Screening and verification method of circMKLN1 circular RNA capable of regulating and controlling triple negative breast cancer

Abstract

The invention provides a method for screening and verifying circMKLN1 circular RNA (Ribonucleic Acid) capable of regulating and controlling triple negative breast cancer, circMKLN1 is formed by transcription and reverse splicing cyclization of 4-7 exons of an MKLN1 gene, and the length of circMKLN1 is 535bp. The primer of the circMKLN1 is used for carrying out PCR (Polymerase Chain Reaction) amplification and then is sent to a living organism company for Sanger sequencing, and the result shows that the specific splicing site of the circMKLN1 is found. The cDNA and the gDNA are used as templates, PCR amplification is carried out by using a circMKLN1 convergence primer and a circMKLN1 divergent primer, and the result shows that the circMKLN1 divergent primer can only be amplified in the cDNA and is not amplified in the gDNA, so that the existence of the circMKLN1 is further proved. The RNase R and actinomycin D experiments show that the circMKLN1 has higher resistance to the RNase R, and the half-life period of the circMKLN1 is far longer than the half-life period of linear MKLN1 mRNA (messenger ribonucleic acid). A nucleoplasm separation experiment shows that circMKLN1 mainly exists in cytoplasm.

Description

Technical Field

[0001] The present invention relates to the technical field of triple-negative breast cancer, and in particular to a screening and verification method for circMKLN1 circular RNA capable of regulating triple-negative breast cancer. Background Art

[0002] The latest cancer statistics for 2020 released by the World Health Organization's International Agency for Research on Cancer show that breast cancer has replaced lung cancer as the world's leading cancer. Triple negative breast cancer (TNBC) is the most invasive and harmful malignant tumor among breast cancers. Although local surgery, systemic chemotherapy and radiotherapy have prolonged the overall survival of TNBC patients, the recurrence rate and mortality rate of TNBC patients are still very high. TNBC is a type of breast cancer that does not express estrogen receptors, progesterone receptors and human epithelial growth factor receptor 2. It has obvious characteristics such as young onset, high malignancy, strong invasiveness, difficult treatment and easy recurrence. At the same time, there are still no tumor markers and molecular targeted therapeutic targets with high specificity and sensitivity. Circular RNA (circRNAs) is a new type of endogenous non-coding RNA and new regulatory molecule, which is widely expressed in the eukaryotic transcriptome of various organisms. Its unique ring structure gives it a more stable structure than linear RNA, insensitivity to nucleases, conservatism, tissue expression specificity and other properties, making it a new hot spot in RNA research in recent years. CircRNAs are closely related to the occurrence and development of many diseases, especially malignant tumors, and play an important regulatory role. They are expected to become new biomarkers and potential therapeutic targets for breast cancer, providing new ideas for the diagnosis and targeted treatment of tumors. In the early stage, we conducted a comprehensive review of the development history, generation mechanism, classification and biological function of circular RNA, as well as the research progress in early screening, diagnosis and mechanism of action of breast cancer, and later provided new directions for early prediction, diagnosis, treatment and research of breast cancer.

[0003] With the continuous development of RNA-seq analysis technology and bioinformatics, more and more circRNAs have been identified in TNBC cell lines. Studies have found that circRNAs have the characteristics of TNBC cell localization and tissue-specific expression, and play an important regulatory role in the occurrence and development of TNBC. At present, the mechanism research of circRNAs in cancer mainly focuses on: (1) acting as a sponge to absorb miRNA and upregulate the expression of its target genes to exert its biological function; (2) interacting with proteins to regulate cancer progression; (3) encoding polypeptides and regulating tumor occurrence and development through polypeptides; (4) regulating the expression of its parent gene at the transcriptional and post-transcriptional levels and participating in cancer occurrence. In addition, in TNBC, circRNAs play a huge regulatory function in cell proliferation, apoptosis, migration, invasion and drug resistance, and circRNAs can be used as TNBC patients. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a screening and verification method for circMKLN1 circular RNA that regulates triple-negative breast cancer.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for screening and verifying circMKLN1 circular RNA that regulates triple-negative breast cancer, the method comprising the following steps:

[0007] 1) Cell culture

[0008] The human breast cancer cell line MDA-MB-436 was cultured in DMEM high glucose medium; the human breast cancer cell line BT-549 was cultured in RPMI 1640 medium; all cells were cultured in a humidified cell incubator at 37°C and 5% CO2;

[0009] 2) Extraction, reverse transcription and real-time fluorescence quantitative PCR of cell RNA;

[0010] 3) PCR amplification and agarose gel electrophoresis

[0011] 3.1) PCR amplification

[0012] The cDNA and gDNA of MDA-MB-436 and BT-549 were used as templates; the required divergent primer (DP) and convergent primer (CP) sequences are shown in the following table:

[0013]

