Molecular target SFXN1 for drugs for diagnosing and treating colon adenocarcinoma and its applications

Abstract

The present invention provides a molecular target SFXN1 for drugs for diagnosing and treating colorectal adenocarcinoma and its applications, relating to the technical field of biomedicine. The technical key points are as follows: the application of SFXN1 as a molecular target in the preparation of products for diagnosing and treating colorectal adenocarcinoma. In this application, it is confirmed that SFXN1 can inhibit the progression of colorectal adenocarcinoma and is related to the prognosis of colorectal adenocarcinoma. Specifically, through specific verification examples, it is confirmed that overexpression of SFXN1 has the effects of inhibiting the proliferation, migration and invasion of colorectal tumor cells, thereby inhibiting the progression of colorectal adenocarcinoma. SFXN1 has the opportunity to become a molecular action target for the treatment of colorectal adenocarcinoma, and the development of drugs targeting SFXN1 can be beneficial to the subsequent treatment of colorectal adenocarcinoma. The present invention provides a new idea for diagnosing and treating colorectal adenocarcinoma, and provides a new strategy for promoting the progression of colorectal adenocarcinoma and improving the prognosis of colorectal adenocarcinoma by means of genetic engineering in the future.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a molecular target SFXN1 for drugs for diagnosing and treating colon adenocarcinoma and its applications. Background Art

[0002] Colon adenocarcinoma (COAD) is a common type of colon cancer, ranking third in incidence among all cancers and second in mortality, posing a serious threat to global health. Despite numerous treatment methods, including endoscopic treatment, surgery, chemotherapy, immunotherapy, and molecular targeted therapy, the 5-year survival rate of COAD patients remains very low. Therefore, improving the pathological molecular mechanism of COAD and exploring new important targets related to disease progression and prognosis have important practical significance for improving the prognosis of COAD patients. Summary of the Invention

[0003] The object of the present invention is to solve the technical problem in the prior art of the lack of treatment molecules and diagnostic means corresponding to COAD.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] Application of SFXN1 as a molecular target in the preparation of products for diagnosing and treating colon adenocarcinoma.

[0006] Preferably, the products include diagnostic reagents and diagnostic kits.

[0007] A kit for diagnosing colon adenocarcinoma, characterized in that: the kit uses SFXN1 as a molecular target.

[0008] Application of SFXN1 as a molecular target in the preparation of drugs for treating colon adenocarcinoma.

[0009] Preferably, the drug includes compounds or molecules that promote the expression of SFXN1.

[0010] Preferably, the drug inhibits cell proliferation, cell migration, and invasion in colon adenocarcinoma by promoting the expression of SFXN1.

[0011] A drug for treating colon adenocarcinoma, the drug uses SFXN1 as a molecular action target for treating colon adenocarcinoma.

[0012] In this application, both in vivo and in vitro experiments show that SFXN1 significantly inhibits the proliferation and metastasis of COAD. Therefore, SFXN1 can be used as a treatment target, a biomarker for diagnosis and prognosis judgment for CRC patients, providing new ideas and new strategies for the research of drugs related to the treatment of COAD patients and diagnostic products. Brief Description of the Drawings

[0013] Figure 1 This shows the expression of SFXN1 in pan-cancer in one embodiment of the present invention and the comparison of its expression between normal tissues and tumors in colorectal adenocarcinoma. Among them, Figure A shows the expression of SFXN1 in pan-cancer; Figure B shows the expression of SFXN1 in unpaired tumor and normal tissue samples; Figure C shows the expression of SFXN1 in paired tumor and normal tissue samples.

[0014] Figure 2 This shows the prognostic differences in overall survival time, recurrence-free survival period, and post-progression survival time between the high and low expression groups of SFXN1 in colorectal adenocarcinoma in one embodiment of the present invention. Among them, Figure A shows the difference in overall survival time between the high and low expression groups of SFXN1 in the TCGA COAD database; Figures B - D show the differences in overall survival time, recurrence-free survival period, and post-progression survival time between the high and low expression groups of SFXN1 in the KM-plotter database.

[0015] Figure 3 This shows the verification of the RNA levels of knockdown and overexpression of SFXN1 in one embodiment of the present invention. Among them, Figure A shows the verification of the mRNA level of knockdown of SFXN1 in the DLD-1 cell line; Figure B shows the verification of the mRNA level of overexpression of SFXN1 in the HCT 116 cell line.

