Application of FKBP5 gene as a molecular marker of photon radiation resistance in nasopharyngeal carcinoma

Abstract

The present invention provides the use of the FKBP5 gene as a molecular marker for photon radiation resistance in nasopharyngeal carcinoma, belonging to the field of biomedicine. Through protein profiling, gene sequencing, and clinical sample gene chip testing, the present invention analyzes and screens genes differentially expressed in nasopharyngeal carcinoma radiation-resistant and radiation-sensitive cell lines. The findings indicate that FKBP5 expression is significantly higher in radiation-resistant nasopharyngeal carcinoma cells than in radiation-sensitive nasopharyngeal carcinoma cells. Inhibiting FKBP5 expression significantly enhances nasopharyngeal carcinoma radiosensitivity and cell lethality.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to the application of the FKBP5 gene as a molecular marker for photon radiation tolerance of nasopharyngeal carcinoma. Background Art

[0002] Nasopharyngeal carcinoma (NPC) is a malignant tumor that develops on the lateral walls and roof of the nasopharynx. Its incidence ranks first among otolaryngological malignancies, and it is predominantly found in Southeast Asia, particularly in southern China. Radiotherapy is one of the preferred treatments for NPC. With advances in science and technology, the application of stereotactic radiotherapy has become increasingly widespread. The five-year survival rate for early-stage NPC after radiotherapy has reached over 90%, and the survival rate for advanced-stage patients has also reached over 70%. Unfortunately, radioresistance can lead to poor treatment outcomes and early recurrence. Therefore, identifying the genes / proteins that confer photoresistance in NPC is crucial for the future development of NPC treatment.

[0003] In recent years, with economic development and technological advancements, multi-omics analysis techniques have become increasingly widely used in molecular biology research. By performing tandem mass spectrometry (TMT) proteomics analysis and transcriptome sequencing on nasopharyngeal carcinoma cells with varying radiation sensitivities, combined with gene chip analysis of nasopharyngeal carcinoma clinical samples, we have screened molecular markers that may be associated with differences in radiation sensitivity, providing a theoretical basis and scientific foundation for the study of prognostic markers for nasopharyngeal carcinoma. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide the use of the FKBP5 gene as a molecular marker for photon radiation resistance of nasopharyngeal carcinoma.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of the FKBP5 gene as a molecular marker for radiation resistance of nasopharyngeal carcinoma.

[0007] The present invention also provides the use of an inhibitor of the FKBP5 gene in preparing a drug for radiation resistance of nasopharyngeal carcinoma.

[0008] The present invention also provides the use of the FKBP5 gene as a molecular marker for predicting radiation resistance of nasopharyngeal carcinoma.

[0009] The present invention also provides the use of the FKBP5 gene as a molecular marker for radioresistance therapy of nasopharyngeal carcinoma.

[0010] Preferably, the gene ID of the FKBP5 gene is 2289.

[0011] The present invention provides the use of the FKBP5 gene as a molecular marker for radiation resistance in nasopharyngeal carcinoma. Through protein profiling, gene sequencing, and clinical sample gene chip testing, the present invention analyzed and screened genes differentially expressed in radiation-resistant and radiation-sensitive nasopharyngeal carcinoma cell lines. The authors found that FKBP5 expression was significantly higher in radiation-resistant nasopharyngeal carcinoma cells than in radiation-sensitive nasopharyngeal carcinoma cells. Inhibiting FKBP5 expression significantly enhances nasopharyngeal carcinoma radiosensitivity and cell lethality. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Survival curves of different nasopharyngeal carcinoma cells after radiation, showing the clone formation experiment, the dose survival curves of nasopharyngeal carcinoma radiation-resistant cell line (CNE-2R) and radiation-sensitive cell line CNE-2 and nasopharyngeal carcinoma cell line HONE1 under 0, 2, 4, 6, and 8 Gy of gamma ray irradiation;

[0013] Figure 2 The expression level of FKBP5 in three nasopharyngeal carcinoma cells is shown in Figure 1. Left: mRNA; Right: protein, showing the expression of FKBP5 in CNE-2R, CNE-2, and HONE1 in Western blot and qRT-PCR experimental results.

