Breast cancer biomarker ZNF662 gene and its detection method and application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a biomarker for breast cancer, the ZNF662 gene, and its detection method and application. The biomarker is the ZNF662 gene, and the sample to be tested is derived from a patient suspected of breast cancer. The nucleotide of the ZNF662 gene is SEQ ID NO: 1. The present application provides a method for detecting the methylation status of the ZNF662 gene promoter, and the specific steps are: ① extracting a DNA sample; ② designing primers, using a methylation detection reagent, and detecting the methylation level of the ZNF662 gene promoter in the DNA sample by the MSP method to determine whether the ZNF662 gene is methylated, thereby achieving breast cancer diagnosis. The present application is the first to confirm through in vivo and in vitro experiments that ZNF662 is a new tumor suppressor gene, and its expression is downregulated in breast cancer due to hypermethylation of the promoter, providing a new option for the screening of early diagnostic markers for breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biological technology, and specifically relates to a breast cancer biomarker ZNF662 gene and a detection method and application thereof. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women, and its incidence rate has always ranked first among female cancers worldwide. It is also one of the leading causes of cancer death in women worldwide. In my country, the new incidence of breast cancer accounts for 15% of all new malignant tumors in women. In terms of treatment, surgery is still the first choice for breast cancer. At the same time, non-surgical treatments are gradually becoming mature. Although significant progress has been made in treatment strategies in recent years, because breast cancer is a highly heterogeneous disease, the clinical manifestations and treatment responses of patients vary. Therefore, the clinical treatment of breast cancer patients still faces huge challenges. Early detection, early diagnosis and early treatment are of great significance to improving the prognosis of breast cancer patients and improving their quality of life.

[0003] Common early screening techniques for breast cancer include: (1) Breast ultrasound: simple and convenient, non-invasive and radiation-free, with high sensitivity and accuracy, but it is difficult to detect microcalcifications and burr-like changes, and cannot reliably distinguish between benign and malignant tumors. (2) Mammography: It can clearly display structures at all levels and is highly sensitive to calcifications, but its diagnostic accuracy is poor for women under 40 years old and those with dense breasts, and its radiation exposure is also controversial. (3) Breast MRI: It has the highest sensitivity and is good at displaying deep lesions, but the examination time is long and the price is expensive. Due to conditional restrictions, it is generally used as a supplementary measure for the above examinations. (4) Genetic testing: As one of the important achievements of the continuous development of genomics today, its clinical application in the early screening of malignant tumors has also received increasing attention. However, the lack of specific biomarkers for breast cancer has limited it.

[0004] DNA methylation biomarkers are defined as molecular targets that undergo DNA methylation changes during carcinogenesis. Such biomarkers can be used for early cancer detection, prediction and / or monitoring of treatment response, and detection of recurrent cancer. Therefore, the discovery of tumor-specific DNA methylation biomarkers and the evaluation of candidate DNA biomarkers for clinical application are current research and development priorities. Currently, the most extensively studied and validated DNA methylation biomarkers include tissue factor pathway inhibitor 2 (TFPI2), Septin 9 (SEPT9), glutathione S-transferase pi1 (GSTP1), 6-methylguanine-DNA methyltransferase (MGMT), and 5-hydroxymethylcytosine. The discovery of aberrant DNA methylation is a prime candidate for specific markers for cancer diagnosis and treatment. In recent years, with the advancement of clinical diagnostic testing technologies, the specific methylation sites of DNA methylation biomarkers in malignant tumors have garnered increasing attention. However, overall, the clinical application of DNA methylation biomarkers in cancer still requires further basic research and clinical discussion.

[0005] DNA methylation occurs throughout the entire process of tumor development and is expected to become a routine clinical tumor marker due to its sensitivity, specificity, and ease of analysis.

[0006] This application improves the detection sensitivity and specificity of samples through gene DNA methylation detection, thereby increasing the early diagnosis rate. Moreover, the detection method is simple, non-invasive, easy to use, convenient, economical and affordable, and easily accepted by the public.

[0007] DNA methylation testing is more conducive to the early detection of cancer than conventional detection methods for the following reasons: (1) Epigenetic changes, such as changes in DNA methylation, occur early in tumor formation and are tissue and cancer type specific; (2) DNA methylation types are common throughout tumor tissues and tumors of the same type, but somatic mutations are usually only found in subpopulations or clones of cancer cells; (3) DNA methylation remains stable in larger gene regions and can therefore be detected using multiple CpG dinucleotides; (4) Multiple pairs of primers can be designed simultaneously for combined detection; and (5) It is suitable for various body fluid tests and can be used to supplement cytological tests when they are unclear or negative.

[0008] The invention patent with application number CN201780015221.8 discloses that ZNF662 can be used as a biomarker for the diagnosis of gynecological tumors such as endometrial cancer, ovarian cancer, vaginal cancer, vulvar cancer, gestational trophoblastic disease, and primary peritoneal cancer, but does not disclose the relevant applications of zinc finger protein ZNF662 in the field of breast cancer. Summary of the Invention

[0009] In view of this, this patent is based on the finding that the ZNF662 gene is highly expressed in most normal breast tissues, but its expression in breast cancer tissues is significantly reduced and is negatively correlated with breast tumor stage. Combined with public database analysis, the detection of ZNF662 can become a biomarker to assist in the screening, diagnosis and treatment of breast cancer patients.

[0010] One of the objectives of the present invention is to provide a biomarker for breast cancer, which can be used for early detection of breast cancer, prediction of breast cancer incidence, monitoring of treatment response, and detection of breast cancer recurrence.

[0011] To achieve the above object, the present invention adopts the following technical solutions:

[0012] A biomarker for breast cancer, wherein the biomarker is the ZNF662 gene; the sample to be tested is derived from a patient suspected of having breast cancer; the ZNF662 gene nucleotide is SEQ ID NO: 1.

[0013] Furthermore, a biomarker for breast cancer is the ZNF662 gene, and the sample to be tested is an in vitro sample from a suspected breast cancer patient; the ZNF662 gene nucleotide is SEQ ID NO: 1.

