Use of prex2 in predicting patient cancer radiotherapy sensitivity and prognosis

Abstract

The application belongs to the field of biological medicine, and particularly relates to application of PREX2 in predicting radiotherapy sensitivity and prognosis of patients. The application provides application of a reagent for detecting expression level of PREX2 in preparation of a composition for detecting radiotherapy sensitivity and / or predicting radiotherapy effect of cancer, wherein the reagent for detecting expression level of PREX2 comprises a primer for detecting expression level of PREX2 gene or a reagent for detecting content of PREX2 protein. Through research and screening, it is found for the first time that PREX2 is a gene related to radiotherapy resistance of colorectal cancer. In in-vivo and in-vitro functional experiments, it is found that high expression level of PREX2 is obviously related to poor prognosis of tumors, and can be used for detecting radiotherapy sensitivity of colorectal cancer, gastric cancer and the like, and providing an evaluation means for patients to receive radiotherapy treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically involving the application of PREX2 in predicting the radiotherapy sensitivity and prognosis of cancer patients. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common digestive system tumors worldwide, ranking third in both incidence and mortality among all malignant tumors. In 2020, there were approximately 19.293 million new cases of malignant tumors and 9.958 million deaths worldwide. Despite significant progress in cancer diagnosis and treatment, over 90% of patients with advanced CRC still die from metastasis. Surgical resection can be very effective for patients with early-stage malignant tumors, but in reality, the vast majority of cancer patients are diagnosed after the optimal window for surgery has passed.

[0003] Radiation therapy (RT) is one of the most effective local treatments for colorectal cancer. The main challenges to improving the efficacy of RT are tumor radioresistance and radiation-induced complications and toxicities. By identifying biomarkers of radiosensitivity and elucidating the underlying mechanisms of radioresistance, radiation-induced toxicity, and complications, the efficacy of RT for rectal cancer may be improved. Therefore, the main goal of clinical and basic research is to understand the molecular mechanisms leading to chemoradiotherapy resistance, identify new targets to improve treatment efficiency, and thus promote personalized treatment for CRC patients. Summary of the Invention

[0004] The purpose of this invention is to overcome the limitations of existing technologies that use pre-treatment clinical staging to predict radiotherapy sensitivity, and to provide a kit for detecting and assessing cancer radiotherapy sensitivity. By detecting the expression of the biomarker PREX2 in vivo, radiotherapy tolerance can be predicted, providing a diagnostic basis for subsequent treatment planning and prognostic assessment.

[0005] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0006] In a first aspect, the present invention provides the use of a reagent for detecting PREX2 expression levels in the preparation of compositions for detecting cancer radiosensitivity and / or predicting the efficacy of cancer radiotherapy.

[0007] This invention, through research and screening, has for the first time discovered that PREX2 is a gene associated with radiotherapy resistance in colorectal cancer. In vitro and in vivo functional experiments revealed a significant correlation between high PREX2 expression and poor tumor prognosis. Detecting PREX2 expression in vivo can be used to assess radiotherapy sensitivity in colorectal cancer, gastric cancer, and other cancers, providing an evaluation tool for patients undergoing radiotherapy.

[0008] Preferably, the reagent for detecting PREX2 expression level includes primers for detecting PREX2 gene expression level or reagents for detecting PREX2 protein content.

[0009] This invention analyzes differentially expressed genes in radiotherapy-resistant and radiotherapy-sensitive cell lines and finds that PREX2 expression is significantly upregulated in radiotherapy-resistant cell lines, indicating that PREX2 can increase the radiotherapy resistance of tumor cells. Therefore, by designing primers to highly specifically target specific regions of the PREX2 gene or by detecting PREX2 protein content, the expression level of PREX2 can be accurately quantitatively analyzed, thereby accurately assessing the radiotherapy sensitivity of cancer patients and prognostic analysis.

