CN111308076A - Application of ubiquitin ligase RNF38 as nasopharyngeal carcinoma marker - Google Patents
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Abstract
The invention provides application of ubiquitin ligase RNF38 as a nasopharyngeal carcinoma marker, belonging to the field of biological monitoring. Basic experiments prove that RNF38 is obviously related to nasopharyngeal carcinoma metastasis, the expression of RNF38 is detected by an immunohistochemical method, the method is simple and convenient, the cost is low, the feasibility and the repeatability are high, and the metastasis of nasopharyngeal carcinoma patients is predicted to a certain extent, so that people with high nasopharyngeal carcinoma metastasis are screened, and the clinical diagnosis and treatment of the nasopharyngeal carcinoma are assisted.
Description
Technical Field
The invention belongs to the field of detection, and particularly relates to application of ubiquitin ligase RNF38 as a nasopharyngeal carcinoma marker, which is used for assisting prognosis prediction and clinical diagnosis and treatment of a nasopharyngeal carcinoma patient by detecting the expression of RNF38 in nasopharyngeal carcinoma tissues.
Background
Nasopharyngeal carcinoma is one of the most common malignant tumors in south China, and the 5-year survival rate of the nasopharyngeal carcinoma can reach more than 80 percent at present. However, more than 70% of patients are in the middle and late stages, and the survival time of the patients is short, and the prognosis is poor. Therefore, the method finds potential targets for predicting nasopharyngeal carcinoma metastasis, screens high-risk groups of nasopharyngeal carcinoma, and has important significance for assisting diagnosis and treatment of nasopharyngeal carcinoma patients. At present, the distant metastasis of nasopharyngeal carcinoma is clinically predicted mainly according to the stage of TNM. However, the biggest problem of TNM staging is to consider only the extent of tumor invasion, and not the biological behavior of tumors and tumor heterogeneity, and relying solely on TNM staging does not accurately distinguish the risk of metastasis in patients, which can lead to under-or over-treatment. Ubiquitination modification is closely related to the occurrence and development of tumors, and abnormal ubiquitination modification is an important factor for carcinogenesis. The ubiquitin-proteasome system comprises ubiquitin activating enzyme, ubiquitin conjugating enzyme, ubiquitin ligase and the like, and mediates 80% -85% of protein degradation in eukaryotes. The ubiquitin ligase is the most abundant type in the ubiquitin-proteasome system mediating ubiquitination modification, can directly determine the activity and the location of protein, participates in various biological behaviors of tumors, and is a hotspot of current tumor research. RNF38 is one of ubiquitin ligases, and cell and mouse experiments show that RNF38 promotes the proliferation of gastric cancer and non-small cell lung cancer cells; RNF38 promotes metastasis of liver cancer cells and non-small cell lung cancer cells; at the same time, RNF38 expression was significantly correlated with prognosis in tumor patients. Therefore, the RNF38 is closely related to the occurrence and development of tumors, can provide a new theoretical basis for predicting nasopharyngeal carcinoma prognosis, clinical diagnosis and treatment and developing targeted drugs, and has important significance.
Disclosure of Invention
The invention aims to provide application of ubiquitin ligase RNF38 as a nasopharyngeal carcinoma marker.
In order to achieve the purpose, the invention adopts the following technical scheme:
application of ubiquitin ligase RNF38 in preparing reagents for detecting nasopharyngeal carcinoma is provided.
The ubiquitin ligase RNF38 is applied to the preparation of auxiliary diagnosis and prognosis prediction reagents for nasopharyngeal carcinoma.
Nasopharyngeal paraffin wrapped blocks of patients with nasopharyngeal carcinoma and healthy people are pathologically diagnosed are collected, the expression of RNF38 is detected by an immunohistochemical method, and the expression rule of RNF38 in nasopharyngeal carcinoma tissues and normal nasopharyngeal tissues is analyzed; also, the correlation of RNF38 with the prognosis of patients with nasopharyngeal carcinoma was analyzed in combination with clinical and prognostic information from the patients. Constructing a stable transgenic cell strain overexpressed by RNF38 and a stable transgenic cell strain silenced by RNF38, and discussing the influence of RNF38 on the nasopharyngeal carcinoma cell transfer capacity at a cell level through a scratch experiment, a cell invasion and a cell migration experiment; constructing a nude mouse tail vein transfer model, and discussing the influence of RNF38 on the transfer capability of nasopharyngeal carcinoma cells in nude mice.
