CN115966254A - Paclitaxel drug-resistant molecular marker for epithelial ovarian cancer and application thereof - Google Patents

Abstract

The invention discloses a paclitaxel drug-resistant molecular marker for epithelial ovarian cancer and application thereof. 488 genes which are differentially expressed in A2780 cells and A2780-Taxol cells are screened firstly, and then 26 of the genes are found to be closely related to the total survival time of patients with ovarian cancer taking paclitaxel. Through KEGG and GO enrichment analysis, 3 paclitaxel drug resistance related genes are obtained through screening. The 3 paclitaxel drug resistance related genes show significant differential expression in A2780 cells and A2780-Taxol cells. Particularly, UNC5C gene has significant expression reduction in A2780 cells and A2780-Taxol cells. This means that UNC5C is expected to provide meaningful biological indexes for paclitaxel drug-resistant diagnosis and treatment of clinical ovarian cancer, and becomes a down-regulated molecular therapeutic target of paclitaxel drug-resistant patients with ovarian cancer and a marker for survival prognosis of patients.

Description

Paclitaxel drug-resistant molecular marker for epithelial ovarian cancer and application thereof

Technical Field

The invention relates to the field of diagnosis and treatment of ovarian cancer, in particular to a paclitaxel drug-resistant molecular marker for epithelial ovarian cancer and application thereof.

Background

Epithelial Ovarian Cancer (EOC) is one of the gynecological malignancies, with a high rate of mortality that ranks third in the gynecological malignancies. Since more than 70% of epithelial ovarian cancer patients are diagnosed in the advanced stage, five-year survival rate is low, only approaching 48%. The currently common treatment modalities are surgery and chemotherapy. However, because the ovary is positioned in the deep position of the female pelvic cavity, the operation difficulty coefficient is increased, and higher risks are brought to the operation safety of patients. In contrast, treatment with drugs can greatly increase the patient's safety factor. Paclitaxel is a common first-line chemotherapy drug for epithelial ovarian cancer patients, but clinical efficacy of paclitaxel is limited due to occurrence of drug resistance, and meanwhile, the drug resistance of paclitaxel can usually cause the epithelial ovarian cancer patients to have multidrug resistance (MDR), which is a main reason for chemotherapy failure and survival rate reduction of the patients. Therefore, the improvement of the paclitaxel drug resistance of the epithelial ovarian cancer patients is beneficial to improving the survival rate of the patients under the condition of ensuring a certain safety factor.

RNA sequencing is a high-throughput next-generation sequencing technology, can simultaneously carry out large-scale parallel sequencing on hundreds of millions of DNA/RNA chains, and generates abundant data information in a short time. With the development of technologies, the content of databases related to various biological information increases, and the maturity of the RNA sequencing technology also increases the data in the sequencing databases. The massive information provided by the databases brings great convenience for high-throughput gene screening, and can be used for researching the taxol resistance of the epithelial ovarian cancer and related new targets.

The super enhancer in the genome transcription regulation and control region can greatly activate the expression of genes, has higher tissue specificity and plays an important role in the processes of development, tumor and the like. Based on the principle that gene control elements, super enhancers, regulate genes, regulators for controlling gene expression can be developed. Solves the problem of diseases by regulating gene expression, and creates a new treatment opportunity for patients.

At present, the research on drug resistance of epithelial ovaries focuses on platinum drugs rather than paclitaxel, but the toxicity of platinum drugs to human bodies is far higher than that of paclitaxel, and in all aspects, paclitaxel is one of the first-line chemotherapy drugs. A few studies on paclitaxel resistance in epithelial ovaries have selected a single gene for study, or selected drugs with indicated indications for use in combination with paclitaxel to reverse its resistance. The research of a single gene is not integral and is relatively one-sided; however, the existing adaptive drugs lack specificity and are accompanied by certain adverse reactions. There are also few studies screening drug resistance related genes of chemotherapeutic drugs for epithelial ovarian cancer by high throughput sequencing and biological analysis, but no experimental results demonstrate.