[0014] 3.2) Agarose gel electrophoresis

[0015] Place the agarose precast gel into the electrophoresis tank, then add the corresponding amount of 1×TAE buffer to immerse the precast gel; add 5ul 100bp DNA Marker to the first loading hole of the precast gel, set the voltage to 150V, and start electrophoresis; stop electrophoresis when the indicator reaches about 1cm from the bottom of the gel; use a gel imager to observe the band position of the amplified product;

[0016] 4) Sanger sequencing

[0017] Cut the gel at the target product band under the gel imager, and then extract the DNA of the target product in the gel; send the eluted DNA to Sangon Biotech for Sanger sequencing;

[0018] 5) RNase R digestion and actinomycin D experiment

[0019] 5.1) RNase R digestion experiment

[0020] (1) To verify that circMKLN1 is insensitive to the exonuclease RNase R, total RNA of TNBC cells was extracted and divided into an experimental group and a control group to prepare the reaction system. 0.1 uL RNase R was added to the experimental group.

[0021] (2) Place both groups in a PCR instrument and set the program to 37°C for 10 min and 70°C for 10 min;

[0022] (3) reverse transcription and RT-qPCR experiments were performed to detect changes in RNA levels;

[0023] 5.2) Actinomycin D experiment

[0024] ActD was used to treat cells to inhibit the synthesis of intracellular RNA to detect the half-life of circMKLN1 and its linear RNA to verify whether circMKLN1 has higher stability;

[0025] 6) Verification of subcellular localization of circMKLN1

[0026] 6.1) Nuclear-cytoplasmic separation experiment

[0027] Cytoplasmic and nuclear RNA extraction kit

[0028] (1) Preparation:

[0029] a) Prepare an appropriate amount of Lysis Buffer J by adding 10 μL of β-mercaptoethanol to every 1 mL of Lysis Buffer J required; place the solution on ice or at 4°C before use;

[0030] b) Prepare the appropriate amount of Buffer SK by adding 10 μL of β-mercaptoethanol to 1 mL of the required Buffer SK;

[0031] c) Prepare a working concentration of Wash Solution A by adding 90 mL of absolute ethanol to each bottle containing Wash Solution A; the final volume is 128 mL;

[0032] d) Before starting, make sure to place Lysis Buffer J in a -20°C freezer for 10 to 15 minutes and remove it before use;

[0033] (2) Preparation of cell components

[0034] a) Transfer the cell suspension to an RNase-free tube and centrifuge at no more than 200 x g or 2000 RPM for 10 minutes to pellet the cells;

[0035] b) Carefully pour off the supernatant; a few microliters of medium may remain in the pellet to ensure that the pellet does not fall off.

[0036] c) Add 200uL of ice-cold Lysis Buffer J to the precipitate;

[0037] d) Lyse cells by vortexing for 15 seconds; make sure the entire pellet is completely dissolved before proceeding to the next step; use a pipette to transfer the lysate to an RNase-free microcentrifuge tube;

[0038] e) Spin the lysate at maximum speed in a benchtop centrifuge for 10 minutes;

[0039] f) Using a pipette with a small tip, transfer the supernatant containing the cytoplasmic RNA to another RNase-free tube;

[0040] (3) Binding cytoplasmic RNA to the column

[0041] a) Add 200uL Buffer SK to the supernatant (cytoplasmic RNA fraction) from the previous step; vortex to mix for 10 seconds;

[0042] b) Add 200uL 96-100% ethanol to the mixture; vortex mix for 10 seconds;

[0043] c) Add the mixture to a spin column assembled with a collection tube and centrifuge at 3500 g or 6000 RPM for 1 minute;

[0044] d) discard the flow-through and reassemble the spin column and its collection tube;

[0045] (4) Binding nuclear RNA to the column

[0046] a) Add 400uL Buffer SK to the pellet (nuclear RNA fraction); vortex for 10 seconds;

[0047] b) Add 200u of anhydrous ethanol to the mixture; vortex mix for 10 seconds;

[0048] c) Add the mixture to a second spin column assembled with a collection tube and centrifuge at 3500 g or 6000 RPM for 1 minute;

[0049] d) discard the flow-through and reassemble the spin column and its collection tube;

[0050] (5) Column washing

[0051] a) Add 400 μL Wash Solution A to the spin column and centrifuge for 1 minute;

[0052] b) Discard the flow-through; repeat this step twice for a total of three washes;

[0053] c) Spin the column for 2 minutes to completely dry the resin; discard the collection tube;

[0054] (6) RNA elution

[0055] a) Place the spin column into a new 1.7 mL elution tube provided in the kit; add 50 uL Elution Buffer E to the spin column;

[0056] b) centrifugation at 200 x g or 2000 RPM for 2 minutes, then at 14000 g or 14000 RPM for 1 minute;

[0057] c) After the cytoplasmic and nuclear RNA extraction is completed, reverse transcription and RT-qPCR are performed to detect its distribution in the cytoplasm and nucleus;

[0058] 6.2) RNA FISH experiments

[0059] The circRNA FISH probe kit of Genema was used for

[0060] (1) According to 2×10 4 The adherent cells were seeded into 24-well plates at a density of cells / well and cultured in an incubator overnight;

