Test method for verifying influence of circMKLN1 on TNBC cell proliferation, migration and invasion ability

Abstract

The invention provides a test method for verifying the influence of circMKLN1 on the proliferation, migration and invasion ability of TNBC cells. The method comprises the following tests: 1) siRNA transfection and knock-down efficiency verification experiment; (2) carrying out a CCK-8 experiment; 3) performing an EDU experiment; 4) performing a clone formation experiment; 5) performing a scratch experiment; and (6) carrying out a Transwell experiment. CCK-8, EDU and clone formation experiments show that the multiplication capacity of TNBC cells can be remarkably inhibited by knocking down the circMKLN1. Scratch and Transwell experiments show that by knocking down the circMKLN1, the migration and invasion ability of TNBC cells can be remarkably inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of triple-negative breast cancer, and in particular to an experimental method for verifying the effects of circMKLN1 on the proliferation, migration and invasion ability of TNBC cells. Background Art

[0002] Worldwide, breast cancer is the most common female malignancy, and its incidence is rising year by year, seriously threatening women's lives and health. According to the results of pathological diagnosis, breast cancer can be divided into different subtypes, among which triple negative breast cancers (TNBC) is one of the most invasive breast cancer subtypes, which has the characteristics of rapid growth, early metastasis, high recurrence rate and lack of effective diagnostic and therapeutic targets. Therefore, the prognosis of TNBC patients is poorer than that of other breast cancer types. Therefore, in-depth research on the molecular mechanism of the occurrence and development of TNBC will help to find early diagnosis, prognostic biomarkers and therapeutic targets for TNBC, which is of great significance for improving the treatment effect and survival prognosis of TNBC patients.

[0003] Circular RNA (circRNA) is a covalently closed circular RNA formed by reverse splicing of precursor mRNA. It has a stable structure, abundant content, and tissue-specific expression, and is of great value in the diagnosis, prognosis, and treatment of cancer. It is reported that circRNA has a variety of biological functions, including acting as a microRNA "sponge", interacting with RNA-binding proteins, and regulating gene transcription. CircRNA has become a research hotspot in recent years due to its unique structure and multiple biological functions. In recent years, the important biological functions of circRNA in multiple processes such as proliferation, metastasis, apoptosis, and invasion of malignant tumors have attracted widespread attention from researchers. Therefore, using circRNA as an entry point to elucidate the molecular mechanism of the occurrence and development of TNBC is expected to open up new avenues for the diagnosis and treatment of TNBC.

[0004] In order to find circRNAs closely related to the occurrence and development of TNBC, circular RNA QPCR chip was used to select circMKLN1, which is highly expressed in breast cancer tissues and cells, as the research target, and the biological function of circMKLN1 was deeply explored through in vitro experiments. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an experimental method to verify the effect of circMKLN1 on the proliferation, migration and invasion ability of TNBC cells.

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

[0007] An experimental method for verifying the effect of circMKLN1 on TNBC cell proliferation, migration and invasion, comprising the following experiments:

[0008] 1) siRNA transfection and knockdown efficiency verification experiments;

[0009] 2) CCK-8 assay;

[0010] 3) 2EDU experiment;

[0011] 4) Clone formation assay;

[0012] 5) Scratch test;

[0013] 6) Transwell assay.

[0014] Further, the steps of step 1) siRNA transfection and knockdown efficiency verification experiment are as follows:

[0015] (1) The day before transfection, cells are routinely digested and centrifuged, and cell counts can be performed. Cells can be plated as required for transfection and subsequent experiments.

[0016] (2) Before transfection, discard the original culture medium of the cells, gently rinse the cells twice with PBS, and then add serum-free culture medium (add 1 ml of culture medium to the 12-well plate, and calculate the required volume of culture medium for other culture dishes / well plates based on the bottom area);

[0017] (3) Take out the sterile EP tube in the biosafety cabinet, prepare two EP tubes for each siRNA, and add 150ul of serum-free culture medium to each EP tube;

[0018] (4) Add Lipofectamine 3000 to one of the EP tubes and gently tap the tube wall or turn it upside down to mix; then add the required amount of siRNA to another EP tube and gently tap the tube wall or turn it upside down to mix;

[0019] (5) Add the EP tube containing siRNA dropwise into the EP tube containing Lipofectamine 3000, invert to mix, and let stand at room temperature for 15 min;

[0020] (6) Place the cells in a 37°C incubator and incubate for 6-8 hours. Discard the culture medium and replace with complete culture medium for another 48 hours before conducting subsequent experiments.

[0021] (7) After 48 h, cellular RNA was extracted and reverse transcribed, and the efficiency of siRNA knockdown of circMKLN1 was determined by RT-qPCR.