[0014] Figure 3 The figure shows the expression level of FKBP5 gene after CNE2 cells were irradiated with different doses of γ-rays, showing the relative expression of FKBP5 in CNE-2 cells after photon irradiation (4 Gy) in qRT-PCR results;

[0015] Figure 4 The expression level of FKBP5 in nasopharyngeal carcinoma and adjacent tissues is shown, showing that the expression of FKBP5 in nasopharyngeal carcinoma tissues is higher than that in normal nasopharyngeal tissues in the GEO dataset GSE12452;

[0016] Figure 5 The figure shows the decreased expression level of FKBP5 gene in nasopharyngeal carcinoma cells after FKBP5 siRNA interference, showing the expression of FKBP5 in CNE-2R and CNE2 in qRT-PCR results after siRNA interference;

[0017] Figure 6 This figure shows the decrease in FKBP5 protein expression in nasopharyngeal carcinoma cells after FKBP5 siRNA interference, showing the expression of FKBP5 in CNE-2R and CNE-2 cells after siRNA interference and the changes in FKBP5 after cells were irradiated;

[0018] Figure 7FKBP5 siRNA enhances the radiosensitivity of nasopharyngeal carcinoma cells. The colony formation experiment after siRNA interference shows the dose-survival curves of the negative control group (Negative control group) and FKBP5 knockdown group (siRNA group) of CNE-2R and CNE-2 under 0, 2, 4, and 6 Gy of γ-ray irradiation.

[0019] Figure 8 The figure shows that FKBP5 siRNA reduces the number of autophagosomes in nasopharyngeal carcinoma cells, showing the changes in autophagosomes in the negative control group (Negative control group) and FKBP5 knockdown group (siRNA group) of CNE-2R in the mRFP-GFP-LC3 detection experiment after siRNA interference;

[0020] Figure 9 FKBP5 siRNA inhibited the expression of autophagy-related protein LC3 in nasopharyngeal carcinoma cells. The Western blot detection experiment showed the changes of autophagy marker protein LC3Ⅱ / Ⅰ in the negative control group (Negative control group) and FKBP5 knockdown group (siRNA group) of CNE-2R after siRNA interference. DETAILED DESCRIPTION

[0021] The present invention provides the use of the FKBP5 gene as a molecular marker for radiation resistance of nasopharyngeal carcinoma.

[0022] The present invention also provides the use of an FKBP5 gene inhibitor in the preparation of a drug for nasopharyngeal carcinoma radiation resistance. The present invention has no particular limitation on the inhibitor, as long as it can inhibit the expression of the FKBP5 gene and is suitable for nasopharyngeal carcinoma patients.

[0023] The present invention also provides the use of the FKBP5 gene as a molecular marker for predicting radiation resistance of nasopharyngeal carcinoma.

[0024] The present invention also provides the use of the FKBP5 gene as a molecular marker for radioresistance therapy of nasopharyngeal carcinoma.

[0025] In the present invention, the gene ID of the FKBP5 gene is 2289.

[0026] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] 1. Methods

[0029] 1.1 Cell lines

[0030] Human nasopharyngeal carcinoma cell line (NPC) CNE2 cells were obtained from the Affiliated Cancer Hospital of Fudan University, and human nasopharyngeal carcinoma cell line HONE1 cells were obtained from the Nanfang Hospital of Southern Medical University.

[0031] 1.2 Main Reagents

[0032] Fetal bovine serum was purchased from Gibco, USA; RPMI1640 culture medium was purchased from Gibco, USA; FastQuant cDNA first-strand synthesis kit and fluorescent quantitative detection kit (SuperReal PreMix (SYBR Green)) were purchased from Beijing Tiangen Biotechnology Co., Ltd.; qRT-PCR primers were synthesized by Shanghai Shengong Technology Co., Ltd. and purified by UNIPAGE; FKBP siRNA (siFKBP5) was purchased from Guangzhou Ruibo Biotechnology Co., Ltd.; β-actin and FKBP antibodies were purchased from CST Co., Ltd.; goat anti-rabbit IgG (H+L) and goat anti-mouse IgG (H+L) (fluorescent secondary antibodies) were purchased from Jiangsu Biyuntian Biotechnology Co., Ltd.; prestained three-color protein marker was purchased from Shanghai Yisheng Biotechnology Co., Ltd.; mRFP-GFP-LC3 was purchased from Shanghai Hanheng Biotechnology Co., Ltd.