[0014] This patent uses the MSP method to detect the methylation status of the ZNF662 gene in the breast tissue of suspected breast cancer patients to determine whether the suspected patients have breast cancer. When the ZNF662 gene promoter is not methylated, the patient is judged to have non-breast cancer. When the ZNF662 gene promoter is methylated, the patient is judged to have breast cancer.

[0015] This patent uses the TCGA database, UALCAN database and Kaplan-Meier Plotter database for analysis and finds that compared with normal breast tissue, the promoter methylation status of ZNF662 in breast tumor tissue is increased and positively correlated with the breast tumor stage. At the same time, the expression of the ZNF662 gene in breast cancer tissue is significantly reduced, and the expression of the ZNF662 gene is negatively correlated with the breast tumor stage, which means that the higher the methylation status, the lower the expression level.

[0016] Under normal circumstances, the CpG island in the promoter region of the ZNF662 gene is unmethylated, while in breast cancer patients, the CpG island in the promoter region of the ZNF662 gene becomes methylated.

[0017] Furthermore, the biomarker is an unmethylated ZNF662 gene and / or a methylated ZNF662 gene.

[0018] Furthermore, the biomarker is the promoter of the ZNF662 gene, and the nucleotide sequence of the promoter is SEQ ID NO: 2.

[0019] A second object of the present invention is to provide a primer for detecting the biomarker.

[0020] To achieve the above object, the present invention adopts the following technical solutions:

[0021] The primers for detecting the biomarker include three pairs of primers, namely: forward primer 1 having the nucleotide shown in SEQ ID NO: 3, and reverse primer 1 having the nucleotide shown in SEQ ID NO: 4; forward primer 2 having the nucleotide shown in SEQ ID NO: 5, and reverse primer 2 having the nucleotide shown in SEQ ID NO: 6; forward primer 3 having the nucleotide shown in SEQ ID NO: 7, and reverse primer 3 having the nucleotide shown in SEQ ID NO: 8.

[0022] Furthermore, primer pair 1 is an expression primer (181 bp); primer pair 2 is a methylation primer (133 bp); and primer pair 3 is a non-methylation primer (136 bp).

[0023] A third object of the present invention is to provide a reagent containing the above primer.

[0024] To achieve the above object, the present invention adopts the following technical solutions:

[0025] A reagent containing the above primers, the reagent also includes a methylation detection reagent and a reagent for extracting sample DNA.

[0026] A fourth object of the present invention is to provide a method for detecting the methylation status of the ZNF662 gene promoter using the primers and / or the reagents.

[0027] To achieve the above object, the present invention adopts the following technical solutions:

[0028] A method for detecting the methylation status of the ZNF662 gene promoter using the primers and / or the reagents specifically comprises the following steps:

[0029] (1) extracting a DNA sample using the reagent for extracting sample DNA;

[0030] (2) Using the primers and / or the reagents, the methylation level of the ZNF662 gene promoter in the DNA sample is detected by the MSP method to obtain the MSP electrophoresis result.

[0031] Furthermore, MethlTarget sequencing was used to further verify the MSP results, and the obtained results were compared with the promoter methylation sites of the normal group to determine their methylation status.

[0032] Furthermore, the method is applicable to various body fluid tests, and the samples include peripheral blood, feces, tissues and / or cells.

[0033] A fifth object of the present invention is to provide a use of the biomarker, the primer and / or the reagent in preparing a kit for diagnosing breast cancer.

[0034] To achieve the above object, the present invention adopts the following technical solutions:

[0035] Use of the biomarker, the primer and / or the reagent in preparing a kit for diagnosing breast cancer.

[0036] Furthermore, the biomarker, the primer and / or the reagent are used in preparing a kit for early diagnosis of breast cancer.

[0037] Furthermore, the biomarker, the primer and / or the reagent are used in the preparation of a kit for predicting the risk of breast cancer.

[0038] Furthermore, the biomarker, the primer and / or the reagent are used in preparing a kit for judging the prognosis of breast cancer.

[0039] The sixth object of the present invention is to provide a methylation inhibitor of the ZNF662 gene for use in the preparation of a drug for treating breast cancer. The methylation inhibitor inhibits the methylation of the ZNF662 gene, thereby inhibiting the proliferation and clone formation ability of breast cancer cells, inhibiting the migration and invasion ability of breast cancer cells, and inducing breast cancer cell cycle arrest in the G0-G1 phase / G2-M phase, thereby inhibiting the growth of breast cancer cells in vivo.

[0040] This patent study found that after breast cancer cells were treated with the demethylating drug Aza (5-aza-2'-deoxycytidine) and the histone deacetylase inhibitor TSA (trichomoniasisin A), the expression of the ZNF662 gene was upregulated to varying degrees, and the abnormally high methylation state of the ZNF662 gene promoter would lead to the silencing of the expression of the ZNF662 gene.

[0041] The beneficial effects of the present invention are:

[0042] (1) This patent uses the TCGA database, UALCAN database, and Kaplan-Meier Plotter database for analysis. Compared with normal breast tissue, the promoter methylation status of ZNF662 in breast tumor tissue is increased and positively correlated with breast tumor stage. At the same time, the expression of ZNF662 gene in breast cancer tissue is significantly reduced, and the expression of ZNF662 gene is negatively correlated with breast tumor stage, which means that the higher the methylation status, the lower the expression level; and the expression of ZNF662 gene is low in certain types of breast cancer cell lines such as MB231 and YCCB1;

[0043] (2) This patent study found that patients with high expression of the ZNF662 gene had a better prognosis and a longer survival period than those with low expression; the high-risk group with methylated CpG islands of the ZNF662 gene in breast cancer had a worse prognosis than the low-risk group; and it was found that abnormal high methylation of the ZNF662 gene promoter would lead to silencing of ZNF662 gene expression;

[0044] (3) This patent study found that by inhibiting the occurrence of ZNF662 gene methylation, the proliferation and clone formation ability of breast cancer cells can be inhibited, the migration and invasion ability of breast cancer cells can be inhibited, and the breast cancer cell cycle can be induced to arrest at the G0-G1 phase / G2-M phase, thereby inhibiting the growth of breast cancer cells in vivo. This provides new thinking and methods for the treatment of breast cancer, namely, using ZNF662 gene methylation inhibitors to inhibit the methylation of ZNF662 gene, thereby achieving the treatment of breast cancer;