[0022] Further, the steps of step 2) CCK-8 experiment are as follows:

[0023] (1) Digest, centrifuge and resuspend the transfected cells, and then count the cells;

[0024] (2) Take a clean, autoclaved centrifuge tube, add a certain amount of culture medium, and then add the required volume of cell suspension to the culture;

[0025] (3) Use a pipette to mix the cell suspension, draw 100 μl of the cell suspension and add it to a 96-well transparent cell culture plate, with a seeding density of 1500-2000 cells per well, and set up 6 replicate wells; add 100 μl of PBS to each well in the outermost circle of the 96-well plate to reduce the edge effect caused by the evaporation of the cell culture medium; each cell type was tested continuously for 4 days;

[0026] (4) The cells were cultured in an incubator for 4 days. At the same time each day, a 96-well plate was taken out, 10 μl of CCK-8 solution was added to each well, and the plate was placed back into the incubator, keeping it away from light.

[0027] (5) After 2 h, take out the 96-well plate, remove the cover of the 96-well plate, and measure the absorbance value of each well at 450 nm using an ELISA reader;

[0028] (6) After collecting absorbance values ​​for 4 days, draw a cell proliferation curve.

[0029] Further, the steps of step 3) 2EDU experiment are as follows:

[0030] 3.1) Self-prepared reagents for the experiment

[0031] Fixative 4% paraformaldehyde Washing liquid 1× PBS buffer Permeabilization fluid PBS containing 0.3% Triton X-100

[0032] 3.2) EdU labeling of cells

[0033] (1) Culture an appropriate amount of cells in a 6-well plate; after the cells have been cultured overnight and have returned to normal, perform transfection;

[0034] (2) Dilute EDU (10 mM) with culture medium at 1:500 to obtain 2× EDU working solution (20 μM);

[0035] (3) Add 2× EdU working solution preheated at 37°C to the 6-well plate at the same volume as the culture medium, so that the final EdU concentration in the 6-well plate becomes 1×. Place in the incubator and incubate for 5 h;

[0036] (4) After the EdU labeling of cells is completed, the culture medium is discarded and 1 mL of 4% paraformaldehyde is added and fixed at room temperature for 15 min;

[0037] (5) Remove the fixative solution and add 1 mL of PBS to each well to wash the cells three times, each time for 5 min;

[0038] (6) Remove the washing solution and add 1 mL of PBS containing 0.3% Triton X-100 to each well and incubate at room temperature for 15 min;

[0039] (7) Remove the permeabilization solution and wash the cells twice with 1 mL of PBS per well, each time for 5 min;

[0040] 3.3) EDU detection

[0041] (1) Dissolve one tube of Click Additive in 1.5 mL of deionized water and mix until completely dissolved to obtain the Click Additive Solution.

[0042] (2) Prepare Click reaction solution: add 4.3 mL Click Reaction Buffer, 200 μL CuSO4, 10 μL 555 Azide, and 500 μL Click Additive Solution to a 15 mL centrifuge tube in sequence;

[0043] (3) Remove PBS from the previous step;

[0044] (4) Add 0.5 mL of Click reaction solution to each well and gently shake the cell culture plate to ensure that the reaction mixture evenly covers the sample;

[0045] (5) Incubate at room temperature in the dark for 50 min;

[0046] (6) Remove the Click reaction solution and wash with PBS three times, 5 min each time;

[0047] 3.4) Nuclear staining

[0048] (1) Preparation of 1× Hoechst 33342 solution: dilute Hoechst 33342 in PBS at a ratio of 1:1,000;

[0049] (2) Continue with EdU detection step (6). After removing PBS, add 1 mL of 1× Hoechst 33342 solution to each well and incubate at room temperature in the dark for 30 min.

[0050] (3) Aspirate the 1× Hoechst 33342 solution and wash with PBS three times, 5 min each time;

[0051] (4) Take photos, observe, and conduct statistical analysis.

[0052] Furthermore, the experimental steps of step 4) clone formation experiment are as follows:

[0053] (1) Digest, centrifuge and resuspend the cells, followed by cell counting;

[0054] (2) The cells to be tested are plated into a 6-well plate at a density of 1000 cells / well, and three replicate wells are set up. The cells in the plate are mixed by the cross-over method and placed in an incubator for culture;

[0055] (3) Change the cell culture medium every 3-4 days and closely observe the size of cell clone growth;

[0056] (4) After about 2 weeks of culture, cell colonies in the plate are visible to the naked eye and the culture can be stopped;

[0057] (5) Take the culture plate out of the incubator, discard the culture medium in the plate, and gently add PBS buffer along the side wall to wash three times, being careful not to blow up the cells. Then add 1 mL of 4% paraformaldehyde to each well, making sure it covers the bottom of the plate, and fix at room temperature for 30 minutes;

[0058] (6) Aspirate the 4% paraformaldehyde in the plate and add crystal violet solution to it for staining for 30 minutes;

[0059] (7) Use a mobile phone to take photos of the clones in the plate and conduct subsequent analysis.

