Method for researching influence of biomarker MND1 on colorectal cancer

Abstract

The invention discloses a method for researching the influence of a biomarker MND1 on colorectal cancer, and relates to the technical field of biological information. Comprising the following steps: S1, preparing materials, determining a cell strain, selecting a reagent, preparing instrument equipment and an antibody, determining a primer sequence, and preparing the reagent; s2, cell culture: selecting a culture medium, setting culture conditions and operation conditions, and carrying out cell resuscitation, cell liquid change, cell passage and cell cryopreservation; s3, extracting cell RNA (Ribonucleic Acid); s4, RNA reverse transcription: preparing a reverse transcription reaction system, performing reverse transcription in a PCR instrument, measuring the cDNA concentration, diluting, and storing in a refrigerator at-20 DEG C; according to the invention, not only is the expression condition of MND1 in colorectal cancer analyzed, but also the expression difference of MND1 in different cell populations and the positioning condition of MND1 in cell nucleus are deeply discussed through a single cell sequencing technology.

Description

Technical Field

[0001] The present invention relates to the field of bioinformatics, and in particular to a method for studying the effect of a biomarker MND1 on colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is a malignant disease that endangers human health. As of 2024, the incidence and mortality of colorectal cancer have increased year by year, making it the third largest cancer in the world after lung cancer and breast cancer. The disease often develops into cancer in the crypts due to inflammatory stimulation of polyps. Once it progresses to the late stage of intestinal cancer, the five-year survival rate of patients after treatment is greatly reduced. Although endoscopic diagnosis and treatment of colorectal cancer, surgery, immunotherapy and other means have been continuously developed in recent years, recurrence and re-progression are still inevitable for colorectal cancer patients, which remains a difficult clinical problem.

[0003] In recent years, the technologies of genome detection and transcriptome detection have enabled us to better understand the key factors that affect colorectal cancer, and to explore the functions and mechanisms of many oncogenes in colorectal cancer at the genetic level. For example, genes are integrated and processed through data information such as The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), and differential gene analysis and screening are performed according to the required conditions to reveal more key genes that affect colorectal cancer. At the same time, prognosis can be evaluated through online analysis and related pathways that affect the disease can be explored. By comparing the changes in genes in different types of cancer, the degree of association with the disease is indicated, providing important molecular markers for predicting the occurrence, development and recurrence of the disease.

[0004] The present invention downloads and analyzes the differential expression of the MND1 gene in colorectal cancer patients through a public platform database, and further verifies the expression of MND1 in normal colorectal epithelial cell lines and colorectal cancer cell lines through experiments. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a research method for the effect of the biomarker MND1 on colorectal cancer.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The research method of the effect of the biomarker MND1 on colorectal cancer includes the following steps:

[0008] S1: Material preparation, cell line determination, reagent selection, instrument and antibody preparation, primer sequence determination, and reagent configuration;

[0009] S2: Cell culture, selection of culture medium, setting of culture conditions and operating conditions, cell recovery, cell medium replacement, cell passaging, and cell cryopreservation;

[0010] S3: Cell RNA extraction;

[0011] S4: RNA reverse transcription, prepare the reverse transcription reaction system, perform reverse transcription in a PCR instrument, measure the cDNA concentration, and store it in a -20℃ refrigerator after dilution;

[0012] S5: Real-time fluorescence quantitative PCR: prepare the reaction system of real-time fluorescence quantitative PCR, add eight tubes and react in RT-qPCR instrument, set the reaction conditions and put it on the instrument;

[0013] S6: Cell protein extraction and protein concentration determination;

[0014] S7: Western blotting experiments;

[0015] S8: Statistical analysis.

[0016] Preferably: in S1, the pan-cancer expression of MND1 in the TCGA database is analyzed using the online analysis website UALCAN, and then the expression of MND1 in normal tissues and tumor tissues in the TCGA database is analyzed using the GEPIA online analysis website;

[0017] Use single-cell sequencing scRNA-seq method to analyze the relationship between MND1 and colorectal cancer and the cell populations in which it is expressed;

[0018] The localization of MND1 in cells was analyzed using the Genecards online website to determine the expression location of the gene.

[0019] Preferably: in said S1, the cell lines include human normal colorectal mucosal epithelial cells NCM-460, human colorectal cancer cells HCT-116, SW-620, SW-480, RKO, HT-29;

[0020] Primers used were MND1 and β-actin, where the sequence of MND1 was:

[0021] Forward:5'-TGTGAGAGGATCGGAACTTCT-3';

[0022] Reverse:5'-CACATCGGCCAATTTTAGCTTC-3';

[0023] In S1, the main reagents are configured as follows:

[0024] Cell culture medium: 5 ml FBS, 500 μl P / S, dilute to 50 ml with DMEM / 1640 medium;

[0025] 5× phosphate buffer: according to the formula, 40.95g sodium chloride, 1.01g potassium chloride, 7.09g anhydrous disodium hydrogen phosphate, 1.22g potassium dihydrogen phosphate, and triple distilled water are diluted to 1L to make 5× PBS, and stored in a refrigerator at 4℃;

[0026] 1×PBST buffer: 200 ml 5×PBS, 800 ml triple distilled water, 500 μl Tween-20;

[0027] Western blot electrophoresis buffer: according to the formula, glycine 72g, Tris 15.15g, SDS 5g, and triple distilled water are diluted to 1L to make 5× electrophoresis buffer, which is dissolved on a magnetic stirrer and stored in a refrigerator at 4℃;

[0028] Western blot transfer buffer: According to the recipe, add 72 g of glycine, 15.15 g of Tris, and dilute to 1 L with triple-distilled water to make 5× transfer buffer, dissolve on a magnetic stirrer, and store in a refrigerator at 4°C.

