Evaluation model for efficacy of inhibiting growth of cervical cancer stem cells and establishment method of evaluation model

Abstract

The invention provides a drug effect evaluation model for inhibiting growth of cervical cancer stem cells and an establishment method of the drug effect evaluation model. The method comprises the following steps: (1) preparing cervical cancer stem cells or stem cell-like cells; (2) preparing total flavonoids of galangal; (3) medicine intervention; and (4) verifying the drug effect evaluation model.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug evaluation, and relates to a drug efficacy evaluation model for inhibiting the growth of cervical cancer stem cells and a method for establishing the model. Background Art

[0002] Alpinia officinarum (Galanga officinarum) is the single herbal ingredient in the Uighur anticancer preparation "Compound Xiao Ai Fei (also known as White Flower Snake Honey Paste)." It comes from the dried rhizome of Alpinia officinarum Hance, a plant of the Zingiberaceae family. During screening for active anticancer components in Compound Xiao Ai Fei, total flavonoids from Alpinia officinarum were identified as the primary active antitumor component.

[0003] There are tumor stem cell populations in cervical cancer tissues or cervical cancer cell lines, and total flavonoids from Alpinia officinarum may inhibit the growth of cervical cancer stem cells, thereby exerting anti-tumor pharmacological effects. However, related research is not sufficient to reveal the above mechanism of action. Summary of the Invention

[0004] The present invention provides a drug efficacy evaluation model for inhibiting the growth of cervical cancer stem cells and a method for establishing the model, which can effectively solve the above problems.

[0005] The present invention is achieved in that:

[0006] The present invention provides a method for establishing a drug efficacy evaluation model for inhibiting the growth of cervical cancer stem cells, comprising the following steps:

[0007] (1) Preparation of cervical cancer stem cells or stem cell-like cells:

[0008] HPV-positive cervical cancer cells are cultured to isolate cervical cancer stem cells or stem cell-like cells;

[0009] (2) Preparation of total flavonoids from Alpinia officinalis:

[0010] The dried medicinal root of Alpinia officinalis is used as a raw material to prepare a crude extract of total flavonoids from Alpinia officinalis, which is then dissolved in DMSO to prepare a total flavonoids solution from Alpinia officinalis.

[0011] (3) Drug intervention:

[0012] The cervical cancer stem cells or stem cell-like cells in step (1) are inoculated into a culture plate, and the galangal total flavonoids solution in step (2) is used for intervention, and a blank control group is set up;

[0013] (4) Validation of drug efficacy evaluation model:

[0014] The efficacy of drug interventions was evaluated using at least one of the following indicators:

[0015] a. Detect cell proliferation activity using MTT or CCK-8 assay;

[0016] b. Flow cytometry was used to detect cell apoptosis and cell cycle regulation;

[0017] c. Transwell assay or cell scratch test to detect cell invasion and migration ability;

[0018] d. Detection of stem cell marker activity by immunofluorescence or flow cytometry;

[0019] e. ELISA method was used to detect the expression levels of stem cell markers.

[0020] Furthermore, the present invention provides a drug efficacy evaluation model for inhibiting the growth of cervical cancer stem cells established by the above method.

[0021] Furthermore, the present invention provides application of the above model in whole genome expression profiling analysis.

[0022] The beneficial effects of the present invention are:

[0023] The present invention provides a method for establishing a drug efficacy evaluation model for inhibiting the growth of cervical cancer stem cells. The method comprises separating and preparing cervical cancer stem cells or stem cell-like cells, intervening with total flavonoids from galangal, and establishing a drug intervention and drug efficacy evaluation cell model, thereby providing a basis for revealing the anti-cancer mechanism of total flavonoids from galangal.

[0024] Furthermore, based on the above-mentioned drug efficacy evaluation cell model, the present invention clarifies the differences in common mRNA expression profiles before and after drug intervention through whole-genome expression profile analysis before and after drug intervention; and explores the relationship between the intervention effect of total flavonoids of Alpinia officinarum and the intracellular gene expression regulatory network through bioinformatics analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 This is a flow chart of a method for establishing a drug efficacy evaluation model for inhibiting the growth of cervical cancer stem cells provided by an embodiment of the present disclosure.