[0060] Further, the experimental steps of step 5) the scratch test are as follows:

[0061] (1) digesting and centrifuging the cells to be tested, then resuspending the cells in culture medium and counting the cells;

[0062] (2) The resuspended cells were plated into a 6-well plate to a cell density of 50-60%, and three replicate wells were set up, and then placed in a 37°C incubator for overnight culture;

[0063] (3) The next day, transfection was performed when the cell density reached 60-70%;

[0064] (4) 48 hours after transfection, when the cell density reaches 100%, take out the culture plate and use the pipette tip to quickly draw three straight lines at appropriate positions in the wells;

[0065] (5) Gently wash the cells in the wells with PBS to completely wash away the floating cells;

[0066] (6) Add serum-free medium and take pictures under an optical microscope (0 h);

[0067] (7) After incubation in the incubator for 24 h, take photos again to observe the healing of scratches on different cells.

[0068] Further, step 6) the experimental steps of the Transwell experiment are as follows;

[0069] 6.1) Transwell assay to detect cell migration ability

[0070] (1) Perform routine digestion and centrifugation on the cells in the logarithmic growth phase after transfection, resuspend the cells in serum-free medium, count the cells, and adjust the cell concentration to 1×10 5 cells / ml; complete medium containing 20% ​​FBS;

[0071] (2) On a sterile operating table, add 700 μL of complete culture medium containing 20% ​​FBS to the lower chamber of a 24-well plate, add the upper chamber to the lower chamber, and add 200 μL of cell suspension to the upper chamber; do not generate bubbles during the operation; carefully place the plate in an incubator and culture for 48 h;

[0072] (3) Remove the chamber, wash the upper and lower chambers of the 24-well plate twice with PBS, and then add 4% paraformaldehyde to fix for 15 min;

[0073] (4) The fixed chambers were removed from the 4% polymethanol and added to a new 24-well plate, and then crystal violet solution was added to stain the cells for 20 min;

[0074] (5) Wash away the residual crystal violet with PBS, gently wipe off the cells in the chamber that have not passed through the chamber membrane into the lower chamber with a cotton swab, and dry the chamber;

[0075] (6) Place the chamber on a glass slide and observe and take photos under a microscope;

[0076] 6.2) Transwell assay to detect cell invasion ability

[0077] (1) The day before the experiment, take out the matrix gel from the -20℃ refrigerator and thaw it in a 4℃ refrigerator overnight. Place the pipette tip in a 4℃ refrigerator to cool down.

[0078] (2) On the second day of the experiment, take out the Matrigel and pipette tips from the refrigerator and place them on an ice box to keep the temperature; mix the Matrigel with serum-free medium at a ratio of 1:8 and pipette to mix well;

[0079] (3) Pipette 60 μL of the mixed solution and add it vertically into the chamber so that it is evenly spread on the bottom;

[0080] (4) Place in an incubator and incubate for 3 h to allow the matrix gel to polymerize into a thin film (do not create bubbles during the process);

[0081] (5) After incubation, the excess liquid in the upper chamber was aspirated, and 100 μL of serum-free culture medium was added to each well and placed in the incubator for 30 min to hydrate the basement membrane;

[0082] (6) Aspirate the liquid in the upper chamber, check whether there is liquid passing through the chamber into the lower chamber, and inoculate cells;

[0083] (7) The subsequent method is the same as the Transwell migration assay.

[0084] The beneficial effects of the present invention are as follows: CCK-8, EDU and clone formation experiments show that knocking down circMKLN1 can significantly inhibit the proliferation ability of TNBC cells. Transwell and scratch experiments show that knocking down circMKLN1 can significantly inhibit the migration and invasion ability of TNBC cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 This is a graph showing the detection of circMKLN1 knockdown efficiency;

[0086] Figure 2 This is a graph of cell proliferation detected by CCK-8 assay after knocking down circMKLN1;

[0087] Figure 3 This is the cell proliferation diagram detected by EDU experiment after knocking down circMKLN1;

[0088] Figure 4 This is a diagram showing the effect of knocking down circMKLN1 on cell clone formation ability;

[0089] Figure 5 The image shows the migration ability of TNBC cells detected by scratch assay;

[0090] Figure 6 This is a diagram of the Transwell experiment to detect the migration and invasion ability of cells. DETAILED DESCRIPTION

[0091] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments.