[0029] Preferably: in S2, the cell culture is specifically as follows:

[0030] Culture medium: Use 1640 culture medium containing 10% FBS and 1% P / S double antibody to culture RKO and HT-29 cell lines; use DMEM culture medium containing 10% FBS and 1% P / S double antibody to culture NCM-460, HCT-116, SW620, and SW480 cell lines;

[0031] Culture conditions: 37°C, 5% CO 2 Cell culture incubator;

[0032] Operating conditions: Before each experiment, clean the clean bench between cells with 75% alcohol and irradiate with ultraviolet light for 30 minutes before starting the experiment. Prepare the prepared cell culture medium, trypsin, and 1× PBS solution to reheat in advance.

[0033] The cell recovery in S2 is specifically as follows: taking out the cell cryopreservation tube from a -80°C refrigerator or a liquid nitrogen tank, and rapidly thawing it in a 37°C water bath for 1 min; after thawing, transferring it to a 15 ml centrifuge tube and adding 2 ml of the culture medium corresponding to the cell line, marking the wall of the centrifuge tube and placing it in a centrifuge, and centrifuging at 750 rpm for 5 min; after centrifugation, discarding the supernatant, adding 1 ml of the corresponding culture medium to resuspend the cells, and adding them to a cell culture dish that has been added with the cell culture medium, shaking evenly, and culturing in a cell incubator.

[0034] Preferably, the cell medium replacement in S2 is specifically as follows: when metabolic waste is generated during cell growth but the cells have not grown all over the culture dish, the cell medium needs to be replaced; the cells are carefully taken out of the incubator, the cell morphology is observed under a microscope, and after the observation, the cells are placed in a clean bench to discard the waste medium, 1×PBS is added to wash the culture dish three times and then discarded, and fresh culture medium is added to complete the medium replacement operation;

[0035] The cell passage in S2 is specifically as follows:

[0036] When the cells grow to 90% of the culture dish, subculture them at a ratio of 1:2 or 1:3;

[0037] Discard the old culture medium, add 1×PBS to wash the culture dish three times and then discard it, add 2ml of trypsin to the 10cm culture dish and 1ml of trypsin to the 6cm culture dish, put it in a 37℃ incubator for digestion for 1-3min, and add culture medium to terminate digestion after digestion is complete;

[0038] Use a pipette to gently blow off the adherent cells, collect the digested cell suspension into a 15 ml centrifuge tube, and centrifuge at 750 rpm for 5 min;

[0039] After centrifugation, discard the supernatant, add 1 ml of the corresponding culture medium to resuspend the cells, add them to the cell culture dish that has been added with cell culture medium, shake evenly and culture in a cell incubator.

[0040] Preferably, the cells in S2 are cryopreserved as follows:

[0041] When the cells grow to 90% of the culture dish, freeze them in a -80℃ refrigerator or liquid nitrogen tank for storage;

[0042] Discard the culture medium, add 1×PBS to wash the culture dish three times and then discard it, add trypsin to digest, and after digestion is complete, add the corresponding culture medium to terminate digestion;

[0043] Use a pipette to gently blow off the adherent cells, collect the digested cell suspension into a 15 ml centrifuge tube, and centrifuge at 750 rpm for 5 min;

[0044] After centrifugation, discard the supernatant, add 1 ml of cell freezing solution, add it to the cell culture dish that has been added with cell culture medium, shake it evenly and culture it in a cell incubator.

[0045] Preferably, in S3, the cell RNA is extracted as follows:

[0046] S31: Wash the cells three times with 1×PBS and discard, add trypsin for digestion, centrifuge at 750 rpm for 5 min, add 500 μl TRIzol to the cell pellet, mix well by pipetting, and place on ice. Add 500 μl TRIzol and 100 μl chloroform, and shake vigorously until there is no stratification;

[0047] S32: After being placed on ice for 5 min, stratification occurred and centrifuged at 12,000 rpm for 15 min at 4 °C;

[0048] S33: Prepare a new enzyme-free EP tube, write the corresponding sample name and time, and aspirate the supernatant transparent liquid after centrifugation;

[0049] S34: Add isopropanol in equal volume according to the volume of supernatant, mix gently and place on ice for 20 min, centrifuge at 12000 rpm for 15 min in a 4°C centrifuge, and precool 75% ethanol in a -20°C refrigerator;

[0050] S35: After centrifugation, the supernatant was aspirated and discarded, leaving the white precipitated RNA, and 1 ml of 75% ethanol was added slowly to adhere to the wall, and centrifuged at 7500 rpm for 5 min at 4°C;

[0051] S36: Discard the supernatant, turn the EP tube upside down with filter paper to dry, wait until the white precipitated RNA becomes translucent, add 20μl of high-pressure triple-distilled water and mix evenly, test the RNA concentration and purity, and store in a -80℃ refrigerator.