[0061] (2) Aspirate the culture medium and wash twice with PBS, 5 min each time;

[0062] (3) Aspirate and discard PBS, add 200 μL of 4% paraformaldehyde to each well, and fix at room temperature for 15 min;

[0063] (4) Aspirate and discard 4% paraformaldehyde, add 200 μL 0.1% Buffer A to each well and treat the cells for 15 min at room temperature;

[0064] (5) Aspirate and discard 0.1% Buffer A, and wash twice with PBS, 5 min each time;

[0065] (6) Add 200 μL of 1× blocking solution to each well and incubate at 37°C for 30 min;

[0066] (7) Aspirate and discard the 1× blocking solution, add 200 μL of 2× Buffer C to each well, and place in a 37°C incubator for 30 min; incubate Buffer E in a 73°C water bath for 30 min until clear and translucent;

[0067] (8) Probe dilution: Add 41.7 μL of sterile DEPC water to each OD probe dry powder product, mix well to obtain a storage solution with a concentration of approximately 100 μM. It is recommended to store it at -20°C in the dark after aliquoting to avoid multiple freeze-thaw operations;

[0068] (9) Prepare the probe working solution: dilute the probe stock solution to 1 μM, place it in a 75°C water bath for denaturation for 10 min, and then add it to PBS in proportion with SA-Cy3;

[0069] (10) Mix the above 20 μL probe working solution and 180 μL Buffer E;

[0070] (11) Aspirate and discard 2× Buffer C, add 200 μL of denatured probe mixture to each well, and place in a 37°C incubator for hybridization overnight after taking measures to protect from light;

[0071] (12) On the second day of hybridization, remove the samples from the 37°C incubator, discard the probe mixture, add 200 μL of 0.1% Buffer F to each well and wash at 37°C for 10 min.

[0072] (13) Aspirate and discard 0.1% Buffer F, add 200 μL of 2× Buffer C to each well, wash at 60°C for 10 min, and wash three times;

[0073] (14) Aspirate and discard 2× Buffer C, add 200 μL of 2× Buffer C to each well, wash at 37°C for 10 min, and wash three times;

[0074] (15) Aspirate and discard 2× Buffer C, add 200 μL of diluted DAPI working solution to each well, and stain for 10-20 min in the dark;

[0075] (16) Aspirate and discard the DAPI working solution and wash twice with PBS, 5 min each time;

[0076] (17) Add an antifade agent, cover with a coverslip, and observe and photograph under a confocal microscope.

[0077] Further, in step 5.2), ActD was used to treat cells to inhibit the synthesis of intracellular RNA, so as to detect the half-life of circMKLN1 and its linear RNA, so as to verify whether circMKLN1 has higher stability; specifically:

[0078] (5.2.1) TNBC cells were seeded into 6-well culture plates, with the seeding volume per well being approximately 80,000 cells;

[0079] (5.2.2) On the second day, after the cells have adhered, add a certain volume of ActD to each well to make the drug working concentration 2 μg / mL;

[0080] (5.2.3) At 0 h, 6 h, 12 h, and 24 h after drug treatment, 300 μL of TRK lysis buffer was added to the cells and the cells were allowed to lyse for 5 min. The cell lysate was then transferred to a clean EP tube and temporarily stored in a -80 °C refrigerator for subsequent RNA extraction and RT-qPCR experiments to detect changes in the expression levels of circMKLN1 and its control linear RNA after ActD treatment for different periods of time.

[0081] The beneficial effects of the present invention are:

[0082] 1. circMKLN1 is transcribed from exons 4-7 of the MKLN1 gene and formed by reverse splicing and circularization, and its length is 535bp. After PCR amplification using primers of circMKLN1, it was sent to Sangon Biotech for Sanger sequencing, and the specific splicing site of circMKLN1 was found. Using cDNA and gDNA as templates, PCR amplification was performed using convergent primers and divergent primers of circMKLN1. The results showed that the divergent primers of circMKLN1 could only be amplified in cDNA, but not in gDNA, which further proved the existence of circMKLN1.

[0083] 2. RNase R and actinomycin D experiments showed that circMKLN1 had higher resistance to RNase R, and the half-life of circMKLN1 was much longer than that of linear MKLN1 mRNA. Nuclear-cytoplasmic separation experiments showed that circMKLN1 was mainly present in the cytoplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 This is the PCR amplification diagram of TNBC cell cDNA;

[0085] Figure 2 Schematic diagram of chromosome location and Sanger sequencing;

[0086] Figure 3 To detect circMKLN1 using convergent and divergent primers PCR;

[0087] Figure 4 This is a picture of the RNase R digestion experiment;

[0088] Figure 5 This is the experimental diagram of actinomycin D;

[0089] Figure 6 This is a diagram of the nuclear-cytoplasmic separation experiment;

[0090] Figure 7 This is a picture of RNA FISH experiment. 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] 1.2.5 Solution preparation

[0106] (1) 1×TAE buffer

[0107] Taking the preparation of 1000 ml 1×TAE buffer as an example, add 980 ml ultrapure water and 20 ml 50×TAE buffer into the beaker in sequence.

