screening.

Example 3 Correlation between DNA methylation and gene expression in cervical cancer cell lines

To confirm whether the expression of the cervical cancer methylation indicator gene was regulated by DNA methylation, 10 μM DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC) (Sigma Chemical Co.) The HeLa cervical cancer cell line was treated for 4 days, and the demethylation of the above six gene promoters was examined by methylation-specific PCR (MSP); the MSP primers which can specifically recognize the unmethylated gene sequence were respectively U), and MSP primer (M) which can specifically recognize the methylation gene sequence for methylation-specific PCR (MSP), the results are shown in Figure 4A, untreated 5'-aza-2'-deoxycytidine (AZC) In the HeLa cervical cancer cell line (AZC-), all six target genes have methylation (as shown in the first column of Figure 4A), and no unmethylated genes are detected (see figure 4A, column 2); in the 5'-aza-2'-deoxycytidine (AZC) 4 day HeLa cervical cancer cell line (AZC+), unmethylated target genes were detected (As shown in the fourth column of Figure 4A), the above six target genes were identified in cervical cancer cell lines treated with the methyltransferase inhibitor 5'-aza-2'-deoxycytidine (AZC). Methylated section has been removed.

The expression of these six genes in HeLa cervical cancer cell lines was analyzed by RT-PCR. The results are shown in Figure 4B, and the cell lines treated with 5'-aza-2'-deoxycytidine (AZC) were used. The mRNAs of these six target genes can be detected (as shown in column 6 of Figure 4B), but no cells detected by 5'-aza-2'-deoxycytidine (AZC) are detected. The mRNA of a target gene (as shown in the fifth column of Figure 4B), the results show that these six target genes in cervical cancer cells do regulate their gene expression through DNA methylation, when the gene has In the case of methylation, the expression of the gene is inhibited, and after the methylation is removed, the target gene begins to manifest itself.

In addition, sulfite sequencing (BS) was used to analyze whether the target gene has hypermethylation in HeLa cervical cancer cell lines. The results are shown in Figure 5. No 5'-aza-2'-deoxycytidine In the (AZC)-treated cell line (Fig. 5A), the number of highly methylated samples of the target gene was higher than that of the 5'-aza-2'-deoxycytidine (AZC)-treated cell line (Fig. 5B). Cervical squamous cell carcinoma (SCC) and normal samples were also analyzed by sulfite sequencing (BS). The results are shown in Figure 6, in the cervical squamous cell carcinoma (SCC) (Figure 6A) sample. The number of samples with high methylation of the target gene was also significantly higher than that of the normal sample (Fig. 6B).

Example 4 Methylation analysis of target genes in clinical cervical samples

Please refer to Table V. The mean ages of normal samples, low-grade squamous cell intraepithelial lesion (LSIL), high-grade squamous cell intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC) samples were 51.0±11.3, 39.7±, respectively. 9.6, 46.4 ± 14.4 and 53.3 ± 10.9 years ( p <0.05); the ratio of high-risk HPV DNA in the sample was: 21.4% for normal samples, 47.7% for low-grade squamous cell intraepithelial lesions (LSIL), high scale The squamous cell intraepithelial lesion (HSIL) sample was 59.3%, and the squamous cell carcinoma (SCC) sample was 88.9%. The results showed that patients with HPV were more likely to have cervical lesions of different severity (the odds ratios of LSIL, HSIL, and SCC samples were 3.1, 5.2, and 29.9, respectively; 95% confidence intervals were 1.1-8.3, 2.1-13.0, respectively. 11.5-77.7).

Methylation-specific PCR (MSP) analysis of methylation status of target genes, methylation status of target genes, and analysis of the presence or absence of human papillomavirus (HPV) in cervical lesion samples of different severity As shown in Table 5, the six genes SOX1, PAX1, LMX1A, NKX6-1, WT1, and ONECUT1 have high frequency methylation in squamous cell carcinoma (SCC), and each gene is in squamous cell carcinoma (SCC). The proportion of methylation in the samples was 81.5%, 94.4%, 89.9%, 80.4%, 77.8%, and 20.4%, respectively; and the proportion of methylation of each gene in normal cervical samples was 2.2%, 0%, 6.7%, 11.9%, 11.1%, and 0% ( p ≦ 0.001); therefore, these 6 genes are methylated in squamous cell carcinoma (SCC) samples compared to normal cervical samples. A lot higher.