[0052] Preferably: the RNA reverse transcription of S4 is specifically as follows: preparing a reaction system, performing reverse transcription in a PCR instrument, measuring the cDNA concentration, and storing in a -20°C refrigerator after dilution;

[0053] The real-time fluorescence quantitative PCR of S5 is specifically as follows: preparing a reaction system, adding eight tubes and reacting in an RT-qPCR instrument; setting reaction conditions and loading the instrument; and calculating the relative expression level of the target mRNA according to the Ct value and the 2-ΔΔCt formula.

[0054] Preferably, in S6, the cell protein extraction and protein concentration determination are as follows:

[0055] S61: Wash the cells three times with 1× PBS and discard, add trypsin for digestion, centrifuge at 750 rpm for 5 min, and discard the supernatant;

[0056] S62: Prepare protein lysis buffer at a ratio of RIPA:PMSF=100:1;

[0057] S63: Add an appropriate amount of the mixed protein lysis solution to the cell pellet for lysis; after blowing evenly, shatter it with an ultrasonic instrument, setting the ultrasonic on time to 5.0s, the ultrasonic off time to 10s, and the vibration frequency to 40Hz;

[0058] S64: After the shattered protein sample was placed on ice for 30 min, it was centrifuged at 12000 rcf for 15 min at 4°C;

[0059] S65: After centrifugation, the protein supernatant was placed in a new 1.5 ml enzyme-free EP tube and the protein concentration was measured using the BCA method;

[0060] S66: Add 5×SDS-PAGE protein loading buffer to the protein at a volume ratio of 5×SDS-PAGE: protein supernatant = 1:4. Vortex and boil in a 100°C metal bath for 10 min to denature the protein. Cool to room temperature and store in a -20°C refrigerator.

[0061] Preferably: in S7, the protein immunoblotting experiment specifically includes:

[0062] S71: preparing a 10% separation gel at the bottom layer to obtain a separation gel mixed solution;

[0063] S72: Add the separation gel mixture to the gel plate, and flatten the liquid surface with 75% ethanol. Let it stand at room temperature for 20 minutes, and prepare the upper layer of 10% concentrated gel to obtain the concentrated gel mixture;

[0064] S73: Add the concentrated glue mixture to the glue plate, insert a comb and let it stand at room temperature for 30 minutes;

[0065] S74: Thaw the protein sample, marker and SDS-PAGE loading buffer from the -20℃ refrigerator; add 1× electrophoresis buffer to the middle of the gel plate;

[0066] S75: protein loading; constant voltage 80V for 30min, at which time the sample runs through the separation gel, the voltage is adjusted to constant voltage 120V for 80min, and the power is turned off when the target protein band can be separated by the marker display;

[0067] S76: Prepare 1× transfer buffer: 600 ml ddHO 2 O, 200ml 5× transfer buffer + 200ml methanol, pre-cooled in a 4℃ refrigerator; cut the PVDF membrane, activate it in methanol for 1min, and prepare for transfer;

[0068] S77: After the electrophoresis is completed, the electrophoresis gel is removed and the membrane is transferred using a sandwich clamp structure. The membrane is transferred at a constant voltage of 100V for 100min in a 4℃ refrigerator. After the transfer is completed, the PVDF membrane is clamped out and blocked with 5% skim milk for 2h.

[0069] S78: Discard the blocking solution and wash the PVDF membrane with 1×PBST; cut the target band according to the protein marker, add the primary antibody and incubate overnight at 4°C on a shaker, recover the primary antibody the next day, wash the membrane three times with 1×PBST, each time for 10 minutes; incubate with the corresponding secondary antibody, incubate on a shaker at room temperature for 2 hours, recover the secondary antibody, and wash the membrane three times with 1×PBST, each time for 10 minutes;

[0070] S79: Prepare the developer in a 1:1 ratio and add it to the membrane, then take a photo using chemiluminescence imaging; use software to process and analyze the image.

[0071] The beneficial effects of the present invention are:

[0072] 1. This paper not only analyzes the expression of MND1 in colorectal cancer, but also deeply explores the expression differences of MND1 in different cell populations and its localization in the cell nucleus through single-cell sequencing technology.

[0073] 2. The present invention uses bioinformatics tools and single-cell sequencing technology to conduct in-depth research on the expression of MND1, providing a new perspective and method for the study of colorectal cancer; through the study of the high expression of MND1 in colorectal cancer and its relationship with tumors, it provides new targets and ideas for the diagnosis and treatment of colorectal cancer.

[0074] 3. The present invention uses multiple methods to verify the expression of MND1, ensuring the accuracy and reliability of the research results. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 This is a schematic diagram of the pan-cancer expression of MND1 in the TCGA database of the present invention;

[0076] Figure 2 This is a schematic diagram of the expression of MND1 in colorectal cancer tissues of the present invention;

[0077] Figure 3 It is a schematic diagram of the expression of MND1 in a single-cell sequencing data set of the present invention;

[0078] Figure 4 This is a schematic diagram of analyzing the expression of MND1 in colorectal cancer in the GSE161277 dataset in the present invention;

[0079] Figure 5 This is a schematic diagram of the expression of MND1 in B lymphocytes and epithelial cells of the present invention;

[0080] Figure 6 This is a schematic diagram showing that the distribution of MND1 is mainly localized in the cell nucleus according to the Genecards analysis of the present invention;

[0081] Figure 7 This is a schematic diagram of RT-qPCR expression of MND1 in HCT-116, SW-620, SW-480 and RKO cell lines of the present invention;

[0082] Figure 8 It is a schematic diagram of WB expression of MND1 in colorectal cancer cell lines of the present invention. DETAILED DESCRIPTION

[0083] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0084] Embodiment 1:

[0085] The research method of the effect of the biomarker MND1 on colorectal cancer includes the following steps:

[0086] S1: Material preparation

[0087] S11: Analysis of UALCAN, TCGA, and GEPIA databases

[0088] The online analysis website UALCAN was used to analyze the pan-cancer expression of MND1 in the TCGA database, and then the GEPIA online analysis website was used to analyze the expression of MND1 in 349 normal tissues and 275 tumor tissues in the TCGA database.