[0027] Figure 2 It is a roadmap for the application of the model provided in the embodiments of the present disclosure in whole genome expression profiling analysis. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure. Therefore, the following detailed description of the embodiments of the present disclosure provided in the drawings is not intended to limit the scope of the present disclosure for protection, but merely represents the selected embodiments of the present disclosure. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure.

[0029] The embodiment of the present invention provides a method for establishing a drug efficacy evaluation model for inhibiting the growth of cervical cancer stem cells.

[0030] Specifically, such as Figure 1 As shown, the method includes the following steps:

[0031] (1) Preparation of cervical cancer stem cells or stem cell-like cells:

[0032] HPV-positive cervical cancer cells are cultured and cervical cancer stem cells or stem cell-like cells are isolated.

[0033] In some embodiments, HPV-positive SiHa or HeLa cells are selected as cultured cervical cancer cells.

[0034] HPV-positive cervical cancer cells were cultured in normal culture medium and cell culture conditions.

[0035] In some embodiments, cervical cancer stem cells or stem-like cells are isolated by at least one of a side population cell method, an agarose gel culture method, or a flow cytometer sorting method.

[0036] In some embodiments, stem cell characteristics are evaluated by detecting cell proliferation ability, aggregation ability, bevacizumab sensitivity, tumorigenic ability, and expression of stem cell markers using methods such as CCK-8, flow cytometry, and agarose culture.

[0037] (2) Preparation of total flavonoids from Alpinia officinalis:

[0038] The dried medicinal root of Alpinia officinalis was used as a raw material to prepare a crude extract of total flavonoids from Alpinia officinalis, and the crude extract of total flavonoids from Alpinia officinalis was dissolved in DMSO to prepare a total flavonoids solution from Alpinia officinalis.

[0039] In some embodiments, the method for preparing the crude extract of total flavonoids from Alpinia officinarum comprises:

[0040] The medicinal root of Alpinia officinarum is crushed to obtain Alpinia officinarum root powder, the Alpinia officinarum root powder is mixed with 95% ethanol at a mass-to-volume ratio of 1:1.67, and reflux extraction is performed twice. The ethanol extracts are combined and concentrated using a rotary evaporator to obtain a concentrate. The concentrate is extracted with petroleum ether and chloroform in sequence twice, and the chloroform extracts are combined, concentrated under reduced pressure, and cooled overnight to obtain a brown-yellow sticky solid, which is a crude extract of total flavonoids from Alpinia officinarum.

[0041] In some embodiments, the reflux extraction time is 1.5-2 hours each time.

[0042] In some embodiments, the volumes of petroleum ether and chloroform are each concentrated to twice the volume.

[0043] (3) Drug intervention:

[0044] The cervical cancer stem cells or stem cell-like cells in step (1) are inoculated into a culture plate, and are intervened with the total flavonoids solution of Alpinia officinalis in step (2), while a blank control group is set up.

[0045] The cells without intervention were set as blank control group.

[0046] In some embodiments, a 96-well cell culture plate was used to culture 5×10 3 The cervical cancer stem cells (cervical cancer stem cells or stem-like cells) were subcultured at a density of 10 cells / well for 24 hours.

[0047] In some embodiments, the concentration of the galangal total flavonoids solution is one of 50 μg / mL, 100 μg / mL, and 200 μg / mL.

[0048] In some embodiments, the intervention is performed with the galangal total flavonoids solution, and the intervention time is one of 24, 48, and 72 hours.

[0049] On this basis, various experiments related to cell identification and whole genome expression profiling studies are carried out.

[0050] (4) Validation of drug efficacy evaluation model:

[0051] The efficacy of drug interventions was evaluated using at least one of the following indicators:

[0052] a. Detect cell proliferation activity using MTT or CCK-8 assay;

[0053] b. Flow cytometry was used to detect cell apoptosis and cell cycle regulation;

[0054] c. Transwell assay or cell scratch test to detect cell invasion and migration ability;

[0055] d. Detection of stem cell marker activity by immunofluorescence or flow cytometry;

[0056] e. ELISA method was used to detect the expression levels of stem cell markers.

