Molecular marker for diagnosis and treatment of cervical cancer-upf2 gene and its expression products
A technology for UPF2 and expression products, which can be used in the field of biomedicine to solve the problems of lack of tumor specificity and elevated levels.
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Embodiment 1
[0066] Example 1 Screening for Abnormally Expressed Genes in Cancer Tissues of Patients with Cervical Cancer
[0067] 1. Organization collection
[0068] Four cases of cervical cancer tissues provided by the Department of Obstetrics and Gynecology of the hospital were collected from patients who were pathologically diagnosed with cervical cancer after hysterectomy (conization or total resection), and the surrounding normal tissues were used as the control group.
[0069] 2. Tissue RNA extraction
[0070] Take about 50 mg of cervical cancer patient tissue and surrounding normal cervical tissue cryopreserved at -80°C, put it into liquid nitrogen for grinding, transfer it to a 1.5mL EP tube when there are no large particles of tissue, add 1mL Trizol for total RNA extraction and real-time quantitative PCR analysis, the specific process is as follows:
[0071] 1) Add 200 μL of chloroform to the above-mentioned tissue suspension containing 1 mL of Trizol, shake and mix thoroughly ...
Embodiment 2
[0090] Example 2 Large sample verification of expression of differentially expressed genes
[0091] 1. Detection at the transcript level
[0092] 1. Organization collection
[0093] According to the standard of Example 1, 40 cases of cervical cancer tissues and corresponding normal control tissues were collected.
[0094] 2. Tissue RNA extraction and identification
[0095] Step is with embodiment 1.
[0096] 3. Design and preparation of primers
[0097] The sequences of real-time quantitative PCR primers used in this application are as follows (designed and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.):
[0098] UPF2 gene primers:
[0099] Upstream primer: 5'-GAAGAAGAAGAAGAAGGT-3' (SEQ ID NO.1);
[0100] Downstream primer 5'-TTATTGGAATCTGTAAGGTA-3' (SEQ ID NO.2),
[0101] GAPDH gene primers:
[0102] Upstream primer: 5'-GACCTGACCTGCCGTCTA-3' (SEQ ID NO.3);
[0103] Downstream primer: 5'-AGGAGTGGGTGTCGCTGT-3' (SEQ ID NO.4).
[0104] 4. Reverse transcript...
Embodiment 4
[0159] Effect of the expression of embodiment 4UPF2 gene on Hela cell proliferation
[0160] Cell proliferation was detected by CCK-8 method
[0161] (1) transfect according to the method of embodiment 3;
[0162] (2) For Hela cells 24 hours after transfection, discard the medium, digest the cells with 0.25% trypsin, count the cells with crystal violet staining solution, resuspend the cells in 1640 medium containing 0.5% FBS and adjust the cell concentration to 2x 10 4 / ml;
[0163] (3) Add 100 μl of cell suspension to each well of a new 96-well culture plate under sterile conditions, that is, 2x10 3 cells, 37°C, 5% CO 2 Stand still for 24 hours;
[0164] (4) Add 10 μl of CCK solution to each well and incubate for 2 hours;
[0165] (5) At a wavelength of 450nm, the absorbance of each experimental group was measured by an ultraviolet spectrophotometer.
[0166] result:
[0167] The results showed that the OD value of the cells in the negative control group (transfected ...
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