[0089] S12: Single-cell sequencing analysis scRNA-seq

[0090] The colorectal cancer scRNA-seq dataset comes from GSE161277, which includes 3 normal colorectal samples, 4 colorectal cancer samples, and 4 colorectal adenoma samples, totaling 22,392 cells. The single-cell sequencing scRNA-seq method was used to analyze the relationship between MND1 and colorectal cancer and the cell populations in which it is expressed.

[0091] S13: Genecards database analysis

[0092] The localization of MND1 in cells was analyzed using the Genecards online website to determine the expression location of the gene.

[0093] S14: Determine the cell line

[0094] Cell lines: including human normal colorectal mucosal epithelial cells (NCM-460) and human colorectal cancer cells (HCT-116, SW-620, SW-480, RKO, HT-29).

[0095] S15: Selection of main reagents

[0096]

[0097]

[0098] S16: Instrument and Equipment Preparation

[0099]

[0100] S17: Antibody preparation

[0101]

[0102] S18: Primer sequence

[0103]

[0104] S19: Main reagent configuration

[0105] (1) Cell culture medium: 5 ml FBS + 500 μl P / S, dilute to 50 ml with DMEM / 1640 medium.

[0106] (2) 5× phosphate buffered saline (5× PBS): Prepare 5× PBS according to the following formula. Dilute to 1× PBS according to a certain ratio when needed and store in a refrigerator at 4°C.

[0107]

[0108] (3) 1×PBST buffer: 200 ml 5×PBS + 800 ml triple distilled water + 500 μl Tween-20.

[0109] (4) Western blot electrophoresis buffer: Prepare 5× electrophoresis buffer according to the following formula, dissolve on a magnetic stirrer, and store in a refrigerator at 4°C.

[0110]

[0111] (5) Western blot transfer buffer: Prepare 5× transfer buffer according to the following formula, dissolve on a magnetic stirrer, and store in a refrigerator at 4°C.

[0112]

[0113] S2: Cell culture

[0114] (1) Culture medium: Use 1640 culture medium (containing 10% FBS + 1% P / S dual antibody) to culture RKO and HT-29 cell lines; use DMEM culture medium (containing 10% FBS + 1% P / S dual antibody) to culture NCM-460, HCT-116, SW620, and SW480 cell lines.

[0115] (2) Culture conditions: 37°C, 5% CO 2 Cell culture incubator.

[0116] (3) Operating conditions: Before each experiment, the clean bench between cells was wiped clean with 75% alcohol and irradiated with ultraviolet light for 30 min before the experiment began. The cell culture medium, trypsin, and 1× PBS solution were prepared in advance and rewarmed.

[0117] (4) Cell recovery:

[0118] ① Take out the cell cryopreservation tube from the -80℃ refrigerator or liquid nitrogen tank, and thaw it quickly in a 37℃ water bath for 1 minute. After thawing, transfer it to a 15ml centrifuge tube and add 2ml of the culture medium corresponding to the cell line. After marking the wall of the centrifuge tube, put it in a centrifuge and centrifuge it at 750rpm for 5 minutes.

[0119] ② After centrifugation, discard the supernatant, add 1 ml of the corresponding culture medium to resuspend the cells, add them to the cell culture dish that has been added with cell culture medium, shake evenly and culture in the cell incubator.

[0120] (5) Cell culture medium replacement:

[0121] ① When metabolic waste is produced during cell growth but the cells have not yet filled the culture dish, the cell culture medium needs to be replaced.

[0122] ② Carefully remove the cells from the incubator and observe the cell morphology under a microscope. After observation, place the cells in a clean bench to discard the waste culture medium. Add 1×PBS to wash the culture dish three times and then discard it. Add fresh culture medium to complete the liquid replacement operation.

[0123] (6) Cell passaging:

[0124] ① When the cells grow to about 90% of the culture dish, they can be passaged at a ratio of 1:2 or 1:3.

[0125] ②Discard the old culture medium, add 1×PBS to wash the culture dish three times and then discard it, add trypsin (specific method: add 2ml trypsin to 10cm culture dish, 1ml trypsin to 6cm culture dish) and put it into 37℃ incubator for digestion for 1-3min (the specific time depends on the degree of cell adhesion, and you can also observe when the cells become round under a microscope). After digestion is complete, add corresponding culture medium to terminate digestion.

[0126] ③ Use a pipette to gently blow off the adherent cells, then collect the digested cell suspension into a 15ml centrifuge tube and centrifuge at 750rpm for 5min.

[0127] ④ After centrifugation, discard the supernatant, add 1 ml of the corresponding culture medium to resuspend the cells, add them to the cell culture dish that has been added with cell culture medium, shake evenly and culture in the cell incubator.