The frequency of NKX6-1 gene methylation was 53.3% in LSIL samples, 55.1% in HSIL samples, and 80.4% in SCC samples; statistical results showed that patients with methylation of NKX6-1 gene There is a higher risk of developing squamous cell carcinoma (SCC) (odds ratio is 29.8, 95% confidence interval is 10.4-85.2).

The frequency of methylation of PAX1 gene was 2.3% in LSIL samples, 42.1% in HSIL samples, and 94.4% in SCC samples. Statistical results showed that patients with methylation of PAX1 gene had high scale The risk of squamous cell intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC) was higher (the odds ratios for HSIL and SCC samples were >999.9; the 95% confidence interval was <0.1->999.9).

The frequency of methylation of SOX1, LMX1A and ONECUT1 genes in samples of precancerous lesions was very low, but the frequency of methylation in HSIL and SCC samples increased significantly, 9.3% and 81.5, respectively. %, 16%, and 89.9%, 7.4%, and 20.4%; statistical results show that patients with methylation of SOX1, LMX1A, or ONECUT1 have a higher risk of developing squamous cell carcinoma (SCC) (the odds of the three) The ratios are 20.2, 124.5, and 7.3; the 95% confidence intervals are 25.8-999.9, 33.0-470.1, and 2.0-25.9, respectively.

The frequency of methylation of the WT1 gene increases with the severity of the lesion. The frequency of WT1 gene methylation in normal samples is 11.1%, 20.0% in LSIL samples, and 42.1% in HSIL samples, in SCC samples. In the middle, it was 77.8%; the statistical results showed that patients with methylation of WT1 gene had higher risk of high-grade squamous cell intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC). They are 6.7 and 27.9 respectively; the 95% confidence intervals are 2.2-19.8 and 9.8-78.9 respectively.

Diagnostic performance of DNA methylation indicators

Analyze the sensitivity and specificity of DNA methylation to determine whether the target gene can be used as a biomarker for cervical cancer screening and cervical cancer screening. The results are shown in Table 6; HPV test The sensitivity and specificity of screening for squamous cell carcinoma (SCC) were 83.1% and 85.5%, respectively (the 95% confidence interval was 77.6-88.5 and 79.6-91.4, respectively); and SOX1, PAX1, LMX1A, The methylation status of the five genes NKX6-1 and WT1 was screened for the presence or absence of squamous cell carcinoma (SCC), and the sensitivity of each gene methylation status to squamous cell carcinoma (SCC) was 77.8%-94.4%. The specificity is 88.1%-100%; when combined with HPV test and individual methylation index gene to detect disease (combined parallel testing, CPT), that is, as long as HPV test or test of any methylation indicator gene If the result is positive, it is determined that the cervical cancer screening result of the test sample is positive, the sensitivity is between 97.2% and 98.2%, and the specificity is between 66.7% and 79.5%; when combined sequential testing (CST) When HPV tests with individual methylation indicator genes, HPV is first tested, and the samples were tested positive for HPV methylation status of each indicator gene detection, the sensitivity is between 69.4% -85.0%, while all the tests were 100% specificity.

When using high-grade squamous cell intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC) diagnostic criteria, the sensitivity and specificity of screening for HPIL testing with or without HSIL or SCC were 75.0% and 85.5%, respectively. The 95% confidence interval is 70.2-79.8 and 79.6-91.4 respectively; and the methylation status of the five genes SOX1, PAX1, LMX1A, NKX6-1 and WT1 are analyzed to screen for the presence or absence of HSIL or SCC in the sample. The sensitivity of the basic state to HSIL or SCC is between 57.4% and 76.2%, and its specificity is between 88.1% and 100%. When HPV test and individual methylation index genes are combined to detect disease (CPT), The sensitivity can be increased to 85.8%-94.9%; and when the sequential combination (CST) HPV test and individual methylation index genes, the specificity of all tests is 100%; when simultaneous (CPT) HPV test and SOX1, PAX1 The methylation status of the three genes of LMX1A is 100% sensitive when screening samples for squamous cell carcinoma (SCC), and the sensitivity is determined by the same method for screening for HSIL or SCC in the sample. It is 93.4%.

