CN107540736B - Biomacromolecule NHERF1 related to property compliance of cervical cancer and application thereof - Google Patents
Biomacromolecule NHERF1 related to property compliance of cervical cancer and application thereof Download PDFInfo
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- CN107540736B CN107540736B CN201610459491.5A CN201610459491A CN107540736B CN 107540736 B CN107540736 B CN 107540736B CN 201610459491 A CN201610459491 A CN 201610459491A CN 107540736 B CN107540736 B CN 107540736B
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Abstract
The invention discloses a PDZ protein molecule NHERF1 related to cisplatin resistance and application thereof, belonging to the field of tumor biotherapy. The invention discloses a PDZ protein molecule NHERF1 related to cisplatin resistance, which has low expression in a cisplatin-resistant cervical cancer cell line and is a novel cervical cancer cisplatin resistance marker. Therefore, the invention provides a new target for designing the cervical cancer cisplatin resistance drug, and is used for designing and developing a new drug directly aiming at the cervical cancer cisplatin resistance based on the new target. The NHERF1 disclosed by the invention has biological functions and action mechanisms for promoting cervical cancer cell drug resistance, and provides an effective way for effectively reversing cervical cancer cisplatin resistance and improving the clinical chemotherapy effect of cervical cancer.
Description
Technical Field
The invention belongs to the field of tumor biotherapy, and particularly relates to a PDZ protein molecule NHERF1 related to cisplatin drug resistance and application thereof.
Background
Cervical cancer is one of the common malignant tumors of women, and the incidence rate seriously threatens the physical and psychological health of women. At present, the incidence rate of cervical cancer malignant tumors in China is higher than the death rate in foreign countries and is the fourth of the death rate of all malignant tumors.
The early treatment of the cervical cancer is still mainly performed by the operation and is assisted by chemotherapy comprehensive treatment, and a combined chemotherapy scheme with platinum as the main part and other chemotherapy medicaments is provided before the operation, so that the killing efficiency of the cancer cells is obviously improved. Cisplatin is widely used in clinical treatment of various types of solid tumors, and binding of the drug to tumor cell DNA causes DNA damage and induces apoptosis. However, the secondary drug resistance of tumor cells also appears while ensuring the curative effect of cisplatin, and the secondary drug resistance has a great influence on the curative effect of tumors.
Disclosure of Invention
The invention aims to provide a biological macromolecule NHERF1 related to cisplatin resistance and application thereof.
The invention is realized by the following technologies:
the invention discloses a biological macromolecule NHERF1 related to cisplatin drug resistance
The invention also discloses application of the biomacromolecule NHERF1 protein or mRNA as a cisplatin drug marker for cervical cancer.
Biomacromolecule NHERF1 protein or mRNA is underexpressed in cervical cancer resistant cells.
The invention also discloses application of the biomacromolecule NHERF1 protein or mRNA in preparing a drug target aiming at the cisplatin resistance of cervical cancer.
The invention also discloses application of the biomacromolecule NHERF1 protein or mRNA in evaluating the curative effect of the cisplatin medicine for cervical cancer.
Compared with the prior art, the invention has the following beneficial technical effects:
NHERF1 has low expression in cervical cancer drug-resistant cells, low expression in patients with cervical cancer cisplatin chemotherapy and the characteristic of reduced expression with the increase of the concentration of chemotherapeutic drugs, and is a novel cervical cancer cisplatin-resistant marker. Therefore, the invention provides a new target for designing the medicine for resisting the cervical cancer cisplatin resistance, and is used for designing and developing a new medicine for resisting the cervical cancer cisplatin resistance based on the new target. The NHERF1 disclosed by the invention has biological functions and action mechanisms for inhibiting the drug resistance of cervical cancer cells, and provides an effective way for effectively reversing the drug resistance of cervical cancer cisplatin and improving the diagnosis and treatment effects of clinical chemotherapy of cervical cancer.
The following examples are provided to illustrate specific embodiments of the present invention.
Detailed Description
The present invention is further described in detail below with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.
Cisplatin plays an important role in the treatment process of cervical cancer, but the existing drug resistance phenomenon is a main reason for limiting the clinical curative effect of cisplatin. The biological function and mechanism of the PDZ protein NHERF1 in the cisplatin resistance of the cervical cancer cells are determined, and a new biological target selection is provided for effectively improving the clinical curative effect of the cervical cancer.