[0045] (4) The method disclosed in this patent can simultaneously design multiple pairs of primers for joint detection of the ZNF662 gene;

[0046] (5) The method disclosed in this patent is applicable to various body fluid tests;

[0047] (6) In breast cancer, research on the zinc finger protein ZNF662 is still blank. This application is the first to confirm through in vivo and in vitro experiments that ZNF662 is a new tumor suppressor gene. Its expression is downregulated in breast cancer due to promoter hypermethylation, providing a new option for the screening of early diagnostic markers for breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The TCGA database and UALCAN database were used to analyze the expression differences of ZNF662 in breast cancer tissues of different stages and normal breast tissues;

[0049] Figure 2 The Kaplan-Meier Plotter database was used to analyze the relationship between ZNF662 expression and the prognosis of breast cancer patients;

[0050] Figure 3 The expression of ZNF662 mRNA in breast cancer cell lines and normal mammary epithelial cells was detected by RT-PCR;

[0051] Figure 4 The UALCAN database was used to analyze the promoter methylation status of ZNF662 in breast cancer tissues of different stages and normal breast tissues;

[0052] Figure 5 The cbioportal database was used to analyze the correlation between ZNF662 promoter methylation status and expression level in breast tumor tissues;

[0053] Figure 6 The SurvivalMeth database was used to analyze the prognostic differences between high-risk and low-risk groups of ZNF662 methylated CpG islands in breast cancer.

[0054] Figure 7 RT-PCR was used to verify the expression of ZNF662 in breast cancer cell lines after treatment with demethylating drugs. ①④ represent the ZNF662 mRNA expression levels of MB231 and YCCB1 cell lines without demethylating drug treatment, ②⑤ represent the ZNF662 mRNA expression levels of MB231 and YCCB1 cell lines after treatment with Aza (5-aza-2'-deoxycytidine), and ③⑥ represent the ZNF662 mRNA expression levels of MB231 and YCCB1 cell lines after treatment with TSA (trichomonoside A); (** indicates p < 0.01)

[0055] Figure 8 The methylation status of ZNF662 in breast tumor tissues was detected by MSP;

[0056] Figure 9 is methtarget to detect the methylation status of ZNF662 in breast tumor tissues;

[0057] Figure 10-11 The ZNF662-overexpressing breast cancer cell line was constructed and verified by RT-PCR and Western blot (*** indicates p < 0.001);

[0058] Figure 12 The cell proliferation experiment verified that ZNF662 inhibited breast cancer cell proliferation, among which, Figure 12 -A represents the absorbance of MB231 from 0 to 27 hours, Figure 12 -B indicates the absorbance of YCCB10-27h (** indicates p < 0.01);

[0059] Figure 13-14 The inhibition of ZNF662 on breast cancer cell colony formation was verified by clone formation assay (*** indicates p < 0.001);

[0060] Figure 15-17 Flow cytometry was used to verify that ZNF662 induced G0-G1 / G2-M arrest in breast cancer cells (*** indicates p<0.001);

[0061] Figure 18-19 Transwell assay was used to verify the ability of ZNF662 to inhibit the migration of breast cancer cells (*** indicates p < 0.001);

[0062] Figure 20-21 Transwell assay was used to verify the ability of ZNF662 to inhibit the invasion of breast cancer cells (*** indicates p < 0.001);

[0063] Figure 22-23 The ability of ZNF662 to inhibit the tumor formation of breast cancer cells in vivo was verified through nude mouse subcutaneous tumor formation experiments. DETAILED DESCRIPTION

[0064] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0065] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0066] Example 1. Extraction of cell tissue DNA

[0067] The following steps were all performed under enzyme-free conditions.

[0068] 1. Cell Extraction

[0069] (1) When the cells have grown to an appropriate density, discard the old culture medium, add 1 ml of pre-cooled PBS, and shake the cells horizontally to wash them. Repeat the above steps twice, shake off the residual liquid in the bottle, add 1 ml of trypsin, place in an incubator to digest for 5 minutes, and then transfer the liquid in the bottle to a centrifuge tube;

[0070] (2) Place the centrifuge tube in a standard centrifuge, pay attention to balancing, and set the parameters to 800 rpm for 5 minutes;

[0071] (3) After centrifugation, discard the top supernatant, add 180 μL of Buffer ATL to the centrifuge tube, mix gently by pipetting, and transfer to an EP tube;

[0072] (4) Add 20 μL of proteinase K to the EP tube, mix gently by pipetting, and incubate in a 55°C thermostat for 30 min;

[0073] (5) Add 20 μL of 20 mg / μL RNase A to the EP tube, shake and mix for 15 seconds, and incubate at room temperature for 2-5 minutes;

[0074] (6) Add 200 μL of Buffer AL to the EP tube, shake and mix for 15 seconds, and incubate in a 70°C thermostat for 10 minutes;

[0075] (7) Add 200 μL of anhydrous ethanol to the EP tube and shake to mix for 15 seconds;

[0076] (8) Transfer the liquid in the EP tube to the sleeve column, place the sleeve column in a conventional centrifuge, pay attention to the balance, and set the parameters to 8000 rpm, 1 min;

[0077] (9) Remove the waste liquid from the lower layer and add 500 μL of Buffer AW1 to the upper centrifuge column. Place the centrifuge column in a standard centrifuge at 8000 rpm for 1 min.

[0078] (10) Remove the waste liquid from the lower layer and add 500 μL of Buffer AW2 to the upper centrifuge column. Place the centrifuge column in a conventional centrifuge and set the parameters to 12,000 rpm for 3 min.

[0079] (11) Remove the waste liquid from the bottom layer and place the centrifuge column in a conventional centrifuge with the parameters set to 12000 rpm and 60 s centrifugation.

[0080] (12) Insert a new enzyme-free EP tube and add 30 μl of Buffer AE / triple-distilled water to the upper centrifuge column. Let it stand for 3 min. Place the centrifuge column in a conventional centrifuge and set the parameters to 8000 rpm for 60 s.

[0081] (13) After centrifugation, liquid can be seen in the EP tube, indicating that the DNA has been extracted. The concentration is measured using a NanoDrop 2000 spectrophotometer and the DNA is stored in a -20°C refrigerator.

