Molecular target SFXN1 for medicine for diagnosing and treating colonic adenocarcinoma and application of molecular target SFXN1

Abstract

The invention provides a molecular target SFXN1 for a medicine for diagnosing and treating colonic adenocarcinoma and application of the molecular target SFXN1, relates to the technical field of biomedicine, and is characterized in that the molecular target SFXN1 is applied to preparation of a product for diagnosing and treating colonic adenocarcinoma as a molecular target. The application proves that the SFXN1 can inhibit the progress of the colon adenocarcinoma and is related to the prognosis of the colon adenocarcinoma. Specifically, a specific verification embodiment proves that the overexpressed SFXN1 has the effects of inhibiting proliferation, migration and invasion of colon tumor cells, so that the progress of colon adenocarcinoma is inhibited. SFXN1 has an opportunity to become a molecular action target for treating the colonic adenocarcinoma, and the development of the targeted SFXN1 medicine can be beneficial to the subsequent treatment of the colonic adenocarcinoma. The invention provides a new thought for diagnosing and treating the colon adenocarcinoma, and provides a new strategy for promoting the progress of the colon adenocarcinoma and promoting the prognosis of the colon adenocarcinoma by using a genetic engineering means in future.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a molecular target SFXN1 of a drug for diagnosing and treating colon adenocarcinoma and an application thereof. Background Art

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

[0003] The purpose of the present invention is to solve the technical problem that the prior art lacks corresponding therapeutic molecules and diagnostic methods for COAD.

[0004] In order 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 diagnosis and treatment of 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 the treatment of colon adenocarcinoma.

[0009] Preferably, the drug comprises a compound or molecule that promotes the expression of SFXN1.

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

[0011] A drug for treating colon adenocarcinoma, wherein the drug uses SFXN1 as a molecular 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 biomarker for the treatment of CRC patients, diagnosis and prognosis, and provides new ideas and strategies for the research of drugs related to the treatment of COAD patients and diagnostic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Figure 1 is a comparison of the pan-cancer expression of SFXN1 in one embodiment of the present invention and the expression in normal tissue and tumor of colon adenocarcinoma, wherein Figure A shows the pan-cancer expression of SFXN1, Figure B shows the expression of SFXN1 in unpaired tumor and normal tissue samples, and Figure C shows the expression of SFXN1 in paired tumor and normal tissue samples.

[0014] Figure 2 The figure shows the prognostic differences in overall survival time, recurrence-free survival time and post-progression survival time between the high and low expression groups of SFXN1 in colon adenocarcinoma in one embodiment of the present invention, wherein Figure A shows the difference in overall survival time between the high and low expression groups of SFXN1 in the TCGACOAD database, and Figure BD shows the difference in overall survival time, recurrence-free survival time and post-progression survival time between the high and low expression groups of SFXN1 in the KM-plotter database.

[0015] Figure 3 Figure 1 is the verification of RNA level of knockdown and overexpression of SFXN1 in one embodiment of the present invention, wherein Figure A is the verification of mRNA level of knockdown of SFXN1 in DLD-1 cell line, and Figure B is the verification of mRNA level of overexpression of SFXN1 in HCT 116 cell line.

[0016] Figure 4 In one embodiment of the present invention, knocking down SFXN1 promoted the proliferation ability of colon adenocarcinoma cells, while overexpressing SFXN1 inhibited the proliferation ability of colon adenocarcinoma cells, wherein Figure A shows the effect of knocking down or overexpressing SFXN1 on the proliferation ability in colon adenocarcinoma cell lines, and Figure B shows a statistical graph of the effect on the proliferation ability.

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

[0018] Figure 6 In one embodiment of the present invention, knocking down SFXN1 promoted the migration and invasion ability of colon adenocarcinoma cells, while overexpressing SFXN1 inhibited the migration and invasion ability of colon adenocarcinoma cells, wherein Figure A shows the effect of knocking down or overexpressing SFXN1 on the migration and invasion ability in colon adenocarcinoma cell lines, and Figure B shows the statistical graph of the effect on the migration and invasion ability. DETAILED DESCRIPTION

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

[0020] Application of SFXN1 as a molecular target in the preparation of products for diagnosis and treatment of 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, wherein the kit uses SFXN1 as a molecular target.

[0023] Application of SFXN1 as a molecular target in the preparation of drugs for the treatment of 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 SFXN1 expression.

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

[0027] The following is an explanation of the above content in combination with specific verification experiments:

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

[0029] The verification experiment is to obtain the molecule SFXN1 through bioinformatics analysis; specifically, the verification experiment comprises 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 / ), we conducted a comprehensive analysis of the expression differences of SFXN1.

[0032] (2) Survival analysis was performed on the TCGA_COAD database using the Kaplan-Meier algorithm. The “surv_cutpoint” algorithm in the survminer package in R was used to divide the patient information in TCGA_COAD into two groups based on the expression of SFXN1, and the prognostic difference between the high and low expression groups was evaluated.

