WTAP as a prognostic marker for nasopharyngeal carcinoma and its detection primers and kits

Abstract

The present invention discloses WTAP as a prognosis prediction marker for nasopharyngeal carcinoma, a detection primer and a kit, and relates to the field of molecular biotechnology of nasopharyngeal carcinoma. The present invention uses WTAP as a prognosis prediction marker for recurrent nasopharyngeal carcinoma, develops molecular primers for detecting WTAP expression, detects WTAP expression of recurrent nasopharyngeal carcinoma through molecular primers expressing WTAP, predicts the prognosis of this type, strengthens the post-treatment management or treatment of recurrent NPC patients with poor prognosis, and is expected to increase the 2-year survival of recurrent NPC patients with poor prognosis by 65% ​​and 55% respectively; the present invention is aimed at the population with relatively poor prognosis of recurrent NPC, and through the prediction of the prognosis of this type of patients by this kit, it is expected to significantly increase the 2-year or even 5-year survival rate of such patients.

Description

Technical Field

[0001] The invention relates to WTAP as a prognosis prediction marker for nasopharyngeal carcinoma, a detection primer and a kit, and relates to the technical field of molecular biology of nasopharyngeal carcinoma. Background Art

[0002] Nasopharyngeal carcinoma (NPC) is a type of squamous cell carcinoma of the head and neck with great geographical distribution and ethnic heterogeneity in the world. It mainly occurs in humans in Southeast Asia, with the highest incidence in southern China. Radiotherapy is recommended as the main treatment for primary NPC. With the development of radiotherapy combined with chemotherapy, targeted therapy and other comprehensive treatment technologies, the 5-year OS rate of NPC can reach 50-64%, but 25% of patients still relapse after the first treatment and improvement of the disease. For resectable recurrent NPC, the 2-year and 5-year overall survival (OS) rates of salvage nasopharyngectomy are 48.6%-100.0% and 38.3%-88.9%, respectively. If re-radiotherapy is implemented, the 2-year and 5-year survival rates are 44.3-77.7% and 27.5%-57.2%. It can be seen that compared with primary NPC, the prognosis of recurrent patients is poor regardless of the treatment mode they receive.

[0003] Patients with recurrent NPC have all received multiple treatments (surgery or radiotherapy), so monitoring their prognosis is actually very important. For patients with recurrent NPC who may have a poor prognosis, the principle of early prevention, early diagnosis, and early treatment should be followed to try to prolong the survival of these patients. However, there is currently no good way to predict the prognosis of recurrent NPC.

[0004] In summary, there is no mature method in the prior art for predicting the prognosis of recurrent NPC patients. How to predict recurrent NPC patients with poor prognosis as early as possible and give them corresponding examinations or treatments as early as possible to improve their 2-year or 5-year OS is a technical problem that urgently needs to be solved in this field. The present invention develops a kit to predict the prognosis of recurrent NPC patients after early treatment, and gives patients with potentially poor prognosis more frequent reexaminations or strengthens their treatment, thereby better improving their 2-year or 5-year OS. Summary of the invention

[0005] The purpose of the present invention is to provide a kit using WTAP as a prognosis prediction marker for nasopharyngeal carcinoma, develop WTAP molecular primer sequence, detect the expression of WTAP in recurrent NPC patients, predict the prognosis of recurrent NPC, and serve as a basis for post-treatment management and enhanced treatment of recurrent NPC, thereby improving the survival rate of such patients.

[0006] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0007] Application of WTAP as a prognostic predictor for recurrent nasopharyngeal carcinoma.

[0008] Furthermore, the application is to detect the expression of WTAP in patients with recurrent nasopharyngeal carcinoma and predict the prognosis of the patients. The expression of WTAP in biopsy samples of recurrent NPC is quantitatively detected using qRT-PCR detection technology. Patients with high WTAP expression have significantly worse prognosis than those with low expression.

[0009] Another object of the present invention is to provide a molecular primer for detecting WTAP expression for use in preparing a product for predicting the prognosis of recurrent nasopharyngeal carcinoma.

[0010] Furthermore, the molecular primers for detecting WTAP expression are:

[0011] WTAP upstream primer: 5'-GCAACCAAGGAACAAGAGATG-3'; downstream primer: 5'-CAAGTTGATCGCTGGGTCTAC-3'.