[0082] 2. DNA Extraction from Peripheral Blood, Feces, and Tissues

[0083] (1) Remove the tissue from the liquid nitrogen tank and grind it thoroughly in a mortar with a sterilized pestle. Place an appropriate amount of tissue powder in a 1.5 ml EP tube, add 600 μl TNES Buffer and 20 μl proteinase K, shake and mix, and incubate at 55°C overnight.

[0084] (2) After cooling to room temperature, add 200 μl of NaCl solution (6 mol / L) and shake to mix;

[0085] (3) Place the EP tube in a conventional centrifuge, pay attention to balancing, and set the parameters to 12000 rpm for 10 minutes;

[0086] (4) After centrifugation, aspirate the top supernatant and place it in a new 1.5 ml EP tube. Add 500 μl of pre-cooled isopropanol, gently invert the tube several times to mix, and let it stand at room temperature for 10 min.

[0087] (5) Place the EP tube in a conventional centrifuge, pay attention to balancing, and set the parameters to 6000 rpm for 5 minutes;

[0088] (6) After centrifugation, a small amount of white precipitate can be seen in the EP tube, which is DNA. Carefully aspirate and discard the supernatant, taking care not to aspirate the precipitate.

[0089] (7) Add 1 ml of pre-chilled 70% ethanol and gently mix by inverting several times;

[0090] (8) Place the EP tube in a conventional centrifuge, pay attention to balancing, and set the parameters to 6000 rpm, 5 min;

[0091] (9) Repeat steps (6)(7)(8)(6);

[0092] (10) Open the EP tube cap on ice for 5 minutes to dry the residual liquid in the tube;

[0093] (11) Add 30 μl of TE buffer, mix well, measure the concentration using a NanoDrop 2000 spectrophotometer, and store in a −80°C refrigerator.

[0094] Example 2. Sulfite-modified extracted DNA and methylation detection

[0095] 1. DNA Bisulfite Modification

[0096] The following steps were all performed under enzyme-free conditions.

[0097] Table 1

[0098] Mixture ingredients volume M-Dilution Buffer 300 μl M-Dissolving Buffer 500 μl Enzyme-free water 900 μl

[0099] Prepare the mixture according to Table 1 and shake to mix.

[0100] (1) Take 130 μl of the above mixture and add 20 μl of the DNA extracted in Example 1, centrifuge briefly to mix, and incubate in a thermostat at 98°C for 10 min, 64°C for 150 min, and 4°C for 20 h.

[0101] (2) After incubation, add 600 μl of M-Binding Buffer, centrifuge briefly to mix, and then pass the liquid through the column. Place the centrifuge column in a conventional centrifuge and set the parameters to 12,000 rpm for 30 seconds.

[0102] (3) Remove the waste liquid from the lower layer and add 200 μl of M-Wash Buffer to the upper centrifuge column. Place the centrifuge column in a conventional centrifuge and set the parameters to 12,000 rpm for 30 seconds.

[0103] (4) Remove the waste liquid from the lower layer and add 200 μl of M-Desulphonation Buffer to the upper centrifuge column. Let it stand at room temperature for 20 min. Place the centrifuge column in a conventional centrifuge and set the parameters to 12,000 rpm for 30 s.

[0104] (5) Remove the waste liquid from the lower layer and add 200 μl of M-Wash Buffer to the upper centrifuge column. Place the centrifuge column in a conventional centrifuge and set the parameters to 12,000 rpm for 30 seconds.

[0105] (6) Repeat step (6);

[0106] (7) Insert a new enzyme-free EP tube, add 10 μl of M-Elution Buffer to the upper spin column, place the spin column in a conventional centrifuge, and set the parameters to 12,000 rpm for 30 seconds;

[0107] (8) After centrifugation, the enzyme-free EP tubes were stored in a -20°C refrigerator.

[0108] 2. Tissue sequencing and public database analysis to screen potential methylation sites as diagnostic biomarkers for breast cancer

[0109] To identify meaningful molecular markers in breast cancer, the applicant initially sequenced breast cancer and normal breast tissues to screen for abnormally methylated and differentially expressed genes. Genes with significant trends were then selected from the screened genes based on fold change and corrected P-value ranking. The TCGA database, UALCAN database, and Kaplan-Meier Plotter database (http: / / kmplot.com / analysis / ) were then used to screen for potential methylation sites as diagnostic biomarkers for early breast cancer. Figure 1As shown in the results, the expression of ZNF662 in breast cancer tissue was significantly lower than that in normal breast tissue, and the expression of ZNF662 was negatively correlated with breast tumor stage. Figure 2 It can be seen that patients with high ZNF662 expression have better prognosis and longer survival period than those with low ZNF662 expression. The expression of ZNF662 mRNA in breast cancer cell lines and normal breast epithelial cells was detected by RT-PCR. The results are as follows Figure 3 shown.

[0110] This patent study found that the methylation level of ZNF662 in breast tumor tissue was significantly higher than that in normal breast tissue. Figure 4 As shown in Figure 2, compared with normal breast tissue, the promoter methylation status of ZNF662 in breast tumor tissue is increased and positively correlated with breast tumor stage. Further analysis combined with the cbioportal database (http: / / www.cbioportal.org / ) online, as shown in Figure 2 Figure 5 As shown in Figure 3, the promoter methylation status of ZNF662 in breast tumor tissues is negatively correlated with its expression level, that is, the higher the methylation status, the lower the expression level.

[0111] 3. Methylation-specific PCR (MSP)

[0112] To simplify the methylation detection method and promote its clinical application, methylation-specific PCR was used to further validate and optimize it based on previous sequencing results and public database analysis so that the detection method can be promoted and applied. The specific steps are as follows:

[0113] The following steps were all performed under enzyme-free conditions.

[0114] (1) Prepare the mixed solution according to Table 2.

[0115] Table 2

[0116] Mixture ingredients volume 10×PCR Buffer 1.25 μl <![CDATA[Mg Cl2]]> 1 μl Primer 1 0.75 μl Primer 2 0.75 μl dNTPs 1 μl Taq-Gold enzyme 0.09375μl

[0117] Prepare the mixture according to Table 2, dilute to 12 μl with enzyme-free water, and centrifuge briefly to mix.

