CN118028466A - Application of SLC6A6 gene as tumor marker and tumor treatment target - Google Patents
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Abstract
The invention discloses application of SLC6A6 gene as a tumor marker and a tumor treatment target, in particular to new discovery of high expression of solute carrier family 6 member 6 gene SLC6A6 in solid tumor tissues and tumor progress and recurrence and metastasis, wherein the gene or/and protein is used as a target for prognosis evaluation and medicine preparation of solid tumors.
Description
Technical Field
The invention relates to a solid tumor prognosis evaluation and drug treatment technology, in particular to application of a solute carrier family 6member 6 gene (SLC 6A6, solute CARRIER FAMILY membrane 6) and a coded protein thereof as a target point in solid tumor prognosis evaluation and drug preparation.
Background
Tumor biomarkers are a series of substances reflecting the occurrence, development and prognosis of diseases, and are widely applied to early diagnosis, treatment monitoring and prognosis evaluation of diseases clinically. The development of biomarkers for diagnosing tumors and monitoring the efficacy of disease treatment in tumor patients is critical to improving survival in patients. In addition, the research of tumor key gene targets is a common means for improving the effect of tumor treatment. Among them, solute carrier is a kind of cell membrane expression protein, which is found to be closely related to tumorigenesis and development recently, and has diagnostic and therapeutic value.
Disclosure of Invention
Based on the research of the invention, the invention provides the application of SLC6A6 gene or the coded protein thereof as a target in preparing a reagent or a kit for prognosis evaluation, curative effect judgment and recurrence monitoring of solid tumors; the solid tumor is gastric cancer, liver cancer, pancreatic cancer, glioblastoma, brain low-grade glioma or mesothelioma.
The invention also provides application of the detection reagent of the SLC6A6 gene or the coded protein thereof in preparing a reagent or a kit for prognosis evaluation, curative effect judgment and recurrence monitoring of solid tumors; the solid tumor is gastric cancer, liver cancer, pancreatic cancer, glioblastoma, brain low-grade glioma or mesothelioma.
Alternatively, the reagent or kit detects transcribed messenger RNA of SLC6A6 gene or protein encoded by SLC6A6 gene by RT-PCR, real-time quantitative PCR, digital PCR, fluorescent dye method, resonance light scattering method, sequencing or biological mass spectrometry, in situ hybridization, northern blotting, chip, high throughput sequencing platform, immunohistochemistry or enzyme-linked immunosorbent assay. Further alternatively, the reagent or the kit contains a specific primer for amplifying the SLC6A6 gene, a probe hybridized with the nucleotide sequence of the SLC6A6 gene or an antibody fragment specifically combined with SLC6A 6. Alternatively, the antibody is a monoclonal or polyclonal antibody.
Alternatively, the test sample of the reagent or the kit is serum, plasma, cells, cell culture supernatant, urine, tissue or tissue lysate.
The invention also provides application of the SLC6A6 gene or the coded protein thereof serving as a target spot in preparing a medicine for treating solid tumors, wherein the solid tumors are gastric cancer, liver cancer, pancreatic cancer, glioblastoma, brain low-grade glioma or mesothelioma.
The invention further provides application of the SLC6A6 gene expression inhibitor in preparing medicines for treating solid tumors; the SLC6A6 gene expression inhibitor is selected from CRISPR gene editing therapeutic drugs or antisense nucleic acid drugs for blocking the normal transcription or posttranscriptional translation process of SLC6A6 genes by CRISPR/Cas9 gene editing technology or RNA interference technology; the solid tumor is gastric cancer, liver cancer, pancreatic cancer, glioblastoma, brain low-grade glioma or mesothelioma.
The invention also provides a construction method of the solid tumor cell research model, which comprises the following steps: knocking out the SLC6A6 gene in the solid tumor cells; the solid tumor is gastric cancer, liver cancer, pancreatic cancer, glioblastoma, brain low-grade glioma or mesothelioma. Alternatively, solid tumor cells are cultured in a culture medium, and then SLC6A6 genes in the solid tumor cells are knocked out to obtain the solid tumor cells. Alternatively, the CRISPR/Cas9 technology is adopted to knock out the SLC6A6 gene in the solid tumor cells to obtain the solid tumor cells.
The invention further provides a construction method of the solid tumor animal model, which comprises the following steps: and injecting the solid tumor cells constructed by the method into an animal model to construct a solid tumor animal model.
Drawings
FIG. 1 is a statistical analysis of SLC6A6 protein expression level and clinical significance thereof in gastric cancer tissues and paracancerous normal tissues of gastric cancer patients in example 1: FIG. 1A is an immunohistochemical staining micrograph of SLC6A6 protein in gastric cancer tissue in patients with gastric cancer of representative clinical stage; FIG. 1B is a quantitative statistical plot of SLC6A6 protein expression in gastric cancer tissue and paracancerous normal gastric tissue of 167 gastric cancer patients; FIG. 1C is a graph showing analysis of total survival of 167 gastric cancer patients divided into SLC6A6 low expression and SLC6A6 high expression groups according to the median of quantitative values of SLC6A6 protein expression in gastric cancer tissues; FIG. 1D is a graph of the results of one-and multi-factor Cox regression analysis affecting the postoperative survival of gastric cancer patients.