[0128] (7) Cell cryopreservation:

[0129] ① When the cells grow to about 90% of the culture dish, they can be frozen in a -80℃ refrigerator or liquid nitrogen tank for preservation.

[0130] ②Discard the culture medium, add 1×PBS to wash the culture dish three times and then discard it, add trypsin to digest, and after digestion is complete, add corresponding culture medium to terminate digestion.

[0131] ③ Use a pipette to gently blow off the adherent cells, then collect the digested cell suspension into a 15ml centrifuge tube and centrifuge at 750rpm for 5min.

[0132] ④ After centrifugation, discard the supernatant, add 1 ml of cell freezing solution, add it to the cell culture dish containing cell culture medium, shake evenly and culture in a cell incubator.

[0133] S3: Cell RNA extraction

[0134] S31: Wash the cells three times with 1×PBS and discard, add trypsin for digestion, centrifuge at 750 rpm for 5 min, add 500 μl TRIzol to the cell pellet, mix well by pipetting, and place on ice, then add 500 μl TRIzol + 100 μl chloroform, shake vigorously until there is no stratification;

[0135] S32: After being placed on ice for 5 min, stratification occurred and centrifuged at 12,000 rpm for 15 min at 4 °C;

[0136] S33: At this time, prepare several new enzyme-free EP tubes, write the corresponding sample names and times, and aspirate the supernatant transparent liquid after centrifugation. Pay attention to ensure the purity when aspirating and do not aspirate the pink protein part below, about 200μl-300μl;

[0137] S34: Add isopropanol in equal volume according to the volume of supernatant, mix gently and place on ice for 20 min, centrifuge at 12000 rpm for 15 min in a 4°C centrifuge, and precool 75% ethanol in a -20°C refrigerator;

[0138] S35: After centrifugation, the supernatant was aspirated and discarded, leaving the white precipitated RNA, and 1 ml of 75% ethanol was added slowly to adhere to the wall, and centrifuged at 7500 rpm for 5 min at 4°C;

[0139] S36: Discard the supernatant and use filter paper to turn the EP tube upside down to dry it. When the white precipitated RNA becomes translucent, add 20μl of high-pressure triple-distilled water and mix evenly. Test the RNA concentration and purity (A260 / 280) and store it in a -80℃ refrigerator.

[0140] S4: RNA reverse transcription

[0141] Prepare the reaction system and perform reverse transcription in a PCR instrument:

[0142]

[0143] Reverse transcription procedure:

[0144]

[0145] The cDNA concentration was measured and stored in a -20°C refrigerator after dilution.

[0146] S5: Real-time quantitative PCR (RT-qPCR)

[0147] Prepare the reaction system, add the following system into eight tubes and perform the reaction in RT-qPCR instrument:

[0148]

[0149]

[0150] Set up the reaction conditions and go on the machine:

[0151]

[0152] Result calculation: According to the Ct value, the relative expression level of the target mRNA was calculated according to the 2-ΔΔCt formula.

[0153] S6: Cell protein extraction and protein concentration determination

[0154] S61: Wash the cells three times with 1× PBS and discard, add trypsin for digestion, centrifuge at 750 rpm for 5 min, and discard the supernatant;

[0155] S62: Prepare protein lysis buffer according to the ratio of RIPA:PMSF=100:1, and prepare it before use;

[0156] S63: adding an appropriate amount of the mixed protein lysis solution to an appropriate amount of cell pellet for lysis (e.g., adding 300 μl of the mixed protein lysis solution to the pellet in a 6 cm cell culture dish); after blowing evenly, shattering with an ultrasonic instrument, setting the ultrasonic on time to 5.0 s, the ultrasonic off time to 10 s, and the vibration frequency to 40 Hz;

[0157] S64: After the shattered protein sample was placed on ice for 30 min, it was centrifuged at 12000 rcf for 15 min at 4°C;

[0158] S65: After centrifugation, the protein supernatant was placed in a new 1.5 ml enzyme-free EP tube and the protein concentration was measured using the BCA method;

[0159] S66: Add 5×SDS-PAGE protein loading buffer to the protein at a volume ratio of 5×SDS-PAGE: protein supernatant = 1:4. Vortex and boil in a 100°C metal bath for 10 min to denature the protein. Cool to room temperature and store in a -20°C refrigerator.

[0160] S7: Western blot

[0161] S71: Prepare the lower layer 10% separation gel according to the following formula to obtain a separation gel mixture:

[0162]

[0163] S72: Add the separation gel mixture to the gel plate and flatten the liquid surface with 75% ethanol. Let it stand at room temperature for about 20 minutes, prepare the upper layer of 10% concentrated gel to obtain the concentrated gel mixture, the specific ingredients are as follows:

[0164]

[0165] S73: Add the concentrated glue mixture to the glue plate, insert a comb and let it stand at room temperature for about 30 minutes;

[0166] S74: Thaw the protein sample, marker and SDS-PAGE loading buffer from the -20℃ refrigerator; add 1× electrophoresis buffer (100ml 5× electrophoresis buffer + 400ml ddH 2 O);

[0167] S75: protein loading (if the internal reference is not uniform, adjust the loading amount later); run at a constant voltage of 80V for 30 minutes. At this time, the sample runs through the separation gel. Adjust the voltage to a constant voltage of 120V and run for about 80 minutes. When the target protein band can be separated by the marker display, turn off the power;

[0168] S76: Prepare 1× transfer buffer (600 ml ddHO 2 O+200ml 5× transfer buffer+200ml methanol), pre-cool in a 4°C refrigerator; cut the PVDF membrane, activate it in methanol for 1 min, and prepare for transfer;

[0169] S77: After the electrophoresis is completed, the electrophoresis gel is removed and the membrane is transferred using a sandwich clamp structure. The membrane is transferred at a constant voltage of 100V for 100min in a 4℃ refrigerator. After the transfer is completed, the PVDF membrane is clamped out and blocked with 5% skim milk for about 2h.