[0118] (2) Add 0.5 μl of the sulfite-modified DNA to the EP tube and centrifuge briefly to mix.

[0119] (3) Place the EP tube in a PCR instrument and set the parameters according to Table 3, with the number of cycles being 40.

[0120] Table 3

[0121] Reaction temperature Reaction time 95℃ 10min 95℃ 30s 58℃ 30s(U) 60℃ 30s(M) 72℃ 30s 72℃ 10min

[0122] (4) While amplifying on the PCR instrument, prepare an agarose gel: prepare a 2% agarose gel (e.g., 20 ml 1×TAE + 0.4 g agarose powder); microwave on high until boiling, add 1 μl nucleic acid stain, swirl back and forth to mix, then quickly pour into the gel mold and wait for 1 hour for it to solidify;

[0123] (5) After the amplification is completed, gently and slowly pull out the comb vertically upward, transfer the agarose gel into the electrophoresis tank, and load the DNA marker and the amplified PCR product into the wells together;

[0124] (6) Set the power supply parameters to: 120V, 25min;

[0125] (7) After electrophoresis is completed, the gel is exposed to an image analyzer, and the results are analyzed and saved.

[0126] Survival Meth database analysis found that ZNF662 methylated CpG islands in breast cancer had a poorer prognosis than low-risk groups. Figure 6 After treating breast cancer cells with the demethylating drug Aza (5-aza-2'-deoxycytidine) and the histone deacetylase inhibitor TSA (trichomoniasisin A), ZNF662 expression was upregulated to varying degrees, suggesting that the silencing of ZNF662 expression may be due to abnormal hypermethylation of the promoter. Figure 7 .

[0127] This patent selected tissue samples from 160 breast tumor patients (near the cancerous area was confirmed to be pathologically normal by a pathologist). Figure 8 As shown, the ZNF662 gene was highly methylated in breast tumor tissues, with a methylation rate of 86.9% (21 / 160).

[0128] To verify the results of MSP, the applicant performed MethlTarget sequencing on 22 breast cancer tissues and 10 normal breast tissues. Figure 9 As shown, the promoter methylation sites of ZNF662 in breast cancer tissues were significantly higher than those in normal breast tissues.

[0129] Example 3. Screening of human breast cancer cell lines stably expressing ZNF662

[0130] 1. Materials

[0131] (1) Cell lines: The breast cancer cell lines used in this patent are MB231 and YCCB1; the complete cell culture medium is RPMI-1640 + 10% fetal bovine serum + 1% PS double antibody; the culture conditions are 37°C, 5% CO2 incubator.

[0132] (2) Plasmid: The M98-ZNF662 plasmid constructed in this patent is constructed by inserting the labeled full-length ZNF662 gene sequence into the M98 framework plasmid, and then transforming the recombinant plasmid into Escherichia coli DH5a, and has been sequenced and verified; the control plasmid is the M98 framework plasmid.

[0133] 2. Screening of human breast cancer cell lines stably expressing ZNF662

[0134] (1) The day before transfection, cells were plated and evenly seeded in a six-well plate after digestion. The next day, cells were observed or transfected when the cell density in the plate reached approximately 70%.

[0135] (2) The transfection reagent is Lip2000 liposomes, which are configured as follows:

[0136] ① Solution A: Dilute 4 μg / well of control plasmid and target ZNF662 plasmid in 250 μl / well RPMI-1640(-), mix gently by pipetting back and forth, and let stand at room temperature for 5 minutes;

[0137] ② Solution B: dilute Lip2000 liposomes in 250 μl / well RPMI-1640(-) and 5 μl / well, mix by gently pipetting back and forth, and let stand at room temperature for 5 minutes;

[0138] ③After 5 minutes, gently pipette and mix solution A and solution B, and let it stand at room temperature for 20 minutes;

[0139] (3) Take out the six-well plate to be transfected, discard the old culture medium, add an appropriate amount of PBS, shake horizontally to wash the cells, discard the PBS, and add 500 μl / well RPMI-1640(-);

[0140] (4) After standing for 20 min, aspirate the transfection mixture of solution A and solution B into the RPMI-1640(-) in the well plate, gently shake back and forth to mix, and return to the incubator;

[0141] (5) After 4-6 hours, aspirate the culture medium in the wells and replace with 2 ml / well of complete culture medium, shake horizontally, and return to the incubator;

[0142] (6) After 48 hours, G418 can be used for stable strain screening (preliminary research by the research group has shown that the screening concentration of MB231 G418 is 24 μl / ml and the screening concentration of YCCB1 G418 is 8 μl / ml), or according to experimental requirements;

[0143] (7) After 10-14 days of G418 screening, half the dose of G418 can be used as a maintenance dose;

[0144] (8) Cell RNA / protein were extracted from the stable strain, and the overexpression of ZNF662 was verified by RT-PCR / Western blotting.

[0145] like Figure 10-11 As shown, this patent shows that the transcription and translation levels of ZNF662 in MB231 and YCCB1 cells transfected with ZNF662 plasmids are increased compared with the control cells, which means that the ZNF662 overexpressing breast cancer cell line was successfully constructed.

[0146] Example 4. Cell proliferation assay

[0147] (1) Conventional cell digestion and centrifugation. After centrifugation, discard the supernatant and add an appropriate amount of complete culture medium to resuspend the cells as needed. Mix thoroughly by pipetting and transfer an appropriate amount of cell suspension to a sterile EP tube.

[0148] (2) Pipette 10 μl of the cell culture medium from the EP tube and evenly apply it to a cell counting plate. Count the cells under a microscope. There are four quadrants in total. Count the cells in the four quadrants according to the basic principle of "count the top, not the bottom; count the left, not the right."

[0149] (3) Calculate the concentration of the cell suspension according to the formula. Calculate the required volume of cell suspension based on 1000-2000 cells / 100 μl / well (96-well plate). Add the required complete medium and mix thoroughly by pipetting.

[0150] (4) Plate 96-well plates with 100 μl of culture medium per well. Repeat 3-5 replicates for each time period. Transfer the plates to a cell culture incubator and let them sit for 2-4 hours to allow the cells to adhere.