[0012] Furthermore, the product for predicting the prognosis of recurrent nasopharyngeal carcinoma is a WTAP detection kit.

[0013] Another object of the present invention is to provide a kit for predicting the prognosis of recurrent nasopharyngeal carcinoma, wherein the kit is the above-mentioned molecular primer kit for detecting WTAP expression.

[0014] Beneficial effects of the present invention:

[0015] The present invention uses WTAP as a prognosis prediction marker for recurrent nasopharyngeal carcinoma, develops molecular primers for detecting WTAP expression, and detects the WTAP expression of recurrent nasopharyngeal carcinoma by using the molecular primers for detecting WTAP expression, so as to predict the prognosis of the patient. The principle is the basic principle of qrt-PCR, and the expression level of WTAP in the patient sample is detected and compared with the median of the WTAP expression level in an established database. The higher one can be judged as a patient with a relatively poor prognosis, and vice versa. For patients with a likely poor prognosis, a more intensive treatment plan and a more stringent reexamination principle are given to prolong their survival as much as possible. The present invention is simple to operate and easy to promote, and the detection uses patient biopsy or surgical samples without the patient suffering from secondary invasive injury.

[0016] The present invention predicts the prognosis of recurrent NPC patients by detecting the expression of WTAP in the patients, strengthens the post-treatment management or treatment of recurrent NPC patients with poor prognosis, and is expected to increase the 2-year survival of recurrent NPC patients with poor prognosis by 55%;

[0017] The present invention is aimed at the group of patients with recurrent NPC who have a relatively poor prognosis. By predicting their prognosis through the kit, it is expected to significantly improve the 2-year or even 5-year survival rate of such patients.

[0018] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The KM curve diagram of the expression level in the biopsy tissue of the recurrent NPC patient detected by the WTAP molecular primers described in the embodiment of the present invention and the patient's survival period after treatment;

[0020] Figure 2 To verify the effect of siRNA silencing WTAP expression in NPC cells described in the examples of the present invention;

[0021] Figure 3 A schematic diagram of the healing status after 24 hours of a scratch test using control group cells and cells whose WTAP expression level is reduced by siRNA as described in the examples of the present invention;

[0022] Figure 4 A statistical diagram of the scratch test described in the embodiment of the present invention;

[0023] Figure 5 This is a comparison chart of the Transwell invasion experiment described in the embodiment of the present invention after 24 hours;

[0024] Figure 6 This is a statistical diagram of the Transwell invasion experiment described in the embodiment of the present invention; DETAILED DESCRIPTION

[0025] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the present invention will be described in detail below with reference to the accompanying drawings.

[0026] Example 1

[0027] The target gene primers of the present invention are designed online through the NCBI Blast website and sent to Sangon Biotech Co., Ltd. for synthesis via e-mail.

[0028] Molecular primer design for detecting WTAP expression:

[0029] Detection of WTAP upstream primer: 5'-GCAACCAAGGAACAAGAGATG-3'; downstream primer: 5'-CAAGTTGATCGCTGGGTCTAC-3';

[0030] The above primers were developed into a kit product and used in detecting the expression of WTAP in patients with recurrent nasopharyngeal carcinoma.

[0031] Example 2

[0032] WTAP molecular primers were used to detect the expression of WTAP in biopsy tissues of patients with recurrent NPC, and the expression was analyzed with the survival rate of patients after treatment.

[0033] Samples from 24 patients with recurrent NPC who had only undergone surgical treatment were collected and followed up at Xiangya Hospital of Central South University. The median survival time was 16 months, and the shortest survival time was 2 months and the longest was 40 months. RNA was extracted from the samples of the above patients and reverse transcribed into cDNA, and the gene expression was detected by real-time fluorescence quantitative PCR;

[0034] The specific operation is as follows: 1) After collecting fresh tissue samples from patients, they were immediately placed in liquid nitrogen tanks for storage, and then relevant clinical data of all patients were collected. The collection of all experimental tissue samples was authorized by the Ethics Committee of Xiangya Hospital of Central South University and the consent of the patients.

