LncRNA SP100-AS1 and its application

Abstract

The present application relates to LncRNA SP100-AS1 and its application, and belongs to the technical field of biological medicine. The present application provides the application of a reagent for detecting the expression amount of long-chain non-coding RNA SP100-AS1 in the preparation of a colorectal cancer prognosis detection preparation; and the application of long-chain non-coding RNA SP100-AS1 in the preparation of a colorectal cancer treatment preparation; long-chain non-coding RNA SP100-AS1 can be used as a new intervention target in the radiotherapy resistance treatment of clinical colorectal cancer.

Description

Technical Field

[0001] This invention relates to LncRNA SP100-AS1 and its applications, belonging to the field of biomedical technology. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors of the gastrointestinal tract, and its incidence ranks third in the world[1]. A large amount of evidence confirms that postoperative radiotherapy and chemotherapy for rectal cancer can reduce local recurrence and improve overall survival[2]. However, in clinical practice, there is great heterogeneity in the sensitivity of individuals to radiotherapy[3]. Therefore, it is of great clinical significance to find new biomarkers to predict radiotherapy sensitivity and to develop personalized radiotherapy strategies for CRC patients.

[0003] In recent years, long noncoding RNA (lncRNA) has received widespread attention for its role in tumor radiotherapy resistance[4]. LncRNA is a noncoding RNA longer than 200 nucleotides, which mainly participates in radiotherapy resistance of CRC through epigenetic, transcriptional and posttranscriptional regulation of gene expression[5]. Studies have shown that abnormal structure and expression of lncRNA are closely related to the occurrence and development of human diseases such as tumors, neurodegenerative diseases and metabolic diseases[6]. Upregulation or downregulation of specific lncRNAs in tumor cells can directly trigger tumor cell apoptosis or increase their sensitivity to therapeutic methods that induce tumor cell apoptosis, such as radiation-induced tumor cell DNA damage. These provide new ideas for the study of tumor radiotherapy resistance[7]. A recent study found that lncRNA MALAT1 can regulate the radioresistance of colorectal cancer cells through "spongeization" of miR-101-3p. MALAT1 is upregulated in radiotherapy resistant colorectal cancer cells, and downregulation of MALAT1 can inhibit cell proliferation and metastasis and promote cell apoptosis[8]. Another study found that lncRNA H19 affected the radiosensitivity and chemosensitivity of gastric cardia cancer cells by interacting with miR-130a-3p and miR-17-4p[9]. In addition, H19 can affect the progression of liver cancer by targeting PSEN1

[10] .

[0004] In recent years, the crucial role of lncRNAs in tumorigenesis and radioresistance has received increasing attention, but the number of known radiotherapy-regulated lncRNAs is still limited. This invention first uses high-throughput sequencing to screen for the significantly overexpressed lncRNA SP100-AS1 in radiotherapy-resistant CRC patients, and discovers that SP100-AS1 enhances radioresistance in CRC by regulating the autophagy pathway. Simultaneously, knockdown of SP100-AS1 in a mouse xenograft model significantly inhibited tumor development and progression after radiation exposure. These findings elucidate the regulatory mechanism of SP100-AS1 on CRC radiosensitivity, providing experimental evidence for the subsequent development of novel therapeutic targets for radiotherapy-resistant CRC.

[0005] References

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[0013] 8.Guo,J.,et al.,Aberrant expression of lncRNA MALAT1 modulatesradioresistance in colorectal cancer in vitro via miR-101-3psponging.Experimental and Molecular Pathology,2020.115:p.104448.

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[0015] 10.Ma,H.,et al.,The LncRNA H19 / miR-193a-3p axis modifies the radio-resistance and chemotherapeutic tolerance of hepatocellular carcinoma cellsby targeting PSEN1.Journal of Cellular Biochemistry,2018.119.