Disclosure of Invention

The research process of the invention is as follows:

(1) Culturing and subculturing the human epithelial ovarian cancer cell line A2780 and the Taxol-resistant A2780-Taxol cell line. Then collecting A2780 and A2780-Taxol cells in logarithmic growth phase to extract total RNA.

(2) Preparing a library, and performing transcriptome sequencing on an illumina sequencing platform;

(3) The raw sequencing data was filtered to remove bases containing linker (adapter) sequences, very low sequencing quality and non-sequenced bases, and clean reads (clean reads) were mapped to the reference genome using HISAT2 (v2.0.4). Aligning clean reads to the reference coding genome using Bowtie2 (v2.2.5);

(4) Calculating the Gene Expression level of A2780 and A2780-Taxol cells by using software RSEM (v1.2.8), calculating the Gene Expression multiple of A2780 Taxol resistance data in a GEO (Gene Expression Omnibus) database and a corrected P value by using a R language package DESeq2, and screening differential genes under the conditions of | log2 (Fold Change) | > =1 and FDR < =0.001 or Padj < 0.01;

(5) The software packages in R language, survivmini and survival, were used to analyze The relationship between differential gene levels and overall survival of patients with ovarian Cancer dosed with paclitaxel in The TCGA (The Cancer Genome Atlas) database;

(6) Acquiring a gene related Pathway (Pathway) by using a KEGG Pathway database, calculating a P value (P value) by a function of R (password), and performing multiple inspection and correction on the Pvalue by a software package qvalue to obtain a paclitaxel drug resistance related gene contained in a significantly enriched Pathway;

(7) The paclitaxel drug resistance related gene of the epithelial ovarian cancer is verified through Western blotting, cell immunofluorescence experiments and cell immunohistochemical experiments.

Throughout this specific embodiment, we screened 488 genes that were differentially expressed in A2780 cells and A2780-Taxol cells by transcriptome sequencing and bioinformatics analysis, combined with sequencing data in the GEO database. Through Kaplan-Meier survival curve analysis and combined with clinical data of paclitaxel-administered patients with ovarian cancer in a TCGA database, 26 difference genes are found to be closely related to the overall survival time of the paclitaxel-administered patients with ovarian cancer. Through KEGG and GO enrichment analysis, 3 paclitaxel drug resistance related genes are obtained by screening, and the enrichment functions of the paclitaxel drug resistance related genes are related to cell adhesion and migration. The 3 paclitaxel drug resistance related genes show obvious differential expression conditions in A2780 cells and A2780-Taxol cells. Particularly, UNC5C gene has significant expression reduction in A2780 cells and A2780-Taxol cells. This means that UNC5C is expected to provide meaningful biological indexes for paclitaxel drug-resistant diagnosis and treatment of clinical ovarian cancer, and becomes a down-regulated molecular therapeutic target of paclitaxel drug-resistant patients with ovarian cancer and a marker for survival prognosis of patients. We carried out subsequent experimental verification on 3 paclitaxel resistance-related genes UNC5C, MMP1, and ZYX.

In the Western blotting result of our cells, the expression of UNC5C in A2780-Taxol cells is obviously lower than that of A2780 cells. Furthermore, the same results were obtained in the cellular immunofluorescence assay and the immunohistochemical assay. These experimental results were consistent with our previous sequencing data. UNC5C is taken as a paclitaxel drug resistance down-regulation molecular target spot to research and develop a targeted regulator for reversing the drug resistance of epithelial ovarian cancer, which not only can improve the expression and transcription efficiency of genes and quickly generate the treatment effect, but also has tissue specificity and can avoid the adverse reaction generated by a gene silencing therapy. In conclusion, the results of RNA sequencing, western blotting of in vitro cells, immunofluorescence experiments and immunohistochemical experiments of three targets UNC5C, MMP1 and ZYX are mutually well verified, and the multiple verification work of the three targets has stability and accuracy. The screening and discovery of the UNC5C target point reveal a new mechanism of the paclitaxel drug resistance occurrence of the epithelial ovarian cancer, and provide new meaningful clues and bases. UNC5C can provide a new down-regulation drug resistance molecular target and a clinical application thought for treating taxol-induced multidrug resistance, enhancing curative effect and prognosis evaluation of epithelial ovarian cancer patients.