[0035] 2) RNA was extracted from tissues of all patients, and all operations were performed on ice; ① Add a mung bean-sized tissue and 100ul of Trizol to a 1.5ml EP tube, use an electric pestle to grind the tissue to a flesh-colored homogenate, then add 900ul of Trizol and let it stand for 5 minutes. ② Add 200ul of chloroform to each sample, mix and shake for 30s until the solution emulsifies and turns white, let it stand on ice for 5 minutes, and then centrifuge it at 12000g for 15 minutes in a 4℃ low-temperature high-speed centrifuge. ③ After taking out the centrifuge tube, the homogenate has been divided into three layers. Take the upper colorless liquid and place it in a new 1.5ml EP tube. About 400-500ul of colorless liquid can be added to each tube. Add an equal amount of 400-500ul of isopropanol to all samples, mix them upside down, let them stand on ice for 10 minutes, and then centrifuge them at 12000g for 10 minutes in a 4℃ low-temperature high-speed centrifuge. ④ At this time, white precipitate can be seen on the side wall of the centrifuge tube. After discarding the supernatant, add 1ml of freshly prepared 75% alcohol, slowly blow up the precipitate, shake it up and down for 15 seconds, and centrifuge it at 7500g for 5 minutes in a 4℃ low-temperature high-speed centrifuge. ⑤ After discarding the supernatant, repeat step ④, discard the supernatant again, and dry it on ice. ⑥ Add 10-30ul of enzyme-free water to each sample to dissolve RNA. ⑦ Use the NanoDrop 2000 platform to measure the concentration of RNA in each sample and keep a record.

[0036] 3) Use the RT mix with DNase kit from US EVERBRIGHT INC for RNA reverse transcription, and configure the system as follows:

[0037]

[0038] The reaction program on the reverse transcription PCR machine is as follows: incubate at 37°C for 2 minutes to remove genomic contamination; incubate at 55°C for 10 minutes; and 85°C for 10 seconds. After the reaction is completed, store the product at -20°C for later use.

[0039] 4) Real-time fluorescence quantitative PCR was used to determine the expression level of WTAP in each sample; the operation was performed according to the experimental instructions of the Universal SYBR Green qPCR Supermix kit of US EVERBRIGHT INC. The system was configured as follows:

[0040]

[0041]

[0042] The reaction procedure on the real-time fluorescence quantitative PCR machine is as follows:

[0043]

[0044] After the above reaction was completed on the Bio-Rad IQ5 real-time fluorescence quantitative PCR instrument, it was normalized with the internal reference gene GAPDH, and the relative expression of the target gene was displayed as 2-△△CT value to determine the expression difference of WTAP. The unpaired t-test was used to calculate the P value.

[0045] like Figure 1 As shown, the WTAP molecular primers were used to detect the expression level in the biopsy tissue of recurrent NPC patients, and a KM curve was drawn with the patient's survival period after treatment:

[0046] The expression of WTAP in the biopsy tissue of recurrent NPC patients and the survival period after treatment were detected by WTAP molecular primers, and the Kaplan–Meier curve was drawn using IBM SPSS version 25.0 software; the expression of WTAP, survival outcomes and follow-up period of 24 recurrent NPC patients are shown in the following table:

[0047]

[0048]

[0049] Example 3

[0050] The occurrence of recurrent NPC is currently believed to be more closely related to the enhanced migration and invasion ability of tumor cells themselves and their resistance to treatment. Therefore, we designed a series of in vitro experiments to explore the effect of WTAP on NPC cells.

[0051] 1) Verification of the effect of silencing WTAP expression in NPC cell lines; The SI sequence of WTAP was designed based on the splicing site. siRNA is a type of RNA molecule with a length of 21-25 nucleotides that can complementarily bind to homologous RNA and specifically degrade the target RNA, thereby inhibiting its expression. It has developed into an important tool for gene function research. In order to study the role of WTSP in the occurrence and development of NPC tumors, we designed siRNA and used Invitrogen's Lipofectamine RNAiMAX reagent to transfect siRNA and siNC (blank control) into S18 and HK1 cell lines to suppress the expression level of WTSP, and detect the transfection efficiency of siRNA, confirming that the transfection efficiency was reduced to less than 50%. The results are shown in Figure 2 .

