Method for researching influence of ACSL5 on breast cancer cells and influence mechanism of ACSL5

Abstract

The invention discloses a method for researching the influence of ACSL5 on breast cancer cells and an influence mechanism of ACSL5, and relates to the technical field of breast cancer cell research. Comprising the following steps of: culturing and treating cells; preparing a culture medium; preparing a DMEM (Dulbecco Modified Eagle Medium) high-glucose culture medium containing 10% of fetal calf serum and 1% of penicillin-streptomycin, and ensuring a sterile condition; inoculating and adapting cells: inoculating the breast cancer cell line into a sterile culture bottle or a culture plate, and putting the sterile culture bottle or the culture plate into an incubator with the temperature of 37 DEG C and the CO2 content of 5% to adapt to growth; cell observation and maintenance: regularly observing the morphology and growth state of the cells by using a microscope, and replacing a fresh culture solution if necessary. By deeply researching the action mechanism of ACSL5 in breast cancer, a new therapeutic target can be found, and a theoretical basis is provided for developing a new therapeutic strategy.

Description

Technical Field

[0001] The present invention relates to the technical field of breast cancer cell research, and in particular to a research method based on the influence of ACSL5 on breast cancer cells and its influence mechanism. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women worldwide. Its pathogenesis is complex and involves multiple genetic and environmental factors. Long-chain acyl-CoA synthetase 5 (ACSL5) plays a key role in regulating lipid metabolism. Recent studies have found that the expression of ACSL5 in breast cancer is associated with patient prognosis, suggesting that it may play an important role in tumor biology. Therefore, in-depth research on the mechanism of action of ACSL5 in breast cancer is expected to provide new strategies for the diagnosis and treatment of breast cancer.

[0003] Existing technologies often only focus on one aspect of ACSL5, such as its effect on fatty acid synthesis, but do not comprehensively study the role of ACSL5 in breast cancer. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a research method based on the effect of ACSL5 on breast cancer cells and its effect mechanism.

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

[0006] A research method based on the effect of ACSL5 on breast cancer cells, comprising:

[0007] S1: Cell culture and treatment:

[0008] Medium preparation: Prepare DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, ensuring sterile conditions;

[0009] Cell seeding and adaptation: Breast cancer cell lines were seeded in sterile culture flasks or culture plates and placed in an incubator at 37°C and 5% CO2 to adapt to growth;

[0010] Cell observation and maintenance: Regularly observe cell morphology and growth status using a microscope, and replace with fresh culture medium when necessary;

[0011] Cell passaging: When the cells reach 80% density, gently wash and digest the cells with trypsin solution and pass them to new culture vessels;

[0012] S2: ACSL5 gene manipulation:

[0013] Lentiviral vector construction: design shRNA or overexpression sequence according to the target sequence and clone it into the lentiviral vector;

[0014] Lentiviral packaging: The constructed lentiviral vector is transfected into packaging cells to produce infectious lentiviral particles;

[0015] Cell infection experiment: Under optimized conditions, breast cancer cells were infected with lentivirus to achieve knockdown or overexpression of ACSL5;

[0016] Stable cell line screening: Screening of cell lines stably expressing ACSL5 gene modification by puromycin or other selective markers;

[0017] S3: RNA extraction and RT-qPCR analysis:

[0018] Total RNA extraction: Total RNA was extracted from cells using an RNA rapid extraction kit according to the manufacturer's protocol, and its purity and integrity were assessed;

[0019] cDNA synthesis: Use a reverse transcription kit to reverse transcribe the extracted total RNA into cDNA, preferably a reagent with high reverse transcription efficiency;

[0020] Primer design: Design highly specific RT-qPCR primers for ACSL5 and other related genes;

[0021] RT-qPCR reaction: amplify in a real-time fluorescence quantitative PCR instrument, monitor the fluorescence signal, and analyze the mRNA expression level;

[0022] S4: Protein expression analysis:

[0023] Total protein extraction: Cellular proteins were collected using lysis buffer containing protease inhibitors;

[0024] Protein concentration determination: Use Bradford or other protein quantification methods to accurately measure protein concentration;

[0025] Western Blot analysis: Proteins were separated by SDS-PAGE, transferred to PVDF membrane, and then the expression of ACSL5 and other related proteins was detected using specific antibodies;