[0108] (2) 2 mg / ml actinomycin D

[0109] Dissolve actinomycin D in DMSO to prepare a 2 mg / ml stock solution, then dispense into sterile EP tubes and store at -20°C. When using, dilute actinomycin D with the complete cell culture medium at a ratio of 1:1000 to a working solution with a final concentration of 2 μg / ml, which is used to treat cells to inhibit transcriptional activity. Note that it needs to be prepared and used immediately.

[0110] 2 Experimental methods

[0111] 2.1 Cell-related experiments

[0112] 2.1.1 Cell culture

[0113] The human breast cancer cell line MDA-MB-436 was cultured in DMEM high glucose medium (10% fetal bovine serum and 1% penicillin-streptomycin solution were added before use to prepare a complete medium). The human breast cancer cell line BT-549 was cultured in RPMI 1640 medium (10% fetal bovine serum and 1% penicillin-streptomycin solution were added before use to prepare a complete medium). All cells were cultured in a humidified cell incubator at 37°C and 5% CO2. The entire cell culture operation must follow the principle of sterility.

[0114] 2.1.2 Cell passaging

[0115] When the cells grew to a density of 85%-90%, the cells were digested and passaged.

[0116] (1) Use a pipette to discard the original culture medium in the culture bottle, then tilt the culture bottle slightly and slowly add an appropriate amount of sterile 1X PBS along the side wall, and gently shake the culture bottle to wash the cells. Wash 3 times in total.

[0117] (2) Use a pipette to discard 1xPBS, then add 1ml of trypsin digestion solution to the culture flask, and gently shake the culture flask to evenly cover the bottom of the culture flask. The digested cells can be briefly placed in a cell culture incubator until the cells shrink and the distance between them increases under the microscope, and some cells are spherical. At this time, add 1ml of complete culture medium to terminate the digestion.

[0118] (3) Use a pipette to repeatedly and gently blow the bottom of the culture flask until the attached cells are completely detached. Transfer the cell suspension to a sterile 15 ml centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature.

[0119] (4) Discard the supernatant, add 1 ml of new complete culture medium and gently pipette the cell pellet to resuspend the cells. Transfer an appropriate amount of cell suspension to a new culture flask that has been pre-added with 5 ml of new culture medium according to experimental needs.

[0120] (5) Tighten the cap of the culture flask, shake the flask gently using the cross method to evenly distribute the cells, mark the cell type and subculture date, and return it to the cell culture incubator.

[0121] 2.1.3 Cell cryopreservation

[0122] (1) Prepare cell freezing solution. Calculate the total volume of freezing solution required based on the amount of 1 ml of cell freezing solution per tube of cells. Make sure to prepare and use it immediately.

[0123] (2) Take out the required number of cell cryopreservation tubes and fill in the relevant information on the tube wall (including cell name, special treatment, generation, cryopreservation person and time, etc.).

[0124] (3) When the cells grow to 85%-90% density, digest the cells and centrifuge them (see 2.1.2 for specific steps).

[0125] (4) Use a pipette to aspirate and discard the supernatant, add an appropriate amount of freezing solution, and gently pipette to form a uniform cell suspension. Add 1 ml of cell suspension to each cell freezing tube.

[0126] 2.1.4 Cell recovery

[0127] (1) First, heat the water bath to 37°C, then remove the cells from the cryopreservation environment. Place the removed cell cryopreservation tube into a PE glove and immerse it in the prepared water bath. Gently shake the PE glove to allow the cell cryopreservation tube to be heated and melted more evenly.

[0128] (2) After the cryopreservation solution in the cryopreservation tube has completely melted into liquid, take it out of the PE gloves, disinfect it with an alcohol cotton ball, and open the cryopreservation tube in the biosafety cabinet.

[0129] (3) Transfer the cell suspension in the cryopreservation tube into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature.

[0130] (4) After centrifugation, discard the supernatant in the centrifuge tube and keep the cell pellet at the bottom. Then add 1 mL of culture medium to resuspend the cell pellet and mix it by pipetting. Then spread the cells evenly into a culture bottle that has been added with 5 mL of new culture medium in advance and place it in an incubator for overnight culture.

[0131] (5) On the next day, observe the survival of the cells and change the medium.

[0132] 2.2 Cell RNA extraction, reverse transcription and real-time fluorescence quantitative PCR

[0133] The primer sequences involved in the RT-qPCR experiment are as follows:

[0134]

[0135]

[0136] 2.3 PCR amplification and agarose gel electrophoresis

[0137] 2.3.1 PCR amplification

[0138] 1. Use cDNA and gDNA of MDA-MB-436 and BT-549 as templates. The required divergent primer (DP) and convergent primer (CP) sequences are shown in the table below.