[0010] Preferably, the primers for detecting PREX2 gene expression levels are selected from at least one of the following primer pairs:

[0011] Primer pair 1: The upstream primer sequence is shown in SEQ ID NO: 1, and the downstream primer sequence is shown in SEQ ID NO: 2;

[0012] Primer pair 2: The upstream primer sequence is shown in SEQ ID NO: 3, and the downstream primer sequence is shown in SEQ ID NO: 4;

[0013] Primer pair 3: The upstream primer sequence is shown in SEQ ID NO: 5, and the downstream primer sequence is shown in SEQ ID NO: 6.

[0014] Preferably, the reagent for detecting PREX2 protein content is selected from PREX2 monoclonal antibody and / or PREX2 polyclonal antibody.

[0015] Preferably, the reagent for detecting PREX2 protein content is selected from PREX2 Antibody (Affinit, catalog number: DF14258).

[0016] Preferably, the cancer is at least one of colorectal cancer and gastric cancer.

[0017] Preferably, the cancer is colorectal cancer.

[0018] In a second aspect, the present invention provides a kit for detecting cancer radiotherapy sensitivity and / or predicting the efficacy of cancer radiotherapy, including primers for detecting PREX2 gene expression levels or reagents for detecting PREX2 protein content.

[0019] Preferably, the primers for detecting PREX2 gene expression levels are selected from at least one of the following primer pairs:

[0020] Primer pair 1: The upstream primer sequence is shown in SEQ ID NO: 1, and the downstream primer sequence is shown in SEQ ID NO: 2;

[0021] Primer pair 2: The upstream primer sequence is shown in SEQ ID NO: 3, and the downstream primer sequence is shown in SEQ ID NO: 4;

[0022] Primer pair 3: The upstream primer sequence is shown in SEQ ID NO: 5, and the downstream primer sequence is shown in SEQ ID NO: 6.

[0023] Preferably, the reagent for detecting PREX2 protein content is selected from PREX2 monoclonal antibody and / or PREX2 polyclonal antibody.

[0024] Preferably, the reagent for detecting PREX2 protein content is selected from PREX2 Antibody (Affinit, catalog number: DF14258).

[0025] Preferably, the cancer is at least one of colorectal cancer and gastric cancer.

[0026] Preferably, the cancer is colorectal cancer.

[0027] Thirdly, the present invention provides the use of a composition in the preparation of a kit for detecting radiosensitivity and / or predicting the efficacy of cancer radiotherapy, the composition comprising primers for detecting PREX2 gene expression levels and / or reagents for detecting PREX2 protein content.

[0028] Preferably, the primers for detecting PREX2 gene expression levels are selected from at least one of the following primer pairs:

[0029] Primer pair 1: The upstream primer sequence is shown in SEQ ID NO: 1, and the downstream primer sequence is shown in SEQ ID NO: 2;

[0030] Primer pair 2: The upstream primer sequence is shown in SEQ ID NO: 3, and the downstream primer sequence is shown in SEQ ID NO: 4;

[0031] Primer pair 3: The upstream primer sequence is shown in SEQ ID NO: 5, and the downstream primer sequence is shown in SEQ ID NO: 6.

[0032] Preferably, the reagent for detecting PREX2 protein content is selected from PREX2 monoclonal antibodies and / or polyclonal antibodies.

[0033] Preferably, the reagent for detecting PREX2 protein content is selected from PREX2 Antibody (Affinit, catalog number: DF14258).

[0034] Preferably, the cancer is at least one of colorectal cancer and gastric cancer.

[0035] Preferably, the cancer is colorectal cancer.

[0036] Fourthly, this invention provides the application of PREX2 inhibitors in the preparation of products that improve the radiosensitivity of cancer.

[0037] Preferably, the PREX2 inhibitor is selected from shRNA (shPREX2) designed based on PREX2.

[0038] Preferably, the shRNA sequence designed based on PREX2 is selected from at least one of SEQ ID NO: 7-9.

[0039] Preferably, the cancer is at least one of colorectal cancer and gastric cancer.

[0040] Preferably, the cancer is colorectal cancer.

