Molecular mechanism research method regulated and controlled through metabolic reprogramming

Abstract

The invention discloses a molecular mechanism research method regulated and controlled through metabolism reprogramming, and relates to the technical field of molecular biology. The method specifically comprises the following steps: S1, preparing an experimental cell material and an experimental reagent; the experimental cell material is melanoma cells, and the experimental reagent comprises a DMEM high glucose culture medium, PINK1 targeting lentivirus and the like. According to the application, cell infection, RNA (Ribonucleic Acid) extraction, RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) detection, Western blot experiment, cell activity detection and the like are carried out on lentivirus targeting PINK1, a molecular mechanism of melanoma caused by BRAF gene V600E mutation regulated by PINK1 through metabolic reprogramming is researched, and aerobic glycolysis caused by PINK1 down-regulation can be alleviated by inhibiting ENO2, so that PINK1 inhibits aerobic glycolysis of melanoma cells, and the application has the advantages that the application is simple, the application is convenient, and the application has a wide application prospect. The low-expression PINK1 promotes the growth of melanoma cells.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a method for studying molecular mechanisms regulated by metabolic reprogramming. Background Art

[0002] Melanoma, usually referred to as malignant melanoma, is a highly malignant tumor derived from melanocytes. It is often found in the skin, but can also be seen in mucous membranes and internal organs, accounting for about 3% of all tumors. Cutaneous malignant melanoma ranks third among malignant skin tumors (accounting for about 6.8% to 20%). It is more common in adults, with a higher incidence in white people with fair skin, and a lower incidence in dark-skinned Asians and Africans. It is rarely seen in children.

[0003] PINK1 is a protein kinase that is expressed in cells throughout the body, with particular expression in energy-intensive organs such as the heart, muscles, and brain. Within cells, it is primarily localized in the inner membrane of mitochondria. The function of PINK1 is not fully understood. It is hypothesized to protect mitochondria within cells during periods of cellular stress, such as when cells have abnormally high energy demands.

[0004] The expression of PINK1 in melanoma tissue and normal skin tissue was analyzed by searching the GEPIA database (http: / / gepia.cancerpku.cn / ) and the Protein Atlas database (https: / / www.proteinatlas.org / ). The results showed that the expression of PINK1 in melanoma tissue was lower than that in normal skin tissue ( Figure 1 A~1B), but there was no statistically significant difference in the expression of PINK1 in different stages of melanoma ( Figure 1 C), suggesting that PINK1 is involved in the entire occurrence and development of melanoma. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for studying the molecular mechanism regulated by metabolic reprogramming.

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

[0007] A method for studying the molecular mechanism regulated by metabolic reprogramming, comprising the following steps:

[0008] S1: Prepare experimental cell materials and reagents; the experimental cell materials are melanoma cells, and the experimental reagents include DMEM high-glucose medium, lentivirus targeting PINK1, RNA rapid extraction kit, reverse transcription reagent, RT-qPCR, lactate detection kit, protein blotting related reagents, glucose content uptake detection kit, ATP determination kit, 2-DG, rabbit anti-human PINK1, ENO2 antibodies and mouse anti-human β-actin antibody;

[0009] S2: Cell culture; melanoma cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2, and passaged every 2 to 3 days;

[0010] S3: Lentivirus infection: Melanoma cells were seeded in 6-well plates. When the cell fusion rate reached 40% to 60%, cells were infected with lentivirus targeting PINK1. After 72 to 96 hours of infection, the fluorescence expression was observed.

[0011] S4: RNA extraction and RT-qPCR detection; use RNA rapid extraction kit to extract total RNA from cells, and then use reverse transcription reagent to reverse transcribe RNA into cDNA for RT-qPCR detection;

[0012] S5: Western blot experiment; cells were collected and lysed to obtain total protein, and the protein concentration was determined by BCA protein assay kit;

[0013] S6: Cell viability assay; For cell viability assay, cells were seeded in a 96-well plate at a density of 4000 to 6000 cells per well, with 5 wells per group; PBS buffer was added to the surrounding wells to reduce water evaporation; cell activity was measured at the same time point for 5 consecutive days;

[0014] S7: Clone formation experiment; cells were seeded in 6-well plates at a density of 1000 cells / well for more than 14 days; when the number of most monoclonal cells was greater than 50, the culture was terminated;

[0015] S8: RNA sequencing; total RNA was extracted from control cells and stable cells using an RNA rapid extraction kit, followed by RNA sequencing and analysis;

[0016] S9: Detection of cellular glucose consumption, lactate production and ATP production; collect cells in EP tubes, add corresponding reagents in sequence according to the instructions of glucose content detection kit, lactate detection kit and ATP detection kit, and use BioTek Synergy multi-function microplate reader to detect the absorbance at 630nm, 530nm and 340nm, respectively, and calculate the relative levels of glucose consumption, lactate production and ATP content;

[0017] S10: The above experiments were repeated three times independently. All experimental results were statistically analyzed using GraphPad Prism 8.0 software. The t-test was used for pairwise comparison of data within each group, and one-way analysis of variance was used to compare data between two or more groups.