[0139]

[0140] 2. Reaction system:

[0141]

[0142] 3. PCR amplification conditions:

[0143]

[0144] 2.3.2 Agarose gel electrophoresis

[0145] Place the agarose precast gel in the electrophoresis tank, then add the appropriate amount of 1×TAE buffer to submerge the precast gel. Add 5ul of 100bp DNA Marker to the first loading well of the precast gel, set the voltage to 150V, and start electrophoresis. Stop electrophoresis when the indicator reaches about 1cm from the bottom of the gel. Use a gel imager to observe the band position of the amplified product.

[0146] 2.4 RNase R and actinomycin D experiments

[0147] 2.4.1 RNase R experiment

[0148] (1) To verify that circMKLN1 is insensitive to the exonuclease RNase R, we extracted total RNA from TNBC cells and divided the RNA into an experimental group and a control group to prepare the reaction system. The experimental group was added with 0.1 μL RNase R.

[0149] (2) Place the two groups into a PCR instrument and set the program to 37°C for 10 min and 70°C for 10 min.

[0150] (3) Reverse transcription and RT-qPCR experiments were performed to detect changes in RNA levels.

[0151] The RNase R reaction system is as follows:

[0152]

[0153] 2.4.2 Actinomycin D (Act D) experiment

[0154] ActD was used to treat cells to inhibit the synthesis of intracellular RNA to detect the half-life of circMKLN1 and its linear RNA to verify whether circMKLN1 has higher stability.

[0155] (1) TNBC cells were seeded into a 6-well culture plate, with the seeding volume per well being approximately 80,000 cells.

[0156] (2) On the second day, after the cells adhered, a certain volume of ActD was added to each well to make the working concentration of the drug 2 μg / mL.

[0157] (3) At 0 h, 6 h, 12 h, and 24 h after drug treatment, 300 μL of TRK lysis buffer was added to the cells and the cells were allowed to lyse for 5 min. The cell lysate was then transferred to a clean EP tube and temporarily stored in a -80 °C refrigerator for subsequent RNA extraction and RT-qPCR experiments to detect changes in the expression levels of circMKLN1 and its control linear RNA after ActD treatment for different periods of time.

[0158] 2.5 Verification of circMKLN1 subcellular localization

[0159] 2.5.1 Nuclear-cytoplasmic separation experiment

[0160] This experiment was performed using the cytoplasmic and nuclear RNA extraction kit from Aimigenes Technologies.

[0161] (1) Preparation:

[0162] a) Prepare an appropriate amount of Lysis Buffer J by adding 10 μL of β-mercaptoethanol per 1 ml of required Lysis Buffer J. Keep the solution on ice or at 4°C before use.

[0163] b) Prepare an appropriate amount of Buffer SK by adding 10 μL of β-mercaptoethanol to 1 mL of the required Buffer SK.

[0164] c) Prepare a working concentration of Wash Solution A by adding 90 mL of absolute ethanol to each bottle containing Wash Solution A. The final volume is 128 mL.

[0165] d) Before you begin, make sure to place Lysis Buffer J in a -20°C freezer for 10 to 15 minutes and remove it before use.

[0166] (2) Preparation of cell components

[0167] a) Transfer the cell suspension to an RNase-free tube and centrifuge at no more than 200 x g or 2000 RPM for 10 minutes to pellet the cells.

[0168] b) Carefully pour off the supernatant. To ensure that the pellet does not fall off, a few microliters of medium may remain in the pellet.

[0169] c) Add 200uL of ice-cold Lysis Buffer J to the pellet.

[0170] d) Lyse the cells by vortexing for 15 seconds. Make sure the entire pellet is completely dissolved before proceeding to the next step. Use a pipette to transfer the lysate to an RNase-free microcentrifuge tube.

[0171] e) Spin the lysate at maximum speed in a benchtop centrifuge for 10 minutes.

[0172] f) Using a pipette with a small tip, transfer the supernatant containing the cytoplasmic RNA to another RNase-free tube.

[0173] (3) Binding cytoplasmic RNA to the column

[0174] a) Add 200uL Buffer SK to the supernatant (cytoplasmic RNA fraction) from the previous step. Vortex to mix for 10 seconds.

[0175] b) Add 200uL 96-100% ethanol to the mixture. Vortex for 10 seconds.

[0176] c) Add the mixture to a spin column assembled with a collection tube and centrifuge at 3500 g or 6000 RPM for 1 minute.

[0177] d) Discard the flow-through and reassemble the spin column and collection tube.

[0178] (4) Binding nuclear RNA to the column

[0179] a) Add 400uL Buffer SK to the pellet (nuclear RNA fraction). Vortex for 10 seconds.

[0180] b) Add 200 u of anhydrous ethanol to the mixture and vortex for 10 seconds.

[0181] c) Add the mixture to a second spin column assembled with a collection tube and centrifuge at 3500 g or 6000 RPM for 1 minute.

[0182] d) Discard the flow-through and reassemble the spin column and collection tube.

[0183] (5) Column washing

[0184] a) Add 400 μL Wash Solution A to the spin column and centrifuge for 1 minute.

[0185] b) Discard the flow-through. Repeat this step two more times for a total of three washes.