[0041] Unless otherwise specified, in the context of this invention, the primers and / or primer pairs refer to PCR primers used in PCR to synthesize the PREX2 gene cDNA strand, thereby detecting the PREX2 gene expression level.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] This invention, through research and screening, has for the first time discovered that PREX2 is a gene associated with radiotherapy resistance in colorectal cancer. In vitro and in vivo functional experiments revealed a significant correlation between high-level PREX2 expression and poor tumor prognosis. PREX2 can be used to assess radiotherapy sensitivity in colorectal cancer, gastric cancer, and other cancers, and provides an evaluation tool for patients undergoing radiotherapy. Attached Figure Description

[0044] Figure 1 This is a schematic diagram showing the expression of PREX2 in radiotherapy-resistant and radiotherapy-sensitive SW480 cell lines.

[0045] Figure 2 This diagram illustrates the expression of PREX2 in colorectal cancers with high and low radiosensitivity.

[0046] Figure 3 A schematic diagram showing the expression of PREX2 in radiosensitive and radioresistant colorectal cancer tissue specimens after immunohistochemical staining.

[0047] Figure 4 This diagram illustrates the expression of PREX2 in SW480 and IR-SW480 cells.

[0048] Figure 5 Images and survival scores of plate clones after irradiation with different radiation intensities for PREX2 overexpression RKO, PREX2 knockdown of SW480, and IR-SW480.

[0049] Figure 6 This diagram illustrates the expression of the repair protein γH2AX in PREX2 overexpression RKO, PREX2 knockdown of SW480, and their respective controls.

[0050] Figure 7 This diagram illustrates the DNA damage observed in the control groups of PREX2-overexpressing RKO, PREX2-knockdown SW480, and IR-SW480 under different irradiation conditions using comet assays.

[0051] Figure 8 This is a schematic diagram illustrating the effect of PREX2 on tumor growth volume in RKO tumor-bearing nude mice treated with radiotherapy.

[0052] Figure 9 This is a schematic diagram illustrating the effect of PREX2 on tumor growth volume in SW480 tumor-bearing mice after radiotherapy.

[0053] Figure 10 This is a schematic diagram illustrating the effect of PREX2 on tumor growth volume in CMT93 tumor-bearing mice after radiotherapy. Detailed Implementation

[0054] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0055] Unless otherwise specified, all cells listed in this invention are cultured according to existing techniques, frozen at -80°C, and used in subsequent experiments. All reagents used in this invention are commercially available. The experimental methods used in this invention, such as RNA extraction, primer design, cell experiments, animal experiments, immunofluorescence, immunohistochemistry, and Western blot, are conventional methods and techniques in the field.

[0056] Representative results from biological experiments were selected from replicates and presented in the contextual figures. Data were displayed as mean ± SD and mean ± SEM as specified in the figures. All experiments were repeated at least twice. Data were analyzed using GraphPad Prism 8.0 software. Standard medical statistical methods such as chi-square test, t-test, and ANOVA were used to compare differences in means between two or more groups. P < 0.05 was considered statistically significant.

[0057] Example 1. Screening for radiotherapy resistance genes in colorectal cancer

[0058] Specifically, the following steps are included:

[0059] Constructing a radioresistant SW480 cell line: SW480 adherent cell lines were irradiated with a dose of 4 Gy each time, with irradiation occurring every other day. After each irradiation, the cells were cultured again, and the culture medium was replaced with fresh medium the following day. The number of irradiations was marked with a "+". When cells suffered irradiation damage, some cells entered the repair phase and resumed growth, while cells that could not repair died. After trypsin digestion, these cells could not adhere and grow. This irradiation cycle was repeated 8 times to accumulate the total clinical radiotherapy dose of 40 Gy. The surviving cells were stably passaged and expanded for radiotherapy resistance identification and named IR-SW480. Figure 1 ).