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[0023] 18. Ramirez, J., G. G. Romagnoli, and R. Kaneno, Inhibiting autophagy to prevent drug resistance and improve anti-tumor therapy. Life Sciences, 2020.

[0024] 19. Galluzzi, L., et al., Activating autophagy to potentiate immunogenic chemotherapy and radiation therapy. Nature Reviews Clinical Oncology, 2017.

[0025] 20. Yamazaki, T., et al., Mitochondrial DNA Drives Abscopal Responses to Radiation that are Inhibited by Autophagy. Social Science Electronic Publishing, 2020.

[0026] 21. Thorburn, A. and M. J. Morgan, Autophagy and Cancer Therapy. 2013.

[0027] 22. Li, Y., et al., Hydroxychloroquine-Loaded Hollow Mesoporous Silica Nanoparticles for Enhanced Autophagy Inhibition and Radiation Therapy. Journal of Controlled Release, 2020.

[0028] 23. Huang, T., et al., MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma. Cancer Cell, 2017. Summary of the Invention

[0029] The purpose of this invention is to solve the technical problem of how to apply LncRNA SP100-AS1.

[0030] To address the aforementioned problems, the present invention provides a long non-coding RNA SP100-AS1 specifically expressed by colorectal cancer cells, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0031] SEQ ID NO: l is:

[0032]

[0033] This invention provides a reagent for detecting the expression level of long non-coding RNA, SP100-AS1, in the preparation of a prognostic test for colorectal cancer. The sequence of the long non-coding RNA SP100-AS1 is shown in SEQ ID NO: 1.

[0034] Preferably, the prognostic test is used to assess the treatment effect.

[0035] Preferably, the prognostic test is a radiotherapy resistance or treatment sensitivity test.

[0036] This invention provides the application of a long non-coding RNA SP100-AS1 in the preparation of a colorectal cancer therapeutic agent, the sequence of which is shown in SEQ ID NO: 1.

[0037] Preferably, the colorectal cancer treatment preparation includes a colorectal cancer radiotherapy tolerance treatment preparation.

[0038] Preferably, the colorectal cancer treatment formulation includes an agent that enhances the radiosensitivity of colorectal cancer.

[0039] Preferably, the colorectal cancer treatment agent comprises an inhibitor of the long non-coding RNA SP100-AS1.

[0040] This invention provides a system comprising a data processing device and a substance for detecting the expression level of SP100-AS1. The system includes a data processing device comprising a data input module, a data recording module, a data comparison module, and a conclusion output module. The data input module is configured to input the relative expression level of SP100-AS1 in the tumor tissue of a colorectal cancer patient. The data recording module is configured to store the relative expression level of SP100-AS1 in the tumor tissue of the colorectal cancer patient and a judgment threshold. The data comparison module is configured to... To receive the relative expression level of SP100-AS1 in the tumor tissue of the colorectal cancer patient to be tested, sent by the data input module, and to compare the relative expression level of SP100-AS1 in the tumor tissue of the colorectal cancer patient to be tested with the judgment threshold retrieved from the data recording module; the conclusion output module is configured to receive the comparison result sent by the data comparison module, and to judge the comparison result according to the predetermined judgment conditions; to determine whether the colorectal cancer patient to be tested is a radiotherapy resistant patient; the nucleotide sequence of SP100-AS1 is shown in SEQ ID NO: 1.

[0041] Preferably, the substance used to detect the expression level of SP100-AS1 is a reagent and / or instrument for detecting the relative expression level of SP100-AS1.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention provides the application of a reagent for detecting the expression level of long non-coding RNA SP100-AS1 in the preparation of a prognostic test for colorectal cancer; and the application of long non-coding RNA SP100-AS1 in the preparation of a therapeutic agent for colorectal cancer; the long non-coding RNA SP100-AS1 provided by this invention can serve as a new intervention target in the clinical treatment of radiotherapy resistance in colorectal cancer. Attached Figure Description