FIG. 2 is a statistical analysis of the correlation between SLC6A6 protein expression level and disease progression after chemotherapy in gastric cancer patients in example 2: FIG. 2A is a IHC staining micrograph of SLC6A6 protein expression in a representative 4-case Post-chemotherapy gastric cancer tissue (Post) and its paired Pre-chemotherapy gastric cancer tissue (Pre) of 31 cases of gastric cancer patients undergoing Post-chemotherapy disease progression; FIG. 2B is a statistical graph of SLC6A6 protein expression levels in post-chemotherapy gastric cancer tissues and paired pre-chemotherapy gastric cancer tissues of 31 gastric cancer patients who received post-chemotherapy disease progression.
FIG. 3 is a statistical analysis of the correlation of SLC6A6 protein expression levels with post-operative distant metastasis of gastric cancer patients in example 3: FIG. 3A is a IHC staining micrograph of SLC6A6 protein expression in a representative 4 patients Primary focus tissue (Primary), metastasis tissue (metatasis) and their paired paracancerous Normal tissue (Normal) of 16 patients with distant Metastasis from gastric cancer; FIG. 3B is a quantitative statistical plot of SLC6A6 protein expression in primary foci, metastases and paired paracancerous normal tissues of 16 patients with gastric cancer undergoing distant metastasis.
FIG. 4 is a statistical analysis of SLC6A6 protein expression level and clinical significance thereof in liver cancer tissues and paracancestor normal tissues of a liver cancer patient in example 4: FIG. 4A is an immunohistochemical staining micrograph of SLC6A6 protein in cancer tissue in a patient with a representative liver cancer at different clinical stages; FIG. 4B is a quantitative statistical chart of SLC6A6 protein expression in liver cancer tissue and paracancerous normal tissue of 90 liver cancer patients; FIG. 4C is a graph showing total survival analysis of 90 liver cancer patients divided into SLC6A6 low expression and SLC6A6 high expression groups according to the median of quantitative values of SLC6A6 protein expression in liver cancer tissues; FIG. 4D is a quantitative statistical graph of SLC6A6 protein expression in liver cancer tissues of 90 liver cancer patients divided into recurrent and non-recurrent groups according to follow-up data.
FIG. 5 is a statistical analysis of SLC6A6 protein expression level and clinical significance thereof in pancreatic cancer tissues and paracancestor normal tissues of pancreatic cancer patients in example 5: FIG. 5A is an immunohistochemical staining micrograph of the SLC6A6 protein in cancerous tissue in patients with pancreatic cancer of a representative clinical stage; FIG. 5B is a quantitative statistical plot of SLC6A6 protein expression in pancreatic cancer tissue and paracancerous normal tissue of 90 pancreatic cancer patients; FIG. 5C is a graph showing total survival analysis of 90 pancreatic cancer patients divided into SLC6A6 low expression and SLC6A6 high expression according to the median of quantitative values of SLC6A6 protein expression in pancreatic cancer tissues; FIG. 5D is a quantitative statistical plot of SLC6A6 protein expression in pancreatic cancer tissues of 47 pancreatic cancer patients, divided into recurrent and non-recurrent groups according to their follow-up data.
FIG. 6 is a correlation analysis of SLC6A6 gene expression with cancer patient survival by TCGA database analysis in example 6: FIG. 6A is a graph of total survival analysis of patients with SLC6A6 gene low and high expression of gastric cancer, liver cancer, pancreatic cancer, glioblastoma, brain low grade glioma and mesothelioma; FIG. 6B is a graph of progression free survival analysis of patients with SLC6A6 gene low and high expression of gastric cancer, liver cancer, pancreatic cancer, glioblastoma, low-grade glioma and mesothelioma.
FIG. 7 is a graph showing the results of the expression level of SLC6A6 protein and mRNA in human gastric normal epithelial cells GES-1 and human gastric cancer cells AGS, SGC7901, NCl-N87, SNU1, BGC823, MKN45, SGC7901 ADR、SGC7901VCR of example 7.
FIG. 8 is a result of construction of SLC6A6 knockout gastric cancer cells using CRISPR/Cas9 genome editing in example 8; FIG. 8A is a graph showing the protein expression of SLC6A6 in two monoclonal cell lines (KO-1 and KO-2) of human gastric cancer cells SGC7901 VCR and MKN45, in which the SLC6A6 gene was knocked out, as verified by Western Blotting (WB) in example 8; FIG. 8B is a graph of proliferation of SGC7901 VCR and MKN45 cells with SLC6A6 knockdown and control;
FIG. 8C is a statistical plot of migration numbers of SLC6A6 knock-out and control SGC7901 VCR and MKN45 cells; FIG. 8D is a statistical plot of the number of apoptosis in SGC7901 VCR and MKN45 cells under fluorouracil (Fluorouracil) treatment conditions for SLC6A6 knockout and control.