[0033] 1.3 Cell culture

[0034] Human nasopharyngeal carcinoma cell lines HONE1, CNE2 and their radiation-resistant line CNE2R were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37° C. in an incubator containing 5% CO 2 .

[0035] 1.4 Establishment of the radiation-tolerant strain CNE2R

[0036] CNE2 cells were irradiated with a Cs-137γ-ray source (0.73Gy / min) at fractionated doses of 2,2,4,4,4,4,6,6,6,6,8,8Gy, with a cumulative dose of 60Gy.

[0037] 1.5 Cell colony formation assay

[0038] Each cell type was divided into a control group (0 Gy) and radiation groups (2, 4, and 6 Gy). Cells in the logarithmic growth phase of the control and radiation groups were trypsinized, diluted to the appropriate concentration, and added to 2 ml of culture medium in a 6-well culture plate. Three replicates were set up for each treatment group and cultured in a 5% CO2, 37°C incubator for 10 to 14 days. After the incubation period, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, and the number of cell colonies was counted.

[0039] 1.6 Cell transfection

[0040] CNE2 and CNE2R cells were transfected with siFKBP5, and the interference sequences were shown in Table 1.

[0041] Table 1 FKBP5 siRNA sequences

[0042] siRNA Targeted gene sequence (5' to 3') siFKBP5 CCCUCGAAUGCAACUCUCUTT

[0043] The nucleotide sequence of siFKBP5 is shown in SEQ ID No. 1, specifically: CCCUCGAAUGCAACUCUCU, with TT base added at the 3-terminus to increase stability.

[0044] 1.7 qRT-PCR Applications

[0045] qRT-PCR was used to detect FKBP5 expression in transfected cells. Cells were collected from each group 48 hours after transfection, and total RNA was extracted using the Trizol method. The extracted RNA was quantified spectrophotometrically. RNA samples were reverse transcribed into cDNA, and FKBP5 levels in the samples were detected using a SYBR Green PCR kit. The primer sequences are shown in Table 2. The reaction conditions included a pre-denaturation step at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, 55°C for 30 seconds, and 72°C for 1 minute. The basic cycle count (Ct value) for FKBP5 and the internal reference β-actin was obtained for each sample. The relative expression of the target gene, which is the relative expression of FKBP5, was calculated using the formula. The experiment was repeated three times.

[0046] Table 2 RT-PCR primer sequences

[0047]

[0048] 1.8 Western blot experiment

[0049] After receiving the different treatments, discard the culture medium, wash the cells twice with pre-chilled PBS, add 1 mL of PBS, gently scrape the cells and transfer them to an EP tube. Centrifuge at 1000 rpm for 5 minutes at 4°C, and carefully drain the liquid from the EP tube using a pipette. Add RIPA lysis buffer (containing 1% PMSF) to lyse the cells and perform BCA protein quantification. Add 5× loading buffer to the sample, mix well, incubate at 100°C for 5 minutes, remove from the sample, centrifuge, and store on ice until ready to use. SDS-PAGE electrophoresis conditions: Wash the glass plate, prepare a 12% or 10% separating gel and a 5% stacking gel. Load samples sequentially from left to right (the sample volume per well is less than 2 / 3 of the well volume), and add 5 μL of protein marker to the side wells. Turn on the power supply for electrophoresis and set the voltage to 80V. After the sample has been transferred to the separating gel, adjust the voltage to 120V. Stop electrophoresis when bromophenol blue has reached the bottom edge of the glass plate. Transfer the membrane at a current of 350mA for 90 minutes. Blocking conditions: Add blocking buffer prepared in 1× TBST (5% nonfat dry milk) and gently shake on a horizontal shaker at room temperature for 2 hours. Antibody incubation: Prepare the primary antibody dilution according to the antibody manufacturer's instructions, thoroughly submerge the membrane in the primary antibody dilution, and incubate at 4°C for at least 12 hours. The next day, remove the primary antibody and wash three times with TBST (10 minutes each). Dilute the secondary antibody at a 1:5000 dilution and submerge the membrane in the secondary antibody dilution with gentle agitation on a shaker for 60-90 minutes. Afterwards, wash three times with TBST (10 minutes each). Develop: Prepare the chemiluminescent working solution (prepare fresh before use). Before developing, carefully blot any remaining liquid from the PVDF membrane with absorbent paper. With the protein side facing up, evenly apply the developer. Development imaging was performed in a luminescence imager, and the exposure time was adjusted according to the strength of the collected signal. The grayscale value was analyzed by QuantityOne software.