[0044] Figure 1 A graph showing the significant increase in SP100-AS1 expression levels in radiotherapy-resistant CRC patients;

[0045] Figure A shows a high-throughput sequencing volcano plot of 8 patients in the radiotherapy-resistant group and 8 patients in the radiotherapy-sensitive group. Red represents high expression, green represents low expression, and gray represents no difference. Figure B shows the expression level of SP100-AS1 in tumor tissues of 44 CRC patients detected by RT-qPCR. Figure C shows the expression level of SP100-AS1 in normal epithelial cell line NCM460 and CRC cell lines HCT116, SW480, LS174T, CT26, HT29, and LoVo detected by RT-qPCR. Figure D shows the SP100-AS1 expression level versus survival curve of CRC patients from the TCGA database.

[0046] Figure 2 Figure 1 shows experimental results related to SP100-AS1 knockdown significantly enhancing radiosensitivity to CRC.

[0047] Figure A shows the sensitivity of HCT116 cells to radiotherapy after SP100-AS1 knockdown, as detected by a colony formation assay; Figure B shows the sensitivity of HCT116 cells to radiotherapy after SP100-AS1 knockdown, as detected by a cell viability assay; Figure C shows the sensitivity of SW480 cells to radiotherapy after SP100-AS1 knockdown, as detected by a colony formation assay; Figure D shows the sensitivity of SW480 cells to radiotherapy after SP100-AS1 knockdown, as detected by a cell viability assay; Figure E shows the expression level of γ-H2AX detected by Western blot; and Figure F shows the apoptosis level of cells detected by flow cytometry.

[0048] Figure 3 The figure shows the experimental results of SP100-AS1 enhancing radiotherapy resistance in CRC by regulating the autophagy pathway.

[0049] Figure A shows the expression levels of autophagy-related proteins LC3 and p62 in HCT116 cells after 4 Gy irradiation, as detected by Western blot. Figure B shows the immunofluorescence images of HCT116 cells infected with LC3-GFP-RFP overexpressing lentivirus and under 4 Gy irradiation.

[0050] Figure 4 Figure showing the experimental results related to SP100-AS1 knockdown significantly enhancing the radiosensitivity of xenograft mice.

[0051] Figure A shows SP100-AS1 stably knocked-out HCT116 cells subcutaneously injected into the axilla of nude mice. Radiation of 2 Gy was administered every other day for 10 days until the tumor reached 90 mm. 3 Figure 1 shows the size of xenografts in nude mice under different groups; Figure 2 shows the growth curve of the xenografts in nude mice; Figure 3 shows the weight of the xenografts in nude mice; Figure 4 shows the immunohistochemical detection of cell apoptosis. Detailed Implementation

[0052] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings:

[0053] like Figure 1-4 As shown; the present invention provides a long non-coding RNA SP100-AS1 specifically expressed in colorectal cancer cells, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0054] SEQ ID NO: l is:

[0055]

[0056] This invention provides a reagent for detecting the expression level of long non-coding RNA, SP100-AS1, in the preparation of a prognostic test for colorectal cancer. The sequence of the long non-coding RNA SP100-AS1 is shown in SEQ ID NO: 1. The prognostic test is used to assess the treatment effect and to detect radiotherapy resistance or treatment sensitivity.

[0057] This invention provides the application of long non-coding RNA SP100-AS1 in the preparation of colorectal cancer therapeutic agents, wherein the sequence of the long non-coding RNA SP100-AS1 is shown in SEQ ID NO: 1; the colorectal cancer therapeutic agent includes a colorectal cancer radiotherapy tolerance treatment agent; the colorectal cancer therapeutic agent includes an agent that enhances the radiosensitivity of colorectal cancer; the colorectal cancer therapeutic agent includes an inhibitor of the long non-coding RNA SP100-AS1.