FIG. 9 is a graph showing the effect of down-regulating SLC6A6 gene on gastric cancer cell MFC in example 9; FIG. 9A is a diagram showing the protein expression of SLC6A6 in two monoclonal cell lines (KO-1 and KO-2) from which the SLC6A6 gene was knocked out in the WB-verified mouse gastric cancer cell MFC of example 9; FIG. 9B is a graph showing tumor growth of MFC cells from the Slc6a6 knockout and control after subcutaneous tumor formation in 615 mice; fig. 9C is a statistical plot of tumor weights of MFC cells from the Slc6a6 knockout and control after subcutaneous tumor formation in 615 mice.
Detailed Description
Unless otherwise indicated, the terms or methods herein are understood or implemented using existing methods based on knowledge of one of ordinary skill in the relevant art.
Solute carrier family 6 member 6 gene SLC6A6 is a specific transporter of taurine on a cytoplasmic membrane, consists of 620 amino acid residues, and comprises 12 hydrophobic transmembrane domains. SLC6A6 uses the potential energy of sodium ion concentration gradients across the cytoplasmic membrane established by sodium pumping activity to transport taurine into cells. The SLC6A6 gene resides on human chromosome 3 and has a sequence region of about 86kbp, comprising 19 exons, and 3 transcriptional variants encoding 3 different protein subtypes.
The clinical staging basis of solid tumors is as follows: TNM staging System published by the United states cancer Congress tumor staging Manual (AJCC, american Joint Committee on Cancer) is divided into I, II, III, IV stages.
The invention relates to application of a detection reagent of SLC6A6 gene or coded protein thereof in preparation of a reagent or a kit for prognosis evaluation, curative effect judgment and recurrence monitoring of solid tumors, wherein the increase of the expression level of the SLC6A6 gene or coded protein thereof is an indication of poor prognosis, poor curative effect and easy recurrence of the solid tumors. Wherein the meaning of the prognostic evaluation includes or is understood to be an estimate of the future time to live or likely outcome of the patient; the meaning of a therapeutic benefit judgment includes or can be understood as assessing the effectiveness of a patient for a treatment regimen; the meaning of recurrence monitoring includes or can be understood as assessing the likelihood of disease progression or recurrence in a patient after treatment.
The term "detection reagent for SLC6A6 gene or a protein encoded thereby" in the present invention shall not be construed as merely being a detection reagent for SLC6A6 gene or a protein encoded thereby, but shall include detection reagents known to those skilled in the art which reflect the expression level of the detection reagent for SLC6A6 gene or a protein encoded thereby. For example, the amount of mRNA expression can be indirectly detected by quantitatively detecting cDNA obtained by reverse transcription of the SLC6A6 gene.
The detection reagent may be any reagent known to those skilled in the art, for example, a nucleic acid which hybridizes to the RNA and is labeled with a fluorescent label; the detection agent of RNA can be selected from primers of RT-PCR and primers for amplifying cDNA, which is the product of RT-PCR; in some embodiments, the detection reagent comprises a reagent suitable for use in at least one of the following methods: real-time quantitative PCR, digital PCR, fluorescent dye method, resonance light scattering method, sequencing or biological mass spectrometry.
The term "antibody" includes polyclonal antibodies as well as monoclonal antibodies, and the term "antibody fragment" includes antigen compound binding fragments of such antibodies, including Fab, F (ab') 2, fd, fv, scFv, bispecific antibodies, and antibody minimal recognition units, as well as single chain derivatives of such antibodies and fragments, such as scFv-Fc, and the like. The type of antibody may be selected from IgG1, igG2, igG3, igG4, igA, igM, igE, igD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional), humanized (humanized) antibodies, and human antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".
In some embodiments, the measurement sample comprises blood (whole blood), serum, plasma, cell culture supernatant, urine, tissue, or tissue lysate.
The SLC6A6 gene expression inhibitor refers to an agent, a preparation or a medicament for blocking the normal transcription or posttranscriptional translation process of SLC6A6 gene based on CRISPR/Cas9 gene editing technology or RNA interference technology, such as CRISPR gene editing therapeutic medicament, antisense nucleic acid medicament, siRNA medicament, miRNA medicament and the like.
The SLC6A6 protein expression inhibitor refers to an agent, a preparation or a medicament which influences the post-translational modification process of SLC6A6 protein or the stability of SLC6A6 protein and influences the expression level, activity or function of SLC6A6 protein, such as an protein glycosylation inhibitor, a protein phosphorylation inhibitor, a neutralizing antibody and the like.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
Gastric cancer is a cancer that occurs in the gastric mucosa, and most gastric cancers belong to adenocarcinoma. In the following examples, stomach cancers not specifically described are gastric adenocarcinoma. However, the research result of the present invention is not limited to gastric cancer, and according to the conventional knowledge of those skilled in the art, the related applications can be related to solid tumors including gastric cancer, liver cancer, pancreatic cancer, glioblastoma, brain low-grade glioma, mesothelioma, etc.