The invention has the following advantages:

(1) The UNC5C gene is screened, found and verified for the first time as a new down-regulated molecular marker of the drug resistance of the taxol of the epithelial ovarian cancer, and the method has wide application prospect.

(2) The remarkable expression down-regulation of the UNC5C gene is reported to influence the acquired paclitaxel resistance of the epithelial ovarian cancer patients and the survival prognosis for the first time. UNC5C can be used as a new down-regulation molecular target of a paclitaxel drug resistance mechanism of an epithelial ovarian cancer patient, and can provide a new action target and a new idea for researching and developing a regulator to reverse the treatment of the epithelial ovarian cancer multi-drug resistance.

(3) The research combines the second generation sequencing technology, the bioinformatics method and the clinical sample data of paclitaxel drug-taking patients of TCGA ovarian cancer, and provides new clues and basis for clinically revealing the paclitaxel drug-resistance mechanism of epithelial ovarian cancer. And the bioinformatics analysis results are verified and matched through in vitro cell Western blotting experiments, immunofluorescence experiments and immunohistochemical experiments.

(4) Three molecular markers, namely UNC5C, MMP1 and ZYX, are screened for common difference genes in data of sequencing and GEO databases. The sequencing result shows that UNC5C is differentially expressed and downregulated in A2780 and A2780-Taxol, and meanwhile, the results of in vitro cell Western blotting experiments, immunofluorescence experiments and immunohistochemical experiments are remarkably differentially downregulated, so that the UNC5C can be used as the most preferable molecular marker for designing a tumor targeting regulator.

(5) The invention can provide a new paclitaxel drug resistance target for treating the epithelial ovarian cancer multi-drug resistance, enhancing the curative effect, performing prognosis evaluation and improving the population health in the future, and has guiding significance for clarifying future research and development directions and practical clinical application.

Drawings

FIG. 1 is a diagram showing the expression of differential genes in DGEs 1;

FIG. 2 is a heat map of the expression of differential genes in DGEs 2;

FIG. 3 is a KEGG significant enrichment pathway map of differential genes in DGEs 1;

fig. 4 is a GO-significantly enriched entry for the differential genes in DGEs 1. Wherein, A: the first fifteen entries of DEG1 differential genes were significantly enriched in CC analysis. B: the first fifteen entries of DEG1 differential gene were significantly enriched in MF analysis. C: the first fifteen entries of DEG1 differential genes significantly enriched in BP analysis;

FIG. 5 is a correlation diagram of the survival-related differential genes and the KEGG pathway;

FIG. 6 shows that differential expression of UNC5C, MMP1 and ZYX in A2780 cells and A2780-Taxol cells is verified by Western blotting experiment. A: UNC5C, MMP1, ZYX express bands in A2780 cells and A2780-Taxol cells. B: expression profiles of UNC5C, MMP1, ZYX in a2780 and a2780-Taxol cells;

FIG. 7 shows the differential expression levels of UNC5C, MMP1, and ZYX in A2780 cells and A2780-Taxol cells in the sequencing data (UNC 5C is expressed as 0 in A2780-Taxol cells);

FIG. 8 shows the differential expression of UNC5C, MMP1, and ZYX in A2780 and A2780-Taxol cells as verified by cellular immunofluorescence assay. A: immunofluorescence hybridization images and fluorescence intensity statistical plots of UNC5C in A2780 and A2780-Taxol cells. B: immunofluorescence hybridization images and fluorescence intensity statistics of MMP1 in A2780 and A2780-Taxol cells. C: immunofluorescence hybridization images and fluorescence intensity statistical images of ZYX in A2780 and A2780-Taxol cells;

FIG. 9 shows that the differential expression of UNC5C, MMP1, and ZYX in A2780 cells and A2780-Taxol cells was confirmed by a cytoimmunohistochemical experiment. A: immunohistochemical staining images of UNC5C, MMP1, ZYX in A2780 and A2780-Taxol cells. B: statistical plots of immunohistochemical staining intensity for UNC5C, MMP1, ZYX in a2780 and a2780-Taxol cells.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto. In addition, the specific technical operation steps are not indicated in the examples or the apparatus is not indicated by manufacturers, and all the conventional products are commercially available.