In the results of screening for squamous cell carcinoma (SCC) with individual methylation index genes, the sensitivity of screening for the presence or absence of squamous cell carcinoma (SCC) in the sample by the methylation status of PAX1 gene alone was the highest. Up to 94.4% (95% confidence interval is 90.0-98.8). Similarly, the sensitivity of screening for samples with PAX1 gene methylation status of HSIL or SCC can reach 76.2% (the 95% confidence interval is 69.7-) 82.7), while the specificity of both tests is 100%.

Example 5 Methylation analysis of target genes in ovarian tumor samples

Methylation-specific PCR (MSP) was used to analyze the methylation status of target genes in ovarian tumor samples. The methylation status analysis results of the target genes are shown in Table 7. The SOX1, PAX1 and SOX1 were analyzed in each ovarian tumor sample. The methylation status of the three genes of LMX1A showed that the three genes, SOX1, PAX1 and LMX1A, were not methylated in all ovarian benign tumors and ovarian marginal tumor samples; The frequency of methylation of these three genes increased significantly. The frequency of methylation of SOX1 gene was 55.7%, the frequency of methylation of PAX1 gene was 49.2%, and the frequency of methylation of LMX1A gene was 32.8%.

Example 6 Methylation analysis of target genes in hepatocyte samples

Methylation-specific PCR (MSP) was used to analyze the methylation status of the target gene in hepatocyte samples. The methylation status analysis of the target gene is shown in Table 8. The SOX1 gene methylation in normal liver cell samples. The frequency of the SOX1 gene methylation was significantly increased in hepatocyte samples with abnormal lesions. In the chronic hepatitis samples, liver cirrhosis samples, and liver cancer samples, the SOX1 gene was methylated. The frequencies were 33.3%, 27.5%, and 53.7%, respectively. In addition, the frequency of NKX6-1 gene methylation in normal liver cell samples (10%) was also significantly lower than the frequency of NKX6-1 gene methylation (57%) in liver cancer samples.

The method for cancer diagnosis provided by the present invention has the following advantages when compared with the conventional techniques described above:

1. The method for screening cancer according to the present invention is to use the degree of methylation of a specific gene in a sample as a diagnostic indicator for the presence or absence of cancer, and to compare with a conventional Pap smear and HPV testing method. The sensitivity and specificity of the cancer diagnostic method of the present invention are both higher than the foregoing.

2. The method for screening cancer provided by the present invention can be combined with or assisted by HPV testing as a second-line cervical cancer in addition to screening for first-line cervical cancer. Screening to achieve a more accurate screening of cervical cancer.

3. The method for cancer diagnosis provided by the present invention can be applied to the detection of cervical cancer, and can also be applied to the detection of other cancers (such as ovarian cancer and liver cancer) to assist in the diagnosis of abnormal samples.

The detailed description above is a detailed description of a possible embodiment of the present invention, but is not intended to limit the scope of the invention, and the equivalents or modifications, such as: Equivalent embodiments of the manner in which the degree of methylation of each target gene in the sample is determined, etc., should be included in the patent scope of the present application.

In summary, the method of cancer diagnosis provided in this case is indeed innovative, and can improve the above-mentioned multiple functions compared with the conventional cervical cancer screening method. It should fully meet the novelty and progressive statutory invention patent requirements. To apply, you are requested to approve the application for this invention patent to encourage the invention.

Figure 1 shows the CpG sequence analysis of each target gene used in the cancer screening method of the present invention. The CpG sequence in each gene is indicated by "∣"; the position of the synthetic fragment of each gene MSP primer is indicated by "-"; Salt sequencing (BS) primers synthesize fragment positions with " Mark

Figure 2 is a diagram showing the target genes used in the cancer screening method of the present invention, in a mixed cervical cancer tissue sample (mixed with 30 samples) and a mixed normal Pap smear sample (mixed with 10 samples). Results of the base-specific PCR (MSP) analysis; column 1 is a mixed normal Pap smear sample (10 samples mixed), and column 2 is a mixed cervical cancer tissue sample (30 samples mixed), Column 3 is the negative control (negative control), column 4 is the positive control group (positive control), and column 5 is the blank control group (water);