The following examples were conducted under conventional conditions or conditions recommended by the manufacturer, without specifying the specific conditions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: multicolor immunohistochemical analysis of SLC6A6 protein expression in cancer tissue and paracancerous Normal tissue of gastric cancer patient 1. Subject
167 Cases of gastric cancer clinical samples are taken from the Beijing digestive disease hospital, and the study is passed through the ethical committee of the Beijing hospital at the air force medical university, and the patients and the family members sign informed consent.
2. Experimental method
The expression and spatial distribution of SLC6A6 protein on GC tissues are detected by a multicolor immunohistochemical (multiplex-IHC) method, and the specific steps are as follows:
Placing the pathological paraffin sections into an oven at 60-65 ℃ for 2 hours, and then dewaxing; placing the sections in 10% formalin to fix the sections for 10min, and increasing the adhesion of the tissue sections; placing the fixed slice in antigen retrieval liquid for retrieval for 15min, cooling to room temperature, and cleaning a slide by using ddH 2 O; placing the trimmed slice in 3% H 2O2 water solution for 15min, and removing endogenous peroxidase; dripping blocking buffer (Akoya Biosciences) in the tissue sample area, ensuring that the tissue area can be completely covered, and placing in a moisturizing box for incubation for 10min; dripping diluted primary antibody working solution (rabbit anti-human SLC6A6 monoclonal antibody, abcam,1:200; mouse anti-human pan-cytokeratin, CELL SIGNALING Technology, 1:200), immersing a tissue region, and placing the tissue region into a moisturizing box at room temperature for incubation for 1h; removing residual liquid after TBST cleaning, dripping an opal polymer horseradish peroxidase (HRP) coupled donkey anti-rabbit IgG (Akoya Biosciences) into a slide, and incubating for 10min in a moisturizing mode; dropwise adding a chromogenic working solution (Opal 520,Opal 570,Opal 620,and Opal 690) on the glass slide to submerge the tissue region for incubation for 10min; microwave treatment, namely removing the primary antibody and the secondary antibody, ending the single dyeing till the end, and counterstaining the subsequent indexes until all antigens are marked; the slides were washed with TBST solution, washed with ddH 2 O, the wash solution was discarded, DAPI (Akoya Biosciences, 1:5) working solution was added dropwise to the slides, incubated for 5min with moisture, then fluorescent anti-quenching coverslipping was added dropwise for coverslipping, coverslipping was performed, images of the tissue sample areas of the slides were collected by a Vectra multispectral imaging system using nail polish, and quantitative statistical analysis was performed on the images using inForm software.
For survival analysis, P-values between groups were calculated using a log rank sum test; and drawing a survival curve by using a Kaplan-Meier method.
For single and multi-factor analysis, a Cox regression analysis was used to estimate the risk ratio and was expressed in 95% confidence intervals; univariate analysis found statistically significant variables that were further scrutinized in multivariate analysis.
3. Conclusion of the experiment
FIG. 1 is a graph showing the results of SLC6A6 protein expression in gastric cancer tissue (GC) and paracancerous Normal tissue (Normal) of a gastric cancer patient; FIG. 1A is an immunohistochemical staining micrograph of SLC6A6 protein in cancer tissue in patients with gastric cancer represented by different clinical stages (Stage); FIG. 1B is a statistical chart of quantitative (H-score) expression of SLC6A6 protein in gastric cancer tissue and paracancerous normal tissue of 167 gastric cancer patients; FIG. 1C is a graph showing the analysis of the total survival curve (Overall survivin) of 167 gastric cancer patients, after dividing them into SLC6A6 low expression (SLC 6A6 low) and SLC6A6 high expression (SLC 6A6 high) groups according to the median of the quantitative values of SLC6A6 protein expression in gastric cancer tissues;
FIG. 1D is a graph of the results of one-and multi-factor Cox regression analysis affecting the postoperative survival of gastric cancer patients. The above results indicate that SLC6A6 protein expression is higher in gastric cancer tissues than in paracancerous normal tissues and increases with stage progression of gastric cancer; patients with gastric cancer with high SLC6A6 expression have a shorter overall survival than patients with SLC6A6 expression; the SLC6A6 protein expression level in gastric cancer tissues is an independent factor for judging prognosis of gastric cancer patients.
Example 2: multicolor immunohistochemical analysis of SLC6A6 protein expression in gastric cancer chemotherapy drug resistant tissue
1. Study object
31 Cases of clinical samples of patients with gastric cancer chemotherapy resistance are taken from the Beijing digestive disease hospital, and the study is passed through the ethical committee of the Beijing hospital at the medical university of air force, and the patients and family members sign informed consent.
2. Experimental method
The expression and spatial distribution of SLC6A6 protein on gastric cancer tissue was examined by multiplex-IHC using the procedure described in example 1.