[0092] Experimental Materials

[0093] 1.1 Cell lines

[0094] Human breast cancer cell lines MDA-MB-436 and BT-549 were purchased from ATCC in the U.S. All cells were identified by STR (no mutation or other cell contamination).

[0095] 1.2 Experimental Reagents

[0096] 1.2.1 Cell culture related reagents

[0097]

[0098] 1.2.2 Kit

[0099]

[0100] 1.2.3 Other reagents

[0101]

[0102] 1.2.4 Main instruments and consumables

[0103]

[0104]

[0105] 2 Experimental methods

[0106] 2.1 siRNA transfection and knockdown efficiency verification experiments

[0107] siRNA targeting circMKLN1 (si-circMKLN1-1, si-circMKLN1-2) and its negative control (Negative control siRNA, si-NC) were purchased from Guangzhou Jisai Biotechnology Co., Ltd. The sequences of siRNA are as follows:

[0108]

[0109] The purchased siRNA was freeze-dried and stored at -20℃. Before use, centrifuge at 2000rpm for 3min to concentrate the siRNA at the bottom of the centrifuge tube, then carefully and slowly open the tube cap, add 125ul DEPC water for every 1OD of siRNA, then cover the tube cap and shake to dissolve. The dissolved siRNA was divided and stored at -20℃ according to the experimental needs to avoid repeated freezing and thawing.

[0110] siRNA transfection takes 12-well plate as an example:

[0111] (1) The day before transfection, cells are routinely digested and centrifuged. Cell counts can be performed and cells are plated as required for transfection and subsequent experiments.

[0112] (2) Before transfection, discard the original culture medium of the cells, gently rinse the cells twice with PBS, and then add serum-free culture medium (add 1 ml of culture medium to the 12-well plate, and calculate the required volume of culture medium for other culture dishes / culture well plates based on the bottom area).

[0113] (3) Take out the sterile EP tube in the biosafety cabinet, prepare two EP tubes for each siRNA, and add 150ul of serum-free culture medium to each EP tube.

[0114] (4) Add Lipofectamine 3000 to one of the EP tubes and mix by gently tapping the tube wall or turning it upside down; then add the required amount of siRNA to another EP tube and mix by gently tapping the tube wall or turning it upside down.

[0115] (5) Add the EP tube containing siRNA dropwise into the EP tube containing Lipofectamine 3000, invert to mix, and let stand at room temperature for 15 min.

[0116] (6) Place the cells in a 37°C incubator and incubate for 6-8 hours. Discard the culture medium and replace with complete culture medium for another 48 hours before conducting subsequent experiments.

[0117] (7) After 48 h, cellular RNA was extracted and reverse transcribed, and the efficiency of siRNA knockdown of circMKLN1 was determined by RT-qPCR.

[0118] 2.2 Cell function experiments

[0119] 2.2.1CCK-8 experiment

[0120] (1) Digest, centrifuge and resuspend the transfected cells, then count the cells.

[0121] (2) Take a clean, autoclaved centrifuge tube, add a certain amount of culture medium, and then add the required volume of cell suspension to the culture.

[0122] (3) Use a pipette to mix the cell suspension, and then pipette 100 μl of the cell suspension into a 96-well transparent cell culture plate. The inoculation density is 1500-2000 cells per well, and 6 replicate wells are set. Add 100 μl of PBS to each well in the outermost circle of the 96-well plate to reduce the edge effect caused by the evaporation of the cell culture medium. Each cell type is tested continuously for 4 days.

[0123] (4) Place the cells in an incubator and culture them for 4 days. Take out a 96-well plate at the same time every day, add 10 μl of CCK-8 solution to each well, and then put it back into the incubator, keeping it away from light.

[0124] (5) After 2 h, take out the 96-well plate, remove the cover of the 96-well plate, and use an ELISA reader to detect the absorbance value of each well at 450 nm.

[0125] (6) After collecting absorbance values ​​for 4 days, draw a cell proliferation curve.

[0126] 2.2.2EDU Experiment

[0127] Self-prepared reagents

[0128] Fixative 4% paraformaldehyde Washing liquid 1× PBS buffer Permeabilization fluid PBS containing 0.3% Triton X-100

[0129] EdU labeling of cells

[0130] (1) Culture an appropriate amount of cells in a 6-well plate. After the cells have been cultured overnight and have returned to normal, perform transfection.

[0131] (2) Dilute EDU (10 mM) with culture medium at 1:500 to obtain 2×EDU working solution (20 μM).

[0132] (3) Add 2× EdU working solution preheated at 37°C to the 6-well plate at the same volume as the culture medium, so that the final EdU concentration in the 6-well plate becomes 1×. Place the plate in an incubator and incubate for 5 h.

