A method for identifying liver cancer cells

Abstract

The present invention discloses a method for identifying liver cancer cells, belonging to the fields of life science and medicine. The present invention uses the MBL2 gene as a marker for liver cancer cells. By extracting the RNA of the cells to be identified, reverse transcribing, and PCR amplifying the MBL2 gene, it is possible to accurately identify cells of liver cancer tissue origin from various tumor tissues. The present invention has good specificity. By detecting only one gene, it can be determined whether the cells are derived from liver cancer tissue.

Description

Technical Field

[0001] The present invention belongs to the fields of life science and medicine, and particularly relates to a method for identifying liver cancer cells. Background Art

[0002] The World Health Organization's 2018 Global Cancer Report indicates that there were 18.1 million new cancer cases and 9.6 million deaths worldwide. China has the highest cancer incidence rate in the world, with liver cancer ranking fifth among malignant tumors in men and ninth among women. Liver cancer causes 600,000 deaths annually, with approximately 82% of cases occurring in developing countries, 55% of which are caused by the hepatitis B virus (HBV) and occur in China. Research on tumor treatment has always been a priority in medical research. Most patients with hepatocellular carcinoma have an insidious onset, with no typical symptoms in the early stages and few physical changes. The disease progresses rapidly, and by the time symptoms appear and medical attention is sought, the disease is often already in the middle or late stages, missing the optimal treatment window.

[0003] Currently, the clinical methods for examining liver cancer generally use the serum marker alpha-fetoprotein or ultrasound imaging analysis technology. The former has poor specificity and is also expressed in gastrointestinal tumors, while the latter is mainly based on histological and cellular characteristics and has low sensitivity, making it difficult to make a clear diagnosis of pathological liver cancer through imaging methods, and it is very easy to miss or misdiagnose.

[0004] With the development of cell identification technology, the problem of quickly identifying diseased cells and identifying microscopic indicators to predict liver cancer cells needs to be overcome urgently.

[0005] From an experimental perspective, cross-contamination and identification of cell lines have long been issues often overlooked by researchers. Cases of HeLa cells contaminating other cell lines have been reported as early as the 1960s. The International Cell Line Authentication Committee (ICLAC) published data (Version 7.2) in October 2014, listing 438 false cell lines and 37 misidentified cell lines. The ICLAC has been updating this data annually to remind researchers to avoid using erroneous cell lines. Various surveys indicate that 18% to 36% of cell lines are incorrectly labeled with their provenance, and new false cell lines are likely to continue to be used without knowledge of their misidentification. These contaminated cell lines continue to be used by researchers and may be used in clinical trials, further complicating cell identification. This presents numerous challenges for various cell-related experiments. The development of simple and rapid cell authentication technologies could significantly reduce the scientific and industrial losses, as well as the human and material resources, caused by the inability to distinguish cell provenance. Current cell identification techniques, including cytological morphology, HLA typing, karyotyping, isozyme analysis, and STR analysis, primarily focus on identifying cells between species or individuals, but rarely address the identification of cell tissue origin. A comprehensive review of existing identification methods reveals that they fall short of meeting the needs for identifying cell and tissue origin in scientific research. To date, there are no unified standards for identifying cell and tissue origin. Summary of the Invention

[0006] The purpose of the present invention is to provide a molecular marker MBL2 and a method for identifying liver cancer cells.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A molecular marker for identifying liver cancer cells is the MBL2 gene.

[0009] A method for identifying liver cancer cells comprises the following steps: extracting RNA from cells to be identified, obtaining cDNA by reverse transcription, and amplifying the MBL2 gene by PCR using the cDNA as a template. If MBL2 can be amplified, the cells to be identified are liver cancer cells.

[0010] The molecular marker is used in the preparation of a reagent for identifying liver cancer cells, wherein the reagent comprises a primer for amplifying the MBL2 gene.

[0011] A reagent for identifying liver cancer cells, comprising primers for amplifying the MBL2 gene.