3. Conclusion of the experiment
FIG. 2A is a IHC staining micrograph of SLC6A6 protein expression in Post-chemotherapy gastric cancer tissue (Post) and its paired Pre-chemotherapy gastric cancer tissue (Pre) of a representative 4 gastric cancer patients (Pt#1-4) undergoing Post-chemotherapy disease progression;
FIG. 2B is a quantitative statistical graph of SLC6A6 protein expression in post-chemotherapy gastric cancer tissue and paired pre-chemotherapy gastric cancer tissue of 31 gastric cancer patients who received post-chemotherapy disease progression. The results show that SLC6A6 protein expression in gastric cancer tissues is higher after chemotherapy resistance than before chemotherapy resistance.
Example 3: multicolor immunohistochemical analysis of SLC6A6 protein expression in stomach cancer metastasis clinical tissue
1. Study object
Clinical samples of patients with metastatic disease were taken from the Beijing digestive disease hospital, and the study was approved by the ethics committee of the Beijing hospital at the air force university of medicine, and the patients and family members signed informed consent.
2. Experimental method
The expression and spatial distribution of SLC6A6 protein on gastric cancer tissue was examined by multiplex-IHC using the procedure described in example 1.
3. Conclusion of the experiment
FIG. 3A is a multiplex-IHC staining micrograph of SLC6A6 protein expression in Primary tissue (Primary), metastatic tissue (metatasis) and paracancerous Normal tissue (Normal) of a representative 4 patients with gastric cancer with distant Metastasis, wherein Pt#1colon Metastasis, pt#2liver Metastasis, pt#3ovary Metastasis and Pt#4adnexa Metastasis represent: gastric cancer patients with colon metastasis, liver metastasis, ovarian metastasis and uterine accessory metastasis; FIG. 3B is a semi-quantitative statistical plot of SLC6A6 protein expression in primary foci, metastases and paired paracancerous normal tissues of 16 patients with distant metastasis, wherein Negative, weak, moderate and Strong represent negative, weak-positive, medium-positive and Strong-positive SLC6A6 expression, respectively. The above results indicate that SLC6A6 protein expression is increased in stomach cancer primary tissue and further increased in metastatic tissue, as compared to paracancerous normal stomach tissue.
Example 4: multicolor immunohistochemical analysis of SLC6A6 protein expression in cancer tissue and paracancerous normal tissue of liver cancer patient
1. Study object
Microarray chips containing 90 cases of liver cancer tissue and paracancerous normal liver tissue were purchased from Shanghai core Biotechnology Inc.
2. Experimental method
The expression and spatial distribution of SLC6A6 protein on liver cancer tissue and normal liver tissue beside cancer were detected by multiplex-IHC method, and survival curve was drawn by Kaplan-Meier method, and the specific procedure was the same as in example 1.
3. Conclusion of the experiment
FIG. 4 is a graph showing the results of SLC6A6 protein expression in liver cancer tissue (LIHC) and paracancerous Normal tissue (Normal) of a liver cancer patient; FIG. 4A is an immunohistochemical staining micrograph of SLC6A6 protein in cancer tissue in patients with liver cancer represented by different clinical stages (Stage); FIG. 4B is a statistical chart of quantitative (H-score) expression of SLC6A6 protein in liver cancer tissue and paracancerous normal tissue of 90 liver cancer patients; FIG. 4C is a graph showing total survival (over survival) curves of 90 liver cancer patients, after dividing them into SLC6A6 low-expression (SLC 6A6 low) and SLC6A6 high-expression (SLC 6A6 high) groups according to the median of quantitative values of SLC6A6 protein expression in liver cancer tissues;
FIG. 4D is a quantitative statistical graph of SLC6A6 protein expression in liver cancer tissues of 90 liver cancer patients divided into recurrent (Recurrence Yes) and non-recurrent (Recurrence No) groups according to follow-up data. The result shows that the expression of SLC6A6 protein in liver cancer tissue is higher than that of adjacent normal tissue, and the expression of SLC6A6 protein is increased along with the tumor stage progress; the total survival time of SLC6A6 high expression liver cancer patients is shorter than that of SLC6A6 low expression patients; SLC6A6 protein expression is higher in liver cancer tissues of recurrent patients than in liver cancer tissues of non-recurrent patients.
Example 5: multicolor immunohistochemical analysis of SLC6A6 protein expression in pancreatic cancer patient cancer tissue and paracancerous normal tissue
1. Study object
Microarray chips containing 90 cases of pancreatic cancer tissue and paracancerous normal pancreatic tissue were purchased from Shanghai core Biotechnology Inc.
2. Experimental method
The expression and spatial distribution of SLC6A6 protein on pancreatic cancer tissues and paracancerous normal pancreatic tissues were detected by multiplex-IHC, and survival curves were drawn by Kaplan-Meier method, and the specific procedure was the same as in example 1.