[0016] Figure 4 This shows that knockdown of SFXN1 promotes the proliferation ability of colorectal adenocarcinoma cells, while overexpression of SFXN1 inhibits the proliferation ability of colorectal adenocarcinoma. Among them, Figure A shows the effect of knockdown or overexpression of SFXN1 on the proliferation ability in the colorectal adenocarcinoma cell line; Figure B shows the statistical chart of the effect on the proliferation ability.

[0017] Figure 5 This shows the effect of overexpression of SFXN1 on the growth of tumors in a nude mouse subcutaneous tumor-bearing model in one embodiment of the present invention. Among them, Figure A shows the visualization of the growth of subcutaneous tumors in nude mice; Figure B shows the statistical chart of the growth of subcutaneous tumors in nude mice. Figure C shows that the positive rate of Ki-67 in the tumors of the overexpression SFXN1 group is lower than that of the control group.

[0018] Figure 6 This shows that knockdown of SFXN1 promotes the migration and invasion abilities of colorectal adenocarcinoma cells, while overexpression of SFXN1 inhibits the migration and invasion abilities of colorectal adenocarcinoma. Among them, Figure A shows the effect of knockdown or overexpression of SFXN1 on the migration and invasion abilities in the colorectal adenocarcinoma cell line; Figure B shows the statistical chart of the effect on the migration and invasion abilities. Detailed Embodiments

[0019] The present invention will be further described in detail below in conjunction with specific embodiments.

[0020] Use of SFXN1 as a molecular target in the preparation of products for diagnosing and treating colon adenocarcinoma.

[0021] The products include diagnostic reagents and diagnostic kits.

[0022] Based on the above application, the present application provides a kit for diagnosing colon adenocarcinoma, and the kit uses SFXN1 as a molecular target.

[0023] Use of SFXN1 as a molecular target in the preparation of drugs for treating colon adenocarcinoma.

[0024] The drug includes compounds or molecules that promote the expression of SFXN1.

[0025] In one embodiment, the drug inhibits cell proliferation, cell migration and invasion in colon adenocarcinoma by promoting the expression of SFXN1.

[0026] The present application also provides a drug for treating colon adenocarcinoma, and the drug uses SFXN1 as a molecular target for treating colon adenocarcinoma.

[0027] The above content is elaborated below in combination with specific verification experiments:

[0028] Example 1: Screening and validation analysis of biomarkers related to colorectal cancer progression and prognosis

[0029] The verification experiment obtained the molecule SFXN1 through bioinformatics analysis; specifically, the verification experiment includes the following steps:

[0030] 1. Screening method

[0031] (1) By collecting transcriptomic and clinical data of colon adenocarcinoma tissues and adjacent normal colon tissues from the TCGA database (https: / / portal.gdc.cancer.gov / ) and the TIMER database (https: / / cistrome.shinyapps.io / timer / ), a comprehensive analysis of the expression difference of SFXN1 was carried out.

[0032] (2) Survival analysis was performed on the TCGA_COAD database by the Kaplan-Meier algorithm, and the "surv_cutpoint" algorithm in the survminer package in R was used to divide the patient information in TCGA_COAD into two groups according to the high and low expression of SFXN1, and the prognostic differences between the high and low expression groups were evaluated.

[0033] (3) Meanwhile, the Kaplan-Meier Plotter online database was used to supplement and verify the impact of the high or low expression level of SFXN1 on the overall survival time, recurrence-free survival period, and post-progression survival time in COAD.

[0034] 2. Statistical methods

[0035] Student's t-test (for normally distributed variables) or Wilcoxon rank-sum test (for non-normally distributed variables) was used for between-group difference analysis, and the Log-rank test was used for prognostic analysis. All statistical tests were performed using R (version 4.1.2), and the significance threshold was set to less than 0.05.