[0060] RNA-seq was performed on radiotherapy-resistant and radiotherapy-sensitive SW480 cell lines. Differentially expressed genes were analyzed, and heatmaps and volcano plots were used for screening. The results showed that PREX2 expression was most significantly upregulated in the radiotherapy-resistant SW480 cell line. Figure 2 Further analysis using colorectal cancer microarray atlases and the TCGA database revealed that PREX2 expression was upregulated in radiotherapy-resistant colorectal cancer. Figure 2 ).

[0061] Example 2. Effect of PREX2 expression on tumor radiosensitivity

[0062] 1. Preoperative samples were collected from 54 patients with colorectal cancer who underwent radiotherapy. Postoperative pathological specimens were evaluated using the AJCC Tumor Regression Grade (TRG) scale, which was divided into four grades: 0, 1, 2, and 3. Based on the postoperative pathological TRG grade, patients were divided into a neoadjuvant radiotherapy-sensitive group (TRG 0+1) and a radioresistant group (TRG 2+3). Immunohistochemical staining was performed on preoperative samples to detect PREX2 expression in colorectal cancers with different radiotherapy sensitivities. Figure 3 The specific steps are as follows:

[0063] 1) Dewaxing and rehydration: Baking: Take out the slices stored at low temperature and bake them in a 60℃ oven for 1-2 hours to dry the moisture and melt the wax. Xylene dewaxing and alcohol gradient hydration: Immerse the slices in xylene I, II, and III for 10-15 minutes each, anhydrous ethanol I and II for 5 minutes each, 95% ethanol, 90% ethanol, 85% ethanol, and 70% ethanol for 1-2 minutes each, tap water for 2 minutes, and finally rinse with tap water.

[0064] 2) Antigen Retrieval: Preparation of Citrate Antigen Retrieval Solution: Dissolve one packet of citrate buffer powder in 2 L ddH2O, with a pH of 6.0. Pressure Cooker Water-Based Retrieval: Pour the prepared retrieval solution into a pressure cooker, ensuring the liquid level covers the tissue sections. Tighten the lid and reset the vent valve. Cook on an induction cooker using the water boiling mode until the pressure cooker emits steam. Then, time the retrieval for 3-5 minutes. After retrieval, gently open the vent valve to slowly release the pressure. After opening the lid, allow the entire submerged tissue section in the retrieval solution to cool naturally at room temperature. Inactivation of Endogenous Peroxidase: Wash the cooled tissue sections three times with gentle shaking in PBS, 5 minutes each time. Then immerse them in 3% hydrogen peroxide working solution and incubate at room temperature in the dark for 10 minutes. Afterward, wash the sections three times with gentle shaking in PBS, 5 minutes each time.

[0065] 3) Specific binding and blocking: Gently shake off excess water from the slide, add an appropriate amount of blocking goat serum to the intact tissue (carefully circle the tissue with a small pipette tip to maximize and homogenize the blocking area), place it face up in a humidified chamber, and incubate at room temperature for 30 minutes to reduce non-specific staining background. Primary antibody incubation: Shake off excess serum, add 30-50 μL of diluted primary antibody (PREX2, 1:150, Abcam, polyclonal antibody Rabbit) directly to the tissue, place it in a humidified chamber, and incubate overnight at 4°C. Secondary antibody incubation: Remove the humidified chamber, allow it to return to room temperature for 20-30 minutes, then wash the slide three times with PBS, 5 minutes each time. Add an appropriate amount of secondary antibody to the tissue, place it in the humidified chamber, and incubate at room temperature for 45 minutes.

[0066] 4) Colorimetric reaction: DAB staining: Wash slides three times with PBS, 5 min each time. Add the prepared DAB staining working solution (1 ml DAB buffer solution B + 20 μL DAB staining agent C solution). Observe the staining background under a microscope. Once the color turns brown, immediately place the slide in PBS buffer to stop the staining (control the same reaction time for each slide). Hematoxylin staining: Rinse the DAB staining solution with running water. Place the slide in hematoxylin to counterstain the cell nuclei for 3-5 min. Observe the staining intensity against a white background. The staining time can be appropriately extended or shortened. After rinsing off the residual staining solution with running water, differentiate with 1% hydrochloric acid ethanol for 1 second, and immediately re-blue under running water for 20-30 min.