3. Conclusion of the experiment
FIG. 5 is a graph showing the results of SLC6A6 protein expression levels in pancreatic cancer tissue (PAAD) and paracancerous Normal tissue (Normal); FIG. 5A is an immunohistochemical staining micrograph of SLC6A6 protein in cancerous tissue in patients with pancreatic cancer represented by different clinical stages (Stage); FIG. 5B is a statistical plot of the quantitative (H-score) expression of SLC6A6 protein in 90 pancreatic cancer patient cancer tissues and paracancerous normal tissues; FIG. 5C is a graph showing total survival analysis of 90 pancreatic cancer patients, divided into SLC6A6 low expression (SLC 6A6 low) and SLC6A6 high expression (SLC 6A6 high) according to the median of quantitative values of SLC6A6 protein expression in pancreatic cancer tissues; FIG. 5D is a quantitative statistical plot of SLC6A6 protein expression in pancreatic cancer tissues of 47 pancreatic cancer patients, divided into recurrent (Recurrence Yes) and non-recurrent (Recurrence No) groups, according to their follow-up data. The result shows that the expression of SLC6A6 protein in pancreatic cancer tissues is higher than that of adjacent normal tissues, and the expression of SLC6A6 protein is increased along with the tumor stage progression; the total survival time of SLC6A6 high-expression pancreatic cancer patients is shorter than that of SLC6A6 low-expression patients; SLC6A6 protein expression is higher in pancreatic cancer tissue in relapsed patients than in pancreatic cancer tissue in non-relapsed patients.
Example 6: relationship between expression and survival of SLC6A6 Gene in different tumor tissues
1. Study object
And analyzing the relation between the expression and the survival time of the SLC6A6 gene in stomach cancer, liver cancer, pancreatic cancer, glioblastoma, brain low-grade glioma and mesothelioma tissues according to the related data in the TCGA database.
2. Analysis method
For survival analysis, P-values between groups were calculated using a log rank sum test; and drawing a survival curve by using a Kaplan-Meier method.
3. Conclusion of the experiment
FIG. 6 is a graph showing the overall and progression free survival of patients with low and high expression of the SLC6A6 gene (STAD), liver cancer (LIHC), pancreatic cancer (PAAD), glioblastoma (GBM), low Grade Glioma (LGG) and Mesothelioma (MESO) as a result of TCGA data analysis. FIG. 6A is a graph of total survival analysis of patients with SLC6A6 gene low expression (SLC 6A6 low) and SLC6A6 gene high expression (SLC 6A6 high) of gastric cancer, liver cancer, pancreatic cancer, glioblastoma, low-grade glioma and mesothelioma; fig. 6B is a graph of progression free survival analysis of patients with low and high expression of the SLC6A6 gene in gastric cancer, liver cancer, pancreatic cancer, glioblastoma, low-grade brain glioma and mesothelioma, wherein the partitioning basis for low and high expression of the SLC6A6 gene is: score value given in best score algorithm. The results show that the total survival and progression free survival of SLC6A6 highly expressed tumor patients are shorter than those of SLC6A6 low expressed tumor patients.
Example 7: detection of SLC6A6 expression in different gastric cancer cell lines by Western Blotting (WB) and real-time fluorescent quantitative PCR (Quantitative real-time PCR, RT-qPCR)
1. Study object
The gastric cancer cell strains MKN45, AGS, GES, NCl-N87, BGC823 and SNU1 are all introduced from a Chinese microbial strain cell library and are preserved by the national key laboratory for digestive system tumor integration prevention and control of air force medical university; SGC7901 cells are purchased from Shanghai research biochemistry reagent limited company, and drug resistant cells SGC7901 ADR and SGC7901 VCR are selected and constructed by exposing SGC7901 cells to chemotherapeutic drugs by the national key laboratory for digestive system tumor integration control of air force university of medical science.
2. Experimental method
The WB is adopted to detect the expression level of SLC6A6 protein in MKN45, AGS, GES, NCl-N87, BGC823, SNU1, SGC7901 and SGC7901 ADR、SGC7901VCR cells, and the specific steps are as follows:
Extracting proteins from cells with RIPA buffer containing protease inhibitor (Roche) when the 9 cell densities increased to 80%; SDS-PAGE gel electrophoresis is adopted, and then the mixture is transferred to a nitrocellulose membrane; following hybridization with a primary antibody to β -actin and SLC6A6 (rabbit anti-human SLC6A6 mab, abcam,1:1000; mouse anti-human β -actin mab, santa Cruz Biotechnology, 1:2000) and a secondary enzyme-labeled antibody (donkey anti-rabbit, sheep anti-mouse IgG, GE Healthcare Life Sciences, 1:5000) according to manufacturer's recommended dilutions, development was performed as described by chemiluminescent detection kit (Pierce) using a Bio-Rad ChemiDoc xrs+ imaging system to display protein bands.