[0012] The sequences of the primers for amplifying the MBL2 gene are preferably as follows:

[0013] MBL2-S: 5'-GATAGTAGCCTGGCTGCCTCAG-3',

[0014] MBL2-A: 5'-TGTTGGGTTCACCTCGTTC-3'.

[0015] Advantages and beneficial effects of the present invention: The present invention only needs to detect one gene (MBL2) to determine whether a cell originates from liver cancer tissue. The present invention has good specificity and can accurately identify liver cancer tissue-derived cells from various tumor tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the agarose gel electrophoresis diagram of different tumor cells detected by PCR amplification of MBL2 gene.

[0017] Figure 2 It is an agarose gel electrophoresis diagram of detecting different tumor cells by PCR amplification of F2 and CFHR2 genes. DETAILED DESCRIPTION

[0018] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0019] Example Identification of Hepatocellular Carcinoma Cells

[0020] 1. Liver cancer cell culture. Resuscitation of human hepatoma cells Huh-7.5.1, human colon cancer cells HCT 116, human gastric cancer cells NCI-N87, human myeloma cells NCI-H929, human neuroblastoma cells IMR-32, human renal adenocarcinoma cells ACHN, human lung cancer cells A549, and human breast cancer cells MCF7. All cells were obtained from the China Center for Type Culture Collection. The culture medium used was MEM supplemented with 10% serum. The culture conditions were 37°C and 5% CO2. After cell recovery, the cells were passaged three times before use in subsequent experiments.

[0021] 2. Primer Design: Log in to the NCBI official website at https: / / www.ncbi.nlm.nih.gov / tools / primer-blast, enter the gene sequences of MBL2, F2, and CFHR2, and design primers online. Primer information is shown in Table 1 below. The MBL2, F2, and CFHR2 genes are specific genes with high expression in liver cancer cells, as identified by the present invention through extensive analysis and screening of databases such as the HPA database and the CCLE database.

[0022] Table 1

[0023]

[0024] 3. To extract total RNA, remove the cultured cells, discard the supernatant, add 1 mL of RNAiso Plus to lyse the cells, and vortex until thoroughly mixed. Add 200 μL of chloroform to each EP tube and vortex until the solution becomes turbid. Let stand at room temperature until the layers are completely separated. Centrifuge at 12,000 rpm at 4°C for 15 min. Transfer the upper layer to a new RNase-free EP tube using a pipette, add an equal volume of isopropanol, invert and mix thoroughly, let stand for 20 min, and centrifuge at 12,000 rpm at 4°C for 15 min. Discard the supernatant and wash the pellet with pre-chilled 75% ethanol (3:1 anhydrous ethanol:DEPC water) to each EP tube. Invert the tube until the pellet falls off the bottom of the tube and floats in the ethanol like a feather. Observe the amount of pellet and mark the tube with the least and most pellets. Centrifuge at 12,000 rpm at 4°C for 5 min. Use a pipette to discard the supernatant. Place the uncapped EP tube in a clean bench to dry. When the RNA precipitate in the EP tube turns from white to a dry powder invisible to the naked eye, place the tube in an ice box. According to the markings, add 25 μL of RNase-free ddH2O to the EP tube with less precipitate, 35 μL of RNase-free ddH2O to the EP tube with a medium amount of precipitate, and 50 μL of RNase-free ddH2O to the EP tube with a large amount of precipitate. Mix the dissolved RNA solution thoroughly, measure the concentration using a Nandrop 2000C spectrophotometer, and store at -80°C.

[0025] 4. Perform the RT reaction and remove the total RNA stored at -80°C. Add 11.5 μL of template RNA and 1 μL of Oligo dT to each PCR tube, label it, and incubate it at 72°C for 5 minutes. Remove the PCR tubes and add 5 μL of RT PCR buffer, 1.5 μL of dNTPs, 0.5 μL of RRI, and 0.5 μL of reverse transcriptase to each tube. Place the tubes in the PCR instrument again and incubate at 42°C for 60 minutes, 70°C for 15 minutes, and terminate at 4°C. Store the resulting cDNA at -20°C.