1. Cell culture

Culturing the human epithelial ovarian cancer A2780 cell line in a 1640 culture medium containing 10% fetal calf serum, 1% double antibody and 2% L-glutamine; the paclitaxel resistant cell line A2780-Taxol of human epithelial ovarian cancer is cultured in DMEM medium containing 10% fetal calf serum, 1% double antibody and 60ng/ml paclitaxel, and placed in 5% CO ₂ Incubate at 37 ℃ in an incubator every 2 to 3 days.

RNA sequencing

1) Extraction of Total RNA

Collect about 1 × 10 ⁷ After adding 1.5ml Trizol, mix well on the vortex shaker, and let stand for 5min at room temperature. Adding 300 μ l chloroform into 1.5ml Trizol, shaking and mixing on a vortex oscillator, standing at room temperature for 3-5min, and centrifuging at 12000 Xg for 10min at 4 deg.C. The upper colorless aqueous phase (about 400 to 500. Mu.l) was collected, an equal volume of chloroform/isoamyl alcohol (24. To the transferred supernatant was added an equal volume of isopropanol, and the mixture was left at-20 ℃ for 1h,4 ℃ and 13600rpm for 20min, and the supernatant was discarded. The RNA precipitate was rinsed twice with 1.0ml of 75% ethanol, left to stand for 3min each time, washed by inversion during this period, and centrifuged at 13600rpm at 4 ℃ for 3min; dissolving the RNA precipitate with 30-50 mul of nucleic-free water, and incubating for 10min at 55-60 ℃ to assist dissolving if necessary.

2) Preparation of RNA sequencing library

Total RNA concentration, RIN value, 28S/18S and fragment size were measured using an Agilent 2100 Bioanalyzer. A certain amount of total RNA was purified using oligo-dT beads, and the finally prepared mRNA was dissolved in 10. Mu.l of Elution Buffer. To the above product was added 3. Mu.L of First Strand Buffer and incubated at 94 ℃ for 8min. Preparing a reverse transcription reaction system, and performing reverse transcription at 25 ℃ for 10min;42 ℃ for 50min; after 70 ℃ for 15min, the two-chain reaction reagent was added, incubated at 16 ℃ for 1 hour, and then magnetic bead purification was performed. And (3) preparing a terminal repairing reaction, and incubating at 20 ℃ for 30min and then carrying out magnetic bead purification. Preparing an A reaction system, incubating at 37 ℃ for 30min, adding a connector (adapter) to connect the reaction system, incubating at 20 ℃ for 15min, and performing magnetic bead purification after the reaction is finished. And (3) configuring a PCR reaction system, amplifying according to a PCR reaction program, and purifying a PCR product by using magnetic beads. The size and concentration of fragments of the library were measured using an Agilent 2100 Bioanalyzer. Adding NaOH into the library which is qualified by detection to denature the library into a single chain, and diluting the single chain to a certain concentration according to the expected computer data amount. And adding the denatured and diluted library into the FlowCell, hybridizing with a joint on the FlowCell, completing bridge PCR amplification on a cluster generation platform cBot, and performing on-machine sequencing.

3) Sequencing data processing

Sequencing data were filtered by SOAPnuke (v1.5.2): (1) deleting reads comprising sequencing adaptors; (2) Deleting reads with a low mass basis ratio (basis weight less than or equal to 5) greater than 20%; (3) Reads with unknown basis ('N' basis) ratios greater than 5% are removed, and then clean reads are taken and stored in FASTQ format. Clean reads were mapped to the reference genome using HISAT2 (v 2.0.4). Clean reads were aligned to the reference coding gene set (GCF-000001405.39 _GRCh38. P13) using Bowtie2 (v2.2.5).