[0052] 2) In vitro experiments, silencing WTAP expression inhibited the migration of NPC cells; ① Place 1000ul / 200ul Tip, ruler, pipette, maker pen, etc. in the ultra-clean workbench and sterilize under ultraviolet irradiation for at least 30 minutes. Take a 6-well cell plate with a cell confluence of 50%-70% and transfect siRNA and control-siRNA. ② After the cells cover the bottom of the culture dish, scratch the next day. Align the 200ul Tip with the ruler and scratch the bottom of the 6-well plate quickly and quickly, keeping the strength consistent to ensure the same scratch width as much as possible. ③ Absorb the original culture medium in the culture dish, gently wash with PBS water 3 times to remove cell debris as much as possible, and then add 1% double antibody and serum-free 1640 medium. ④ Take pictures under the microscope and record them as the scratch width of each group at 0h. Then put the 6-well plate back into the cell culture incubator, take it out and take pictures after 12 hours. Take it out after another 12 hours, that is, 24 hours after the scratch, take it out and take pictures and keep records. ⑤ Arrange all pictures, measure the scratch spacing at each time period, and use Graphpad8.4.3 software for statistical analysis. Figure 3 and Figure 4 , in the same time period, the smaller the scratch spacing is, the stronger the migration ability of this group of cells is.

[0053] 3) In vitro cell transwell invasion assay; ① Place the BD Matrigel gel frozen at -20℃ in a 4℃ ice chamber to thaw overnight one day in advance, and pre-cool the Tip head and EP tube used to prepare the matrix gel at 4℃. ② Dilute the matrix gel at a ratio of 1:7, that is, add 20ul of matrix gel to 140ul of 1640 medium. Add the diluted matrix gel to the hydrated transwell chamber with a pore size of 8um, and add 50ul of matrix gel to each chamber. ③ Place the chamber with matrix gel in a 37℃ constant temperature cell culture incubator and incubate for 3 hours. When the matrix gel layer is white, it indicates that the colloid has solidified. ④ Digested and resuspended in serum-free medium, counted transfected cells, and then seeded at 25,000 cells / 200ul serum-free or low-concentration serum medium per chamber. Then add 800ul of 1640 medium containing 10% fetal bovine serum to the lower chamber, and carefully place the upper chamber into the lower chamber to avoid bubbles between the two. ⑤ Place the 24-well cell plate back into the cell culture incubator and incubate for 24-48 hours depending on the cell status and invasion speed. ⑥ Take out the 24-well plate after 36 hours, wash it twice with PBS water, and then fix the cells with 4% paraformaldehyde for 15 minutes, and then wash it lightly with clean water for 3 times. ⑦ Place the transwell chamber with fixed cells into a 24-well plate containing 0.1% crystal violet stain, let it stand at room temperature for 10 minutes, then wash it with PBS water 2-3 times, and gently remove the matrix gel at the bottom of the upper chamber with a medical cotton swab. ⑧ Add 200ul of distilled water to the upper chamber and 800ul of distilled water to the 24-well plate, place it under an inverted microscope and take pictures of each field of view. ⑨ Count the photos under the microscope using image J software, and use Graphpad software for statistics and graphic analysis. Specific results such as Figure 5 and Figure 6 shown.

[0054] The present invention uses WTAP as a prognosis prediction marker for recurrent nasopharyngeal carcinoma, develops molecular primers for detecting WTAP expression, detects WTAP expression in recurrent nasopharyngeal carcinoma by using molecular primers for detecting WTAP expression, predicts the prognosis of this type, strengthens the post-treatment management or treatment of recurrent NPC patients with poor prognosis, and is expected to increase the 2-year survival period of recurrent NPC patients with poor prognosis by 55%.

[0055] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0056] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Use of a molecular primer for detecting WTAP expression in a sample in the preparation of a product for predicting the prognosis of recurrent nasopharyngeal carcinoma, characterized in that: The molecular primers for detecting WTAP expression are: WTAP upstream primer: 5′-GCAACCAAGGAACAAGAGATG-3′; downstream primer: 5′-CAAGTTGATCGCTGGGTCTAC-3′.

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