The invention has the advantages that:
the clinical significance of the ubiquitin ligase RNF38 in nasopharyngeal carcinoma is not clear at present, the application proves that RNF38 is obviously related to nasopharyngeal carcinoma metastasis through basic experiments, the expression of RNF38 is detected through an immunohistochemical method, the method is simple and convenient, the cost is low, the feasibility and the repeatability are high, and the metastasis of nasopharyngeal carcinoma patients is predicted to a certain extent, so that people with high nasopharyngeal carcinoma metastasis are screened, and the clinical diagnosis and treatment of the nasopharyngeal carcinoma are assisted.
Drawings
FIG. 1 shows RNF38 expression in immunohistochemistry.
FIG. 2 is a graph showing the effect of RNF38 expression level on the prognosis of nasopharyngeal carcinoma.
FIG. 3 shows the in vitro and in vivo scratch test results of RNF 38.
FIG. 4 shows the results of the invasion and metastasis experiments of RNF 38.
FIG. 5 shows the result of RNF38 transfer to the tail vein of nude mice.
Detailed Description
Example 1
First, experiment method
1. Patient organization and data collection
Collecting nasopharyngeal paraffin blocks of patients with nasopharyngeal carcinoma and healthy persons; collecting all patient pre-treatment basic clinical information including sex, age, pathology type, ECOG score, T stage, N stage, M stage, plasma EBV DNA level, etc.; treatment-related information including radiation dose, chemotherapy regimen, etc.; and post-treatment follow-up information including total survival time, survival time without distant metastasis, survival time without local recurrence, etc. to be built into a database for later analysis.
Immunohistochemical staining procedure (S-P method)
3.0% H is added dropwise to each 4mm paraffin section2O2Incubating the solution at room temperature for 10 min to block endogenous peroxidase activity, washing with PBS for 3X 3 min; the sections were added with RNF38 antibody, l: 50 concentration diluted RNF38 antibody, respectively incubating at 37 deg.C for 60 min, and washing with PBS for 3 × 3 min; dripping 50.0 ul of second antibody into each section, incubating at room temperature for 30min, and washing with PBS for 3X 3 min;
dripping 50.0 ul of streptavidin-peroxidase solution into each slice, incubating at room temperature for 30min, and washing with PBS for 3 × 3 min; dripping 100.0 ul of DAB color development solution (3, 3' diaminobenzidine hydrochloride) prepared freshly on each slice, and observing for 3-5 min under a microscope;
fully washing tap water, counterstaining with hematoxylin, differentiating with 0.1% hydrochloric acid alcohol, washing with PBS and turning blue; dehydrating and drying by gradient alcohol, then, transparent dimethylbenzene and sealing by neutral gum; known testis tissues are used as positive controls, and IgG antibodies matched with homology are used for replacing primary anti-staining NPC tissues to be used as negative controls; the microscope was used for 100X and 200X photographs.
Judgment standard for immunohistochemical staining result
The interpretation criteria were as follows: RNF38 is mainly expressed on the nucleus, and expression of RNF38 is determined by staining intensity and positive cells: the dyeing intensity is graded as 0 point (none), 1 point (weak), 2 points (medium) and 3 points (strong); the percentage of positive cells was 0min (0%), 1 min (<25%), 2 min (25% -50%), 3 min (50% -75%) and 4 min (>75%), respectively. Multiplying the staining intensity score by the percentage score of staining positive cells, and taking the average score as a critical value for judging the positivity of RNF38, wherein the value smaller than the critical value is the expression negativity of RNF 38; otherwise, the product is positive.
Analysis of expression and clinical significance of
Analysis and mapping was performed using SPSS 20.0 and Graphpad based on RNF38 expression, in combination with patient clinical and prognostic information.
Establishment and validation of overexpressing and downregulating cell lines
6.1 transfection and construction of cell lines: RNF38 overexpression and down-regulation lentivirus is purchased from Shanghai Jikai Gene Co., Ltd, and is transfected with SUNE-1, CNE-2, 6-10B according to the operating instructions, fluorescence of nasopharyngeal carcinoma cells is observed, and screening and expanded culture are performed by using antibiotic resistance for identification and cryopreservation.