[0026] Image analysis: The immunoblot results were digitally scanned and quantitatively analyzed using image analysis software;

[0027] S5: Functional experimental verification:

[0028] CCK-8 experiment: evaluate the effect of ACSL5 on the proliferation ability of breast cancer cells;

[0029] Scratch healing assay: evaluate the effect of ACSL5 on the migration ability of breast cancer cells;

[0030] Transwell assay: evaluate the effect of ACSL5 on the invasion ability of breast cancer cells;

[0031] Nile red staining: analyze the effect of ACSL5 on the formation of intracellular fatty acid lipid droplets;

[0032] Metabolite detection: FFA and TG detection kits were used to evaluate the effect of ACSL5 on fatty acid synthesis;

[0033] Specifically:

[0034] CCK-8 proliferation assay: The treated cells were inoculated into a 96-well plate. After a certain period of culture, CCK-8 reagent was added and the OD value was measured to evaluate the cell proliferation ability;

[0035] Scratch healing migration assay: make scratches when cells have grown into a monolayer, take pictures at different time points, and compare the speed of scratch closure;

[0036] Transwell invasion assay: Matrigel is placed on the bottom of the transwell chamber to simulate the extracellular matrix and examine the ability of cells to penetrate the matrix;

[0037] Nile red staining experiment: The cells were stained with Nile red, and the accumulation of intracellular lipid droplets was observed and quantified using a fluorescence microscope;

[0038] FFA and TG determination: Free fatty acid test kit and triglyceride test kit were used to determine the intracellular FFA and TG content respectively;

[0039] S6: Bioinformatics analysis:

[0040] Expression data analysis: The expression difference of ACSL5 in breast cancer tissues and normal tissues was analyzed by online tools such as GEPIA2;

[0041] Survival analysis: Kaplan-Meier Plotter was used to analyze the relationship between ACSL5 expression level and patient survival;

[0042] Pathway enrichment analysis: With the help of KEGG database, ACSL5-related signaling pathways were enriched to find potential regulatory networks;

[0043] S7: Statistical processing:

[0044] Data collation: Summarize and organize all experimental data in preparation for subsequent statistical analysis;

[0045] Statistical analysis: GraphPad Prism 8.0 software was used to perform t-test or one-way analysis of variance on the data set to determine whether there were significant differences among the experimental groups. P < 0.05 indicated statistical significance.

[0046] The beneficial effects of the present invention are:

[0047] 1. The present invention can discover new therapeutic targets by deeply studying the mechanism of action of ACSL5 in breast cancer, and provide a theoretical basis for developing new treatment strategies.

[0048] 2. The present invention can promote the application of precision medicine in the treatment of breast cancer by conducting a detailed study on the specific role and mechanism of ACSL5 in different breast cancer subtypes; selecting the most appropriate treatment method based on the patient's specific molecular characteristics can improve treatment efficiency and reduce unnecessary side effects.

[0049] 3. The present invention not only involves research at the molecular level, but also includes cell function experiments and bioinformatics analysis. This multi-angle research method can fully interpret the role of ACSL5 in breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 A schematic diagram of the GEPIA2 database analysis and the correlation between the expression level of ACSL5 and the breast cancer stage in a research method based on the effect of ACSL5 on breast cancer cells proposed by the present invention;

[0051] Figure 2 A schematic diagram of Kaplan-Meier survival analysis of a breast cancer data set in a research method based on the effect of ACSL5 on breast cancer cells proposed in the present invention;

[0052] Figure 3 A schematic diagram of TCGA database analysis and ACSL5 expression in breast cancer cell lines in a research method based on the effect of ACSL5 on breast cancer cells proposed in the present invention;

[0053] Figure 4 A schematic diagram of a lentiviral infection and CCK-8 experiment in a research method based on the effect of ACSL5 on breast cancer cells proposed in the present invention;

[0054] Figure 5 Schematic diagram of a scratch healing experiment and a transwell experiment in a research method based on the effect of ACSL5 on breast cancer cells proposed in the present invention;

[0055] Figure 6 A relationship diagram of the expression of key enzyme genes for de novo fatty acid synthesis in a research method based on the effect of ACSL5 on breast cancer cells proposed by the present invention;

[0056] Figure 7This is a schematic diagram of a research method proposed by the present invention based on the effect of ACSL5 on breast cancer cells, in which ACSL5 inhibits the expression of mRNA of a key enzyme in de novo fatty acid synthesis. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0058] Embodiment 1:

[0059] A research method based on the effect of ACSL5 on breast cancer cells, comprising:

[0060] S1: Cell culture;

[0061] The cells were cultured in a DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2 in an incubator. When the cell density reached about 80%, they were digested with trypsin and then passaged.