[0151] (5) After the cells adhered, add 10 μl of CCK8 to each well, tap the wall of the 96-well plate to help mix, and then return the plate to the cell culture incubator, strictly protecting from light;

[0152] (6) 2 hours after adding CCK8, measure the absorbance of each well at OD value = 450 nm according to the CCK8 parameter setting of the microplate reader, and continuously measure at 0h, 24h, 48h, and 72h, analyze and save the results.

[0153] The results are as follows Figure 12 As shown, the experimental results showed that the growth rate of breast cancer cell lines stably overexpressing ZNF662 slowed down, which means that overexpression of ZNF662 can significantly inhibit the proliferation ability of breast cancer cells.

[0154] Example 5. Cell clone formation experiment

[0155] (1) Conventional cell digestion and centrifugation. After centrifugation, discard the supernatant and add an appropriate amount of complete culture medium to resuspend the cells as needed. Mix thoroughly by pipetting and transfer an appropriate amount of cell suspension to a sterile EP tube.

[0156] (2) Pipette 10 μl of the cell culture medium from the EP tube and evenly apply it to a cell counting plate. Count the cells under a microscope. There are four quadrants in total. Count the cells in the four quadrants according to the basic principle of "count the top, not the bottom; count the left, not the right."

[0157] (3) Calculate the concentration of the cell suspension according to the formula. Calculate the required volume of cell suspension according to 100 / 200 / 400 cells / 2 ml complete medium / well (6-well plate). Then add the required volume of cell suspension to each well, pipette thoroughly to mix, and slowly transfer to the cell culture incubator.

[0158] (4) Intermittently observe the cell growth in the 6-well plate. Generally, after 10-14 days, when cell colonies are clearly visible under the naked eye or microscope, discard the old culture medium, add 1 ml of PBS, and wash the cells by horizontal shaking. Repeat the above steps once, shake off the residual liquid in the bottle, and then add an appropriate amount of 4% paraformaldehyde to each well and fix at room temperature for 30 minutes.

[0159] (5) Recover 4% paraformaldehyde and add an appropriate amount of crystal violet stain to each well, and stain at room temperature for 30 min;

[0160] (6) Recover the crystal violet staining solution, place the 6-well plate in a foam box filled with running water, and slowly rinse the residual crystal violet back and forth, constantly changing the running water until the color turns light purple;

[0161] (7) Invert the 6-well plate on absorbent paper overnight to absorb the moisture, observe the staining results, analyze and scan for storage.

[0162] The results are as follows Figure 13-14 As shown, the experimental results showed that the number of colonies formed by breast cancer cell lines stably overexpressing ZNF662 was reduced, which means that overexpression of ZNF662 significantly inhibited the clone-forming ability of breast cancer cells.

[0163] Example 6. Cell cycle experiment

[0164] (1) Conventional cell digestion and centrifugation: After centrifugation, discard the supernatant, add 5 ml of PBS, mix thoroughly by pipetting, and place the centrifuge tube in a centrifuge with the setting of 1000 rpm / 5 min.

[0165] (2) After centrifugation, pour off the supernatant, add 100 μl of PBS, and mix gently by pipetting slowly. Then add 5 ml of pre-chilled 75% ice ethanol to fix the tube, and mix gently by pipetting slowly. Mark the tube, seal the tube with sealing glue, and store in a freezer at 4°C overnight.

[0166] (3) After overnight, place the centrifuge tube in a centrifuge and set the parameters to 2000 rpm / 5 min. Note that ice ethanol is very slippery. After the centrifugation is completed, carefully aspirate and discard the supernatant with a pipette tip;

[0167] (4) Add 5 ml of PBS, mix thoroughly by pipetting, and place the tube in a centrifuge at 2000 rpm / 5 min.

[0168] (5) Pour off the supernatant, add 1.2 ml of PBS, mix thoroughly by pipetting, and transfer to three EP tubes, 400 μl per tube;

[0169] (6) Add 2.5 μl of RNase to each EP tube and incubate in a 37°C thermostat for 30 min;

[0170] (7) Add 5 μl of propidium iodide (PI) to each EP tube under strict light-proof conditions, let it stand for 30 minutes, and then load it onto the flow cytometer;

[0171] (8) Laboratory technicians detect cell cycle analysis and save data.

[0172] The results are as follows Figure 15-17 As shown, the experimental results showed that stable overexpression of ZNF662 could arrest the cell cycle of MB231 and YCCB1 cells at the G0 / G1 phase and G2-M phase, respectively, suggesting that ZNF662 significantly induced breast cancer cell cycle arrest at the G0-G1 phase / G2-M phase.

[0173] Example 7. Cell migration assay

[0174] (1) Conventional cell digestion and centrifugation. After centrifugation, discard the supernatant and add appropriate amount of RPMI-1640(-) to resuspend the cells as needed. Mix thoroughly by pipetting and transfer appropriate amount of cell suspension to a sterile EP tube.

[0175] (2) Pipette 10 μl of the cell culture medium from the EP tube and evenly apply it to a cell counting plate. Count the cells under a microscope. There are four quadrants in total. Count the cells in the four quadrants according to the basic principle of "count the top, not the bottom; count the left, not the right."

[0176] (3) Select appropriate wells in a 24-well plate, add 800 μl of complete cell culture medium containing 20% ​​FBS to each well, and then place the cells in the chamber;

[0177] (4) Calculate the concentration of the cell suspension according to the formula. Calculate the required volume of cell suspension based on 3 × 10^4 cells in the upper chamber of the chamber. Add RPMI-1640(-) for a total of 200 μl, gently pipette and mix, then add to the upper chamber of the chamber and slowly transfer to the cell culture incubator.

[0178] (5) After an appropriate number of days, usually 1-2 days, remove the 24-well plate, discard the liquid in the plate, aspirate the liquid in the upper chamber, and then add 800 μl of 4% paraformaldehyde to each well of the 24-well plate. Return the plate to the chamber and fix at room temperature for 30 min.

[0179] (6) Recover 4% paraformaldehyde and add an appropriate amount of crystal violet stain to each well of the 24-well plate. Return the plate to the chamber and stain at room temperature for 30 min.