[0036] Experimental results

[0037] The results showed that the expression of SFXN1 varied among different types of tumors. It was highly expressed in tumors such as breast cancer and lung cancer, but lowly expressed in liver cancer and colon adenocarcinoma (see Figure 1 A). Therefore, to further verify its expression in colon cancer, the expression of SFXN1 between unpaired and paired tumor and normal tissues was analyzed through the TCGA database. It was found that the expression of SFXN1 was downregulated in tumor tissues compared to normal tissues (P < 0.001) (see Figure 1 B, C). Subsequently, through KM analysis, it was obtained that the high-expression group of SFXN1 had a better prognosis in COAD (see Figure 2 A). Meanwhile, through the online database Kaplan-Meier Plotter, we found that the high expression of SFXN1 had a better prognosis in COAD, and the same results were obtained in the recurrence-free survival period and post-progression survival time, indicating that SFXN1 could be used as a prognostic evaluation molecule for COAD (see Figure 2 B - D).

[0038] Example 2: Construction of SFXN1 small interfering RNA transfection and overexpression lentiviral stable cell lines

[0039] The verification experiment was carried out by transfecting HCT116 cells with SFXN1 lentivirus. Specifically, the verification experiment included the following steps:

[0040] 1. Cell culture:

[0041] Take the cryopreserved HCT 116 and DLD-1 cells, quickly thaw them in a 37°C water bath, add them to 10 ml of complete medium (RPMI-1640 / F-12K medium + 10% fetal bovine serum + 1% double antibody), centrifuge at 900 rpm / min for 5 min, add 5 ml of complete medium, pipette evenly, and place them in an incubator (37°C and 5% CO2). When the cells reach 80% confluence, perform subculture. Digest with 0.25% trypsin for 2 min, collect the cells and put them into a 15 ml centrifuge tube, centrifuge at 1000 rpm / min for 5 min, and subculture at a ratio of 1:3. Passage the cells 2 times for later use.

[0042] Small interfering RNA transfection

[0043] Plate DLD-1 cells in a 6-well plate and transfect them with Lipofectamine 2000 (Invitrogen, #11668019) and 50 nM small interfering RNA for 48 hours.

[0044] Among them, the small interfering RNA sequences targeting SFXN1 are as follows:

[0045] (1) Small interfering RNA-1

[0046] Sense strand: 5’-GUUGUAUGAUGACGUUUUATT-3’

[0047] Antisense strand: 5’-UAAAACGUCAUCAUACAACTT-3’

[0048] (2) Small interfering RNA-2

[0049] Sense strand: 5’-CCUGUUUCCUCAGAAAAGUTT-3’

[0050] Antisense strand: 5’-ACUUUUCUGAGGAAACAGGTT-3’

[0051] 3. Construction of overexpressing lentiviral stable cell line

[0052] (1) One day before infection, seed HCT116 cells at 250,000 cells / well in 2 ml of culture medium in a 6-well plate, and make the cells reach about 30% - 40% before infection. Equilibrate the complete medium and polybrene to room temperature, and thaw the virus solution on ice for later use. Take 1 ml of the corresponding complete medium, add the virus solution according to different multiplicity of infection (MOI) values, and then add polybrene (final concentration 5 μg / ml), mix well, and set aside. Discard the medium in the 6-well plate, add 1 ml / well of the prepared medium containing polybrene and virus solution, and after 6 - 8 hours of infection, supplement the complete medium to 2 ml.

[0053] (2) After 24 h of infection, discard the culture medium, wash once with PBS, and replace it with fresh culture medium at 2 ml / well; after 48 h, observe the cell status under bright field by fluorescence microscope first, and then detect the fluorescence intensity of cells at each multiplicity of infection in the fluorescence channel to confirm the optimal viral multiplicity of infection.

[0054] (3) Perform drug screening on the cells 72 h after virus infection with 5 μg / ml puromycin, change the medium every 2 - 3 days, and continue to screen with the addition of the drug. Use the cells without virus infection treatment as a control. When all the control cells are killed by drug screening, the entire drug screening process is completed.

[0055] (4) After the drug screening is completed, passage culture the cells. When the cells are fused to about 80%, use qRT-PCR to detect the expression of SFXN1 mRNA in the cells.