[0133] (4) After the EdU labeling of cells was completed, the culture medium was discarded and 1 mL of 4% paraformaldehyde was added and fixed at room temperature for 15 min.

[0134] (5) Remove the fixative solution and add 1 mL of PBS to each well to wash the cells three times, each time for 5 min.

[0135] (6) Remove the washing solution, add 1 mL of PBS containing 0.3% Triton X-100 to each well, and incubate at room temperature for 15 min.

[0136] (7) Remove the permeabilization solution and wash the cells twice with 1 mL of PBS per well, each time for 5 min.

[0137] EDU Detection

[0138] (1) Dissolve one tube of Click Additive in 1.5 mL of deionized water and mix until completely dissolved. This is the Click Additive Solution.

[0139] (2) Prepare Click reaction solution: add 4.3 mL Click Reaction Buffer, 200 μL CuSO4, 10 μL 555 Azide, and 500 μL Click Additive Solution to a 15 mL centrifuge tube in that order.

[0140] (3) Remove the PBS from the previous step.

[0141] (4) Add 0.5 mL of Click reaction solution to each well and gently shake the cell culture plate to ensure that the reaction mixture evenly covers the sample.

[0142] (5) Incubate at room temperature in the dark for 50 min.

[0143] (6) Aspirate the Click reaction solution and wash the cells three times with PBS, each time for 5 min.

[0144] Nuclear staining

[0145] (1) Preparation of 1× Hoechst 33342 solution: Dilute Hoechst 33342 in PBS at a ratio of 1:1,000.

[0146] (2) Continue with EdU detection step (6). After removing PBS, add 1 mL of 1× Hoechst 33342 solution to each well and incubate at room temperature in the dark for 30 min.

[0147] (3) Aspirate the 1× Hoechst 33342 solution and wash three times with PBS, 5 min each time.

[0148] (4) Take photos, observe, and conduct statistical analysis.

[0149] 2.2.3 Clone formation assay

[0150] (1) Digest, centrifuge and resuspend the cells, then count the cells.

[0151] (2) The cells to be tested are plated into a 6-well plate at a density of 1000 cells / well, and three replicate wells are set up. The cells in the plate are mixed by the cross-over method and placed in an incubator for culture.

[0152] (3) Change the cell culture medium every 3-4 days and closely monitor the size of cell clone growth.

[0153] (4) After about 2 weeks of culture, cell colonies in the plate are visible to the naked eye and the culture can be stopped.

[0154] (5) Take the culture plate out of the incubator, discard the culture medium in the plate, and gently add PBS buffer along the side wall to wash three times. Be careful not to blow up the cells. Then add 1 mL of 4% paraformaldehyde to each well, making sure it covers the bottom of the plate, and fix at room temperature for 30 minutes.

[0155] (5) Aspirate the 4% paraformaldehyde in the plate and add crystal violet solution to it and stain for 30 minutes.

[0156] (6) Use a mobile phone to take photos of the clones in the plate and conduct subsequent analysis.

[0157] 2.2.4 Scratch test

[0158] (1) The cells to be tested are digested and centrifuged, then the cells are resuspended in culture medium and the cells are counted.

[0159] (2) The resuspended cells were plated in a 6-well plate to a cell density of 50-60%, and three replicate wells were set up. The plate was then placed in a 37°C incubator for overnight culture.

[0160] (3) The next day, transfection was performed when the cell density reached 60-70%.

[0161] (4) 48 hours after transfection, when the cell density reaches 100%, take out the culture plate and use the pipette to quickly draw three straight lines at appropriate positions in the wells. Then observe under a microscope to ensure that the width of the straight lines meets our requirements.

[0162] (5) Gently wash the cells in the wells with PBS to completely wash away the floating cells.

[0163] (6) Add serum-free culture medium and take pictures under an optical microscope (0 h).

[0164] (7) After incubation in the incubator for 24 h, take photos again to observe the healing of scratches on different cells.

[0165] 2.2.5 Transwell experiment

[0166] Transwell assay to detect cell migration ability

[0167] (1) Perform routine digestion and centrifugation on the cells in the logarithmic growth phase after transfection, resuspend the cells in serum-free medium, count the cells, and adjust the cell concentration to 1×10 5 cells / ml. Prepare complete medium containing 20% ​​FBS.

[0168] (2) On a sterile operating table, add 700 μL of complete culture medium containing 20% ​​FBS to the lower chamber of a 24-well plate, add the upper chamber to the lower chamber, and add 200 μL of cell suspension to the upper chamber. Do not generate bubbles during the operation. Carefully place in an incubator and culture for 48 hours.

[0169] (3) Remove the chamber, wash the upper chamber and lower chamber of the 24-well plate twice with PBS, and then add 4% paraformaldehyde to fix for 15 minutes.