[0170] S78: Discard the blocking solution and wash the PVDF membrane with 1×PBST; cut the target band according to the protein marker, add the primary antibody and incubate overnight at 4°C on a shaker, recover the primary antibody the next day, wash the membrane three times with 1×PBST, each time for 10 minutes; incubate with the corresponding secondary antibody, incubate on a shaker at room temperature for 2 hours, recover the secondary antibody, and wash the membrane three times with 1×PBST, each time for 10 minutes;

[0171] S79: Add the developer in a 1:1 ratio to the membrane, and take photos using chemiluminescence imaging. Use Photoshop software to process and analyze the images.

[0172] S8: Statistical analysis

[0173] GraphPad Prism 9.0 software was used to perform statistical analysis on the data in this study, and the Student's t test was used to evaluate the differences between the two groups. When P < 0.05, the difference was considered significant.

[0174] The results of the study are as follows:

[0175] ①MND1 is highly expressed in colorectal cancer

[0176] The pan-cancer expression of MND1 in the TCGA database was analyzed using the online bioinformatics analysis website UALCAN. The results showed that the expression of MND1 was elevated in the various cancers shown in the figure ( Figure 1 The GEPIA online analysis website was used to analyze the expression of MND1 in 349 normal tissue samples and 275 tumor tissue samples in the TCGA database. The results showed that compared with normal tissues, the expression of MND1 in colorectal cancer tissues was increased (P<0.05, Figure 2 ).

[0177] ②MND1 is highly expressed in colorectal cancer and significantly expressed in B cells and epithelial cells

[0178] The colorectal cancer scRNA-seq dataset comes from GSE161277, which includes 3 normal colorectal samples, 4 colorectal cancer samples, and 4 colorectal adenoma samples. They are divided into two groups, including a normal group and a tumor group, with a total of 22,392 cells. Single-cell sequencing is used to analyze scRNA-seq. After strict quality control and data standardization, the data is subsequently analyzed, and dot plots and violin plots are drawn. The results show that MND1 is still found to be highly expressed in colorectal cancer in the single-cell sequencing dataset ( Figure 3 ). At the same time, the SingleR package was used in combination with manual annotation of single cell types to identify eight major cell populations, including: T lymphocytes, B lymphocytes, macrophages, monocytes, NK cells, epithelial cells, endothelial cells and fibroblasts. The results showed that MND1 was highly expressed in B lymphocytes and epithelial cells ( Figure 5 ).

[0179] ③MND1 is mainly localized in the cell nucleus

[0180] The location of MND1 in the TCGA database was analyzed by Genecards, an online bioinformatics analysis website, to determine its expression. The results showed that the expression level of MND1 in the cell nucleus was relatively high ( Figure 6 ).

[0181] ④MND1 is highly expressed in colorectal cancer cell lines

[0182] RNA and protein were extracted from normal colorectal epithelial cell line NCM-460 and colorectal cancer cell lines HCT-116, SW-620, SW-480, RKO, and HT-29, and the expression of MND1 in colorectal cancer cell lines was detected by RT-qPCR and Western blot. RT-qPCR results showed that compared with normal colorectal cell lines, the expression of MND1 in colorectal cancer cell lines HCT-116 and RKO was significantly increased (P<0.001), while its expression in other cell lines was slightly increased but not as obvious as the former two. Western blot results showed that compared with normal colorectal epithelial cell lines, the expression level of MND1 in HCT-116, SW-620, SW-480 and RKO cell lines was also higher ( Figure 7 , Figure 8 ).

[0183] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for studying the effect of the biomarker MND1 on colorectal cancer, characterized in that: The steps include: S1: Material preparation, cell line determination, reagent selection, instrument and antibody preparation, primer sequence determination, and reagent configuration; S2: Cell culture, selection of culture medium, setting of culture conditions and operating conditions, cell recovery, cell medium replacement, cell passaging, and cell cryopreservation; S3: Cell RNA extraction; S4: RNA reverse transcription, prepare the reverse transcription reaction system, perform reverse transcription in a PCR instrument, measure the cDNA concentration, and store it in a -20℃ refrigerator after dilution; S5: Real-time fluorescence quantitative PCR: prepare the reaction system of real-time fluorescence quantitative PCR, add eight tubes and react in RT-qPCR instrument, set the reaction conditions and put it on the instrument; S6: Cell protein extraction and protein concentration determination; S7: Western blotting experiments; S8: Statistical analysis.