[0056] 4. Detection of the expression level of SFXN1 in cells by RT-qPCR

[0057] According to the manufacturer's instructions (Qiagen, Hilden, Germany), use TRIzol reagent to extract total RNA from the cell line. Use III RT SuperMix (Vazyme, Nanjing, China) to reverse transcribe the extracted total RNA into cDNA by reverse transcription PCR (RT-PCR). Use ChamQ SYBR qPCR MasterMix (Vazyme, Nanjing, China) to detect the gene and its expression level by quantitative real-time PCR (qRT-PCR). Normalize the results to the expression level of GAPDH.

[0058] The primers targeting SFXN1 and the internal reference are as follows:

[0059] SFXN1

[0060] Forward primer, 5'-CAAGCCATCACGCAAGTTGT-3'

[0061] Reverse primer, 5'-GGTGTAGCAAACACCAAACAGA-3';

[0062] GAPDH

[0063] Forward primer, 5'-GGAGCGAGATCCCTCCAAAAT-3',

[0064] Reverse primer, 5'-GGCTGTTGTCATACTTCTCATGG-3';

[0065] Statistical methods

[0066] The Student's t-test (for normally distributed variables) or the Wilcoxon rank-sum test (for non-normally distributed variables) was used for inter-group difference analysis. All statistical tests were performed using GraphPad 9.0, and the significance threshold was set to less than 0.05.

[0067] Experimental results

[0068] The knockdown efficiency detection of DLD-1 cells and the overexpression efficiency of HCT 116 cells are shown in Figure 3 A and B. As shown in the figure, compared with the NC group, the expression levels of SFXN1 were significantly decreased after transfection with small interfering RNAs 1 and 3, while compared with the Vector group, the expression levels of SFXN1 in HCT 116 cells were significantly increased after overexpression of SFXN1.

[0069] Example 3: SFXN1 inhibits cell proliferation in colon adenocarcinoma.

[0070] Colony formation assay

[0071] (1) Seeding: Digest the HCT116 cells stably expressing Lv-NC and Lv-SFXN1 and the DLD-1 cells of Si-NC and Si-1, 3 with good growth status, resuspend the cells in complete medium after centrifugation, count the cells in each group respectively, and inoculate them into 6-well plates.

[0072] (2) Culture under the incubation conditions of 37 °C with 5% CO2, change the medium every 4 days, and take out the 6-well plates from the incubator after 14 days.

[0073] (3) Aspirate the medium, then gently wash twice with 1 ml of PBS from the edge of the well plate, and then add 1 ml of 4% paraformaldehyde fixative to fix the cells for 10 min.

[0074] (4) After aspirating the fixative, add 1 ml of crystal violet solution, stain for 30 min, recover the crystal violet solution, gently wash 3 times with PBS, aspirate the liquid in the well, dry it in a cool and ventilated place, and take pictures to observe and analyze the number and size of cell colonies.

[0075] 2. Statistical methods

[0076] The Student's t-test (for normally distributed variables) or the Wilcoxon rank-sum test (for non-normally distributed variables) was used for inter-group difference analysis. All statistical tests were performed using GraphPad 9.0, and the significance threshold was set to less than 0.05.

[0077] 3. Experimental results

[0078] Through the plate cloning experiment, it can be seen that the knockdown of SFXN1 promoted the proliferation of COAD. However, after overexpressing SFXN1, the proliferation ability of COAD cells was significantly inhibited( Figure 4 ).

[0079] Example 4: SFXN1 inhibits the growth of tumors in nude mice bearing xenografts.

[0080] (1) Expand and culture the two stable transfected cell lines of HCT116 Lv-SFXN1 and HCT116 Lv-NC.

[0081] (2) After 4-week-old female BALB / C nude mice adapted to the breeding environment for 1 week, subcutaneous xenograft experiments were carried out.

[0082] (3) Digest, centrifuge the stable transfected cell lines of HCT116 in the experimental group and the control group, wash once with PBS, count, and resuspend 1×10 7 stable transfected HCT116 cells in 100 μl of serum-free DMEM medium. This is the injection volume for one mouse.

[0083] (4) Mix the resuspended cells with Matrigel at a volume ratio of 1:1. Inject the mixed solution subcutaneously into male nude mice.

[0084] (5) Observe the mice. After the tumors grew out, use vernier calipers to measure the tumors grown on the nude mice. Raise them under SPF conditions for 4 weeks. During the breeding period, measure the long diameter and short diameter of the tumors outside the nude mice using vernier calipers, and use the formula: volume = length × width2 × 1 / 2 to calculate the volume of the tumors, and draw a growth curve graph.