Figure 3 shows the results of methylation-specific PCR (MSP) analysis of individual cervical cancer tissue samples and individual normal Pap smear samples for each target gene used in the cancer screening method of the present invention. T1, T2, T3, and T4 represent four individual cervical cancer tissue samples. N1, N2, N3, and N4 represent four individual normal samples, and the column labeled U indicates that the non-methylated gene sequence can be specifically identified. The results of methylation-specific PCR (MSP) of MSP primer (U) indicate that the M column indicates methylation-specific PCR (MSP) with MSP primer (M) which can specifically recognize the methylation gene sequence. result;

Figure 4A shows the target genes used in the cancer screening method of the present invention in HeLa cervical cancer cell lines without treatment of 5'-aza-2'-deoxycytidine (AZC-, columns 1, 2), and In the HeLa cervical cancer cell line treated with 5'-aza-2'-deoxycytidine (AZC+, columns 3 and 4), the results of methylation-specific PCR (MSP) analysis were performed; The result of methylation-specific PCR (MSP) can be specifically identified by the MSP primer (U) of the unmethylated gene sequence, and the column indicating M indicates the MSP primer (M) which can specifically recognize the methylation gene sequence. Results of methylation-specific PCR (MSP); Figure 4B shows the target genes used in the cancer screening method of the present invention in HeLa cervical cancer cell lines without treatment of 5'-aza-2'-deoxycytidine (AZC-, column 5), and HeLa cervical cancer cell line treated with 5'-aza-2'-deoxycytidine (AZC+, column 6), the results of RT-PCR analysis; Figure 5A is for Each target gene used in the cancer screening method of the present invention is carried out in a HeLa cervical cancer cell line which is not treated with 5'-aza-2'-deoxycytidine. Results of sulfite sequencing (BS) analysis; Figure 5B shows the target genes used in the cancer screening method of the present invention in HeLa cervical cancer cell lines treated with 5'-aza-2'-deoxycytidine , the results of sulfite sequencing (BS) analysis; Figure 6A is the sulfite sequencing in cervical squamous cell carcinoma (SCC) for each target gene used in the cancer screening method of the present invention (BS) analysis results; and Figure 6B shows the results of sulfite sequencing (BS) analysis in normal samples for each target gene used in the cancer screening method of the present invention.

Claims (6)

A method for screening for high risk of liver cancer by detecting the state of methylation of a target gene in a test subject cell as a screening index for the presence or absence of liver cancer, the method comprising the following steps: Step 1 provides the same sample, wherein The sample sample comprises a liver cell sample sample or blood; step 2 detects a methylation status of a CpG sequence of at least one target gene in the liver cell sample sample, at least one of which is selected from SOX1, NKX6-1 The group formed; and step 3 if the target gene is methylated, indicating that the sample has the possibility of liver cancer. The method for screening for high risk of liver cancer according to claim 1, wherein the liver cell sample comprises a normal liver cell sample, a chronic hepatitis sample, a cirrhosis sample, and a liver cancer sample test. body. The method for screening for high risk of liver cancer according to the first aspect of the patent application, wherein the methylation-specific PCR (MSP) of the CpG sequence methylation state of the target gene is methylated-specific PCR (MSP) Quantitative methylation-specific polymerase chain reaction (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid Denaturing high-performance liquid chromatography (DHPLC), pyrosequencing. The method for screening for high risk of liver cancer according to claim 1, wherein the target gene SOX1 has a nucleotide sequence as shown in SEQ ID No: 1; and the NKX6-1 has SEQ ID No: The nucleotide sequence shown in 4. The method for screening for high-risk screening of liver cancer according to the fourth aspect of the patent application, wherein the primer for identifying the methylation sequence of the target gene sulfite is: the SOX1 primer is the core represented by SEQ ID No: 31-32. Glycosylation sequence; methylated target gene NKX6-1 primer is the nucleotide set forth in SEQ ID No: 41-44 sequence. The method for screening for high risk of liver cancer according to the first aspect of the patent application is characterized in that the primer pair sequence for identifying the methylated target gene is: SOX1 primer is SEQ ID No: 7-10, 47-48 The nucleotide sequence shown; the methylated target gene NKX6-1 primer is the nucleotide sequence shown in SEQ ID Nos: 23-26, 55-56.