[0033] (3) At the same time, the online database Kaplan-Meier Plotter was used to supplement and verify the effect of SFXN1 expression on overall survival, recurrence-free survival, and post-progression survival in COAD.

[0034] 2. Statistical methods

[0035] The Student's t test (normally distributed variables) or the Wilcoxon rank sum test (non-normally distributed variables) were used to analyze the differences between the groups, 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 breast cancer, lung cancer, and other tumors, but was lowly expressed in liver cancer and colon adenocarcinoma (see Figure 1 A), therefore, in order 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, and it was found that SFXN1 was downregulated in tumor tissues compared with normal tissues (P < 0.001) (see Figure 1 B, C). Subsequently, KM analysis showed that the SFXN1 high expression group had a better prognosis in COAD (see Figure 2 A). At the same time, we found through the online database Kaplan-Meier Plotter that high expression of SFXN1 has a better prognosis in COAD, and the same results are also found in recurrence-free survival and post-progression survival time, indicating that SFXN1 can be used as a prognostic assessment molecule for COAD (see Figure 2 BD).

[0038] Example 2: SFXN1 small interfering RNA transfection and overexpression lentiviral stable transfection strain construction

[0039] The verification experiment is performed by transfecting HCT116 cells with SFXN1 lentivirus; specifically, the verification experiment comprises the following steps:

[0040] 1. Cell culture:

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

[0042] Small interfering RNA transfection

[0043] DLD-1 cells were plated in 6-well plates and transfected using Lipofectamine 2000 (Invitrogen, #11668019) and 50 nM small interfering RNA for 48 hours.

[0044] Among them, the small interfering RNA sequence targeting SFXN1 is as follows:

[0045] (1) Small interfering RNA-1

[0046] The positive strand is 5'-GUUGUAUGAUGACGUUUUATT-3'

[0047] The antisense strand is 5'-UAAAACGUCAUCAUACAACTT-3'

[0048] (2) Small interfering RNA-2

[0049] The positive strand is 5'-CCUGUUUCCUCAGAAAAGUTT-3'

[0050] The antisense strand is 5'-ACUUUUCUGAGGAAACAGGTT-3'

[0051] 3. Construction of stable cell lines with overexpression lentivirus

[0052] (1) The day before infection, inoculate HCT116 cells, 250,000 cells / well, and inoculate 2 ml of culture medium in a 6-well plate. Allow the cells to reach about 30% to 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 MOI values, and then add polybrene (final concentration 5 μg / ml), mix well, and set aside. Discard the culture medium in the 6-well plate, add 1 ml / well of the prepared culture medium containing polybrene and virus solution, and replenish the complete medium to 2 ml after 6 to 8 hours of infection.

[0053] (2) 24 hours after infection, discard the culture medium, wash with PBS, and replace with 2 ml / well of fresh culture medium; 48 hours later, observe the cell status under bright field under fluorescence microscopy, and then detect the fluorescence intensity of the cells at various infection multiplicity in the fluorescence channel to confirm the optimal virus infection multiplicity.

[0054] (3) After 72 hours of virus infection, cells were screened with 5 μg / ml puromycin, and the medium was changed every 2-3 days. The screening was continued by adding drugs, and cells that were not infected with the virus were used as controls. When all control cells were screened to death, the entire screening process was completed.

[0055] (4) After the drug screening is completed, the cells are subcultured, and when the cell fusion reaches about 80%, qRT-PCR is used to detect the expression of SFXN1 mRNA in the cells.

[0056] 4. RT-qPCR detection of SFXN1 expression level in cells

[0057] Total RNA was extracted from cell lines using TRIzol reagent according to the manufacturer's instructions (Qiagen, Hilden, Germany). The extracted total RNA was reverse transcribed into cDNA by reverse transcription PCR (RT-PCR) using III RT SuperMix (Vazyme, Nanjing, China). Genes were detected and their expression levels were measured by quantitative real-time PCR (qRT-PCR) using ChamQ SYBR qPCR MasterMix (Vazyme, Nanjing, China). The results were normalized to the expression level of GAPDH.

[0058] Primers targeting SFXN1 and internal control 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 (normal distribution variables) or Wilcoxon rank sum test (non-normal distribution variables) were used to analyze the differences between the groups. 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 of DLD-1 cells and the overexpression efficiency of HCT 116 cells are shown in Figure 3 A, B, as shown in the figure, compared with the NC group, the expression level of SFXN1 was significantly decreased after transfection with small interfering RNA1 and 3, while compared with the Vector group, the expression level of SFXN1 in HCT 116 cells was significantly increased after overexpression of SFXN1.

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

[0070] Plate cloning experiment

[0071] (1) Plating: HCT116 cells with good growth and stable expression of Lv-NC and Lv-SFXN1, as well as DLD-1 cells with Si-NC and Si-1, 3, were digested and centrifuged before adding complete medium to resuspend the cells. The cells in each group were counted and seeded into 6-well plates.

[0072] (2) Cultivate at 37°C in an incubator containing 5% CO2, change the culture medium every 4 days, and remove the 6-well plate from the incubator after 14 days.