[0186] c) Spin the column for 2 minutes to completely dry the resin. Discard the collection tube.

[0187] (6) RNA elution

[0188] a) Place the spin column into a new 1.7 mL elution tube provided in the kit. Add 50 uL Elution Buffer E to the spin column.

[0189] b) Centrifuge at 200 x g or 2000 RPM for 2 minutes, then at 14000 g or 14000 RPM for 1 minute.

[0190] c) After the cytoplasmic and nuclear RNA extraction was completed, reverse transcription and RT-qPCR were performed to detect the distribution of the cytoplasmic and nuclear RNA.

[0191] 2.5.2 RNA FISH experiments

[0192] This experiment was performed using the circRNA FISH probe kit from Jima Gene.

[0193] (1) According to 2×10 4 The adherent cells were seeded into a 24-well plate at a density of 10 cells / well (the wells were pre-placed with processed cell slides of appropriate size) and cultured in an incubator overnight.

[0194] (2) Aspirate the culture medium and wash twice with PBS, 5 min each time.

[0195] (3) Aspirate and discard PBS, add 200 μL of 4% paraformaldehyde to each well, and fix at room temperature for 15 min.

[0196] (4) Aspirate and discard 4% paraformaldehyde, add 200 μL 0.1% Buffer A (prepared immediately before use) to each well and treat the cells for 15 min at room temperature (cell permeabilization).

[0197] (5) Aspirate and discard 0.1% Buffer A, and wash twice with PBS, 5 min each time.

[0198] (6) Add 200 μL of 1× blocking solution to each well and incubate at 37°C for 30 min.

[0199] (7) Aspirate and discard the 1× blocking solution, add 200 μL of 2× Buffer C to each well, and place in a 37°C incubator for 30 min; incubate Buffer E in a 73°C water bath for 30 min until it becomes clear and translucent.

[0200] (8) Probe dilution: Add 41.7 μL of sterile DEPC water to each OD probe dry powder product and mix well to obtain a storage solution with a concentration of approximately 100 μM. It is recommended to store it in aliquots at -20°C in the dark to avoid multiple freeze-thaw operations.

[0201] (9) Prepare the probe working solution: dilute the probe stock solution to 1 μM, place it in a 75°C water bath for denaturation for 10 min, then add it to PBS in proportion with SA-Cy3 (for example, a 20 μL system, i.e., 8 μL 1 μM biotin-probe + 2 μL 1 μM SA-Cy3 + 10 μL PBS), and incubate at 37°C for 30 min.

[0202] (10) Mix the above 20 μL probe working solution and 180 μL Buffer E.

[0203] (11) Aspirate and discard 2× Buffer C, add 200 μL of denatured probe mixture to each well, and place in a 37°C incubator for hybridization overnight (12-16 h) after protecting from light.

[0204] (12) On the day after hybridization, remove the samples from the 37°C incubator, discard the probe mixture, add 200 μL of 0.1% Buffer F to each well and wash at 37°C for 10 min.

[0205] (13) Aspirate and discard 0.1% Buffer F, add 200 μL of 2× Buffer C to each well, and wash at 60°C for 10 min. Wash three times.

[0206] (14) Aspirate and discard 2× Buffer C. Add 200 μL of 2× Buffer C to each well and wash at 37°C for 10 min. Wash three times.

[0207] (15) Aspirate and discard 2× Buffer C, add 200 μL of diluted DAPI working solution to each well, and stain for 10-20 min in the dark.

[0208] (16) Aspirate and discard the DAPI working solution and wash twice with PBS, 5 min each time.

[0209] (17) Add an antifade agent, cover with a coverslip, and observe and photograph under a confocal microscope.

[0210] 3 Experimental results

[0211] 3.1 Sanger sequencing confirmed the existence of circMKLN1

[0212] To verify the genomic origin of circMKLN1, PCR was performed using cDNA of TNBC cells as template and specific primers of circMKLN1. The PCR products were subjected to agarose gel electrophoresis ( Figure 1 ) and Sanger sequencing ( Figure 2 ), by combining the circBase database and sequencing results, the genomic origin and reverse splicing site of circMKLN1 were confirmed. As shown in the figure, circMKLN1 is transcribed and reversely spliced ​​from exons 4-7 of the MKLN1 gene, and its length is 535bp.

[0213] Since circMKLN1 is transcribed from the MKLN1 gene and reversely cyclized, the divergent primers of circMKLN1 should be able to be amplified in cDNA but not in gDNA, while the convergent primers of circMKLN1 should be able to be amplified in both. Therefore, cDNA and gDNA of TNBC cells were extracted, and PCR amplification and agarose gel electrophoresis experiments were performed using convergent primers and divergent primers, respectively. As shown in the figure ( Figure 3 ), the results were consistent with the expected results, verifying the existence of circMKLN1.

