Method for screening thyroid myeloid carcinoma para-follicular cell marker molecules

Abstract

The invention discloses a method for screening myeloid thyroid carcinoma para-follicular cell marker molecules, which comprises the following steps: carrying out single cell transcriptome sequencing on myeloid thyroid carcinoma para-follicular cells to obtain gene sequence data; obtaining a gene expression matrix of each cell; filtering the gene number lt; 200, the number of single-molecule tags is less than 400, and the mitochondrial gene proportion is gt; 20% of cells; carrying out dimension reduction processing on the gene expression matrix of the cell sample, and carrying out clustering analysis to obtain a plurality of cell subgroups with tumor specificity; respectively carrying out differential expression analysis on cells of each cell subset and cells of other subpopulations by adopting wilcoxon inspection, and screening out a gene with the most specific expression in each cell subset. According to the invention, follicular cell subgroups with tumor specificity in follicular para-cells of the myeloid thyroid carcinoma can be screened, and specific high-expression genes of the cell subgroups can be found, so that a more reliable basis can be provided for personalized precise treatment of the myeloid thyroid carcinoma.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and mainly to a method for screening parafollicular cell marker molecules of medullary thyroid carcinoma. Background Art

[0002] Medullary thyroid carcinoma (MTC) is a relatively rare neuroendocrine malignancy that originates from calcitonin-producing parafollicular cells derived from the thyroid neural crest. MTC accounts for 1.8% of all thyroid malignancies, with a malignancy between differentiated and undifferentiated types, high invasiveness, poor prognosis, and an increased likelihood of extensive nodal metastasis and local advanced invasion. The mortality rate accounts for about 13% of all deaths from thyroid cancer. Compared with differentiated thyroid cancer, the histopathological manifestations of MTC vary greatly, and the tumor is highly heterogeneous, with significant differences in clinical manifestations, prognosis, diagnosis, and treatment. The treatment of medullary thyroid carcinoma is not uniform, with surgical resection as the priority treatment, and radiotherapy and chemotherapy as supplementary postoperative treatment. Radiotherapy and chemotherapy are only considered when the disease progresses to the point where surgery is not possible. In terms of targeted therapy, the reticulocyte (RET) gene and vascular endothelial growth factor are currently used as therapeutic targets for medullary thyroid carcinoma.

[0003] At present, the commonly used tumor gene detection methods in clinical practice are fluorescence in situ hybridization (FISH), followed by polymerase chain reaction (PCR), first-generation sequencing (Sanger sequencing) and high-throughput sequencing (NGS). FISH is most commonly used to detect whether a gene has large-fragment translocation or rearrangement, but it cannot determine its partner gene; PCR and Sanger sequencing are mainly used to detect gene mutations, and RT-PCR products can also be sequenced to determine gene fusion sites. NGS, or next-generation sequencing, can detect a large number of target genes at one time; but these methods have certain limitations. They detect gene states in a relatively single manner and can detect various forms of variation including point mutations, insertions and deletions, but they have great limitations for the amplification, fusion, expression level of intracellular genes and the detection of rare non-coding RNAs, and cannot meet clinical needs.

[0004] Tumor heterogeneity poses a serious challenge to cancer treatment and patient survival, especially intratumor heterogeneity, which is increasingly considered a key determinant of treatment failure, drug resistance, and disease recurrence. Therefore, exploring the unique cell populations of medullary thyroid cancer tissue and their corresponding highly specific molecular targets can provide a biological basis for personalized medicine. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for screening parafollicular cell marker molecules of medullary thyroid carcinoma, so as to solve the technical problem of exploring the cell populations unique to medullary thyroid carcinoma tissue and their corresponding highly specific molecules.

[0006] The method for screening parafollicular cell marker molecules of medullary thyroid carcinoma of the present invention comprises the following steps:

[0007] 1) performing single-cell transcriptome sequencing on parafollicular cells of medullary thyroid carcinoma, wherein the parafollicular cells of medullary thyroid carcinoma include medullary thyroid carcinoma tissue cells and adjacent normal tissue cells, to obtain gene sequence data of several single cells;

[0008] 2) Perform gene sequence alignment, identify cell barcodes and unique molecular identifiers (UMI) in the gene sequence, and count each specific molecular identifier of each cell to obtain the gene expression matrix of each cell;

[0009] 3) Filter cells with gene number <200, single molecule tag number <400, and mitochondrial gene ratio >20% to obtain cell samples;

[0010] 4) Perform dimensionality reduction on the gene expression matrix of the cell samples, screen for highly variable genes, and obtain several tumor-specific cell subpopulations through cluster analysis;

[0011] 5) The Wilcoxon test was used to perform differential expression analysis between each cell subpopulation and cells in other subpopulations to obtain the specific expression genes of each cell subpopulation. The two statistical indicators of log2 (FC) and corrected P value in the differential expression analysis results were combined to finally screen out the most specifically expressed genes in each cell subpopulation.