Detection of expression of RNF 38: collecting cells in logarithmic growth phase, cracking lysate and collecting supernatant, carrying out protein quantification by Bradford method, adding a loading buffer solution, boiling the sample for 5min for denaturation, carrying out SDS-PAGE electrophoresis, carrying out protein loading of 50-100 mu g, carrying out electrotransfer by a semi-dry method, sealing with 10% skimmed milk powder for 2h, adding a primary antibody, incubating overnight at 4 ℃, washing a membrane with TTBS for 3 times, 5min each time, adding a corresponding secondary antibody marked by HRP, incubating for 2h at room temperature, developing by ECL in a dark room, scanning the result, and taking GAPDH as an internal reference.
7. Functional experiments with RNF38
7.1 scratch test: the cell strains of the no-load control and experimental groups are inoculated in 6-well plates, three wells are respectively arranged, and the plates are placed at 37 ℃ and 5% CO2The culture box is used for forming a monolayer of adherent cells. After culturing for 24h by using the serum-free culture solution, discarding the culture solution, and marking uniform scratches by using a 200ul plastic gun head; serum-free medium is used for washing floating cells or fragments; 2 ml/well of culture medium was added again. Cell scratch healing and scratch width were observed and measured under a microscope at 0h, 12h or 24 h.
7.2 invasion and metastasis experiments: mu.l of fibronectin (1 mg/ml) was applied uniformly to the bottom membrane of a Transwell chamber (no Matrigel gel spreading) and an Invasion chamber (Matrigel gel spreading) and 100. mu.l of a cell suspension (containing 2.5X 10 cells) in logarithmic growth phase was applied5Cells) was added to the upper chamber of the chamber, 600. mu.l of 1640 medium containing 20% FBS was added to the lower chamber,three multiple holes are respectively arranged. After incubation for 24h, the Transwell and Invasion chambers were removed, cells that did not migrate on the membrane were gently wiped off with a cotton swab, washed twice with PBS, the filter membrane was fixed with methanol for 30min, air dried naturally, and then 600. mu.l/well of 0.1% crystal violet stain was added to the 24-well plate and stained for 10-20 min. The dye solution was discarded and the PBS was used for two washes. Counting and comparing the number of cells passing through the membrane under a microscope, counting the total number of cells in 5 different fields of view in the upper, lower, left, right and middle of each membrane, and taking the average value to reflect the migration and invasion capacities of the tumor cells to a certain extent.
7.3 nude mouse tail vein transfer model: cells from control and experimental groups in logarithmic growth phase were trypsinized, washed 2 times with PBS, pelleted by centrifugation, and thoroughly pipetted into PBS for suspension. Injecting 10^ into each nude mouse through tail vein6Each cell (0.1ml suspension) is 10, the transfer condition of the mouse is observed by regular living body imaging, the fluorescence condition of the lung of the nude mouse is detected about 8 weeks, the nude mouse is killed by a cervical removal method, the lung at two sides is cut, and the weight detection is carried out.
Statistical method
Statistical analysis was performed using SPSS 24.0 statistical software. The differences of the different groups were compared using the t-test. Analyzing the influence of RNF38 on survival prognosis by using a Kaplan-Meier method; correlations of RNF38 with clinical characteristic parameters and prognosis were analyzed using one-way, multi-way analysis. p <0.05 was considered statistically significant for the differences.
Second, experimental results
RNF38 expression and its correlation analysis with clinical characteristics
129 nasopharyngeal carcinoma patients were collected after pathological diagnosis and intensity modulated radiation therapy (see Table 1). Median age of patients was 46 years (12-78 years), 24% female, 76% male; phase I patients 10 cases (7.8%), phase II 23 cases (17.8%), phase III 69 cases (53.5%), phase IV 27 cases (20.9%). There were no statistical differences between RNF38 expression and clinical characteristics, including gender, age, T classification, N classification, TNM staging and whether chemotherapy was received. Immunohistochemistry showed that: RNF38 is mainly expressed in the nucleus; the positive rate and the negative rate of the nasopharyngeal carcinoma patients are 41.86 percent and 58.14 percent respectively; compared with normal nasopharyngeal tissues, the expression of RNF38 in nasopharyngeal carcinoma tissues is remarkably low (see figure 1 and table 2), and the expression of RNF38 in normal nasopharyngeal epithelial cells is high, and the positive rate is as high as 80%.