[0170] (4) The fixed chambers were removed from the 4% polymethanol and placed in a new 24-well plate, followed by the addition of crystal violet solution to stain the cells for 20 min.

[0171] (5) Wash off the residual crystal violet with PBS, gently wipe off the cells in the chamber that have not passed through the chamber membrane into the lower chamber with a cotton swab, and dry the chamber.

[0172] (6) Place the chamber on a glass slide and observe and take pictures under a microscope.

[0173] Transwell assay to detect cell invasion ability

[0174] (1) The day before the experiment, take out the matrix gel from the -20℃ refrigerator and thaw it in a 4℃ refrigerator overnight. Place the pipette tip in a 4℃ refrigerator to cool down.

[0175] (2) On the second day of the experiment, take out the Matrigel and pipette tips from the refrigerator and place them on an ice box to keep the temperature. Mix the Matrigel with serum-free medium in a ratio of 1:8 and pipette to mix thoroughly.

[0176] (3) Pipette 60 μL of the mixed solution and add it vertically into the chamber so that it spreads evenly on the bottom.

[0177] (4) Place the plate in an incubator and incubate for 3 h to allow the matrix gel to polymerize into a thin film (do not create bubbles during the process).

[0178] (5) After incubation, remove excess liquid from the upper chamber and add 100 μL of serum-free culture medium to each well. Place the wells in the incubator for 30 min to hydrate the basement membrane.

[0179] (6) Aspirate the liquid in the upper chamber, check whether there is liquid passing through the chamber into the lower chamber, and inoculate the cells.

[0180] (7) The subsequent method is the same as the Transwell migration assay.

[0181] 3 Experimental results

[0182] In vitro experimental verification of the biological function of circMKLN1

[0183] 3.1 Detection of circMKLN1 knockdown efficiency

[0184] In order to explore the biological function of circMKLN1, we first transfected circMKLN1 siRNA si-circMKLN1-1, si-circMKLN1-2 and its negative control si-NC into TNBC cells MDA-MB-436 and BT-549 cells, respectively. After 48 h of transfection, total RNA was extracted from the cells and RT-qPCR was used to detect the knockdown efficiency of circMKLN1. Figure 1 It was found that both siRNAs could effectively knock down the expression level of circMKLN1, and the expression level of the host gene MKLN1 was detected in the same samples, and it was found that the expression level of MKLN1 did not change significantly, indicating that knocking down circMKLN1 did not exert its corresponding function by affecting the host gene MKLN1.

[0185] 3.2 Knockdown of circMKLN1 can significantly inhibit the proliferation of TNBC cells

[0186] In order to explore the biological function of circMKLN1 in TNBC cells, we first detected the effect of knocking down circMKLN1 on the proliferation ability of TNBC cells. Through CCK-8 experiments, we found that ( Figure 2 ), after knocking down circMKLN1, the cell proliferation rate was significantly inhibited.

[0187] The EDU cell proliferation assay was further used to detect the changes in TNBC cell proliferation activity after knocking down circMKLN1. As shown in the figure ( Figure 3 ), after knocking down circMKLN1, the proportion of EDU-positive cells was significantly reduced, indicating that the proliferation activity of TNBC cells was inhibited, which was consistent with the results of the CCK-8 experiment. Subsequently, a clone formation experiment was performed, and the results showed that ( Figure 4 ), knocking down circMKLN1 can significantly inhibit the clone formation of TNBC single cells, which is consistent with the previous results. Figure 2-4 It can be seen that knocking down circMKLN1 can significantly inhibit the proliferation ability of TNBC cells.

[0188] 3.3 Knockdown of circMKLN1 can significantly inhibit the migration and invasion ability of TNBC cells

[0189] The effects of knockdown of circMKLN1 on the migration and invasion of TNBC cells were examined in vitro. First, a scratch assay was used to evaluate the effect of circMKLN1 on the migration ability of TNBC cells. Figure 5 As shown in the figure, knocking down circMKLN1 significantly inhibited the cell's "wound repair" ability, indicating that its cell migration ability was impaired. At the same time, the Transwell migration assay found that knocking down circMKLN1 could significantly inhibit the migration ability of TNBC cells, and the same result was obtained using the Transwell invasion assay ( Figure 6 ). The above results confirmed that circMKLN1 could significantly inhibit the migration and invasion ability of TNBC cells in vitro.

[0190] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An experimental method for verifying the effect of circMKLN1 on TNBC cell proliferation, migration and invasion, characterized in that: The method includes the following tests: 1) siRNA transfection and knockdown efficiency verification experiments; 2) CCK-8 assay; 3) EDU experiment; 4) Clone formation assay; 5) Scratch test; 6) Transwell assay.