2. The method for studying the effect of the biomarker MND1 on colorectal cancer according to claim 1, characterized in that: In S1, the pan-cancer expression of MND1 in the TCGA database was analyzed using the online analysis website UALCAN, and then the expression of MND1 in normal tissues and tumor tissues in the TCGA database was analyzed using the GEPIA online analysis website; Use single-cell sequencing scRNA-seq method to analyze the relationship between MND1 and colorectal cancer and the cell populations in which it is expressed; The localization of MND1 in cells was analyzed using the Genecards online website to determine the expression location of the gene.

3. The method for studying the effect of the biomarker MND1 on colorectal cancer according to claim 1, characterized in that: In the S1, the cell lines include human normal colorectal mucosal epithelial cells NCM-460, human colorectal cancer cells HCT-116, SW-620, SW-480, RKO, and HT-29; Primers used were MND1 and β-actin, where the sequence of MND1 was: Forward:5'-TGTGAGAGGATCGGAACTTCT-3'; Reverse:5'-CACATCGGCCAATTTTAGCTTC-3'; In S1, the main reagents are configured as follows: Cell culture medium: 5 ml FBS, 500 μl P / S, dilute to 50 ml with DMEM / 1640 medium; 5× phosphate buffer: according to the formula, 40.95g sodium chloride, 1.01g potassium chloride, 7.09g anhydrous disodium hydrogen phosphate, 1.22g potassium dihydrogen phosphate, and triple distilled water are diluted to 1L to make 5× PBS, and stored in a refrigerator at 4℃; 1×PBST buffer: 200 ml 5×PBS, 800 ml triple distilled water, 500 μl Tween-20; Western blot electrophoresis buffer: according to the formula, glycine 72g, Tris 15.15g, SDS 5g, and triple distilled water are diluted to 1L to make 5× electrophoresis buffer, which is dissolved on a magnetic stirrer and stored in a refrigerator at 4℃; Western blot transfer buffer: According to the recipe, add 72 g of glycine, 15.15 g of Tris, and distilled water to make a volume of 1 L to prepare 5× transfer buffer, dissolve on a magnetic stirrer, and store in a refrigerator at 4°C.

4. The method for studying the effect of the biomarker MND1 on colorectal cancer according to claim 3, characterized in that: In S2, the cell culture is specifically as follows: Culture medium: Use 1640 culture medium containing 10% FBS and 1% P / S double antibody to culture RKO and HT-29 cell lines; use DMEM culture medium containing 10% FBS and 1% P / S double antibody to culture NCM-460, HCT-116, SW620, and SW480 cell lines; Culture conditions: 37°C, 5% CO2 cell culture incubator; Operating conditions: Before each experiment, clean the clean bench between cells with 75% alcohol and irradiate with ultraviolet light for 30 minutes before starting the experiment. Prepare the prepared cell culture medium, trypsin, and 1× PBS solution to reheat in advance. The cell recovery in S2 is specifically as follows: taking out the cell cryopreservation tube from a -80°C refrigerator or a liquid nitrogen tank, and rapidly thawing it in a 37°C water bath for 1 min; after thawing, transferring it to a 15 ml centrifuge tube and adding 2 ml of the culture medium corresponding to the cell line, marking the wall of the centrifuge tube and placing it in a centrifuge, and centrifuging at 750 rpm for 5 min; after centrifugation, discarding the supernatant, adding 1 ml of the corresponding culture medium to resuspend the cells, and adding them to a cell culture dish that has been added with the cell culture medium, shaking evenly, and culturing in a cell incubator.

5. The method for studying the effect of the biomarker MND1 on colorectal cancer according to claim 4, characterized in that: The cell medium replacement in S2 is specifically as follows: when metabolic waste is generated during cell growth but the cells have not grown all over the culture dish, the cell medium needs to be replaced; the cells are carefully taken out of the incubator, the cell morphology is observed under a microscope, and after observation, the cells are placed in a clean bench to discard the waste medium, 1×PBS is added to wash the culture dish three times and then discarded, and fresh culture medium is added to complete the medium replacement operation; The cell passage in S2 is specifically as follows: When the cells grow to 90% of the culture dish, subculture them at a ratio of 1:2 or 1:3; Discard the old culture medium, add 1×PBS to wash the culture dish three times and then discard it, add 2ml of trypsin to the 10cm culture dish and 1ml of trypsin to the 6cm culture dish, put it in a 37℃ incubator for digestion for 1-3min, and add culture medium to terminate digestion after digestion is complete; Use a pipette to blow off the adherent cells, then collect the digested cell suspension into a centrifuge tube and centrifuge; After centrifugation, discard the supernatant, add 1 ml of the corresponding culture medium to resuspend the cells, add them to the cell culture dish that has been added with cell culture medium, shake evenly and culture in a cell incubator.

6. The method for studying the effect of the biomarker MND1 on colorectal cancer according to claim 5, characterized in that: The cells in S2 are cryopreserved as follows: When the cells grow to 90% of the culture dish, freeze them in a -80℃ refrigerator or liquid nitrogen tank for storage; Discard the culture medium, add 1×PBS to wash the culture dish three times and then discard it, add trypsin to digest, and after digestion is complete, add the corresponding culture medium to terminate digestion; Use a pipette to blow off the adherent cells, collect the digested cell suspension into a 15 ml centrifuge tube, adjust the parameters to 750 rpm, and centrifuge for 5 minutes; After centrifugation, discard the supernatant, add 1 ml of cell freezing solution, add it to the cell culture dish that has been added with cell culture medium, shake it evenly and culture it in a cell incubator.