[0058] This invention provides a system comprising a data processing device and a substance for detecting the expression level of SP100-AS1. The system includes a data processing device comprising a data input module, a data recording module, a data comparison module, and a conclusion output module. The data input module is configured to input the relative expression level of SP100-AS1 in the tumor tissue of a colorectal cancer patient. The data recording module is configured to store the relative expression level of SP100-AS1 in the tumor tissue of the colorectal cancer patient and a judgment threshold. The data comparison module is configured to... The system receives the relative expression level of SP100-AS1 in the tumor tissue of a colorectal cancer patient from the data input module, and compares it with the relative expression level of SP100-AS1 in the tumor tissue of the colorectal cancer patient from the data recording module. The conclusion output module is configured to receive the comparison result sent by the data comparison module and judge the comparison result according to predetermined judgment conditions to determine whether the colorectal cancer patient is radiotherapy resistant. The nucleotide sequence of SP100-AS1 is shown in SEQ ID NO: 1. The substance for detecting the expression level of SP100-AS1 is a reagent and / or instrument for detecting the relative expression level of SP100-AS1.

[0059] Example

[0060] 1. Experimental Materials and Methods

[0061] 1.1. Research Subjects:

[0062] This study collected tissue specimens from 44 patients with chronic red blood cell (CRC). All specimens were obtained from CRC patients who underwent pathological diagnosis at Changzhou First People's Hospital between January 2018 and December 2019, including CRC tissue and adjacent normal tissue. This study was approved by the Ethics Committee of Changzhou First People's Hospital. Patients were divided into a radiotherapy-resistant group and a radiotherapy-sensitive group. CRC tissue from 8 patients in the radiotherapy-resistant group and 8 patients in the radiotherapy-sensitive group were used for high-throughput sequencing.

[0063] 1.2. Cell Culture and Transfection:

[0064] CRC cell lines HCT116, SW480, LS174T, CT26, HT29, and LoVo were purchased from the ATCC cell bank and cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. Cells were transfected into SW480 and HCT116 cells according to the Lipofectamine 2000 reagent instructions, and collected 48 hours after transfection for subsequent experiments.

[0065] 1.3. Cell irradiation:

[0066] CRC cells in the logarithmic growth phase were selected for irradiation. The radiation source was a VARIAN 2300EX linear accelerator with 6MV X-rays and a source-to-skin distance of 100cm. Different irradiation doses were selected for different groups.

[0067] 1.4. RNA extraction, reverse transcription, and real-time quantitative PCR (RT-qPCR):

[0068] Total RNA was extracted from cells using the Trizol kit (Invitrogen) according to the instruction manual. After determining the concentration, reverse transcription was performed using the Superscript First-Strand cDNA Synthesis Kit (Invitrogen). RT-qPCR (LightCycle 480 Real-Time PCR system, Roche) was performed using the Power SYBR Green PCR master mix.

[0069] 1.5. Cell viability assay:

[0070] Each group of cells was seeded into 96-well plates (4 × 10⁶ cells / well). 3 Add 10 μL of CCK-8 solution to each well ( / well), incubate at 37°C and 5% CO2 for 2 hours, and measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0071] 1.6. Apoptosis detection:

[0072] Cells from each group were collected, washed three times with pre-cooled PBS, mixed with 500 μL of binding buffer, and 10 μL of Annexin V / FITC was added. Then, 5 μL of PI was added, mixed, and incubated in the dark for 10 minutes. Finally, the cells were analyzed by flow cytometry.

[0073] 1.7. Western blot:

[0074] Cells were washed with pre-cooled PBS, and total cellular protein was extracted. 50 μL of protein was separated by polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and then incubated overnight at 4°C with antibody. After washing the membrane 3 times, secondary antibody was added and incubated at room temperature for 2 hours. After washing the membrane 3 times, the membrane was developed and fixed. The gray value of the protein bands was measured with Quantity One, and the relative expression level was calculated with GAPDH as a reference.