[0180] (7) Recover the crystal violet dye solution, place the chamber in a foam box filled with running water, and slowly rinse the remaining crystal violet back and forth, constantly changing the running water until the color turns light purple;

[0181] (8) Use a cotton swab to gently wipe off the cells on the inner surface of the upper chamber of the chamber, and invert the chamber on absorbent paper to absorb moisture overnight;

[0182] (9) Observe the results under the microscope and the Institute of Life Sciences will make an appointment to take photos, analyze and save them.

[0183] The results are as follows Figure 18-19 As shown, the experimental results showed that the number of migrating cells in breast cancer cell lines that stably overexpressed ZNF662 was reduced, suggesting that overexpression of ZNF662 significantly inhibited the migration ability of breast cancer cells.

[0184] Example 8. Cell invasion assay

[0185] (1) Place Matrigel in a 4°C refrigerator overnight to melt into a gel;

[0186] (2) Dilute Matrigel with RPMI-1640(-) at a ratio of 1:7 and mix thoroughly by pipetting;

[0187] (3) Add an appropriate amount of diluted Matrigel into the upper chamber of the cell and place it in a cell culture incubator for an appropriate time until it solidifies into a solid gel;

[0188] (4) The remaining steps are the same as the Transwell chamber migration experiment.

[0189] The results are as follows Figure 20-21 As shown, the experimental results showed that the number of invasive cells in breast cancer cell lines that stably overexpressed ZNF662 was reduced, suggesting that overexpression of ZNF662 significantly inhibited the invasive ability of breast cancer cells.

[0190] Example 9. Nude mouse tumor formation experiment

[0191] (1) Order five 4-6 week old female BALB / c nude mice;

[0192] (2) Conventional cell culture: When the cells grow to an appropriate number, digest and centrifuge the cells. After centrifugation, discard the supernatant and add an appropriate amount of PBS to resuspend the cells as needed. Mix thoroughly by pipetting and transfer an appropriate amount of cell suspension to a sterile EP tube.

[0193] (3) Draw an appropriate amount of cell suspension from the EP tube, dilute it 10-fold, and transfer it to a new sterile EP tube; draw 10 μl from the second EP tube and evenly apply it to the cell counting plate. Count the cells under a microscope. There are four quadrants in total. Count the cells in the four quadrants according to the basic principle of "count the top, not the bottom, count the left, not the right";

[0194] (4) Calculate the concentration of the cell suspension according to the formula and multiply it by 10 to get the initial undiluted cell suspension concentration. 4 Calculate the required volume of cell suspension, add PBS for a total of 150ul, gently pipette to mix, and transfer to a new sterile EP tube for use;

[0195] (5) The same number and volume of control group / cells overexpressing ZNF662 were subcutaneously injected into the left and right sides of the buttocks of nude mice;

[0196] (6) After a hard tumor mass appears to the naked eye, the length and width of the tumor are measured every 3 days to calculate the tumor volume;

[0197] (7) If the tumor volume exceeds 1 cm 3 Afterwards, the nude mice were killed according to the ethical principles of experimental animals;

[0198] (8) The nude mouse tumor tissue was dissected and separated layer by layer, and placed in the order of the upper and lower corresponding overexpression groups of the same nude mouse control group, photographed, and weighed;

[0199] (9) After the photography is completed, the tumor tissue is fixed in 4% paraformaldehyde and the next step of the experiment is planned according to the experimental requirements.