[0062] S2: infection and drug screening;

[0063] Lentivirus infection: cells were inoculated in a 24-well plate. When the cell density reached 60%, transfection reagent and lentivirus were added according to the instructions. The medium was changed after 8 hours. After observing the infection under a fluorescence microscope, puromycin was used to select stably infected cell lines.

[0064] S3: RNA extraction and RT-qPCR;

[0065] Total RNA was extracted from cells using an RNA rapid extraction kit, and the total RNA concentration was determined using NanoDropLite (Thermo Fisher Scientific, 200c). 1 μg of target RNA was taken for reverse transcription, and the RNA was reverse transcribed into cDNA using a reverse transcription kit. (Reverse transcription procedure: step 1: 42°C 2min; step 2: 37°C 15min, 85°C 5s, 4°C∞), and cDNA was amplified using a real-time fluorescence quantitative PCR instrument CFXConnectTM (Bio-Rad, 1855200) to detect the expression level of the target mRNA, with β-actin as the internal reference.

[0066] S4: Protein expression analysis;

[0067] Collect and lyse cells to obtain total protein, and use the NanoDropLite instrument to detect protein concentration. Add buffer according to the volume ratio of protein volume: SDS-PAGE protein loading buffer (5X) = 4:1 (the above steps are placed on ice throughout the process), boil in boiling water for 10 minutes to denature, and finally perform Western blot experiments with 25μg protein loading amount. Load the sample for SDS-PAGE, and transfer to PVDF membrane with a current of 210mA after electrophoresis. Block with 5% skim milk powder for 2h, add the corresponding primary antibody and incubate overnight at 4℃. After washing with TBST, incubate with HRP-labeled secondary antibody at room temperature for 1h, wash the membrane and develop it with a developer, and analyze the results with ImageJ (β-actin is the internal reference).

[0068] S5: Functional experimental verification;

[0069] Transwell experiment:

[0070] The chamber was placed in a 24-plate, and the cells were digested with trypsin, resuspended in serum-free medium, and 600 μl of complete medium was added to the lower chamber of each chamber. In the migration experiment, cells were added to the upper chamber at a cell suspension of 4000 cells / 350 μl, and placed in an incubator for 24 hours (in the invasion experiment, 30 μl of diluted matrix gel was added to the bottom of the chamber 1 hour in advance and placed at 37°C), fixed with 4% paraformaldehyde for 15 minutes, stained with crystal violet for 15 minutes, washed, dried naturally, and photographed with an inverted microscope. At least 3 fields of view were randomly selected and counted for statistical analysis.

[0071] Scratch healing assay:

[0072] The cells were inoculated into a 6-well cell culture plate. After the cells were fully grown, a cross was drawn in the middle of each well with a 10 μL pipette. The floating cells were washed with phosphate-buffered saline (PBS) to remove them. A culture medium without penicillin and streptomycin was added. Pictures were taken under an optical microscope and counted as 0 h. After 24 h, pictures were taken at the same position. Each group of experiments was repeated 3 times.

[0073] Nile red staining experiment:

[0074] Nile red staining Place the slide in a 24-well plate, inoculate the cells in the well plate, and place it in an incubator overnight. The next day, take out the 24-well plate, wash it three times with PBS, five minutes each time, and stain it with Nile red working solution for 10 minutes. Wash it three more times with PBS, five minutes each time, and observe and take pictures under a confocal microscope.

[0075] RNA sequencing Three samples of each control and stable overexpression ACSL5 cell line were taken, RNA was extracted, and transcriptome sequencing was performed.

[0076] S6: Statistical analysis;

[0077] The experimental results were statistically analyzed using GraphPad Prism 8.0 software. All experiments were repeated three times independently. The Student's t test was used for pairwise comparison within the group, and one-way analysis of variance was used for comparison of means among multiple groups. P < 0.05 was considered statistically significant.