[0075] 1.8. Construction of mouse xenograft model:

[0076] All animal experiments have been approved by the Animal Experimentation Ethics Committee of Changzhou First People's Hospital. (5×10) 6 Transfected HCT116 cells were subcutaneously injected into 6-week-old male nude mice. Mice were randomly divided into a radiation-free group and a radiation-treated group. Tumors were classified as radiation-free when they reached 90 mm in size. 3 During this period, the transplanted tumors in the radiation group received local tumor radiation every other day at a dose of 2 Gy for 10 consecutive days. Tumor volume was measured every 5 days and calculated using the formula: (length × width) 2 ) / 2.

[0077] 1.9. Statistical Analysis:

[0078] All data are expressed as mean ± standard deviation (SEM) of at least three independent trials. Differences between groups were assessed using Student's t-test and one-way ANOVA. Graphs were generated using GraphPad Prism software. A p-value < 0.05 was considered statistically significant.

[0079] 2. Experimental Results:

[0080] 2.1. SP100-AS1 was significantly overexpressed in radiotherapy-resistant CRC patients:

[0081] Tumor tissues and adjacent normal tissues from radiosensitive and radioresistant CRC patients collected at Changzhou First People's Hospital were subjected to high-throughput sequencing. The results showed that SP100-AS1 was significantly elevated in radioresistant CRC patients (e.g., ...). Figure 1 A), RT-qPCR assays also confirmed this result (e.g. Figure 1B). Meanwhile, SP100-AS1 was found to be significantly more highly expressed in CRC cell lines compared to normal epithelial cell lines (e.g., ...). Figure 1 C) Among them, the expression level was highest in HCT116 and SW480, so these two cell lines were selected for subsequent experiments.

[0082] Furthermore, results from The Cancer Genome Atlas (TCGA) database also show that high expression of SP100-AS1 predicts shorter survival in CRC patients (e.g., Figure 1 D). The above results indicate that high expression of SP100-AS1 is significantly associated with radiotherapy resistance and shorter survival in CRC patients, and can serve as one of the biomarkers for the prevention, diagnosis, and treatment of radiotherapy resistance.

[0083] like Figure 1 Figure 1 shows the correlation between SP100-AS1 expression levels and radiotherapy-resistant CRC patients. Figure A is a high-throughput sequencing volcano plot of 8 radiotherapy-resistant patients and 8 radiotherapy-sensitive patients, where red represents high expression, green represents low expression, and gray represents no difference. Figure B is a plot of SP100-AS1 expression levels in tumor tissues of 44 CRC patients detected by RT-qPCR. Figure C is a plot of SP100-AS1 expression levels in normal epithelial cell line NCM460 and CRC cell lines HCT116, SW480, LS174T, CT26, HT29, and LoVo detected by RT-qPCR. Figure D is a curve showing the relationship between SP100-AS1 expression levels and CRC patient survival in the TCGA database.

[0084] 2.2. SP100-AS1 knockdown model significantly enhances radiosensitivity in CRC:

[0085] In CRC cell lines HCT116 and SW480, knockdown of SP100-AS1 using SP100-AS1 siRNA significantly enhanced the radiosensitivity of CRC cells (e.g., Figure 2 A and Figure 2 C). Simultaneously, the viability of HCT116 and SW480 cells under a 4 Gy radiation dose was investigated, and the results showed that SP100-AS1 knockdown significantly inhibited cell proliferation and viability (e.g., Figure 2 B and Figure 2 D). Blocking DNA damage repair is a common clinical strategy for treating CRC. By detecting the expression of the DNA damage marker γ-H2AX, it was found that SP100-AS1 knockdown significantly increased the expression level of γ-H2AX (e.g., Figure 2E). Furthermore, apoptosis results showed that SP100-AS1 knockdown significantly increased apoptosis in HCT116 and SW480 cells (e.g., Figure 2 F).