The invention discloses that the expression level of NHERF1 expression in cisplatin-resistant cervical carcinoma cells is reduced, and the NHERF1 expression in patients undergoing cisplatin chemotherapy has low expression and the characteristic of reduced expression along with the increase of the concentration of chemotherapeutic drugs, and is a novel cervical carcinoma cisplatin-resistant marker.
The invention also discloses application of NHERF1 as a target for designing a drug resistant to the cervical cancer cisplatin, application of NHREF1 in clinical diagnosis of the drug resistant to the cervical cancer cisplatin, and a drug designed according to the target molecule.
1. Construction of HeLa cisplatin-resistant cell line (HeLa-R)
In the process of constructing the Hela cell drug-resistant cell line, a screening method for gradually increasing the drug concentration is adopted to culture the Hela drug-resistant cell line. Inducing cell drug resistance by adopting a method of gradually increasing intermittent action of cisplatin concentration, acting for 48 h from low concentration of 0.1 mu g/ml, discarding the culture solution containing the drug, adding fresh culture solution, continuing to treat for 48 h with the drug concentration of 0.1 mu g/ml after the culture solution recovers normal growth, and repeating for 4 times. The Hela cells are full in shape, tightly arranged, uniform in size, clear in boundary and grow adherent under an inverted microscope; after the medicine is added, the cells are obviously changed, part of the cells die 2-3 hours after the medicine is added, the dead cells float in the culture solution, the adherent cells are different in size, irregular in shape, some cells are fusiform, some protrusions are increased, and a large number of bubbles appear in cytoplasm. After the cell screened by the medicine reaches the stable form and growth speed, the concentration of the cis-platinum is gradually increased, and the medicine screening step is repeated. At present, the drug-resistant Hela cell line which is constructed and cultured in a laboratory and can normally proliferate at the cisplatin drug concentration of 1.0 mu g/ml. The growth status of the drug-resistant Hela cell line was microscopically observed at a cisplatin drug concentration of 1.0. mu.g/ml as shown in FIG. 1.
2. Detecting the expression level of NHERF1 in HeLa parent cells and HeLa-R cells
The treated cells were removed, the medium was discarded, and the cells were washed 3 times with cold PBS. Cells were collected with a cell scraper by adding 500. mu.L of PBS,centrifuge at 2,000 rpm at 4 ℃ for 5 min. At 500. mu.L/10 6 Adding ice to pre-cool cell lysate in proportion of each cell, and repeatedly blowing, sucking and uniformly mixing. Sonicate 5 times for 3 sec/time on ice. The cell protein sample was mixed with 2Xlodding buffer and sonicated at 50hz for 12 times for 3 seconds each. After 5 min of denaturation at the later 100 ℃, the mixture was immediately placed on ice for 5 min of cooling and centrifuged instantaneously at 1500 rpm.
1X 106 cells were collected, washed twice with pre-chilled PBS, lysed on ice and total protein extracted. And (3) performing polyacrylamide gel electrophoresis on the extracted protein at a loading amount of 30-100 mu g, transferring the protein onto a PVDF membrane, sealing the protein with TBST buffer solution containing 5% skimmed milk powder at room temperature for 1 h, respectively incubating the protein with NHERF and GAPDH antibodies for 2 h, washing the membrane, and incubating the membrane with HRP-mouse anti-rabbit antibody (1: 3000) at room temperature for 1 h. And (3) after the exposure of the X-ray film in the dark room, analyzing the gray value of the protein band by adopting Image-J Image analysis software, and performing semi-quantitative analysis by taking the gray value ratio of the target protein to the control GAPDH as a relative expression level.
3. Cell proliferation-apoptosis experiment drug resistance study after detection of cell transfection NHERF1
Cells were seeded into 96-well plates at 3000 cells per well, and 20ul CCK-8 (Homopoly Japan) was added on days 1, 3, 5 and 7 for incubation for 1 hour, followed by measurement of the absorbance at 450nm using an enzyme reader. HeLa cells that interfered with NHERF expression were treated with cisplatin drug and the effect of cisplatin treatment on cell viability was analyzed. The results are shown in table 2 and fig. 3, and the relative cell viability of HeLa cells is increased in the cisplatin-resistant viability. HeLa cells interfering NHERF expression are utilized to research the change of apoptosis caused by the whole cell tolerance medicament under the action of different chemotherapy medicaments of cis-platinum, paclitaxel and methotrexate. As a result, the NHERF expression is reduced, and the cisplatin and paclitaxel treatment HeLa group cells have reduced apoptosis.