[0214] 3.2circMKLN1 has high stability and long half-life

[0215] CircRNA is an RNA molecule with a circular structure formed by covalently linking the 5' and 3' ends. Therefore, compared with linear RNA transcripts, it has the characteristics of high stability and long half-life. In order to explore whether circMKLN1 also has these circRNA characteristics, the extracted total RNA samples were first digested with RNaseR to detect the stability of RNA. The RNaseR experiment showed ( Figure 4), compared with the linear transcript, circMKLN1 has a higher resistance to RNase R exonuclease, confirming the stability of circMKLN1. Next, TNBC cells were treated with actinomycin D to detect the half-life of circMKLN1 in cells by inhibiting the synthesis of mRNA. The results showed that ( Figure 5 ), the half-life of circMKLN1 was significantly longer than that of the linear transcript, indicating that circMKLN1 does have the characteristics of higher stability and longer half-life.

[0216] 3.3circMKLN1 is mainly present in the cytoplasm

[0217] Since the function of circRNA is closely related to its intracellular sublocalization, the sublocalization of circMKLN1 in tumor cells was detected and analyzed. First, the cytoplasmic RNA and nuclear RNA of TNBC cells were extracted for nuclear-cytoplasmic separation experiments, and the distribution of circMKLN1 in different components was detected by RT-qPCR. The results are shown in the figure ( Figure 6 ), circMKLN1 was expressed at a higher level in the cytoplasm. We then performed RNA FISH experiments, and the results were consistent with the above. circMKLN1 was mainly present in the cytoplasm ( Figure 7 ).