The expression level of the SLC6A6 gene in MKN45, AGS, GES, NCl-N87, BGC823, SNU1, SGC7901 and SGC7901 ADR、SGC7901VCR cells is detected by adopting RT-qPCR, and the specific steps are as follows:
Extracting total RNA when the density of 9 cells is increased to 80%; then reverse transcribed into cDNA using PRIMESCRIPT RT kit (Takara); qPCR was performed on a real-time PCR detection system (Bio-Rad) using SYBR Premix Ex-Taq kit (Takara). The primer sequences for each PCR reaction are shown in Table 1.
TABLE 1 RT-qPCR primer sequences
3. Conclusion of the experiment
FIG. 7 is a graph showing the results of the expression levels of SLC6A6 protein and mRNA in human gastric normal epithelial cells GES-1 and human gastric cancer cells AGS, SGC7901, NCl-N87, SNU1, BGC823, MKN45, SGC7901 ADR、SGC7901VCR. As can be seen from fig. 7, SLC6A6 expression was elevated in most gastric cancer cells compared to the normal gastric epithelial cell line GES-1; in particular, higher SLC6A6 expression levels were observed in two multidrug resistant cell lines SGC7901 ADR and SGC7901 VCR, whereas both cell lines were chemoresistant and strongly invasive.
Example 8: construction of SLC6A6 gene knockout gastric cancer cells by CRISPR/Cas9 genome editing technology and evaluation of malignant biological behaviors thereof
1. Study object
SGC7901 VCR and MKN45 gastric cancer cells were as in example 7.
2. Experimental method
The gastric cancer cells SGC7901 VCR and MKN45 are respectively used as target cells, a CRISPR/Cas9 technology is adopted to construct a cell line for stably knocking out SLC6A6 gene and verify, two successfully constructed monoclonal cell lines (KO-1 and KO-2) are adopted to detect the proliferation, migration and the change of chemotherapy drug sensitivity of the cells, and the specific experimental steps are as follows:
Using CRISPRDIRECT to design the sgRNA target sequence, cloning the sgRNA target sequence into the grna_gfp-T1 plasmid to construct a Cas9 single vector plasmid, transfecting and sorting the designated cells, and validating knockout efficiency by WB. Control cells and lentivirus transfected cells were plated in 96-well plates, medium containing CCK8 reagent (Dojindo) was changed at 12, 24, 36, 48, 60h, and after incubation for 2h, absorbance values of the supernatants were measured using an enzyme-labeled instrument at 450nm to determine proliferation of the cells. Cells grown to the logarithmic phase were resuspended to 5×10 5/ml and inoculated into a Transwell chamber having 8 μm microwells (Millipore), the bottom chamber was filled with a medium containing 20% fetal bovine serum, and after culturing for 12 hours, the cells remaining on the upper surface of the membrane were removed, the cells adhered to the lower surface of the membrane were fixed, stained with crystal violet, and counted under a microscope to examine the cell migration ability. Spreading control group cells and lentivirus transfected cells into a culture dish, adding fluorouracil chemotherapeutic drugs for continuous culture, collecting the treated gastric cancer cells, re-suspending and detecting by using an Annexin V-FITC apoptosis detection kit (Invitrogen), detecting fluorescence intensities of Annexin V-FITC and propylene iodide by using a Coulter Epics XL-MCL flow cytometer (Beckman), and performing data analysis by using EXPO32 ADC software; annexin V-FITC-stained positive cells are considered to undergo apoptosis. The target sequence of sgrnas is shown in table 2.
TABLE 2 sgRNA sequence targeting SLC6A6 Gene
3. Conclusion of the experiment
FIG. 8A is a graph showing the verification of SLC6A6 protein expression in two monoclonal cell lines (KO-1 and KO-2) from SLC6A6 knockdown in human gastric cancer cells SGC7901 VCR and MKN45 by WB; FIG. 8B is a graph of proliferation of SGC7901 VCR and MKN45 cells with SLC6A6 knockdown and control; FIG. 8C is a statistical plot of migration numbers of SLC6A6 knock-out and control SGC7901 VCR and MKN45 cells; FIG. 8D is a statistical plot of the number of apoptosis in SGC7901 VCR and MKN45 cells under fluorouracil (Fluorouracil) treatment conditions for SLC6A6 knockout and control. The result shows that the sgRNA successfully induces SLC6A6 gene cutting and defective repair, resulting in SLC6A6 protein expression deletion; down-regulating the SLC6A6 gene (SLC 6A6 KO1, SLC6A6 KO 2) can inhibit cell proliferation, metastasis and invasion capacity, and can increase sensitivity to chemotherapeutic drugs.
Example 9: down-regulating influence of SLC6A6 gene on gastric cancer cell MFC (MFC) neoplasia
1. Study object
MFC was drawn from the american type culture collection (AMERICAN TYPE culture collection, ATCC) and deposited by the national emphasis laboratory for digestive system tumor control at the air force medical university. Female 615 mice, 4-6 weeks old, were purchased from Shanghai laboratory animal research centers.