Following the procedures of the apoptosis test kit (BD Co., UK), HeLa cells were digested with EDTA-free pancreatin, washed twice with PBS, suspended in a binding buffer, and cell density was adjusted to 1X 10 6 And/ml. Adding 400 μ L cell suspension into flow tube, adding 5 μ L Annexin V-FITC, incubating at 4 deg.C for 15 min, incubating at 4 deg.C for 5 min, and performing flow cytometryDetecting the apoptosis rate of the cells.
4. Study on expression level of NHERF1 after clinical cisplatin chemotherapy of cervical cancer patient
mRNA and prognostic data from cervical cancer patients in the TCGA database were collected and analyzed, and NHERF1 was found to be expressed at lower levels in the presence of high concentrations of cisplatin than in patients treated with low concentrations of cisplatin.
The results show that:
1) detecting and screening out cisplatin-resistant capacity of HeLa cisplatin-resistant cell line through cell proliferation-toxicity experiment
Cell proliferation-toxicity test results show that, compared with the HeLa parent cell, the HeLa-R cell can survive under high-concentration cis-platinum treatment, and the drug resistance is obviously enhanced, see figure 1.
2) The expression level of NHERF1 in the cervical cancer parent cell and the cervical cancer drug-resistant cell is detected by western blot, which is shown in figure 2.
3) The expression level of NHERF1 decreased after high-concentration cisplatin chemotherapy in clinical patients as shown in FIG. 3.
4) Cell proliferation assay the effect of NHERF1 transfection on the proliferation capacity of HeLa cisplatin-resistant cells of cervical cancer was examined as shown in fig. 4.
5) Apoptosis test the effect of NHERF1 transfection on the apoptosis ability of cervical cancer HeLa cisplatin-resistant cells is shown in fig. 5.
In view of the above, it is desirable to provide,
with the continuous and deep research of PDZ protein in the process of tumor occurrence development and tumor drug resistance acquisition, the invention discovers that the expression level of NHERF1 in a drug-resistant cell HeLa is reduced through the detection of a cervical cancer drug-resistant cell line, and discovers that the NHERF1 is low in expression in patients undergoing cervical cancer cisplatin chemotherapy and has the characteristic of expression reduction along with the increase of the concentration of chemotherapeutic drugs, so that the NHERF1 is a novel cervical cancer cisplatin-resistant marker, and is further analyzed and verified to inhibit the biology, functions and action mechanisms of the cervical cancer cell cisplatin resistance, thereby providing an effective way for effectively reversing the cervical cancer cisplatin resistance and improving the clinical chemotherapeutic effect of the cervical cancer.
Claims (2)
- Application of a detection product of NHERF1 in preparation of a product for detecting cisplatin resistance of cervical cancer.
- 2. An application of a biological macromolecule NHERF1 protein or mRNA related to cisplatin resistance in preparing a medicament for treating the cisplatin resistance of cervical cancer.
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| CN111249465B (en) * | 2020-01-16 | 2022-03-01 | 山东大学齐鲁医院 | Application of TOPK as cervical cancer cisplatin resistance treatment target |
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| CN102656458A (en) * | 2009-10-26 | 2012-09-05 | 雅培制药有限公司 | Diagnostic methods for determining prognosis of non-small cell lung cancer |
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Non-Patent Citations (4)
| Title |
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| EBP50影响HeLa细胞微丝骨架的分布和定位;郑君芳等;《中国肿瘤临床》;20081231;第20卷;1192-1195 * |
| EBP50抑制Hela细胞增殖及基质金属蛋白酶表达;郑君芳等;《基础医学与临床》;20090331;第29卷(第3期);314-315 * |
| Homo sapiens SLC9A3 regulator 1 (SLC9A3R1), mRNA, NCBI Reference Sequence: NM_004252.4;Loureiro CA等;《GenBank Database》;20160220;NM_004252.4 * |
| NHERF1 regulates actin cytoskeleton organization through modulation of α-actinin-4 stability;Sun Licui等;《FASEB J》;20160228;第30卷(第2期);578-589 * |
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