[0050] 1.9 Detection of autophagic flux

[0051] Cells were transfected with mRFP-GFP-LC3 labeled adenovirus and autophagy was detected by analyzing the formation of autophagosome fluorescent spots. (1) Cell preparation: Prepare a clean 24-well plate and seed each well with approximately 3×10 5 nasopharyngeal carcinoma cells, cultured for 24 hours. (2) Adenovirus infection: dilute the virus with double-antibody-free medium so that the amount of virus added is 20, and change the medium 2 hours after transfection. 48 hours after infection, wash the cells twice with PBS, add 500μl of 4% paraformaldehyde to each well for 15 minutes, wash once with PBS, stain with DAPI for 40 seconds, wash away the residual dye with PBS, and add 400ml of PBS to each well. (3) Image acquisition: Use a high-content cell imaging system to take pictures of the cells before and after transfection. Yellow spots indicate the fusion of GFP and RFP signals, and red spots (only RFP signal) indicate late autolysosomes.

[0052] 2. Statistical processing

[0053] SPSS 20.0 statistical software was used for analysis. The experimental results in the paper were the average values ​​of 3 to 5 repeated experiments. The Student's t test was used for statistical analysis, and P < 0.05 was considered to be statistically significant.

[0054] The result is:

[0055] After constructing the radiation-resistant strain of nasopharyngeal carcinoma CNE2 cell line, the radiation sensitivity of CNE2-R was verified using a γ-source (Cs-137, 0.73 Gy / min). Figure 1 ),from Figure 1 The survival rates of HONE1 cells after irradiation at different doses (0, 2, 4, 6, 8 Gy) were (1; 0.32; 0.085; 0.019; 0.00037); the survival rates of CNE2 cells after irradiation at different doses (0, 2, 4, 6, 8 Gy) were (1; 0.48; 0.16; 0.034; 0.0086); and the survival rates of CNE2R cells after irradiation at different doses (0, 2, 4, 6, 8 Gy) were (1; 0.68; 0.28; 0.10; 0.035). The dose-survival curves show that CNE2R exhibits significant radiation resistance compared to its parental cell line, CNE2, and both cells exhibit higher radiation resistance than the nasopharyngeal carcinoma cell line HONE1. CNE2R / CNE2 TMT mass spectrometry sequencing and transcriptome sequencing analysis revealed that FKBP5 is upregulated in nasopharyngeal carcinoma (NPC)-resistant cells (Tables 3-4). Gene chip analysis of clinical NPC samples revealed that FKBP5 expression was significantly upregulated in NPC tumor tissue after radiotherapy in patients with NPC remission who underwent radiotherapy compared with before radiotherapy (Table 5). Therefore, FKBP5 can serve as a marker for NPC radiation resistance.