2. The experimental method for verifying the effect of circMKLN1 on TNBC cell proliferation, migration and invasion according to claim 1, wherein step 1) the siRNA transfection and knockdown efficiency verification experimental steps are as follows: (1) The day before transfection, cells are routinely digested and centrifuged, and cell counts can be performed. Cells can be plated as required for transfection and subsequent experiments. (2) Before transfection, discard the original culture medium of the cells, gently rinse the cells twice with PBS, and then add serum-free culture medium; (3) Take out the sterile EP tube in the biosafety cabinet, prepare two EP tubes for each siRNA, and add 150ul of serum-free culture medium to each EP tube; (4) Add Lipofectamine 3000 to one of the EP tubes and gently tap the tube wall or turn it upside down to mix; then add the required amount of siRNA to another EP tube and gently tap the tube wall or turn it upside down to mix; (5) Add the EP tube containing siRNA dropwise into the EP tube containing Lipofectamine 3000, invert to mix, and let stand at room temperature for 15 min; (6) Place the cells in a 37°C incubator and incubate for 6-8 hours. Discard the culture medium and replace with complete culture medium for another 48 hours before conducting subsequent experiments. (7) After 48 h, cellular RNA was extracted and reverse transcribed, and the efficiency of siRNA knockdown of circMKLN1 was determined by RT-qPCR.

3. The experimental method for verifying the effect of circMKLN1 on TNBC cell proliferation, migration and invasion according to claim 1, characterized in that: Step 2) The steps of CCK-8 experiment are as follows: (1) Digest, centrifuge and resuspend the transfected cells, and then count the cells; (2) Take a clean, autoclaved centrifuge tube, add a certain amount of culture medium, and then add the required volume of cell suspension to the culture; (3) Use a pipette to mix the cell suspension, draw 100 μl of the cell suspension and add it to a 96-well transparent cell culture plate, with a seeding density of 1500-2000 cells per well, and set up 6 replicate wells; add 100 μl of PBS to each well in the outermost circle of the 96-well plate to reduce the edge effect caused by the evaporation of the cell culture medium; each cell type was tested continuously for 4 days; (4) The cells were cultured in an incubator for 4 days. At the same time each day, a 96-well plate was taken out, 10 μl of CCK-8 solution was added to each well, and the plate was placed back into the incubator, keeping it away from light. (5) After 2 h, take out the 96-well plate, remove the cover of the 96-well plate, and measure the absorbance value of each well at 450 nm using an ELISA reader; (6) After collecting absorbance values ​​for 4 days, draw a cell proliferation curve.

4. The experimental method for verifying the effect of circMKLN1 on TNBC cell proliferation, migration and invasion according to claim 1, characterized in that: Step 3) The steps of the 2EDU experiment are as follows: 3.1) Self-prepared reagents for the experiment 3.2) EdU labeling of cells (1) Culture an appropriate amount of cells in a 6-well plate; after the cells have been cultured overnight and have returned to normal, perform transfection; (2) Dilute EDU (10 mM) with culture medium at 1:500 to obtain 2× EDU working solution (20 μM); (3) Add 2× EdU working solution preheated at 37°C to the 6-well plate at the same volume as the culture medium, so that the final EdU concentration in the 6-well plate becomes 1×. Place in the incubator and incubate for 5 h; (4) After the EdU labeling of cells is completed, the culture medium is discarded and 1 mL of 4% paraformaldehyde is added and fixed at room temperature for 15 min; (5) Remove the fixative solution and add 1 mL of PBS to each well to wash the cells three times, each time for 5 min; (6) Remove the washing solution and add 1 mL of PBS containing 0.3% Triton X-100 to each well and incubate at room temperature for 15 min; (7) Remove the permeabilization solution and wash the cells twice with 1 mL of PBS per well, each time for 5 min; 3.3) EDU detection (1) Dissolve one tube of Click Additive in 1.5 mL of deionized water and mix until completely dissolved to obtain the Click Additive Solution. (2) Prepare Click reaction solution: add 4.3 mL Click Reaction Buffer, 200 μL CuSO4, 10 μL 555 Azide, and 500 μL Click Additive Solution to a 15 mL centrifuge tube in sequence; (3) Remove PBS from the previous step; (4) Add 0.5 mL of Click reaction solution to each well and gently shake the cell culture plate to ensure that the reaction mixture evenly covers the sample; (5) Incubate at room temperature in the dark for 50 min; (6) Remove the Click reaction solution and wash with PBS three times, 5 min each time; 3.4) Nuclear staining (1) Preparation of 1× Hoechst 33342 solution: dilute Hoechst 33342 in PBS at a ratio of 1:1,000; (2) Continue with EdU detection step (6). After removing PBS, add 1 mL of 1× Hoechst 33342 solution to each well and incubate at room temperature in the dark for 30 min. (3) Aspirate the 1× Hoechst 33342 solution and wash with PBS three times, 5 min each time; (4) Take photos, observe, and conduct statistical analysis.