[0200] The results of the nude mouse subcutaneous tumor formation experiment showed that the size, weight and volume of the transplanted tumors formed by the ZNF662 target group were significantly lower than those of the control group. Further in vivo verification of the ZNF662 gene's ability to inhibit the growth of breast cancer cells in vivo showed that the ZNF662 gene could inhibit the growth of breast cancer cells in vivo. Figure 22-23 shown. Sequence Listing <110> Chongqing Morsecode Biotechnology Co., Ltd. <120> Breast cancer biomarker ZNF662 gene and its detection method and application <130> 2022.06.01 <141> 2022-06-06 <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1281 <212> DNA <213> Homo sapiens <400> 1 atgctggaga attatggggc tgtggcttcc ctggcagcat ttccatttcc caaaccggct 60 ctgatttccc agctggagcg aggggaaaca ccctggtgct cggttcctcg gggagctctg 120 gatggagagg ccccaagggg catctcctca ggatatccat ttctaaagcc tgctgggatt 180 tcccatcctg agcaggtgga agagccatta aacctgaaac tgcaaggaga gggtccaagc 240 ctgatttgtc cggagggtgt gttgaagagg aagaaagaag attttattct gaaggaggaa 300 attattgagg aagcacagga cctcatggtc ctatcaagtg gaccccagtg gtgtggatcc 360 caggaattat ggtttgggaa aacctgtgaa gagaaaagca ggttagggag atggcctggt 420 tacctcaatg ggggacgtat ggaaagttct acaaatgata ttatagaagt gattgtcaag 480 gatgagatga tctcagtaga agagagttca gggaatactg atgtcaataa cctccttggt 540 atacatcaca aaattctaaa tgagcaaata ttctatatat gtgaggaatg cggcaagtgt 600 tttgatcaaa atgaggactt tgatcaacac cagaaaactc ataatggaga gaaggtctat 660 ggatgtaagg aatgtgggaa ggctttcagt tttcgatcac attgcattgc acatcagaga 720 attcacagtg gggtgaaacc ctatgaatgt caagaatgtg ctaaggcctt tgtttggaag 780 tcaaacctga ttcgtcacca gagaatacat actggagaga aaccctttga atgtaaggaa 840 tgtgggaagg gctttagtca gaacacaagc cttacgcaac atcaacggat ccacactggt 900 gagaaaccat acacatgtaa ggaatgtggg aaaagcttta ctcgaaaccc agcccttctt 960 cgacatcaga gaatgcacac tggggagaag ccttacgaat gtaaggactg tgggaagggc 1020 ttcatgtgga actcagatct ttctcagcac cagagggtcc acactgggga caagcctcat 1080 gaatgtactg actgtgggaa aagcttcttt tgcaaggcac atcttattcg acatcaaaga 1140 atccatactg gggaaagacc ctataaatgt aatgactgtg ggaaggcctt cagtcagaat 1200 tctgtcttaa ttaagcacca gaggcgccat gctagagaca aaccctataa ctgtcagatc 1260 tctcaccttc ttgaacatta g 1281 <210> 2 <211> 2340 <212> DNA <213> Homo sapiens <400> 2 cttccttcct tcttcccttc tccttccttc ctctctccct cccttccctc cttccttccc 60 tccttccttc ctcccttcct tttccttcct tccttcttcc cttctccttc cttcctttct 120 ttttctttct ctctcttttt ctttctttct ctctctctct ttccctcctt cccttccttc 180 cttcgtctct tctttctttc cctctctccc tcccactctt tctttctttc cctccctcct 240 ttccttcctt cctctttcct tctcttctct tttctcttct tctctttctt ctttccaggt 300 tcttgctgga gtgcaatggt gcaatcacag ctcactgcag ccttgacttc tcaggctcaa 360 gtgatcctcc catctcagac ttctgagtag ccaggagctg gctatttttc tttatttctt 420 tctttctttc tttttttatt tttgtagaga tggggtagca atttgattaa aaactatcag 480 [[ID=第21]]attccctact ttttcccata ctacttccaa cttaggacca aacagagaaa gccaaaaatt 540 tccccctacc aattacataa gattccctgc ttctagttag cccgcctgta gtttccccat 600 gccagcaact tccaataagg gaatacctga agccattcct tttttccact gtgaagcttt 660 It should be noted that there seems to be a minor issue in the original text where the "[[ID=第21]]" should probably be just "". This has been adjusted in the translation as much as possible while keeping the overall integrity of the translation task requirements.ccccactctt ctgcctgact ttcagtctgc tgaatgcaag tgatgagggc tgactctctt 720 gtcatatata gcaagctctg aataatagtc tttgttctca tttgaataat cttattttc 780 acacttctta aaaaatttaa tttcctggca tgttgcataa tgttaggtaa gccattttct 840 ctctttgggc cttactttct tcaactgaaa aactggttca acatttattc attcttttat 900 tccgtaagcc aaaaataaaa ttcaaagccc tctgaccata tgaatggatc tctcctcttg 960 gctaagggca ttccaaagtt aacgtgaaaa actggttcag gccataatgg gaaggaggag 1020 ttggacatgg ctcattatgc tcccctacct ttgaaattc aggaccagct gaccagcatt 1080 aacatcaaca cagaccttaa gactgataga acagcctctt taagtctcac agactatccg 1140 gtagtgtgat aatgatgaga aacatttgca gtctattctc tgaagcctgc tacaggctcc 1200 atctgcatga taaaactttg gtctccacaa gcccttatct taactgagac atccctttct 1260 attgattcta agtctttata caataactta actctttcaa ccaattgcca ctcagaaaat 1320 ctttgaatcc acctatgtcc tgaaagcctc ctcttccagt tgtcccacct ttccataccg 1380 aaccaacgta catcttacat gtattaattg atgtcttatg tcttcctaaa atgtgtaaaa 1440 ccaagttgta gcccgaccac gttgggcaca tgttctcagg atctcctggg gctgtggcac 1500 gggccatggt cactcatatt tggctcagga taaatccctt caaatatttt acagtttgac 1560 ttttcgtgga caattctaca aatattcctt ggcagttaaa gaaataactc cccaggaggt 1620 gctagacatc gggcgcatgt tagaggcaca aagatgagaa tgttaaaaga cataaaatac 1680 aggtctgctc tgggaatgca aaaactggta aaaggagaga taaaagagca ggtgcagtct 1740 agtgaagtac ccgctacctc ctatcagcaa atatctgtcc acctagggtg cagcgagctg 1800 gagaggaagg ggtggcctag agcgcagggg aaggattcct ccccagccgc ctgcacccct 1860 accccggtag cggtccctgg gatcgtccgt gtctccagga gaaccggacc gctctcccct 1920 ccctccccga gcgagaaagg aggaccacag agatgcggcg ccctccgccg tcctagagca 1980 accggagcgg cccgagcccc ggcctcccgg atgctggggc ctggcgggtg tggagcacgg 2040 ggagtcgggc gtggggcggg cagggagtgg agtcggggtc ttactccggt ggctgcaggg 2100 cgcagggtag ccgtgtcagg cctgcccagg tgcagagcgc tcttccgcga ccccaacagc 2160 ctctggtccg gtctggcgcg ccctcgcttt cccagagggc gacctgggct atggcggccg 2220 tggcgctggc gagcgggaca cgcctcggcc ttgtcctcga gctgctcccg ggacagcccg 2280 cgctgccccg ggcgcgccgg gtgagtgcgg ggccggagcc tggaagcagt cttggccctg 2340 <210> 3 <211> 22 <212> DNA <213> Human (Homo sapiens) <400> 3 gacctcatgg tcctatcaag tg 22 <210> 4 <211> 23 <212> DNA <213> Human (Homo sapiens) <400> 4 ctactgagat catctcatcc ttg 23 <210> 5 <211> 21 <212> DNA <213> Human (Homo sapiens) <400> 5 gtggagtcgg ggttttattt c 21 <210> 6 <211> 22 <212> DNA <213> Human (Homo sapiens) <400> 6 cctctaaaaa aacgaaaacg cg 22 <210> 7 <211> 23 <212> DNA <213> Human (Homo sapiens) <400> 7 gagtggagtt ggggttttat ttt 23 <210> 8 <211> 24 <212> DNA <213> Human (Homo sapiens) <400> 8 tccctctaaa aaaacaaaaa caca 24

Claims

1. Use of a ZNF662 gene detection reagent in the preparation of a product for diagnosing breast cancer, characterized in that: The sample to be tested is derived from a patient suspected of having breast cancer; the nucleotide sequence of the ZNF662 gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The detection reagent includes primers for detecting ZNF662 gene expression, wherein the primers include a forward primer 1 having a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer 1 having a nucleotide sequence as shown in SEQ ID NO:

4.

3. Use of primers for detecting ZNF662 gene expression in preparing a kit for diagnosing breast cancer, characterized in that: The primers used to detect ZNF662 gene expression include a forward primer 1 having a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer 1 having a nucleotide sequence as shown in SEQ ID NO: 4.

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