[0086] like Figure 2 Figure 1 shows the experimental results related to the significant enhancement of radiosensitivity of CRC by SP100-AS1 knockdown. Figure 2 shows the sensitivity of HCT116 cells to radiotherapy after SP100-AS1 knockdown, as detected by a colony formation assay. Figure 3 shows the sensitivity of HCT116 cells to radiotherapy after SP100-AS1 knockdown, as detected by a cell viability assay. Figure 4 shows the sensitivity of SW480 cells to radiotherapy after SP100-AS1 knockdown, as detected by a colony formation assay. Figure 5 shows the sensitivity of SW480 cells to radiotherapy after SP100-AS1 knockdown, as detected by a cell viability assay. Figure 6 shows the expression level of γ-H2AX detected by Western blot. Figure 7 shows the apoptosis level of cells detected by flow cytometry.

[0087] 2.3. SP100-AS1 enhances CRC radiotherapy resistance by regulating the autophagy pathway:

[0088] Recent studies have shown that activation of the autophagy pathway plays an important role in radiotherapy-resistant CRC

[11] . To further explore the mechanism by which SP100-AS1 promotes radiotherapy resistance, the autophagy status of HCT116 cells was observed. The results showed that after SP100-AS1 knockdown, the autophagy-related protein LC3-II was significantly downregulated and p62 was significantly upregulated (e.g., Figure 3 A). Next, after SP100-AS1 knockdown, HCT116 cells were infected with a lentivirus overexpressing LC3-GFP-RFP and irradiated with 4 Gy. Immunofluorescence results showed that, compared with the control group, SP100-AS1 knockdown significantly reduced autophagy flux (e.g., ...). Figure 3 B). The above results indicate that SP100-AS1 may enhance CRC radiotherapy resistance by regulating the autophagy pathway.

[0089] like Figure 3 The figures shown are experimental results related to SP100-AS1 enhancing radiotherapy resistance in CRC by regulating the autophagy pathway. Figure A shows the expression levels of autophagy-related proteins LC3 and p62 in HCT116 cells after 4 Gy irradiation, as detected by Western blot. Figure B shows the immunofluorescence images of HCT116 cells infected with LC3-GFP-RFP overexpressing lentivirus and irradiated with 4 Gy.

[0090] 2.4. In a mouse CRC xenograft model, SP100-AS1 knockdown significantly enhanced CRC radiosensitivity:

[0091] In a mouse CRC xenograft model, SP100-AS1 in HCT116 cells was knocked down using SP100-AS1 shRNA. The modified cells were then seeded into mice, and the growth of xenografts was observed under a 2 Gy radiation dose. The results showed that compared to the control group, the growth rate of xenografts in mice with SP100-AS1 knockdown was significantly slower after radiation (e.g., ...). Figure 4 A and Figure 4 B), and the tumor volume also decreased significantly (e.g. Figure 4 C). Furthermore, apoptosis experiments showed that SP100-AS1 knockdown promoted apoptosis and exacerbated DNA damage in tumor cells (e.g., Figure 4 D). The above data indicate that SP100-AS1 knockdown significantly enhances radiosensitivity and inhibits the occurrence and development of tumors in mice after radiation.

[0092] like Figure 4 The figure shows the results of an experiment demonstrating the significant enhancement of radiosensitivity in xenograft mice by SP100-AS1 knockdown. Figure A shows the subcutaneous injection of SP100-AS1 stably knocked-out HCT116 cells into the axilla of nude mice. Radiation of 2 Gy was administered every other day for 10 days until the tumor reached 90 mm. 3 Figure 1 shows the size of xenografts in nude mice under different groups; Figure 2 shows the growth curve of the xenografts in nude mice; Figure 3 shows the weight of the xenografts in nude mice; Figure 4 shows the immunohistochemical detection of cell apoptosis.