[0078] like Figure 1-3 As shown, the mechanism of ACSL5's effect on breast cancer cells:

[0079] ACSL5 is lowly expressed in breast cancer and its expression is correlated with patient prognosis:

[0080] A: The GEPIA2 database analyzed the expression of ACSL5 in breast cancer. The results showed that the expression of ACSL5 in breast cancer tissue was significantly lower than that in adjacent adjacent tissues.

[0081] B: The expression level of ACSL5 is correlated with breast cancer stage.

[0082] CD: Kaplan-Meier survival analysis of breast cancer datasets revealed that breast cancer patients with high ACSL5 expression had longer overall survival and recurrence-free survival.

[0083] E: Analysis of the TCGA database showed that patients with high ACSL5 expression had a longer overall survival.

[0084] F: Compared with normal breast epithelial cells MCF-10A, ACSL5 expression is low in breast cancer cell lines.

[0085] like Figure 4 As shown, ACSL5 inhibits breast cancer progression:

[0086] AB. ACSL5 was stably knocked down and overexpressed in MDA-MB-231 and MCF-7 cell lines by lentiviral infection, respectively. RT-qPCR and western blot experiments confirmed successful infection.

[0087] CD.CCK-8 experiments showed that ACSL5 could inhibit the proliferation of breast cancer.

[0088] E. The scratch healing experiment showed that ACSL5 could significantly inhibit the lateral migration ability of breast cancer cells.

[0089] F. The results of transwell experiments showed that ACSL5 could significantly inhibit the invasion and longitudinal migration ability of breast cancer cells.

[0090] ACSL5 affects lipid metabolism in breast cancer:

[0091] Transcriptome sequencing analysis revealed that fatty acid synthesis and degradation pathways were significantly enriched.

[0092] Nile red staining experiments showed that ACSL5 could significantly inhibit the production of cellular fatty acid lipid droplets.

[0093] The FFA kit test showed that ACSL5 could significantly inhibit the production of FFA in breast cancer cells.

[0094] The TG detection kit showed that ACSL5 can significantly reduce the production of triglycerides in breast cancer cells.

[0095] like Figure 5-7 As shown, ACSL5 inhibits de novo fatty acid synthesis in breast cancer cells:

[0096] A. Analysis of the TCGA database revealed that ACSL5 was negatively correlated with the expression of key enzyme genes in de novo fatty acid synthesis.

[0097] BC: ACSL5 significantly inhibited the expression of mRNA of key enzymes in de novo fatty acid synthesis.

[0098] DE: ACSL5 significantly inhibits the expression of key enzyme proteins in de novo fatty acid synthesis.

[0099] Among them, the cell lines used in the experiment were all preserved by the laboratory of the School of Laboratory Medicine of Chongqing Medical University. Fetal bovine serum was purchased from CLARK Bioscience, USA; DMEM culture medium was purchased from Chongqing Saimike Company; knockdown and overexpression of ACSL5 lentivirus was purchased from Zhenjiang Aibimeng Biotechnology Co., Ltd.; RNA rapid extraction kit was purchased from Yishan Biotechnology; reverse transcription reagent was purchased from Takara Company, Japan; real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) primers were designed and synthesized by Shanghai Shenggong Company; FFA detection kit and TG detection kit were purchased from Shanghai Beyotime Company; CCK-8 detection kit was purchased from Biosharp Company; protein blotting (Western blot) related reagents were purchased from Shanghai Beyotime Company; rabbit anti-human ACSL5 antibody and mouse anti-human β-actin antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd., rabbit anti-human ACACA, SCD1, ACLY, FASN antibodies were purchased from Huaan Biotechnology, rabbit anti-human p38, pp38, ERK, p-ERK were purchased from Abmart Company; Nile red dye was purchased from MCE Company.