[0018] Preferably: the RT-qPCR is a real-time fluorescent quantitative polymerase chain reaction primer, the 2-DG is 2-deoxy-D-glucose, and the protein blotting related reagents include an SDS-PAGE gel preparation kit, an electrophoresis solution, an electrotransfer solution, a TBST buffer, a 4XLaemmli sample buffer, a protein marker, a transfer membrane, a blocking solution and an antibody.

[0019] Further: the Western blot experiment was carried out with a protein loading of 30 μg; the protein samples were separated in a 10% SDS-PAGE gel and transferred to a PVDF membrane, blocked with 5% skim milk powder at 37°C for 2 hours, and then the corresponding primary antibody was added and allowed to stand at 4°C overnight. On the second day, the membrane was incubated with HRP-labeled goat anti-rabbit / mouse immunoglobulin G antibody at 37°C for 1 hour, and the membrane was washed and developed with a developer.

[0020] As a preferred embodiment of the present invention: before the cell viability detection, 10 μl of CCK reagent is added to each well and incubated at 37° C. for 2 h; the absorbance value at 490 nm is measured using a universal microplate spectrophotometer; the absorbance value represents cell activity, and the data is represented by a line graph.

[0021] On the basis of the above scheme: in the clone formation experiment, fresh culture medium was replaced every 3 days to observe cell morphology; cells were fixed with 4% paraformaldehyde; cells were stained with crystal violet; staining results were observed under a microscope, and clone formation rate was calculated for statistical analysis.

[0022] Preferably: the RNA sequencing uses an Agilent 2100 bioanalyzer to detect the fragment size and concentration of the library, and uses probe anchor polymerization technology for sequencing.

[0023] The beneficial effects of the present invention are:

[0024] This molecular mechanism research method regulated by metabolic reprogramming uses lentivirus targeting PINK1 for cell infection, RNA extraction and RT-qPCR detection, Western blot experiments, cell viability detection, clone formation experiments, RNA sequencing and cell glucose consumption, lactate production and ATP production detection to study the molecular mechanism of PINK1 regulating BRAF gene V600E mutation leading to melanoma through metabolic reprogramming. It can be found that inhibiting ENO2 can reduce aerobic glycolysis caused by PINK1 downregulation, thereby enabling PINK1 to inhibit aerobic glycolysis of melanoma cells, and low expression of PINK1 promotes the growth of melanoma cells, indicating that ENO2 is an indispensable key factor for PINK1 to regulate glycolysis and promote tumor progression. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a statistical comparison group of abnormally low expression of PINK1 in melanoma of the present invention;

[0026] Figure 2 This is a statistical comparison chart group of the effects of low expression of PINK1 on melanoma cells in the present invention;

[0027] Figure 3 A statistical comparison chart group of the effects of PINK1 on melanoma cells of the present invention;

[0028] Figure 4 A statistical comparison chart group showing the effect of PINK1 on the expression of ENO2 in melanoma cells according to the present invention;

[0029] Figure 5 This is a statistical comparison chart group of the effects of the present invention's inhibition of ENO2 and PINK1 on melanoma cells. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0031] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0032] A method to study the molecular mechanisms regulated by metabolic reprogramming, such as Figures 1 to 5 As shown, the following steps are included:

[0033] S1: Prepare experimental cell materials and reagents; the experimental cell materials are melanoma cells, and the experimental reagents include DMEM high-glucose medium, lentivirus targeting PINK1, RNA rapid extraction kit, reverse transcription reagent, RT-qPCR, lactate detection kit, protein blotting related reagents, glucose content uptake detection kit, ATP determination kit, 2-DG, rabbit anti-human PINK1, ENO2 antibodies and mouse anti-human β-actin antibody;

[0034] The melanoma cell line used in the experiment was preserved by the laboratory of the School of Laboratory Medicine of Chongqing Medical University; DMEM high-glucose medium was purchased from GIBCO; fetal bovine serum was purchased from CLARK Bioscience, USA; lentivirus targeting PINK1 was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.; RNA rapid extraction kit was purchased from Shanghai Yishan Biotechnology Co., Ltd.; reverse transcription reagent was purchased from Takara, Japan.