3. Differential gene analysis

The gene expression levels of A2780 and A2780-Taxol cells were calculated using software RSEM (v1.2.8), and 6226 differential genes (DGEs 1) were screened under conditions of | log2 (Fold Change) | > =1 and FDR < =0.001, as shown in FIG. 1. The GEO database downloads a gene expression level file GSE159791 of a paclitaxel resistant A2780-Taxol cell, calculates gene expression Fold and corrected P value by using an R software package DESeq2 by taking A2780 as a control, and selects 488 common genes (DGEs 2) by comparing differential genes obtained by screening by taking | log2 (Fold Change) | > =1 and Padj <0.01 as conditions with DGEs1, as shown in figure 2. The results were visualized using the R software packages pheatmap and ggplot 2.

Kaplan-Meier survival Curve analysis

Screening out the sequencing sample in TCGA (https:// xenoxybrowser. Net/datapages /) as ovarian cancer, using paclitaxel as clinical information of a patient for main treatment, and analyzing and visualizing the relation between the differential gene expression level of simultaneous up-and-down regulation in DGEs2 and the total life cycle of the patient through software packages surfmer and survival of R language. Of these, only 26 differential genes were associated with overall survival (P < 0.05), as shown in table 1.

TABLE 1 survival-associated Difference genes

KEGG and GO enrichment analysis

Gene-related GO term and Pathway were obtained from the Gene Ontology and KEGG Pathway databases. Pvalue is calculated by the function of R, physer, and is subjected to multiple check correction by the software package qvalue. Significantly enriched GO term and Pathway were screened with Qvalue (corrected Pvalue) <0.05 as a threshold, and results were visualized using the software packages of R language package, gollot and ggplot 2. The genes of DGEs1 are significantly enriched in the pathways such as Focal adhesion (Focal adhesion), a Hippo signaling pathway (Hippo signaling pathway), a MAPK signaling pathway (MAPK signaling pathway), axon guidance (Axon guidance), a PPAR signaling pathway (PPAR signaling pathway) (shown in FIG. 3), and the Biological processes (Biological processes, BP) of Cell adhesion (Cell adhesion) and Cell migration (Cell migration), and the significant enrichment results of Cell localization (CC) and Molecular Function (MF) are mostly related to Cell adhesion and migration (FIGS. 4A-C). Of these, only three survival-related genes, UNC5C, MMP1, and ZYX, were included in the PPAR signaling, focal attachment, and Axon guidance pathways, respectively, that were significantly enriched (see fig. 5). Therefore, UNC5C, MMP1, and ZYX were used as genes related to paclitaxel resistance. KEGG and GO enrichment results show that the development of paclitaxel resistance in ovarian cancer is strongly correlated with tumor cell adhesion and migration.

6.Western Blotting

The A2780 and A2780-Taxol cells were lysed using RIPA buffer to extract total protein. Total protein concentration was quantified using the BCA protein concentration assay kit assay according to the product instructions. Protein samples were separated by SDS-PAGE and transferred to membranes. Antibodies ZYX, MMP1 and UNC5C were diluted in a ratio of 1 to 1000 and the membranes were placed in primary antibody and incubated overnight at 4 ℃. TBST was washed 3 times and incubated with secondary antibody for 2h at room temperature. Visualization was performed using a chemiluminescent kit. The protein abundance results were processed with software Image J. Results As shown in FIGS. 6A-B, UNC5C was reduced in expression of the drug-resistant cell strain A2780-Taxol (P < 0.05), MMP1 and ZYX were increased in expression of the drug-resistant cell strain A2780-Taxol (P <0.05, P < -0.01), consistent with the expression of the sequencing results (FIG. 7).

