CN117599078A - Cancer marker PANX1 and application thereof - Google Patents

Cancer marker PANX1 and application thereof Download PDF

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CN117599078A
CN117599078A CN202311285902.XA CN202311285902A CN117599078A CN 117599078 A CN117599078 A CN 117599078A CN 202311285902 A CN202311285902 A CN 202311285902A CN 117599078 A CN117599078 A CN 117599078A
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panx1
cancer
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谢克平
蒋朦朦
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South China University of Technology SCUT
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Abstract

The invention discloses a cancer marker PANX1 and application thereof, wherein the cancer comprises pancreatic duct adenocarcinoma, and the marker is PANX1 gene and/or an expression product of the PANX1 gene. The PANX1 gene is remarkably and highly expressed in pancreatic duct adenocarcinoma, can be used for early diagnosis of pancreatic duct adenocarcinoma, and has the advantages of high specificity and sensitivity; the high-expression PANX1 gene is closely related to the poor prognosis of the pancreatic duct adenocarcinoma, and the PANX1 gene can be used as a prognosis marker to provide effective information for the prognosis evaluation and treatment effect detection of the pancreatic duct adenocarcinoma. The invention discloses an expression inhibitor of PANX1 genes, which is siRNA of PANX1, has good interference effect on the expression of the PANX1 genes, has application potential of clinical gene therapy, and is beneficial to further research on gene functions.