TABLE 1.129 nasopharyngeal carcinoma clinical features and their correlation analysis with RNF38 expression
TABLE 2 expression of RNF38 in NPC tissue and normal nasopharyngeal tissue
2. Clinical significance analysis of RNF38
Kaplan-Meier analysis showed: total survival (OS) and non-distant metastasis survival (DMFS) were significantly increased in RNF38 positive patients compared to RNF38 negative patients (fig. 2), with P values of 0.0079 and 0.0465, respectively. While RNF38 expression was not statistically different from Local relapse-free survival (LRFS) and Regional relapse-free survival (RRFS), with p values greater than 0.05. Multifactor survival analysis showed: RNF38 expression is OS (HR, 0.21; 95% CI, 0.06 to 0.73;p= 0.010)、PFS(HR, 0.43; 95% CI, 0.16 to0.97;p= 0.042) as an independent prognostic factor. Therefore, ubiquitin ligase RNF38 is a potential biomarker for prognosis in patients with nasopharyngeal carcinoma.
Functional study experiment of
Stable transgenic cell strains (SUNE-1-RNF 38 and CNE-2-RNF 38) with over-expression of RNF38 are established in SUNE-1 and CNE-2, and stable transgenic cell strains (6-10B-in-RNF 38 and SUNE-1-in-RNF 38) with down-regulation of RNF38 are constructed in 6-10B, SUNE-1. The scratch test, cell invasion and cell migration test show that compared with the control group, the over-expression of RNF38 inhibits the invasion and metastasis of NPC cells, and the down-regulation of RNF38 is opposite (see FIG. 3 and FIG. 4). To further evaluate the ability of RNF38 to inhibit metastasis, we injected SUNE-1-RNF38 or SUNE-1-in-RNF38 and its corresponding control group (NC or in-NC) into the tail vein of nude mice to construct a nude mouse lung metastasis model of NPC. The nude mouse experiment shows that: compared with a control group, the over-expression RNF38 inhibits the lung metastasis of nasopharyngeal carcinoma cells, and the fluorescence value and the weight of the lung of a nude mouse in the RNF over-expression group are lower; while down-regulation of RNF38 promoted lung metastasis of nasopharyngeal carcinoma cells (see FIG. 5).
In conclusion, RNF38 is probably an anti-cancer gene of nasopharyngeal carcinoma, plays a role in inhibiting cancer in the occurrence and development of nasopharyngeal carcinoma and inhibits the metastasis of nasopharyngeal carcinoma cells; meanwhile, RNF38 is a prognosis prediction factor for patients with nasopharyngeal carcinoma, and comprises potential biomarkers of total survival time and survival time without distant metastasis, and patients with low RNF38 expression have poorer prognosis and are more likely to have metastasis, so that the method can assist clinical diagnosis and treatment to a certain extent, and provides a new theoretical basis for screening targets for interfering with nasopharyngeal carcinoma metastasis and developing targeted drugs in the future.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (2)
1. Application of ubiquitin ligase RNF38 in preparing reagents for detecting nasopharyngeal carcinoma is provided.
2. The ubiquitin ligase RNF38 is applied to the preparation of auxiliary diagnosis and prognosis prediction reagents for nasopharyngeal carcinoma.
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Citations (3)
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| CN1482256A (en) * | 2002-09-13 | 2004-03-17 | 中南大学 | Nasopharyngeal carcinoma molecule marker----BRD7 reagent kit |
| CN109593852A (en) * | 2018-12-24 | 2019-04-09 | 朱伟 | One kind serum miRNA marker relevant to nasopharyngeal carcinoma auxiliary diagnosis and its application |
| CN110547767A (en) * | 2019-08-29 | 2019-12-10 | 暨南大学 | hypoxic imaging strategy for nasopharyngeal carcinoma in-situ and spontaneous metastasis model by taking CAIX as target point |
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Patent Citations (3)
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|---|---|---|---|---|
| CN1482256A (en) * | 2002-09-13 | 2004-03-17 | 中南大学 | Nasopharyngeal carcinoma molecule marker----BRD7 reagent kit |
| CN109593852A (en) * | 2018-12-24 | 2019-04-09 | 朱伟 | One kind serum miRNA marker relevant to nasopharyngeal carcinoma auxiliary diagnosis and its application |
| CN110547767A (en) * | 2019-08-29 | 2019-12-10 | 暨南大学 | hypoxic imaging strategy for nasopharyngeal carcinoma in-situ and spontaneous metastasis model by taking CAIX as target point |
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| CHENG LIN 等: "EBV-miR-BART8-3p induces epithelialmesenchymal transition and promotes metastasis of nasopharyngeal carcinoma cells through activating NF-κB and Erk1/2 pathways", 《JOURNAL OF EXPERIMENTAL&CLINICAL CANCER RESEARCH》 * |
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Application publication date: 20200619 |