7. Cellular immunofluorescence assay

The slide was placed in a 12-well plate and pre-plated with 0.1% Gelatin for 1 hour at 37 ℃ in an incubator. Subsequently, about 3X 10 of each well was added ⁴ Individual cells were prepared as cell sheets for immunofluorescence staining. After the cells had stabilized growth, the medium was aspirated and the cells were washed 3 times with PBS. Then, the cells were fixed in 4% paraformaldehyde for 30min, washed 3 times with PBS, and permeabilized with a membrane-disrupting solution. The cell slide was then blocked with 5% BSA, washed 3 times with PBS, diluted primary antibody (1. After 3 washes in PBS, the cell fluorescent secondary antibodies were incubated together protected from light. PBS washing 3 times, adding DAPI dye solution to counterstain the nucleus, and incubating for 10min at room temperature in a dark place. Finally, PBS is washed 3 times, sealed with an anti-fluorescence quenching sealing agent in the dark, observed under a fluorescence microscope and images collected. As shown in FIGS. 8A-C, the fluorescence expression intensity of UNC5C in A2780-Taxol cells was significantly lower than that of A2780 (P)<0.05). In addition, MMP1 and ZYX were expressed more in A2780-Taxol cells than in A2780 cells (P)<0.01，P<0.001)。

8. Cell immunohistochemical assay

Putting the climbing film into a 12-hole plateThe cell density of each well is 4 multiplied by 10 ⁴ 1ml of cell suspension per ml was cultured overnight. Cell fixation, BSA blocking, primary antibody incubation and cell immunofluorescence experiment steps. PBS was washed 3 times, secondary antibody (HRP-labeled) was added dropwise, and the mixture was incubated at room temperature for 50min. PBS was washed 3 times. Dripping a DAB color developing solution prepared freshly, controlling the color developing time under a microscope, and flushing the section with water to stop color development. Counter-staining with hematoxylin for about 3min, washing with water, differentiating with hematoxylin differentiation solution for several seconds, washing with water, returning the hematoxylin to blue, and washing with running water. The slide was dehydrated and the side with the cells facing down was mounted on a slide with neutral gum. Microscopic examination and image acquisition and analysis. As shown in FIGS. 9A-B, UNC5C was expressed less in A2780-Taxol cells than in A2780 cells (P)<0.05). While the expression intensity of ZYX and MMP1 in A2780-Taxol cells is higher than that of A2780 (P)<0.001)。

9. Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics 26 statistical software, R language and GraphPad Prism 5, with P <0.05 statistically significant.

The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications to the present invention based on the disclosure of the present invention within the protection scope of the present invention.

Claims (9)

1. The paclitaxel drug-resistant molecular marker for the epithelial ovarian cancer is characterized by comprising UNC5C and/or MMP1 and/or ZYX.

2. Use of a molecular marker according to claim 1 in the manufacture of a medicament for the treatment of epithelial ovarian cancer.

3. The use of claim 2, wherein the epithelial ovarian cancer is a paclitaxel-resistant or multi-drug resistant epithelial ovarian cancer.

4. Use according to claim 2 or 3, characterized in that the medicament promotes the expression of the UNC5C gene and/or inhibits the expression of the MMP1 gene and/or inhibits the expression of the ZYX gene.

5. Use of the molecular marker of claim 1 in the manufacture of a product for assessing the prognosis of a patient with epithelial ovarian cancer.

6. The use of claim 5, wherein the epithelial ovarian cancer is a paclitaxel-resistant or multi-drug resistant epithelial ovarian cancer.

7. The use according to claim 5, wherein the product is a kit comprising a reagent for detecting the UNC5C gene of a patient and/or a reagent for detecting the MMP1 gene expression level of a patient and/or a reagent for detecting the ZYX gene expression level of a patient.

8. The drug for treating the epithelial ovarian cancer is characterized by comprising a reagent I and/or a reagent II and/or a reagent III, wherein the reagent I takes a UNC5C gene as a target point to promote the expression of the UNC5C gene, the reagent II takes an MMP1 gene as the target point to inhibit or silence the expression of the MMP1 gene, and the reagent III takes a ZYX gene as the target point to inhibit or silence the expression of the ZYX gene.

9. A product for prognostic evaluation of a patient with epithelial ovarian cancer, comprising a reagent or a set of reagents for specifically detecting the expression level of UNC5C gene and/or MMP1 gene and/or ZYX gene in a sample.

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