[0057] In some embodiments, the stem cell marker includes at least one of ALDH1A1, OCT4, and Twist1.

[0058] In the efficacy evaluation model, the dose-dependent effect of total flavonoids from Alpinia officinarum was shown as follows: the expression levels of ALDH1A1, OCT4, and Twist1 genes all decreased significantly with increasing drug concentration, suggesting that total flavonoids from Alpinia officinarum may inhibit the growth of cervical cancer stem cells.

[0059] ALDHA1 is a marker of early stem cell differentiation, expressed not only in isolated and cultured cervical cancer cells but also at high rates in cervical cancer and precancerous lesion tissues. OCT4, which exists in multiple isoforms, is also expressed in isolated and cultured cervical cancer stem cells, with the highest expression levels in HPV-positive cells. Previous work, through a combination of basic and clinical research, has demonstrated that stem cell markers such as OCT4 and ALDH1A1 are not only expressed in cervical cancer and precancerous lesion tissues but also have significantly elevated levels in the circulating blood of cancer patients, potentially serving as early warning biomarkers for cervical cancer. This suggests that cancer stem cell populations exist within cervical cancer tissues or cervical cancer cell lines, and that total flavonoids from galangal may inhibit the growth of these stem cells, thereby exerting anti-tumor pharmacological effects.

[0060] Based on the above identification data, evaluate the cell model preparation and drug intervention effects, clarify the establishment of the cell model for drug efficacy evaluation, and based on this, collect cell specimens before and after the intervention. Perform at least three biological replicates, and the collected cell specimens will be used as samples for whole-genome expression profiling analysis.

[0061] The embodiment of the present invention provides a drug efficacy evaluation model for inhibiting the growth of cervical cancer stem cells established by the above method.

[0062] Furthermore, the present invention provides application of the above model in whole genome expression profiling analysis.

[0063] Based on the aforementioned drug efficacy evaluation cell model, three drug doses (high, medium, and low (50 μg / mL, 100 μg / mL, and 200 μg / mL)) and three time gradients were selected for drug intervention trials. The aforementioned drug efficacy evaluation model was established to collect cell specimens with clear drug efficacy, extract total cellular RNA, and prepare mRNA analysis specimens. Whole-genome expression profiling analysis before and after drug intervention can clearly identify differences in common mRNA expression profiles before and after drug intervention.

[0064] Furthermore, through bioinformatics analysis, the relationship between the intervention effect of total flavonoids of Alpinia officinarum and the intracellular gene expression regulatory network was explored, and candidate mRNA (protein) indicators were identified.

[0065] Among them, based on the above-mentioned drug efficacy evaluation model, total cellular RNA was extracted before and after drug intervention, and analyzed by UV spectrophotometer and agarose gel electrophoresis to determine the concentration, integrity and purity. Relying on the high-throughput second-generation sequencing technology provided by Shenzhen BGI Genomics Co., Ltd., the changes in mRNA expression profiles before and after intervention were quantitatively analyzed to identify differentially expressed mRNA groups.

[0066] Relying on IPA@ (Ingenuity Pathway Analysis) and MetaCoreTM software and their online database, we analyzed and clarified the relationship between galangal total flavonoids intervention and the function or abnormal changes of proteins encoded by differentially expressed genes in cancer stem cells. We conducted predictive analysis of protein functions and their regulatory networks, clarified the regulatory networks, typical pathways, biological functions, and biomarkers of differentially expressed protein groups, and explored the mechanism of drug intervention and potential targets of drug action.

[0067] Specifically include:

[0068] (1) Regulatory network analysis: Provides a rapid solution to quickly evaluate the data of interest and the most influential signal regulatory networks, metabolic pathways, molecular action networks and physiological processes.