[0085] (6) After the observation, sacrifice the nude mice, dissect the tumors, weigh the tumor bodies and take pictures( Figure 5 ).

[0086] 2. Statistical methods

[0087] Student's t-test (for normally distributed variables) or Wilcoxon rank sum test (for non-normally distributed variables) was used for inter-group difference analysis. All statistical tests were performed using GraphPad 9.0, and the significance threshold was set to less than 0.05.

[0088] 3. Experimental results

[0089] Twenty-eight days after the cells were successfully injected into the armpits of nude mice, tumors were obtained as shown in Figure 5 A and B. Overexpression of SFXN1 significantly inhibited the growth of tumors. In addition, immunohistochemistry further confirmed that the positive rate of Ki-67 in the tumors of the SFXN1 overexpression group was lower than that of the control group( Figure 5 C).

[0090] Example 5: SFXN1 inhibits cell migration and invasion in colon adenocarcinoma

[0091] Transwell assay for migration and invasion

[0092] Digest the cells with 0.25% trypsin solution, centrifuge at 1000 rpm for 3 min, discard the culture medium, wash with PBS 1 - 2 times, then resuspend with serum - free DMEM medium, count, and adjust the cell density to 1×10 5 / ml.

[0093] Steps for increased invasion: Dissolve Matrigel at 4℃ overnight, dilute Matrigel with pre - cooled serum - free medium at a volume ratio of 1:8, take 50 μl and add it to a pre - cooled Transwell chamber, air - dry at 4℃, incubate at 37℃ for 2 h to solidify Matrigel, aspirate the excess liquid in the chamber, and add 100 μl and 700 μl of serum - free medium to the upper and lower chambers respectively, and equilibrate overnight at 37℃.

[0094] (2) Add 100 μl of the cell suspension in serum - free DMEM medium to the upper chamber of the Transwell chamber, add 700 μl of medium containing 10% FBS to the lower chamber, and then incubate in a 37℃ incubator with 5% CO2 for 24 h.

[0095] (3) Remove the liquid from the upper and lower chambers, add 600 μl of 4% paraformaldehyde to fix the cells for 30 min, remove the paraformaldehyde, wipe off the cells that have not passed through the membrane with a cotton swab, add 600 μl of 0.1% crystal violet to stain for 30 min, wash the chamber 2 times with PBS, randomly select fields of view under the microscope for each group to take pictures, and observe and count the cells.

[0096] 2. Statistical methods

[0097] Student's t - test (for normally distributed variables) or Wilcoxon rank - sum test (for non - normally distributed variables) was used for inter - group difference analysis. All statistical tests were performed using GraphPad 9.0, and the significance threshold was set to less than 0.05.

[0098] 3. Experimental results

[0099] It was found that knocking down SFXN1 promoted the migration and invasion of COAD cells, while overexpressing SFXN1 inhibited the migration and invasion ability of COAD cells ( Figure 6 ).

[0100] In summary, it is confirmed in this application that SFXN1 can inhibit the progression of colon adenocarcinoma and is related to the prognosis of colon adenocarcinoma. Specifically, through specific verification examples, it is confirmed that overexpression of SFXN1 has the effects of inhibiting the proliferation, migration and invasion of colon tumor cells, thereby inhibiting the progression of colon adenocarcinoma. SFXN1 has the opportunity to become a molecular target for the treatment of colon adenocarcinoma, and the development of drugs targeting SFXN1 can be beneficial to the subsequent treatment of colon adenocarcinoma. The present invention provides new ideas for the diagnosis and treatment of colon adenocarcinoma, and provides a new strategy for promoting the progression of colon adenocarcinoma and improving the prognosis of colon adenocarcinoma by means of genetic engineering in the future.

Claims

1. Application of lentivirus or cells overexpressing SFXN1 in the preparation of products for the treatment of colon adenocarcinoma.

2. The use of the lentivirus or cell overexpressing SFXN1 according to claim 1 in the preparation of a product for treating colon adenocarcinoma, characterized in that: The product inhibits the proliferation, migration and invasion of colon adenocarcinoma cells by promoting the expression of SFXN1.

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