[0035] RT-qPCR is a real-time fluorescence quantitative polymerase chain reaction primer designed and synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.; 2-DG is 2-deoxy-D-glucose, purchased from MedChemExpress, USA; the lactate detection kit was purchased from Beijing Solebow Technology Co., Ltd.; rabbit anti-human PINK1, ENO2 antibodies and mouse anti-human β-actin antibody were purchased from Proteintech.

[0036] Western blot reagents, glucose uptake detection kit, and ATP determination kit were all purchased from Shanghai Biotech Co., Ltd.; Western blot reagents included SDS-PAGE gel preparation kit, electrophoresis fluid, electrotransfer fluid, TBST buffer, 4XLaemmli sample buffer, protein marker, transfer membrane, blocking solution, and antibodies.

[0037] S2: Cell culture; melanoma cells were cultured in a DMEM high-glucose medium containing 10% fetal bovine serum in an incubator at 37°C and a CO2 volume fraction of 5%, and passaged approximately every 2 to 3 days.

[0038] S3: Lentivirus infection: Melanoma cells were seeded in 6-well plates. When the cell fusion rate reached 40% to 60%, the cells were infected with lentivirus targeting PINK1 according to the Genema recombinant lentivirus operation manual. After 72 to 96 hours of infection, the fluorescence expression was observed.

[0039] S4: RNA extraction and RT-qPCR detection; use an RNA rapid extraction kit to extract total cellular RNA, and then use a reverse transcription reagent to reverse transcribe the RNA into cDNA for RT-qPCR detection.

[0040] S5: Western blot experiment; cells were collected and lysed to obtain total protein, and the protein concentration was determined by BCA protein assay kit; protein samples were separated in 10% SDS-PAGE gel and transferred to PVDF membrane (PVDF membrane is a type of transfer membrane), blocked with 5% skim milk powder at 37°C for 2h, added with corresponding primary antibody, and allowed to stand at 4°C overnight. On the second day, goat anti-rabbit / mouse immunoglobulin G (IgG) antibody labeled with HRP was incubated at 37°C for 1h, and the membrane was washed and developed with a developer.

[0041] S6: Cell viability assay; For cell viability assay, cells were seeded in 96-well plates at a density of 4000 to 6000 cells per well, with 5 wells per group; PBS buffer was added to the surrounding wells to reduce water evaporation; cell viability was measured at the same time point for 5 consecutive days. Before the cell viability assay, 10 microliters of CCK reagent was added to each well and incubated at 37°C for 2 hours; finally, the absorbance value at 490 nanometers was measured using a universal microplate spectrophotometer. The absorbance value represents cell viability, and the data is represented by a line graph.

[0042] S7: Clone formation experiment; cells were seeded in 6-well plates at a density of 1000 cells / well for more than 14 days; when the number of most monoclonal cells was greater than 50, the culture was terminated. During this period, fresh culture medium was replaced every 3 days and cell morphology was observed. The cells were then fixed with 4% paraformaldehyde and stained with crystal violet. The staining results were observed under a microscope, and the clone formation rate was calculated for statistical analysis.

[0043] S8: RNA sequencing; total RNA was extracted from control cells and stable cells using an RNA rapid extraction kit, followed by RNA sequencing and analysis (sequencing was performed at Shanghai Kangce Biological Company). The digested total RNA samples were separated, interrupted, and cDNA was synthesized. The fragment size and concentration of the library were detected using an Agilent 2100 bioanalyzer, and probe-anchored polymerization technology was used for sequencing. Differential expression analysis was performed in R (version 4.0.5) using the DESeq2 software package (v1.20.0). Genes with an fdr-adjusted p value of less than 0.01 and a log2-fold change of greater than or equal to 1.5 were considered to have significant differential expression. The phatmap package (v1.0.12) was used to generate a heat map of differentially expressed genes (DEGs) using unsupervised hierarchical clustering. GOATOOLS was used to perform graphene oxide functional enrichment analysis on the gene set. Fisher's exact test p value of less than 0.05 indicated significant enrichment of GO functions. KEGG pathway enrichment analysis was performed, and the corrected p-adjusted value was less than 0.05 (refer to the attached manual Figure 3 a, b, supplement Figure 3 a).