[0056] Table 3 The ratio of FKBP5 expression in CNE-2R and CNE-2 cells detected by TMT

[0057]

[0058] Table 4 Ratio of FKBP5 expression in CNE-2R and CNE-2 detected by RNA-seq

[0059] RNA sequencing (RNA-seq) Gene name Fold difference (upregulation) P-value CNE2R / CNE2 FKBP5 4.41024387731 0.0456817517

[0060] Table 5 Changes of FKBP5 detected by gene chip in nasopharyngeal carcinoma tissue samples before and after radiotherapy

[0061] Nasopharyngeal carcinoma tissue Gene name Fold difference (upregulation) P-value After / before radiotherapy FKBP5 3.230326105 0.01750192

[0062] qRT-PCR and Western blot were used to detect protein expression in nasopharyngeal carcinoma cell lines HONE1, CNE2 and their radiation-resistant line CNE2R. It was found that the transcription level and protein level of FKBP5 in CNE2R cells were higher than those in CNE2, which was consistent with the results of transcriptome sequencing, TMT and tumor tissue gene chip detection ( Figure 2 ). RT-qPCR was further used to detect that after CNE2 cells were irradiated with different doses (0, 2, 4, 6, 8Gy) of γ-rays, the expression of FKBP5 was upregulated with increasing doses ( Figure 3 ),from Figure 3 It can be concluded that after irradiation with different doses of 2, 4, 6, and 8 Gy, FKBP5 was upregulated to 2.15, 2.57, 2.51, and 5.32 times that of non-irradiated tissues, respectively. Using the GEO online dataset, it was found that the expression of FKBP5 in nasopharyngeal carcinoma tissues was higher than that in normal tissues ( Figure 4 ),from Figure 4 It can be seen that the average expression level of normal epithelial tissue is 9.606, and the average expression level of nasopharyngeal carcinoma tissue is 10.610. These results further indicate that the high expression level of FKBP5 is associated with the radiation resistance of nasopharyngeal carcinoma cells.

[0063] The expression level of FKBP5 in nasopharyngeal carcinoma cells was knocked down by siFKBP5, and the results were verified by qRT-PCR and Western blot experiments ( Figure 5-6 ),from Figure 5 It can be concluded that FKBP mRNA levels were significantly downregulated in both CNE2R and CNE2 cells. Figure 6 It can be concluded that FKBP mRNA levels were significantly downregulated in both CNE2R and CNE2 cells. Through cell colony formation experiments, it was found that after inhibiting FKBP5 expression, the photon radiation sensitivity of nasopharyngeal carcinoma cells was significantly increased ( Figure 7 ),from Figure 7 It can be concluded that after irradiation with different doses (0, 2, 4, 6, 8 Gy), the survival scores of the cells in the CNE2R siNC group were (1; 0.65; 0.28; 0.11), the survival scores of the cells in the CNE2 siFKBP5 group were (1; 0.53; 0.16; 0.043), the survival scores of the cells in the CNE2siNC group were (1; 0.45; 0.15; 0.042), and the survival scores of the cells in the CNE2 siFKBP5 group were (1; 0.36; 0.10; 0.028), indicating that FKBP5 can play a stabilizing role in regulating cell radiation sensitivity.

[0064] Using mRFP-GFP-LC3 adenovirus to characterize the level of cellular autophagy, it was found that FKBP5 knockdown significantly reduced the number of autophagosomes in CNE2R nasopharyngeal carcinoma cells ( Figure 8 Western blot analysis showed that the ratio of LC3Ⅱ / Ⅰ in CNE2R decreased after siFKBP5 transfection compared with the control group ( Figure 9 These results confirm that FKBP5 can promote autophagy and thus reduce the radiosensitivity of cancer cells.

[0065] Therefore, FKBP5 and the autophagy signaling pathway it regulates can not only provide new ideas for the study of the molecular mechanism of nasopharyngeal carcinoma radiation tolerance, but also provide a new reference for the development and screening of clinical nasopharyngeal carcinoma radiation tolerance drugs. That is, FKBP5 can be used to screen drugs that inhibit tumor radiation tolerance, especially drugs that inhibit nasopharyngeal carcinoma radiation tolerance.

[0066] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of an FKBP5 gene inhibitor in the preparation of a drug for radiation resistance of nasopharyngeal carcinoma, wherein the inhibitor is siFKBP5, and the sequence of the siFKBP5 is CCCUCGAAUGCAACUCUCUTT.

2. The use according to claim 1, characterized in that The gene ID of the FKBP5 gene is 2289.

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