2. Experimental method
Taking gastric cancer cell MFC as a target cell, constructing a cell line (KO-1 and KO-2) for stably knocking out the Slc6a6 gene by using a CRISPR/Cas9 technology, verifying, subcutaneously injecting 1 monoclonal cell line (Slc 6a6 KO1) successfully constructed into a mouse, and detecting the cell tumorigenesis condition, wherein the specific experimental steps are as follows:
The procedure for constructing and verifying a cell line with stable knockdown of the Slc6a6 gene was the same as in example 8. Female 615 mice of 4-6 weeks of age were selected and inoculated subcutaneously with 5-10 x 10 5 Slc6a6 KO1 cells and control MFC cells; after reaching the tumor growth end point, the mice are sacrificed, and the subcutaneous tumor measurement volume is taken out and weighed; the tumor volume is measured along a maximum axis (L) and a diameter (W) at right angles to this axis, calculated as follows: tumor volume= (l×w 2)/2.
3. Conclusion of the experiment
FIG. 9A is a diagram showing the verification of SLC6A6 protein expression in two monoclonal cell lines (KO-1 and KO-2) from which the Slc6A6 gene knocked out in mouse gastric cancer cell MFC; fig. 9B is a tumor growth curve of MFC cells of the Slc6a6 knockout (Slc 6a6 KO1) and control after subcutaneous tumor formation in 615 mice, with differences significant (P < 0.01); fig. 9C is a statistical plot of tumor weights of the MFC cells of the Slc6a6 knockout (Slc 6a6 KO1) and the control after subcutaneous tumor formation in 615 mice, with significant differences (P < 0.01). The result shows that sgRNA successfully induces the cutting and defective repair of the Slc6A6 gene, resulting in the deletion of SLC6A6 protein expression in MFC cells; the Slc6a6 gene knockout inhibited the growth of 615 mouse subcutaneous tumors compared to the non-knocked-out group, manifested as a reduction in tumor volume and tumor weight.
Claims (12)
- Application of SLC6A6 gene or coded protein thereof as target in preparation of reagent or kit for prognosis evaluation, curative effect judgment and recurrence monitoring of solid tumor; the solid tumor is gastric cancer, liver cancer, pancreatic cancer, glioblastoma, brain low-grade glioma or mesothelioma.
- Application of SLC6A6 gene or detection reagent of coded protein in preparing reagent or reagent kit for prognosis evaluation, curative effect judgment and recurrence monitoring of solid tumor; the solid tumor is gastric cancer, liver cancer, pancreatic cancer, glioblastoma, brain low-grade glioma or mesothelioma.
- 3. The use according to claim 1, wherein the reagent or kit detects transcribed messenger RNA of the SLC6A6 gene or a protein encoded by the SLC6A6 gene by RT-PCR, real-time quantitative PCR, digital PCR, fluorescent dye method, resonance light scattering method, sequencing or bio mass spectrometry, in situ hybridization, northern blotting, chip, high throughput sequencing platform, immunohistochemistry or enzyme linked immunosorbent assay.
- 4. The use according to claim 1, wherein said reagent or kit comprises specific primers for amplifying the SLC6A6 gene, probes hybridizing to the nucleotide sequence of the SLC6A6 gene or antibodies or antibody fragments specifically binding to SLC6 A6.
- 5. The use according to claim 4, wherein the antibody is a monoclonal or polyclonal antibody.
- 6. The use according to any one of claims 1 to 5, wherein the test sample of the reagent or kit is serum, plasma, cells, cell culture supernatant, urine, tissue or tissue lysate.
- The application of SLC6A6 gene or its coded protein as target in preparing medicine for treating solid tumor, such as gastric cancer, liver cancer, pancreatic cancer, glioblastoma, brain low-grade glioma or mesothelioma.
- Application of SLC6A6 gene expression inhibitor in preparing medicine for treating solid tumor; the SLC6A6 gene expression inhibitor is selected from CRISPR gene editing therapeutic drugs or antisense nucleic acid drugs for blocking the normal transcription or posttranscriptional translation process of SLC6A6 genes by CRISPR/Cas9 gene editing technology or RNA interference technology; the solid tumor is gastric cancer, liver cancer, pancreatic cancer, glioblastoma, brain low-grade glioma or mesothelioma.
- 9. The method for constructing the research model of the solid tumor cells is characterized by comprising the following steps of: knocking out the SLC6A6 gene in the solid tumor cells; the solid tumor is gastric cancer.
- 10. The method for constructing a research model of solid tumor cells according to claim 9, wherein the solid tumor cells are cultured in a culture medium, and then SLC6A6 genes in the solid tumor cells are knocked out to obtain the solid tumor cells.
- 11. The method of claim 10, wherein the solid tumor cells are obtained by knocking out the SLC6A6 gene from the solid tumor cells using CRISPR/Cas9 technology.
- 12. A method for constructing an animal model of a solid tumor, the method comprising: injecting the solid tumor cells constructed by the method of claim 9, 10 or 11 into an animal model to construct a solid tumor animal model.
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