7. The method for studying the effect of the biomarker MND1 on colorectal cancer according to claim 6, characterized in that: In S3, RNA extraction from cells is performed as follows: S31: Wash the cells three times with 1×PBS and discard, add trypsin for digestion, centrifuge at 750 rpm for 5 min, add 500 μl TRIzol to the cell pellet, mix well by pipetting, and place on ice. Add 500 μl TRIzol and 100 μl chloroform, and shake vigorously until there is no stratification; S32: After being placed on ice for 5 min, stratification occurred and centrifuged at 12,000 rpm for 15 min at 4 °C; S33: Prepare a new enzyme-free EP tube, write the corresponding sample name and time, and aspirate the supernatant transparent liquid after centrifugation; S34: Add isopropanol in equal volume according to the volume of supernatant, mix gently and place on ice for 20 min, centrifuge at 12000 rpm for 15 min in a 4°C centrifuge, and precool 75% ethanol in a -20°C refrigerator; S35: After centrifugation, the supernatant was aspirated and discarded, leaving the white precipitated RNA, and 1 ml of 75% ethanol was added slowly to adhere to the wall, and centrifuged at 7500 rpm for 5 min at 4°C; S36: Discard the supernatant, turn the EP tube upside down with filter paper to dry, wait until the white precipitated RNA becomes translucent, add 20μl of high-pressure triple-distilled water and mix evenly, test the RNA concentration and purity, and store in a -80℃ refrigerator.

8. The method for studying the effect of the biomarker MND1 on colorectal cancer according to claim 7, characterized in that: The reverse transcription of RNA of S4 is specifically as follows: preparing a reaction system, performing reverse transcription in a PCR instrument, measuring the cDNA concentration, and storing in a -20°C refrigerator after dilution; The real-time fluorescence quantitative PCR of S5 is specifically as follows: preparing a reaction system, adding eight tubes and reacting in an RT-qPCR instrument; setting reaction conditions and loading the instrument; and calculating the relative expression level of the target mRNA according to the Ct value and the 2-ΔΔCt formula.

9. The method for studying the effect of the biomarker MND1 on colorectal cancer according to claim 8, characterized in that: In S6, cell protein extraction and protein concentration determination are as follows: S61: Wash the cells three times with 1× PBS and discard, add trypsin for digestion, centrifuge at 750 rpm for 5 min, and discard the supernatant; S62: Prepare protein lysis buffer at a ratio of RIPA:PMSF=100:1; S63: Add an appropriate amount of the mixed protein lysis solution to the cell pellet for lysis; after blowing evenly, use an ultrasonic instrument to shatter the cell pellet, set the ultrasonic on time to 5.0s, the ultrasonic off time to 10s, and the vibration frequency to 40Hz; S64: After the shattered protein sample was placed on ice for 30 min, it was centrifuged at 12000 rcf for 15 min at 4°C; S65: After centrifugation, the protein supernatant was placed in a new 1.5 ml enzyme-free EP tube and the protein concentration was measured using the BCA method; S66: Add 5×SDS-PAGE protein loading buffer to the protein at a volume ratio of 5×SDS-PAGE: protein supernatant = 1:

4. Vortex and boil in a 100°C metal bath for 10 min to denature the protein. Cool to room temperature and store in a -20°C refrigerator.

10. The method for studying the effect of the biomarker MND1 on colorectal cancer according to claim 9, characterized in that: In S7, the protein immunoblotting experiment specifically includes: S71: preparing a 10% separation gel at the bottom layer to obtain a separation gel mixed solution; S72: Add the separation gel mixture to the gel plate, and flatten the liquid surface with 75% ethanol. Let it stand at room temperature for 20 minutes, and prepare the upper layer of 10% concentrated gel to obtain the concentrated gel mixture; S73: Add the concentrated glue mixture to the glue plate, insert a comb and let it stand at room temperature for 30 minutes; S74: Thaw the protein sample, marker and SDS-PAGE loading buffer from the -20℃ refrigerator; add 1× electrophoresis buffer in the middle of the gel plate; S75: protein loading; constant voltage 80V for 30min, at which time the sample runs through the separation gel, the voltage is adjusted to constant voltage 120V for 80min, and the power is turned off when the target protein band can be separated by the marker display; S76: Prepare 1× transfer buffer: 600ml ddH2O, 200ml 5× transfer buffer + 200ml methanol, pre-cool in a 4°C refrigerator; cut the PVDF membrane, activate it in methanol for 1min, and prepare for transfer; S77: After the electrophoresis is completed, the electrophoresis gel is removed and the membrane is transferred using a sandwich clamp structure. The membrane is transferred at a constant voltage of 100V for 100min in a 4℃ refrigerator. After the transfer is completed, the PVDF membrane is clamped out and blocked with 5% skim milk for 2h. S78: Discard the blocking solution and wash the PVDF membrane with 1×PBST; cut the target band according to the protein marker, add the primary antibody and incubate overnight at 4°C on a shaker, recover the primary antibody the next day, wash the membrane three times with 1×PBST, each time for 10 minutes; incubate with the corresponding secondary antibody, incubate on a shaker at room temperature for 2 hours, recover the secondary antibody, and wash the membrane three times with 1×PBST, each time for 10 minutes; S79: Prepare the developer in a 1:1 ratio and add it to the membrane, then take a photo using chemiluminescence imaging; use software to process and analyze the image.

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