[0069] (2) Biological function analysis: Understand the differentially expressed proteins in known well-studied pathways and their interactions, identify proteins that have significant effects on cell and disease phenotypes, and understand how proteins affect phenotypes and whether these proteins enhance or weaken a biological process.

[0070] (3) Typical pathway analysis: Set network size parameters and association parameters to optimize the visualization and biological context analysis of the network, create interaction and regulatory networks of differentially expressed proteins, and focus on the most relevant analysis results of the experimental model.

[0071] After determining the candidate differentially expressed mRNA groups based on the whole-genome expression profile and bioinformatics analysis data, quantitative RT-PCR was used for quantitative screening and analysis. Based on commercially available specific antibodies and multiple reaction monitoring mass spectrometry (MRM-MS), single candidate indicator quantitative analysis was performed on cell specimens. Through data processing and comprehensive evaluation, the feasibility and accuracy of the whole-genome expression profile analysis data were explored.

[0072] (1) Quantitative RT-PCR analysis: First, specific PCR primers were designed based on the mRNA sequence information corresponding to the candidate protein. Professional software analysis was used to determine the feasibility of the primers as quantitative PCR primers. PCR analysis was performed on a small sample to verify the specificity and accuracy of the primers. Then, total RNA was extracted from all cell specimens to be tested. Using the above-mentioned specific primers and quantitative RT-PCR reagents, quantitative RT-PCR screening and analysis were performed to clarify the relationship between the intervention of galangal on cervical cancer stem cells and the changes in the expression level of the gene.

[0073] (2) Protein immunoblotting analysis: In the presence of specific antibodies, the protein immunoblotting method is selected to perform quantitative analysis on the indicators screened by other analysis methods, which can further verify the reliability and accuracy of the above-mentioned method verification and whole genome expression profile data.

[0074] (3) ELISA analysis method: Use a 96-well ELISA plate coated with specific antibodies and its kit, follow the conventional analysis method and the kit instructions, and read the A value at 450nm on the ELISA reader (Bio-Rad, Model 680), which is the reaction result. All plasma samples are tested in 3 replicates, and standard positive and negative controls are set. The average value of each sample read 3 times is used as the value of the ELISA antibody test of the sample. The OD value of each standard and sample is deducted from the OD value of the blank well and plotted (seven-point graph). If duplicate wells are set, the average value should be taken for calculation. Use professional curve making software (Curve Expert1.30) to draw the standard curve and calculate the actual concentration according to the conventional equation calculation method.

[0075] (4) Multiple reaction monitoring mass spectrometry (MRM-MS) analysis: There are no specific antibodies or ELISA reagents available on the market for some proteins encoded by candidate mRNAs. MRM-MS analysis can be performed using only peptide information and high-throughput (high-resolution mass spectrometry) analysis. The sample preparation and process for MRM-MS analysis are essentially the same as those for high-throughput proteomics analysis based on iTRAQ technology, including sample preparation, establishment of specific peptide determination methods, and quantitative analysis of sample analysis.

[0076] A. According to the iTRAQ-based proteomics analysis method, all samples to be tested and the extracted total protein were selected. After trypsin digestion, iTRAQ labeling, SCX chromatography and C18 reverse-phase chromatography purification, single MRM-MS analysis samples or combined analysis samples were prepared.

[0077] B. Based on the information of each protein to be tested, an online database of trypsin-cleavage-specific peptides is collected and analyzed using specialized software to identify peptides specific to the specific target protein. This allows the construction of a protein panel for simultaneous detection of multiple specific peptides (multiple reaction monitoring).

[0078] C. Take a pooled sample and perform MRM-MS simultaneous detection of multiple protein-specific peptides. After data processing and software analysis, exclude peptides that may interfere with crosstalk and determine a method for simultaneous determination of at least 15 proteins, thus establishing an MRM-MS analysis method.

[0079] D. Take all single analysis samples and quantitatively measure them using the above-established method. Through data processing and statistical analysis, clarify the expression differences of candidate proteins before and after intervention, and evaluate the specificity and accuracy of the whole genome expression profiling data.