[0093] Experimental conclusion:

[0094] Different tissues and organs, especially tumor tissues, respond differently to radiation

[12] . As a common malignant tumor in clinical practice, colorectal cancer is generally treated with a combination of surgery, radiotherapy, and chemotherapy

[13] . With the continuous development of personalized treatment, a variety of molecularly targeted drugs have emerged in recent years

[14] . However, before choosing an ideal treatment plan, the various molecular characteristics of colorectal cancer should be fully considered. For example, G proteins of the Ras family are frequently mutated in CRC, with approximately 40% of cases having KRAS mutations and 5%-8% having NRAS mutations

[15] . In addition to drug therapy, radiotherapy is a commonly used method for treating colorectal cancer. Since the efficacy of radiotherapy depends on radiosensitivity and radiotolerance, it is crucial to identify the specific changes in tumor cells after radiotherapy and locate the molecular targets that can enhance the anti-tumor effect of radiotherapy in CRC treatment.

[0095] Autophagy is an important catabolic process that regulates cell proliferation and tumorigenesis

[16] . It depends on a conserved E1-E2-E3 three-enzyme cascade that catalyzes the lipidization of LC3 protein, with the central enzyme being E2 ATG3. ATG3 receives LC3 from E1 to form the ATG3-LC3 intermediate. Then, LC3 is transferred from the ATG3 catalytic site to the phosphatidylethanolamine lipid molecule under the catalysis of E3 enzymes

[17] . This process may promote the survival of tumor cells after chemotherapy or radiotherapy and is therefore associated with radiotherapy resistance[18-21]. Increasing evidence suggests that tumor cells rely on autophagy to avoid radiotherapy-induced DNA damage and reduce apoptosis, so inhibiting autophagy in tumor cells may increase the sensitivity of tumor cells to radiotherapy[22,23].

[0096] This invention is the first to discover that SP100-AS1, a non-coding RNA, is significantly upregulated in radiation-resistant CRC tissues. SP100-AS1 knockdown increases the sensitivity of CRC cells to radiation, further enhances the autophagy phenotype, and significantly enhances radiosensitivity in mice. Therefore, SP100-AS1 may serve as a novel drug intervention target, offering new hope for CRC radiotherapy.

[0097] This invention utilizes high-throughput sequencing technology to screen for the lncRNA SP100-AS1, which is significantly highly expressed in radiotherapy-resistant CRC patients, and discovers that SP100-AS1 enhances radiotherapy resistance in CRC by regulating the autophagy pathway. Simultaneously, knockdown of SP100-AS1 in a mouse xenograft model significantly inhibited tumor development and progression after radiation exposure. These findings elucidate the regulatory mechanism of SP100-AS1 on CRC radiosensitivity, providing a basis for the subsequent development of novel therapeutic targets for radiotherapy-resistant CRC.

[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The application of a reagent for detecting the expression level of long non-coding RNA SP100-AS1 in the preparation of a radiotherapy resistance detection reagent for colorectal cancer, characterized in that, The sequence of the long non-coding RNA SP100-AS1 is shown in SEQ ID NO:

1.

2. A system, characterized in that, The system includes a data processing device and reagents for detecting the expression level of SP100-AS1. The data processing device includes a data input module, a data recording module, a data comparison module, and a conclusion output module. The data input module is configured to input the relative expression level of SP100-AS1 in the tumor tissue of a colorectal cancer patient. The data recording module is configured to store the relative expression level of SP100-AS1 in the tumor tissue of a colorectal cancer patient and a judgment threshold. The data comparison module is configured to receive the relative expression level of SP100-AS1 in the tumor tissue of a colorectal cancer patient sent by the data input module, and retrieve the judgment threshold from the data recording module to compare it with the relative expression level of SP100-AS1 in the tumor tissue of a colorectal cancer patient. The conclusion output module is configured to receive the comparison result sent by the data comparison module and judge the comparison result according to predetermined judgment conditions. To determine whether the colorectal cancer patient to be tested is a radiotherapy resistant patient; the nucleotide sequence of SP100-AS1 is shown in SEQ ID NO: 1.

Images