[0218] 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. A method for screening and validating circRNA circMKLN1 that regulates triple-negative breast cancer, characterized in that: The method comprises the following steps: Cell culture The human breast cancer cell line MDA-MB-436 was cultured in DMEM high glucose medium; the human breast cancer cell line BT-549 was cultured in RPMI 1640 medium; all cells were cultured in a humidified cell incubator at 37°C and 5% CO2; 2) Extraction, reverse transcription and real-time fluorescence quantitative PCR of cell RNA; 3) PCR amplification and agarose gel electrophoresis 3.1) PCR amplification The cDNA and gDNA of MDA-MB-436 and BT-549 were used as templates; the required divergent primer (DP) and convergent primer (CP) sequences are shown in the following table: 3.2) Agarose gel electrophoresis Place the agarose precast gel into the electrophoresis tank, then add the corresponding amount of 1×TAE buffer to immerse the precast gel; add 5ul 100bp DNA Marker to the first loading hole of the precast gel, set the voltage to 150V, and start electrophoresis; stop electrophoresis when the indicator reaches about 1cm from the bottom of the gel; use a gel imager to observe the band position of the amplified product; 4) Sanger sequencing Cut the gel at the target product band under a gel imager, and then extract the DNA of the target product in the gel; The eluted DNA was sent to Sangon Biotech for Sanger sequencing; 5) RNase R digestion and actinomycin D experiments 5.1) RNase R digestion experiment (1) To verify that circMKLN1 is insensitive to the exonuclease RNase R, total RNA of TNBC cells was extracted and divided into an experimental group and a control group to prepare the reaction system. 0.1 μL RNase R was added to the experimental group. (2) Place both groups in a PCR instrument and set the program to 37°C for 10 min and 70°C for 10 min; (3) reverse transcription and RT-qPCR experiments were performed to detect changes in RNA levels; 5.2) Actinomycin D experiment ActD was used to treat cells to inhibit the synthesis of intracellular RNA to detect the half-life of circMKLN1 and its linear RNA to verify whether circMKLN1 has higher stability; 6) Verification of subcellular localization of circMKLN1 6.1) Nuclear-cytoplasmic separation experiment Cytoplasmic and nuclear RNA extraction kit (1) Preparation: (a) Prepare an appropriate amount of Lysis Buffer J by adding 10 μL of β-mercaptoethanol to every 1 mL of Lysis Buffer J required; keep the solution on ice or at 4°C before use. (b) Prepare an appropriate amount of Buffer SK by adding 10 μL of β-mercaptoethanol to 1 mL of the required Buffer SK; (c) Prepare a working concentration of Wash Solution A by adding 90 mL of anhydrous ethanol to each bottle containing Wash Solution A; the final volume is 128 mL. (d) Before starting, make sure to place Lysis Buffer J in a -20°C refrigerator for 10 to 15 minutes and remove it before use. (2) Preparation of cell components a) Transfer the cell suspension to an RNase-free tube and centrifuge at no more than 200 x g or 2000 RPM for 10 minutes to pellet the cells; b) Carefully pour off the supernatant. To ensure that the precipitate does not fall off, a few microliters of culture medium may remain in the precipitate. c) Add 200uL of ice-cold Lysis Buffer J to the precipitate; d) Lyse cells by vortexing for 15 seconds; make sure the entire pellet is completely dissolved before proceeding to the next step; use a pipette to transfer the lysate to an RNase-free microcentrifuge tube; e) Spin the lysate at maximum speed in a benchtop centrifuge for 10 minutes; f) Using a pipette with a small tip, transfer the supernatant containing the cytoplasmic RNA to another RNase-free tube; (3) Binding cytoplasmic RNA to the column a) Add 200uL Buffer SK to the supernatant (cytoplasmic RNA fraction) from the previous step; vortex to mix for 10 seconds; b) Add 200uL 96-100% ethanol to the mixture; vortex mix for 10 seconds; c) Add the mixture to a spin column assembled with a collection tube and centrifuge at 3500 g or 6000 RPM for 1 minute; d) discard the flow-through and reassemble the spin column and its collection tube; (4) Binding nuclear RNA to the column a) Add 400uL Buffer SK to the pellet (nuclear RNA fraction); vortex for 10 seconds; b) Add 200u of anhydrous ethanol to the mixture; vortex mix for 10 seconds; c) Add the mixture to a second spin column assembled with a collection tube and centrifuge at 3500 g or 6000 RPM for 1 minute; d) discard the flow-through and reassemble the spin column and its collection tube; (5) Column washing a) Add 400 μL Wash Solution A to the spin column and centrifuge for 1 minute; b) Discard the flow-through. Repeat this step twice for a total of three washes; c) Spin the column for 2 minutes to completely dry the resin; discard the collection tube; (6) RNA elution a) Place the spin column into a new 1.7 mL elution tube provided in the kit; add 50 uL Elution Buffer E to the spin column; b) centrifugation at 200 x g or 2000 RPM for 2 minutes, then at 14000 g or 14000 RPM for 1 minute; c) After the cytoplasmic and nuclear RNA extraction is completed, reverse transcription and RT-qPCR are performed to detect its distribution in the cytoplasm and nucleus; 6.2) RNA FISH experiments The circRNA FISH probe kit of Genema was used for (1) According to 2×10 4 The adherent cells were seeded into 24-well plates at a density of cells / well and cultured in an incubator overnight; (2) Aspirate the culture medium and wash twice with PBS, 5 min each time; (3) Aspirate and discard PBS, add 200 μL of 4% paraformaldehyde to each well, and fix at room temperature for 15 min; (4) Aspirate and discard 4% paraformaldehyde, add 200 μL 0.1% Buffer A to each well and treat the cells for 15 min at room temperature; (5) Aspirate and discard 0.1% Buffer A, and wash twice with PBS, 5 min each time; (6) Add 200 μL of 1× blocking solution to each well and incubate at 37°C for 30 min; (7) Aspirate and discard the 1× blocking solution, add 200 μL of 2× Buffer C to each well, and place in a 37°C incubator for 30 min; incubate Buffer E in a 73°C water bath for 30 min until clear and translucent; (8) Probe dilution: Add 41.7 μL of sterile DEPC water to each OD probe dry powder product, mix well to obtain a storage solution with a concentration of approximately 100 μM, divide and store in a dark place at -20°C to avoid multiple freeze-thaw operations; (9) Prepare the probe working solution: dilute the probe stock solution to 1 μM, place it in a 75°C water bath for denaturation for 10 min, and then add it to PBS in proportion with SA-Cy3; (10) Mix the above 20 μL probe working solution and 180 μL Buffer E; (11) Aspirate and discard 2× Buffer C, add 200 μL of denatured probe mixture to each well, and place in a 37°C incubator for hybridization overnight after taking measures to protect from light; (12) On the second day of hybridization, remove the samples from the 37°C incubator, discard the probe mixture, add 200 μL of 0.1% Buffer F to each well and wash at 37°C for 10 min. (13) Aspirate and discard 0.1% Buffer F, add 200 μL of 2× Buffer C to each well, wash at 60°C for 10 min, and wash three times; (14) Aspirate and discard 2× Buffer C, add 200 μL of 2× Buffer C to each well, wash at 37°C for 10 min, and wash three times; (15) Aspirate and discard 2× Buffer C, add 200 μL of diluted DAPI working solution to each well, and stain for 10-20 min in the dark; (16) Aspirate and discard the DAPI working solution and wash twice with PBS, 5 min each time; (17) Add an antifade agent, cover with a coverslip, and observe and photograph under a confocal microscope.

2. The screening and verification method for circMKLN1 circular RNA capable of regulating triple-negative breast cancer according to claim 1, characterized in that: In step 5.2), ActD was used to treat cells to inhibit the synthesis of intracellular RNA, so as to detect the half-life of circMKLN1 and its linear RNA, and to verify whether circMKLN1 has higher stability; specifically: (5.2.1) TNBC cells were seeded into 6-well culture plates, with the seeding volume per well being approximately 80,000 cells; (5.2.2) On the second day, after the cells have adhered, add a certain volume of ActD to each well to make the drug working concentration 2 μg / mL; (5.2.3) At 0 h, 6 h, 12 h, and 24 h after drug treatment, 300 μL of TRK lysis buffer was added to the cells and the cells were allowed to lyse for 5 min. The cell lysate was then transferred to a clean EP tube and temporarily stored in a -80 °C refrigerator for subsequent RNA extraction and RT-qPCR experiments to detect changes in the expression levels of circMKLN1 and its control linear RNA after ActD treatment for different periods of time.

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