[0067] 5) Dehydration and mounting: Dehydrate, clear, and mount the sections in the following order: ① 70% ethanol, 3 min; ② 80% ethanol, 3 min; ③ 90% ethanol, 2 min; ④ 95% ethanol, 2 min; ⑤ anhydrous ethanol I, 2 min; ⑥ anhydrous ethanol II, 2 min; ⑦ xylene I, 5 min; ⑧ xylene II, 5 min. Finally, place the sections in a ventilated area to air dry naturally, and then mount them with neutral resin.

[0068] Figure 3 The results showed that the expression level of PREX2 in radiotherapy-resistant colorectal cancer tissue specimens was significantly higher than that in radiotherapy-sensitive colorectal cancer tissue specimens.

[0069] 2. The expression of PREX2 in SW480 cells and radioresistant IR-SW480 cells was detected by RT-PCR / Western-Blot. The results showed that the expression of PREX2 in IR-SW480 cells was higher than that in SW480 cells. Figure 4 ).

[0070] 3. PREX2-overexpressing RKO cells, PREX2-knockdown SW480 and IR-SW480 cells were constructed using overexpression plasmids and shRNA in RKO, SW480, and IR-SW480 cells. Clonogenic assays combined with a multi-target click model were used to fit the survival curves of RKO / Control, RKO / PREX2, SW480 / shControl, SW480 / shPREX2, IR-SW480 / shControl, and SW480-R / shPREX2 cells after irradiation. The results showed that at irradiation doses of 2, 4, 6, and 8 Gy, the survival rate of the RKO / PREX2 group was higher than that of the RKO / Control group (P<0.01), while the survival rate of the SW480 / shPREX2 group was lower than that of the SW480 / shControl group (P<0.001). Similarly, the survival rate of the IR-SW480 / shPREX2 group was lower than that of the IR-SW480 / shControl group (P<0.001). Figure 5 ).

[0071] Example 3. Effect of PREX2 on DNA double-strand break repair after radiotherapy for colorectal cancer

[0072] 1. Radiotherapy can cause DNA double-strand breaks in tumor cells. DNA double-strand break repair (DSBs) involves two mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR) repair. To verify whether PREX2 affects DSBs, the expression of the key protein γH2AX was detected. The results showed that RKO tumors with PREX2 overexpression had lower γH2AX expression than their control group, while SW480 tumors with PREX2 knockdown had higher γH2AX expression than their control group. Figure 6 ).

[0073] 2. The comet assay was used to observe changes in DNA damage in RKO / Control, RKO / PREX2, SW480 / shControl, SW480 / shPREX2, IR-SW480 / shControl, and IR-SW480 / shPREX2 colorectal cancer cells after X-ray irradiation. Independent samples t-tests showed significant differences in the comet tail distance among the RKO, SW480, and IR-SW480 cell groups (P<0.05). The results suggest that compared to the Control group, the PREX2 overexpression group had fewer DNA breaks; compared to the shControl group, the PREX2 interference group had more DNA breaks; similarly, in IR-SW480 cells, compared to the shControl group, the PREX2 interference group in the resistant strain had more DNA breaks and increased radiosensitivity. Figure 7 ).

[0074] Example 4. Study on the radiosensitivity of PREX2 in tumor-bearing mice

[0075] Specifically, the following steps are included:

[0076] 1) Target cell expansion and culture: RKO colorectal cancer stable transgenes overexpressing PREX2 and their control groups (RKO / PREX2 and RKO / Control), and SW480 colorectal cancer stable transgenes with PREX2 interference and their control groups (SW480 / shPREX2 and SW480 / shControl) were expanded and cultured in 15cm dishes. It was planned that each cell line would require 12-16 nude mice for seeding, with each mouse requiring 2 × 10⁶ tumor cells. 6 One, change the cell culture medium the day before the tumor is implanted;

[0077] 2) Cell digestion: On the day of tumor implantation, cells are digested, centrifuged to collect all cells from the expansion culture dishes, diluted, and the total cell count is calculated. Finally, each tumor is injected at a volume of 0.1 ml containing 2 × 10⁶ cells. 6 One tumor cell was inoculated into nude mice. After obtaining the required number of cells, the cells were centrifuged to obtain a cell pellet. The cells were resuspended in sterile PBS and placed on an ice box for later use.