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

[0074] (4) After discarding the fixative, add 1 ml of crystal violet solution and stain for 30 min. After recovering the crystal violet solution, gently wash three times with PBS. Discard the liquid in the wells and place in a cool and ventilated place to dry. Take photos to observe and analyze the number and size of cell clones.

[0075] 2. Statistical methods

[0076] The Student's t test (normal distribution variables) or Wilcoxon rank sum test (non-normal distribution variables) were used to analyze the differences between the groups. 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 plate cloning experiments, it can be seen that knockdown of SFXN1 promoted the proliferation of COAD cells. However, after overexpression of SFXN1, the proliferation ability of COAD cells was significantly inhibited ( Figure 4 ).

[0079] Example 4: SFXN1 inhibits tumor growth in nude mice.

[0080] (1) The two stably transfected cells, HCT116 Lv-SFXN1 and HCT116 Lv-NC, were expanded and cultured.

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

[0082] (3) The HCT116 stable cell lines in the experimental and control groups were digested, centrifuged, washed once with PBS, and counted. 7 Each stably transfected HCT116 cell was resuspended in 100 μl of serum-free DMEM medium, which was the injection volume for one mouse.

[0083] (4) The resuspended cells were mixed with Matrigel at a volume ratio of 1:1. The mixed solution was injected subcutaneously into male nude mice.

[0084] (5) Observe the condition of the mice and measure the tumors grown on the nude mice using a vernier caliper after the tumors grow. The mice were raised under SPF conditions for 4 weeks. During the raising period, the long and short diameters of the tumors in vitro were measured using a vernier caliper, and the volume of the tumors was calculated using the formula: volume = length × width 2 × 1 / 2, and a growth curve was drawn.

[0085] (6) After the observation, the nude mice were killed, the tumors were removed, the tumors were weighed and photographed. Figure 5 ).

[0086] 2. Statistical methods

[0087] The Student's t test (normal distribution variables) or Wilcoxon rank sum test (non-normal distribution variables) were used to analyze the differences between the groups. 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] 28 days after the cells were successfully injected into the armpits of nude mice, tumors were obtained. Figure 5 As shown in A and B, overexpression of SFXN1 significantly inhibited tumor growth. 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] Migration and invasion Transwell assay procedures

[0092] The cells were digested with 0.25% trypsin solution, centrifuged at 1000 rpm for 3 min, the culture medium was discarded, and the cells were washed 1-2 times with PBS, then resuspended in serum-free DMEM medium, counted, and the cell density was adjusted to 1×10 5 / ml.

[0093] Invasion increase step: dissolve Matrigel at 4°C overnight, dilute Matrigel with pre-cooled serum-free medium at a volume ratio of 1:8, take 50 μl and add it to the pre-cooled Transwell chamber, air-dry at 4°C, incubate at 37°C 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 chamber and lower chamber respectively, and equilibrate at 37°C overnight.

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

[0095] (3) Remove the liquid from the upper and lower chambers, add 600 μl of 4% paraformaldehyde to the lower chamber 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 the lower chamber for staining for 30 min, wash the chamber twice with PBS, and randomly select fields of view for each group under a microscope to take pictures, observe and count the cells.

[0096] 2. Statistical methods

[0097] The Student's t test (normal distribution variables) or Wilcoxon rank sum test (non-normal distribution variables) were used to analyze the differences between the groups. 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] The results showed that knockdown of SFXN1 promoted the migration and invasion of COAD cells, whereas overexpression of SFXN1 inhibited the migration and invasion of COAD cells ( Figure 6 ).

[0100] In summary, the present application confirms 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 effect of inhibiting 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 a new idea for the diagnosis and treatment of colon adenocarcinoma, and provides a new strategy for promoting the progression of colon adenocarcinoma and the prognosis of colon adenocarcinoma by genetic engineering in the future.

Claims

1. Application of SFXN1 as a molecular target in the preparation of diagnostic and therapeutic products for colon adenocarcinoma.

2. The use of SFXN1 as a molecular target in the preparation of a product for diagnosis and treatment of colon adenocarcinoma according to claim 1, characterized in that: The products include diagnostic reagents and diagnostic kits.

3. A kit for diagnosing colon adenocarcinoma, characterized in that: The kit uses SFXN1 as a molecular target.

4. Application of SFXN1 as a molecular target in the preparation of drugs for the treatment of colon adenocarcinoma.

5. The use of SFXN1 as a molecular target in the preparation of a drug for treating colon adenocarcinoma according to claim 4, characterized in that: The drug includes compounds or molecules that promote the expression of SFXN1.

6. The use of SFXN1 as a molecular target in the preparation of a drug for treating colon adenocarcinoma according to claim 5, characterized in that: The drug inhibits cell proliferation, cell migration and invasion in colon adenocarcinoma by promoting SFXN1 expression.

7. A drug for treating colon adenocarcinoma, characterized in that: The drug uses SFXN1 as a molecular target for the treatment of colon adenocarcinoma.

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