[0012] Beneficial effects of the present invention:

[0013] The method of screening parafollicular cell marker molecules of medullary thyroid carcinoma in the present invention uses high-throughput single-cell transcriptome sequencing of parafollicular cells of medullary thyroid carcinoma and carefully analyzes each cell subpopulation, identifies tumor-specific follicular cell subpopulations, and discovers highly expressed genes specific to the cell subpopulation. These specific highly expressed genes found are closely related to the prognosis and treatment of patients, and can provide a more reliable basis for personalized and precise treatment of medullary thyroid carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is an expression graph of the results of filtering cell samples.

[0015] Figure 2 This is the cell subpopulation expression diagram obtained by cluster analysis. DETAILED DESCRIPTION

[0016] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0017] The method for screening parafollicular cell marker molecules for medullary thyroid carcinoma in this embodiment comprises the following steps:

[0018] 1) Single-cell transcriptome sequencing is performed on parafollicular cells of medullary thyroid carcinoma, wherein the parafollicular cells of medullary thyroid carcinoma include medullary thyroid carcinoma tissue cells and adjacent normal tissue cells, to obtain gene sequence data of several single cells.

[0019] 2) Perform gene sequence alignment, identify the cell barcodes and unique molecular identifiers (UMI) in the gene sequence, and count each specific molecular identifier of each cell to obtain the gene expression matrix of each cell. This step can be completed using the cellranger software.

[0020] 3) Filter cells with gene number (nFeature_RNA) <200, single molecule tag number (nCount_RNA) <400, and mitochondrial gene ratio (percent.mt) >20% to obtain cell samples. This step can be completed by the software seurat. In this step, 32,544 cells of tumor samples were obtained, and the median number of UMI and genes per cell was 4,349 and 2,250, respectively; 11,751 cells of adjacent cancer samples, and the median number of UMI and genes per cell was 1,274 and 967, respectively. The sequencing saturation of 80.25% indicates a comprehensive sampling of available transcripts. Figure 1 shown.

[0021] 4) The gene expression matrix of the cell sample was processed in a standard way, and then the gene expression matrix was linearly reduced using Principal Component Analysis (PCA) to screen highly variable genes. The cells were clustered using the K-Nearest Neighbors (KNN) algorithm to cluster cells, and cells with the same expression pattern were clustered together to form a cell subpopulation, thus obtaining several tumor-specific cell subpopulations. The cell subpopulations obtained by clustering analysis were further nonlinearly reduced using the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm and displayed on a two-dimensional graph as shown below. Figure 2 shown.

[0022] 5) The Wilcoxon test was used to perform differential expression analysis on each cell subpopulation and other subpopulations of cells, and the specific expression genes of each cell subpopulation were obtained. The two statistical indicators of log2(FC) and corrected P value in the differential expression analysis results ((log2(FC)>0.5 and corrected Pvalue<0.05)) were combined to finally screen out the most specific genes expressed in each cell subpopulation. In the specific implementation, the specific expression labels screened from the parafollicular cells of medullary thyroid carcinoma included: CALCA, BCL2, RICTOR, CHGB and SCG2 / 3 / 5.

[0023] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents, but these essentially the same modifications or equivalent replacements should also be included in the scope of the claims of the present invention.

Claims

1. A method for screening parafollicular cell marker molecules for medullary thyroid carcinoma, characterized in that: The following steps are involved: 1) performing single-cell transcriptome sequencing on parafollicular cells of medullary thyroid carcinoma, wherein the parafollicular cells of medullary thyroid carcinoma include medullary thyroid carcinoma tissue cells and adjacent normal tissue cells, to obtain gene sequence data of several single cells; 2) Perform gene sequence alignment, identify cell barcodes and specific molecular tags in the gene sequence, and count each specific molecular tag of each cell to obtain the gene expression matrix of each cell; 3) Filter cells with gene number <200, single molecule tag number <400, and mitochondrial gene ratio >20% to obtain cell samples; 4) Perform dimensionality reduction on the gene expression matrix of the cell samples, screen for highly variable genes, and obtain several tumor-specific cell subpopulations through cluster analysis; 5) The Wilcoxon test was used to perform differential expression analysis between each cell subpopulation and cells in other subpopulations to obtain the specific expression genes of each cell subpopulation. The two statistical indicators of log2 (FC) and corrected P value in the differential expression analysis results were combined to finally screen out the most specifically expressed genes in each cell subpopulation.

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