[0026] 5. For the PCR reaction, first dilute the primers: Take 1 μL of each primer solution (MBL2, F2, CFHR2) and dilute to 10 μL, resulting in a primer concentration of 10 μM. Label. Then dilute the resulting template cDNA: Take 0.5 μL of each template cDNA (extracted from HCT 116, NCI-N87, NCI-H929, IMR-32, ACHN, A549, and Huh-7.5.1 cells, respectively), add 9.5 μL of enzyme-free water, and mix thoroughly. Prepare the PCR reaction system according to Table 2. Aliquot the prepared system and add 2 μL of the diluted cDNA solution to each aliquot (add 2 μL of enzyme-free water to the negative control). PCR reaction conditions: 95°C pre-denaturation for 10 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles of 72°C extension for 10 min, and termination at 16°C.

[0027] Table 2 PCR reaction system

[0028]

[0029]

[0030] 6. Agarose gel electrophoresis: Prepare a 1.5% agarose gel: Weigh 0.75g of agarose and add it to a conical flask. Using a graduated cylinder, measure 50mL of 1% TAE buffer and add it to the conical flask. Microwave and boil several times until the solution is clear. Allow to cool slightly, then add 5μL of 20000X GelRed dye to the flask. Pour the solution onto a gel-forming plate, insert a comb, and cool to solidify. Place the agarose gel in an electrophoresis tank and add 1× TAE buffer until the gel is just covered. Apply the following sample: Marker, load volume 5μL; Sample: First, take 5μL of the sample, add 1μL of 6× Loading Buffer, mix well, and then apply 5μL of the sample. Wait for the sample to settle before closing the electrophoresis tank, ensuring the sample wells are facing the negative pole. Turn on the power supply, set the electrophoresis voltage to 120V, and stop the electrophoresis when the bromophenol blue band has run 3 / 4 of the way through. Take out the gel, put it into the gel imaging device, photograph the gel, and save the image.

[0031] 7 Results were determined based on the agarose gel electrophoresis pattern ( Figure 1 and 2). The MBL2 gene showed a band only in human liver cancer cells Huh-7.5.1, but no bands were observed in human colon cancer cells HCT 116, human gastric cancer cells NCI-N87, human myeloma cells NCI-H929, or human neuroblastoma cells IMR-32. Faint nonspecific bands were observed in human renal adenocarcinoma cells ACHN, human lung cancer cells A549, and human breast cancer cells MCF7. F2 was expressed in both human myeloma cells NCI-H929 and human liver cancer cells Huh-7.5.1, and CFHR2 was expressed in both human gastric cancer cells NCI-N87, human myeloma cells NCI-H929, and human liver cancer cells Huh-7.5.1. These results demonstrate that the MBL2 gene has high specificity and can effectively identify cells of liver cancer origin.

[0032] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention. Sequence Listing <110> Wuhan University <120> A method for identifying liver cancer cells <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <400> 1 gatagtagcc tggctgcctc ag 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 tgttgggttc accctcgttc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gaggacgcct cgagataagc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 acggtggggt ctgtagtgta 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 tttggactcg cataacgtgc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 agtggacctg catttgggag 20

Claims

1. A method for identifying liver cancer cells, characterized by: The method comprises the following steps: extracting RNA from cells to be identified, obtaining cDNA by reverse transcription, and amplifying the MBL2 gene by PCR using the cDNA as a template. If MBL2 can be amplified, the cells to be identified are liver cancer cells. The method for identifying liver cancer cells is for non-disease diagnosis purposes.

2. Application of MBL2 gene in the preparation of reagents for identifying liver cancer cells.

3. The use according to claim 2, characterized in that: The reagents contain primers for amplifying the MBL2 gene.

4. The use according to claim 3, characterized in that: The sequences of the primers for amplifying the MBL2 gene are as follows: MBL2-S: 5'-GATAGTAGCCTGGCTGCCTCAG-3', MBL2-A: 5'-TGTTGGGTTCACCTCGTTC-3'.

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