5. The experimental method for verifying the effect of circMKLN1 on TNBC cell proliferation, migration and invasion according to claim 1, characterized in that: Step 4) The experimental steps of the clone formation experiment are as follows: (1) Digest, centrifuge and resuspend the cells, followed by cell counting; (2) The cells to be tested are plated into a 6-well plate at a density of 1000 cells / well, and three replicate wells are set up. The cells in the plate are mixed by the cross-over method and placed in an incubator for culture; (3) Change the cell culture medium every 3-4 days and closely observe the size of cell clone growth; (4) After about 2 weeks of culture, cell colonies in the plate are visible to the naked eye and the culture can be stopped; (5) Take the culture plate out of the incubator, discard the culture medium in the plate, and gently add PBS buffer along the side wall to wash three times, taking care not to blow up the cells; then add 1 mL of 4% paraformaldehyde to each well, making sure it covers the bottom of the plate, and fix at room temperature for 30 minutes; (6) Aspirate the 4% paraformaldehyde in the plate and add crystal violet solution to it for staining for 30 minutes; (7) Use a mobile phone to take photos of the clones in the plate and conduct subsequent analysis.

6. The experimental method for verifying the effect of circMKLN1 on TNBC cell proliferation, migration and invasion according to claim 1, characterized in that: Step 5) The experimental steps of the scratch test are as follows: (1) digesting and centrifuging the cells to be tested, then resuspending the cells in culture medium and counting the cells; (2) The resuspended cells were plated into a 6-well plate to a cell density of 50-60%, and three replicate wells were set up, and then placed in a 37°C incubator for overnight culture; (3) The next day, transfection was performed when the cell density reached 60-70%; (4) 48 hours after transfection, when the cell density reaches 100%, take out the culture plate and use the pipette tip to quickly draw three straight lines at appropriate positions in the wells; (5) Gently wash the cells in the wells with PBS to completely wash away the floating cells; (6) Add serum-free medium and take pictures under an optical microscope (0 h); (7) After incubation in the incubator for 24 h, take photos again to observe the healing of scratches on different cells.

7. The experimental method for verifying the effect of circMKLN1 on TNBC cell proliferation, migration and invasion according to claim 1, characterized in that: Step 6) The experimental steps of the Transwell experiment are as follows; 6.1) Transwell assay to detect cell migration ability (1) Perform routine digestion and centrifugation on the cells in the logarithmic growth phase after transfection, resuspend the cells in serum-free medium, count the cells, and adjust the cell concentration to 1×10 5 cells / ml; complete medium containing 20% ​​FBS; (2) On a sterile operating table, add 700 μL of complete culture medium containing 20% ​​FBS to the lower chamber of a 24-well plate, add the upper chamber to the lower chamber, and add 200 μL of cell suspension to the upper chamber; do not generate bubbles during the operation; carefully place the plate in an incubator and culture for 48 h; (3) Remove the chamber, wash the upper and lower chambers of the 24-well plate twice with PBS, and then add 4% paraformaldehyde to fix for 15 min; (4) The fixed chambers were removed from the 4% polymethanol and added to a new 24-well plate, and then crystal violet solution was added to stain the cells for 20 min; (5) Wash away the residual crystal violet with PBS, gently wipe off the cells in the chamber that have not passed through the chamber membrane into the lower chamber with a cotton swab, and dry the chamber; (6) Place the chamber on a glass slide and observe and take photos under a microscope; 6.2) Transwell assay to detect cell invasion ability (1) The day before the experiment, take out the matrix gel from the -20℃ refrigerator and thaw it in a 4℃ refrigerator overnight. Place the pipette tip in a 4℃ refrigerator to cool down. (2) On the second day of the experiment, take out the Matrigel and pipette tips from the refrigerator and place them on an ice box to keep the temperature; mix the Matrigel with serum-free medium at a ratio of 1:8 and pipette to mix well; (3) Pipette 60 μL of the mixed solution and add it vertically into the chamber so that it is evenly spread on the bottom; (4) Place in an incubator and incubate for 3 h to allow the matrix gel to polymerize into a thin film; (5) After incubation, the excess liquid in the upper chamber was aspirated, and 100 μL of serum-free culture medium was added to each well and placed in the incubator for 30 min to hydrate the basement membrane; (6) Aspirate the liquid in the upper chamber, check whether there is liquid passing through the chamber into the lower chamber, and inoculate cells; (7) The subsequent method is the same as the Transwell migration assay.

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