[0080] For a roadmap on the application of the model in whole genome expression profiling, please refer to Figure 2 .

[0081] The embodiments of the present invention clarify the relationship between the intervention of total flavonoids of Alpinia officinarum and the regulation of intracellular gene expression through whole-genome expression profiling research and quantitative verification, and can discover the molecular mechanism and potential targets of the drug in inhibiting the growth of cervical cancer stem cells at the cellular level, providing a basis for the use of traditional Chinese medicine related to Alpinia officinarum in the clinical treatment of cervical cancer and the development of new anti-tumor drugs based on total flavonoids of Alpinia officinarum.

[0082] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for establishing a drug efficacy evaluation model for inhibiting the growth of cervical cancer stem cells, characterized in that: The following steps are involved: (1) Preparation of cervical cancer stem cells or stem cell-like cells: HPV-positive cervical cancer cells are cultured to isolate cervical cancer stem cells or stem cell-like cells; (2) Preparation of total flavonoids from Alpinia officinalis: The dried medicinal root of Alpinia officinalis is used as a raw material to prepare a crude extract of total flavonoids from Alpinia officinalis, which is then dissolved in DMSO to prepare a total flavonoids solution from Alpinia officinalis. (3) Drug intervention: The cervical cancer stem cells or stem cell-like cells in step (1) are inoculated into a culture plate, and the galangal total flavonoids solution in step (2) is used for intervention, and a blank control group is set up; (4) Validation of drug efficacy evaluation model: The efficacy of drug interventions was evaluated using at least one of the following indicators: a. Detect cell proliferation activity using MTT or CCK-8 assay; b. Flow cytometry was used to detect cell apoptosis and cell cycle regulation; c. Transwell assay or cell scratch test to detect cell invasion and migration ability; d. Detection of stem cell marker activity by immunofluorescence or flow cytometry; e. ELISA method was used to detect the expression levels of stem cell markers.

2. The method according to claim 1, characterized in that In step (1), cervical cancer stem cells or stem cell-like cells are isolated by at least one of the side population cell method, agarose gel culture method or flow cytometer sorting method.

3. The method according to claim 1, characterized in that In step (2), the preparation method of the crude extract of total flavonoids from Alpinia officinarum comprises: The medicinal root of Alpinia officinarum is crushed to obtain Alpinia officinarum root powder, the Alpinia officinarum root powder is mixed with 95% ethanol at a mass-to-volume ratio of 1:1.67, and reflux extraction is performed twice. The ethanol extracts are combined and concentrated using a rotary evaporator to obtain a concentrate. The concentrate is extracted with petroleum ether and chloroform in sequence twice, and the chloroform extracts are combined, concentrated under reduced pressure, and cooled overnight to obtain a brown-yellow sticky solid, which is a crude extract of total flavonoids from Alpinia officinarum.

4. The method according to claim 1, wherein In step (3), the concentration of the total flavonoids solution of Alpinia officinalis is one of 50 μg / mL, 100 μg / mL, and 200 μg / mL.

5. The method according to claim 1, wherein In step (3), the galangal total flavonoids solution is used for intervention, and the intervention time is one of 24, 48, and 72 hours.

6. The method according to claim 1, wherein In step (4), the stem cell marker includes at least one of ALDH1A1, OCT4, and Twist1.

7. A drug efficacy evaluation model for inhibiting the growth of cervical cancer stem cells established by the method according to any one of claims 1 to 6.

8. Use of the model according to claim 7 in whole genome expression profiling analysis, characterized in that: Cervical cancer stem cells or stem cell-like cell specimens before and after drug intervention in the model were collected as specimens for whole genome expression profiling analysis.

9. The use according to claim 8, characterized in that The changes in mRNA expression profiles before and after drug intervention in the whole genome expression profiling specimens were quantitatively analyzed to screen for differentially expressed mRNA groups.

10. The use according to claim 9, characterized in that The differentially expressed mRNA populations were validated by at least one of quantitative RT-PCR, western blot, ELISA, or multiple reaction monitoring mass spectrometry.

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