[0100] 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 research method based on the effect of ACSL5 on breast cancer cells, characterized in that: include: S1: Cell culture and treatment: Medium preparation: Prepare DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, ensuring sterile conditions; Cell seeding and adaptation: Breast cancer cell lines were seeded in sterile culture flasks or culture plates and placed in an incubator at 37°C and 5% CO2 to adapt to growth; Cell passaging: When the cells reach 80% density, wash and digest the cells with trypsin solution and pass them to new culture vessels; S2: ACSL5 gene manipulation: Lentiviral vector construction: design shRNA or overexpression sequence according to the target sequence and clone it into the lentiviral vector; Lentiviral packaging: The constructed lentiviral vector is transfected into packaging cells to produce infectious lentiviral particles; Cell infection experiment: Under optimized conditions, breast cancer cells were infected with lentivirus to achieve knockdown or overexpression of ACSL5; Stable cell line screening: Screening of cell lines stably expressing ACSL5 gene modification by puromycin or other selective markers; S3: RNA extraction and RT-qPCR analysis; S4: Protein expression analysis; S5: Functional experimental verification: CCK-8 experiment: evaluate the effect of ACSL5 on the proliferation ability of breast cancer cells; Scratch healing assay: evaluate the effect of ACSL5 on the migration ability of breast cancer cells; Transwell assay: evaluate the effect of ACSL5 on the invasion ability of breast cancer cells; Nile red staining: analyze the effect of ACSL5 on the formation of intracellular fatty acid lipid droplets; Metabolite detection: FFA and TG detection kits were used to evaluate the effect of ACSL5 on fatty acid synthesis; S6: Bioinformatics analysis; S7: Statistical processing.

2. A research method based on the effect of ACSL5 on breast cancer cells according to claim 1, characterized in that: The S3 specifically includes: Total RNA extraction: Total RNA was extracted from cells using an RNA rapid extraction kit according to the manufacturer's protocol, and its purity and integrity were assessed; cDNA synthesis: Use a reverse transcription kit to reverse transcribe the extracted total RNA into cDNA, preferably a reagent with high reverse transcription efficiency; Primer design: Design highly specific RT-qPCR primers for ACSL5 and other related genes; RT-qPCR reaction: Amplify in a real-time fluorescence quantitative PCR instrument, monitor the fluorescence signal, and analyze the mRNA expression level.

3. A research method based on the effect of ACSL5 on breast cancer cells according to claim 1, characterized in that: The S4 specifically includes: Total protein extraction: Cellular proteins were collected using lysis buffer containing protease inhibitors; Protein concentration determination: Use Bradford or other protein quantification methods to accurately measure protein concentration; Western Blot analysis: Proteins were separated by SDS-PAGE, transferred to PVDF membrane, and then the expression of ACSL5 and other related proteins was detected using specific antibodies; Image analysis: The immunoblot results were digitally scanned and quantitatively analyzed using image analysis software.

4. A research method based on the effect of ACSL5 on breast cancer cells according to claim 1, characterized in that: The CCK-8 proliferation experiment: the treated cells were inoculated into a 96-well plate, and after a certain period of culture, CCK-8 reagent was added, and the OD value was measured to evaluate the cell proliferation ability.

5. A research method based on the effect of ACSL5 on breast cancer cells according to claim 4, characterized in that: The scratch healing migration experiment: scratches were made when cells grew fully in the monolayer, and pictures were taken at different time points to compare the speed of scratch closure.

6. A research method based on the effect of ACSL5 on breast cancer cells according to claim 5, characterized in that: The Transwell invasion assay is as follows: Matrigel is spread on the bottom of the transwell chamber to simulate the extracellular matrix and examine the ability of cells to penetrate the matrix.

7. A research method based on the effect of ACSL5 on breast cancer cells according to claim 6, characterized in that: The Nile red staining experiment: cells were stained with Nile red, and the accumulation of intracellular lipid droplets was observed and quantified using a fluorescence microscope.

8. A research method based on the effect of ACSL5 on breast cancer cells according to claim 7, characterized in that: The FFA and TG assays were performed using free fatty acid test kits and triglyceride test kits to measure the intracellular FFA and TG contents, respectively.

9. A research method based on the effect of ACSL5 on breast cancer cells according to claim 1, characterized in that: In the S6, specifically: Expression data analysis: The expression difference of ACSL5 in breast cancer tissues and normal tissues was analyzed by online tools such as GEPIA2; Survival analysis: Kaplan-Meier Plotter was used to analyze the relationship between ACSL5 expression level and patient survival; Pathway enrichment analysis: With the help of KEGG database, ACSL5-related signaling pathways were enriched to find potential regulatory networks.

10. A research method based on the effect of ACSL5 on breast cancer cells according to claim 1, characterized in that: In the S7, specifically: Data collation: Summarize and organize all experimental data in preparation for subsequent statistical analysis; Statistical analysis: GraphPad Prism 8.0 software was used to perform t-test or one-way analysis of variance on the data set to determine whether there were significant differences among the experimental groups.

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