[0044] S9: Cell glucose consumption, lactate production and ATP production detection; collect cells in EP tubes, add corresponding reagents in sequence according to the instructions of glucose content detection kit, lactate detection kit and ATP detection kit, and use Bio-Tek Synergy multi-function microplate reader to detect the absorbance (absorbance, A) at 630nm, 530nm and 340nm respectively, and calculate the relative levels of glucose consumption, lactate production and ATP content. The calculation formula is (A treatment group / treatment group protein concentration) / (A control group / control group protein concentration);

[0045] S10: The above experiments were repeated three times independently. All experimental results were statistically analyzed using GraphPad Prism 8.0 software. The t-test was used for pairwise comparison of data within each group, and one-way analysis of variance was used to compare data between two or more groups.

[0046] like Figure 1 As shown in Figure 2, PINK1 is abnormally low expressed in melanoma. The Protein Atlas database also shows that PINK1 expression varies in different melanoma cell lines ( Figure 1 D), and then RT-qPCR and Western blot were used to detect the expression levels of PINK1 in five melanoma cells ( Figure 1 E~1F).

[0047] like Figure 2 As shown in Figure 2, low expression of PINK1 promotes melanoma cell growth. Based on the PINK1 expression level in melanoma cell lines, a stable model of PINK1 knockdown in A375 and SK-MEL-28 cell lines was established. The efficiency of knockdown was verified by RT-qPCR and Western blot ( Figure 2 A), compared with the control group, the expression of PINK1 in the shPINK1 group was significantly decreased (P<0.01). CCK8 and clone formation assays showed that ( Figure 2 B~2C), knocking down PINK1 in melanoma cells increases cell proliferation. Then we used lentivirus to stably overexpress PINK1 in A375 cells, and the results of RT-qPCR and Western blot showed ( Figure 2 D), A375 successfully overexpressed PINK1. CCK8 and clone formation results showed that, in contrast to knockdown of PINK1, overexpression of PINK1 reduced cell proliferation ( Figure 2 E~2F).

[0048] like Figure 3As shown in Figure 2, PINK1 inhibits aerobic glycolysis in melanoma cells. When PINK1 was successfully knocked down, lactate production, ATP generation levels, and glucose uptake in A375 and SK-MEL-28 cells were significantly increased ( Figure 3 A~3C). However, after A375 overexpressed PINK1, the lactate production level, ATP production level and glucose uptake were significantly reduced ( Figure 3 D~3F). The above results indicate that PINK1 inhibits aerobic glycolysis in melanoma cells. 2-DG is a glucose analog and a widely used glycolysis inhibitor. After the application of 2-DG, the increase in lactate production after knockdown of PINK1 in cells was reversed, and the lactate content decreased in a dose-dependent manner with the increase of 2-DG concentration ( Figure 3 G). CCK8 and clone formation experiments showed that (2-DG can reverse the effect of knocking down PINK1 on promoting cell proliferation, Figure 3 H~3I).

[0049] like Figure 4 As shown in Figure 2, PINK1 downregulates ENO2 expression in melanoma cells. To investigate how PINK1 affects glycolysis, A375 shCTRL cells and A375 shPINK1 cells were subjected to RNA sequencing analysis. A heat map of glycolysis gene expression differences was obtained by intersecting the differentially expressed genes and glycolysis-related genes of the two groups of cells ( Figure 4 A). The heat map showed that the ENO2 gene had the most significant difference between the two groups of cells. RT-qPCR was used to detect the expression of glycolysis-related enzymes in shCTRL / shPINK1 and LvCON / LvPINK1 cells, and the downstream molecules of PINK1 were further identified. ENO2 was most significantly upregulated in the two PINK1 knockdown cells, which was consistent with the sequencing results ( Figure 4 B). RT-qPCR and Western blot showed ( Figure 2 C-2D), compared with the control group, the expression of ENO2 in the shPINK1 group was significantly increased (P<0.01), while the expression of ENO2 was decreased when PINK1 was overexpressed. Based on the above results, ENO2 may be a downstream molecule of PINK1.

[0050] like Figure 5 As shown in Figure 2, inhibition of ENO2 can reduce aerobic glycolysis caused by PINK1 downregulation. POMHEX, a specific inhibitor of ENO2, was used in A375 shPINK1 and SK-MEL-28 shPINK1 cells to specifically inhibit the enzymatic activity of ENO2 in vitro and in vivo. CCK8 and clone formation experiments showed that the application of POHEX to inhibit ENO2 enzymatic activity can attenuate the proliferation of melanoma cells induced by PINK1 downregulation ( Figure 5A~5B). At the same time, inhibition of ENO2 reversed the increase in lactate level, ATP production, and glucose consumption caused by downregulation of PINK1 ( Figure 5 These results suggest that inhibition of ENO2 can attenuate the proliferation of melanoma cells induced by PINK1 downregulation, indicating that ENO2 is an indispensable key factor for PINK1 to regulate glycolysis and promote tumor progression.