[0078] 3) Inoculation of tumor cells: 64 SPF-grade BALB / C nude mice aged 4-6 weeks were randomly divided into 8 groups of 6 mice each. Each cell line was inoculated into 2 tumor groups, divided into irradiated and unirradiated groups. The back of the nude mice was disinfected by wiping with 75% alcohol cotton balls. The needle tip was pointed upwards and inserted into the subcutaneous tissue at a temperature of 10-15° with the skin. The skin was gently lifted and the needle was inserted a little further until 2 / 3 of the needle tip was inserted. 0.1 ml of cell suspension was slowly injected. It is advisable to see a small bulge on the skin surface without leakage. After each type of tumor was completed, a group was labeled with the name and date.

[0079] 4) Irradiation: Subcutaneous tumors in nude mice were observed on days 10-15 after injection. Obvious spheroidization was observed. The tumor volume (measured long and short diameters) and weight of nude mice were observed and recorded every 4 days thereafter. When the transplanted tumors grew to 100 mm3, the nude mice in the irradiation group were anesthetized and the subcutaneous tumors of the irradiation group were irradiated with a dose of 6 Gy, 3 times, with an interval of one day between each irradiation. The volume and weight of all transplanted tumors were observed and recorded.

[0080] 5) Tumor Removal: After approximately 4 weeks of tumor transplantation, the trends among the groups were relatively stable and the differences were significant. In each group of BALB / c nude mice, the cervical spine was dislocated, the skin was peeled back, and the subcutaneous tumors were completely dissected. The long and short diameters of the tumors were measured using calipers, and the weight of the tumors was measured and recorded. The tumors were arranged in descending order of size, with the control group on top and the experimental group on the bottom, and photographed for preservation. (The CMT93 stable transgenic strain with PREX2 interference and its control group (CMT93 / shPREX2 and CMT93 / shControl) were measured at 1×10⁻⁶ mm.) 6 One tumor cell was inoculated into C57BL / 6 mice, and the rest of the procedure was the same.

[0081] The results are as follows Figure 8-10 As shown, in the non-irradiated group, tumors with PREX2-overexpressing RKO were larger than those in the control group, while tumors with PREX2-knockout SW480 and CMT93 were smaller. In the irradiated group, tumors with PREX2-overexpressing RKO were larger than those in the control group, while tumors with PREX2-knockout SW480 and CMT93 were smaller. These conclusions indicate that PREX2 promotes tumor growth and increases the radioresistance of tumor cells.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The use of a reagent for detecting PREX2 expression levels in the preparation of compositions for detecting cancer radiosensitivity and / or predicting the efficacy of cancer radiotherapy, characterized in that, The cancer in question is colorectal cancer.

2. The application according to claim 1, characterized in that, The reagents for detecting PREX2 expression levels include primers for detecting PREX2 gene expression levels or reagents for detecting PREX2 protein content.

3. The application according to claim 2, characterized in that, The primers used to detect PREX2 gene expression levels are selected from at least one of the following primer pairs: Primer pair 1: The upstream primer sequence is shown in SEQ ID NO: 1, and the downstream primer sequence is shown in SEQ ID NO: 2; Primer pair 2: The upstream primer sequence is shown in SEQ ID NO: 3, and the downstream primer sequence is shown in SEQ ID NO: 4; Primer pair 3: The upstream primer sequence is shown in SEQ ID NO: 5, and the downstream primer sequence is shown in SEQ ID NO:

6.

4. The application according to claim 2, characterized in that, The reagent used to detect PREX2 protein content is selected from PREX2 monoclonal antibody and / or PREX2 polyclonal antibody.

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