[0051] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for studying the molecular mechanism of metabolic reprogramming regulation, characterized in that: The steps include: S1: Prepare experimental cell materials and reagents; the experimental cell materials are melanoma cells, and the experimental reagents include DMEM high-glucose medium, lentivirus targeting PINK1, RNA rapid extraction kit, reverse transcription reagent, RT-qPCR, lactate detection kit, protein blotting related reagents, glucose content uptake detection kit, ATP determination kit, 2-DG, rabbit anti-human PINK1, ENO2 antibodies and mouse anti-human β-actin antibody; S2: Cell culture; melanoma cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2, and passaged every 2 to 3 days; S3: Lentivirus infection: Melanoma cells were seeded in 6-well plates. When the cell fusion rate reached 40% to 60%, cells were infected with lentivirus targeting PINK1. After 72 to 96 hours of infection, the fluorescence expression was observed. S4: RNA extraction and RT-qPCR detection; use RNA rapid extraction kit to extract total RNA from cells, and then use reverse transcription reagent to reverse transcribe RNA into cDNA for RT-qPCR detection; S5: Western blot experiment; cells were collected and lysed to obtain total protein, and the protein concentration was determined by BCA protein assay kit; S6: Cell viability assay; For cell viability assay, cells were seeded in a 96-well plate at a density of 4000 to 6000 cells per well, with 5 wells per group; PBS buffer was added to the surrounding wells to reduce water evaporation; cell activity was measured at the same time point for 5 consecutive days; S7: Clone formation experiment; cells were seeded in 6-well plates at a density of 1000 cells / well for more than 14 days; when the number of most monoclonal cells was greater than 50, the culture was terminated; S8: RNA sequencing; total RNA was extracted from control cells and stable cells using an RNA rapid extraction kit, followed by RNA sequencing and analysis; S9: Detection of cellular glucose consumption, lactate production and ATP production; collect cells in EP tubes, add corresponding reagents in sequence according to the instructions of glucose content detection kit, lactate detection kit and ATP detection kit, and use BioTek Synergy multi-function microplate reader to detect the absorbance at 630nm, 530nm and 340nm, respectively, and calculate the relative levels of glucose consumption, lactate production and ATP content; S10: The above experiments were repeated three times independently, and all experimental results were analyzed using GraphPad Prism Statistical analysis was performed using Statistical Analysis Software 8.

0. The t-test was used for pairwise comparisons of data within each group, and one-way analysis of variance was used to compare data between two or more groups.

2. The method for studying the molecular mechanism regulated by metabolic reprogramming according to claim 1, characterized in that: The RT-qPCR is a real-time fluorescent quantitative polymerase chain reaction primer, the 2-DG is 2-deoxy-D-glucose, and the protein blotting related reagents include an SDS-PAGE gel preparation kit, an electrophoresis solution, an electrotransfer solution, a TBST buffer, a 4XLaemmli sample buffer, a protein marker, a transfer membrane, a blocking solution and an antibody.

3. The method for studying the molecular mechanism regulated by metabolic reprogramming according to claim 1, characterized in that: The Western blot experiment was performed with a protein loading of 30 μg; the protein samples were separated in 10% SDS-PAGE gel and transferred to a PVDF membrane, blocked with 5% skim milk powder at 37°C for 2 hours, and then the corresponding primary antibody was added and allowed to stand at 4°C overnight. On the second day, the membrane was incubated with HRP-labeled goat anti-rabbit / mouse immunoglobulin G antibody at 37°C for 1 hour, and the membrane was washed and developed with a developer.

4. The method for studying the molecular mechanism regulated by metabolic reprogramming according to claim 1, characterized in that: Before the cell viability test, 10 μl of CCK reagent was added to each well and incubated at 37° C. for 2 h. The absorbance value at 490 nm was measured using a universal microplate spectrophotometer. The absorbance value represents cell activity, and the data is represented by a line graph.

5. The method for studying the molecular mechanism regulated by metabolic reprogramming according to claim 1, characterized in that: In the clone formation experiment, fresh culture medium was replaced every 3 days, and cell morphology was observed; cells were fixed with 4% paraformaldehyde; cells were stained with crystal violet; staining results were observed under a microscope, and clone formation rate was calculated for statistical analysis.

6. The method for studying the molecular mechanism regulated by metabolic reprogramming according to claim 1, characterized in that: The RNA sequencing uses an Agilent 2100 bioanalyzer to detect the fragment size and concentration of the library, and uses probe-anchored polymerization technology for sequencing.

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