Description

Cancer marker PANX1 and application thereof
Technical Field
The invention belongs to the field of tumor molecular biology, and particularly relates to a cancer marker PANX1 and application thereof.
Background
Pancreatic ductal adenocarcinoma is a highly fatal malignancy, one of the common causes of death among cancer patients worldwide, with the incidence being the tenth among male tumor patients and the ninth among female tumor patients, however mortality rates are the fourth. It was counted that in 2018, there were about 45 ten thousand new cases of pancreatic ductal adenocarcinoma and 43 dying cases worldwide. In the 5-year survival data of all cancers diagnosed in 2010-2016, the survival rate of pancreatic ductal adenocarcinomas was lowest, less than 10% and the malignancy was extremely high, integrating all stages of onset. Because of the non-specific or occult clinical symptoms of pancreatic ductal adenocarcinoma, about 80% of patients are already in the non-surgical excision or metastasis stage at the time of diagnosis. Realizing early screening and early diagnosis and treatment of pancreatic duct adenocarcinoma and greatly improving the survival rate of patients.
Annexin Pannexin1 (abbreviated PANX 1) was found expressed in vertebrates as early as 2000 and is mainly distributed in the heart, brain, kidneys, thymus, small intestine, skeletal muscle, prostate, testis, ovary and placenta in humans, which constitute multimeric hemichannels on cell membranes, allowing small molecules such as ATP to enter and exit the cells. Under physiological conditions, PANX1 channels mediate intracellular ATP release, intercellular calcium wave transmission, blood flow regulation, taste transmission, immune response, apoptosis, etc.; under pathological conditions, the traditional Chinese medicine composition is involved in the occurrence and development of diseases such as inflammation, tumor, cerebral ischemia, chronic neuropathic pain, epilepsy, hypertension and the like.
Currently, PANX1 has been found to significantly up-regulate protein and mRNA expression levels in a variety of tumors, including diffuse large B-cell lymphomas, esophageal cancers, glioblastomas, brain gliomas, gastric adenocarcinoma, thymus carcinoma, pancreatic ductal adenocarcinoma, and the like, and is directly related to tumor malignancy and worse prognosis.
Shi et al explored the relationship between colon cancer and iron death by belief analysis, found that PANX1 was down-regulated in colon cancer, and verified this phenomenon by Qrt-pcr experiments. It was suggested that PANX1 expression tendencies in different cancers were not all up-regulated (Shi WK, liu YX, qia XY et al construction and validation of a novel Ferroptosis-rel ated gene signature predictive model in rectal Cancer [ J ]. BMC Genomics,2022, 23:764).
CN105572276 a discloses the use of plasma or serum differential metabolites as pancreatic cancer diagnostic markers. However, the patent needs to use high performance liquid detection technology, which is expensive, but the application only needs immunohistochemical technology to detect PANX1, so that the clinical cost is low.
CN115485287 a discloses the treatment of PANX1 related diseases, the invention of specific peptide fragments around 50AA has therapeutic effects on diseases including fibrosis, melanoma, liver cancer, liver disease, bladder cancer, ischemia, hypertension, ophthalmic diseases or disorders, microbial infection, musculoskeletal disorders, huntington's chorea, sepsis and multiple sclerosis. The disadvantage is that the content range is extremely wide, but the experimental content mainly surrounds in-vitro cell experiments, and no further in-vivo experiments exist.
WO 2022/197869 A2 discloses that PANX1 is overexpressed in novel coronavirus infections, PANXI is overexpressed in cells of the immune system, while caspase expression is increased. Directly inhibiting the expression or functional activity of Mra/protein can prevent disease progression. Covering different disease areas than the present application.
Disclosure of Invention
The invention proves that the PANX1 gene expression in cancer cells is down-regulated to obviously inhibit proliferation, migration and tumorigenesis of the cancer cells. PANX1 expression was detected to begin early in cancer and to increase as the disease progressed, compared to normal tissue.
Therefore, PANX1 can be used as a cancer early diagnosis marker.
The invention aims to provide a cancer diagnosis marker for realizing early screening of cancers and early diagnosis and treatment aiming at poor curative effect of malignant tumor treatment.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides the use of a panx1 inhibitor for the preparation of a medicament for the prevention and/or treatment of cancer.
Further, the PANX1 inhibitor is selected from siRNA targeting PANX 1.
Still further, the PANX 1-targeting siRNA is an oligo nucleic acid Human si2, human si3, mouse si1, mouse si2, and/or Mouse si3;
the Human si2 consists of a single-stranded nucleic acid molecule 1 and a single-stranded nucleic acid molecule 2, wherein the single-stranded nucleic acid molecule 1 consists of a single-stranded RNA molecule shown in SE Q ID NO.1 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 2 consists of a single-stranded RNA molecule shown in SEQ ID NO.2 and 2 dGs, wherein the 2 dGs are positioned at the 3' end;
the Human si3 consists of a single-stranded nucleic acid molecule 3 and a single-stranded nucleic acid molecule 4, wherein the single-stranded nucleic acid molecule 3 consists of a single-stranded RNA molecule shown by SE Q ID NO.3 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 4 consists of a single-stranded RNA molecule shown in SEQ ID NO.4 and dAdC, wherein the dAdC is positioned at the 3' -end;
the Mouse si1 consists of a single-stranded nucleic acid molecule 5 and a single-stranded nucleic acid molecule 6, wherein the single-stranded nucleic acid molecule 5 consists of a single-stranded RNA molecule shown by SE Q ID NO.5 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 6 consists of a single-stranded RNA molecule shown in SEQ ID NO.6 and 2 dT, wherein the 2 dT is positioned at the 3' end;
the Mouse si2 consists of a single-stranded nucleic acid molecule 7 and a single-stranded nucleic acid molecule 8, wherein the single-stranded nucleic acid molecule 7 consists of a single-stranded RNA molecule shown by SE Q ID NO.7 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 8 consists of a single-stranded RNA molecule shown in SEQ ID NO.8 and 2 dT, wherein the 2 dT is positioned at the 3' end;
the Mouse si3 consists of a single-stranded nucleic acid molecule 9 and a single-stranded nucleic acid molecule 10, wherein the single-stranded nucleic acid molecule 9 consists of a single-stranded RNA molecule shown in SEQ ID NO.9 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 10 consists of a single-stranded RNA molecule shown in SEQ ID NO.10 and 2 dT, wherein the 2 dT is positioned at the 3' end.
Further, the cancer is one or more of pancreatic duct cancer, diffuse large B cell lymphoma, esophageal cancer, glioblastoma, brain glioma, gastric adenocarcinoma and thymus cancer.
In a second aspect, the present invention provides a pharmaceutical composition for preventing and/or treating cancer, which comprises a PANX1 inhibitor.
Further, the PANX1 inhibitor is selected from siRNA targeting PANX 1.
Still further, the siRNA targeting PANX1 is an oligomeric nucleic acid Human si2, human si3, mouse si1, mouse si2, and/or Mouse si3;
the Human si2 consists of a single-stranded nucleic acid molecule 1 and a single-stranded nucleic acid molecule 2, wherein the single-stranded nucleic acid molecule 1 consists of a single-stranded RNA molecule shown in SEQ ID NO.1 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 2 consists of a single-stranded RNA molecule shown in SEQ ID NO.2 and 2 dGs, wherein the 2 dGs are positioned at the 3' end;
the Human si3 consists of a single-stranded nucleic acid molecule 3 and a single-stranded nucleic acid molecule 4, wherein the single-stranded nucleic acid molecule 3 consists of a single-stranded RNA molecule shown in SEQ ID NO.3 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 4 consists of a single-stranded RNA molecule shown in SEQ ID NO.4 and dAdC, wherein the dAdC is positioned at the 3' -end;
the Mouse si1 consists of a single-stranded nucleic acid molecule 5 and a single-stranded nucleic acid molecule 6, wherein the single-stranded nucleic acid molecule 5 consists of a single-stranded RNA molecule shown in SEQ ID NO.5 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 6 consists of a single-stranded RNA molecule shown in SEQ ID NO.6 and 2 dT, wherein the 2 dT is positioned at the 3' end;
the Mouse si2 consists of a single-stranded nucleic acid molecule 7 and a single-stranded nucleic acid molecule 8, wherein the single-stranded nucleic acid molecule 7 consists of a single-stranded RNA molecule shown in SEQ ID NO.7 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 8 consists of a single-stranded RNA molecule shown in SEQ ID NO.8 and 2 dT, wherein the 2 dT is positioned at the 3' end;
the Mouse si3 consists of a single-stranded nucleic acid molecule 9 and a single-stranded nucleic acid molecule 10, wherein the single-stranded nucleic acid molecule 9 consists of a single-stranded RNA molecule shown in SEQ ID NO.9 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 10 consists of a single-stranded RNA molecule shown in SEQ ID NO.10 and 2 dT, wherein the 2 dT is positioned at the 3' end.
Further, the cancer is one or more of pancreatic duct cancer, diffuse large B cell lymphoma, esophageal cancer, glioblastoma, brain glioma, gastric adenocarcinoma and thymus cancer.
In a third aspect of the present invention, there is provided a cancer marker which is one or more of PANX1 gene, PANX1mRNA and PANX1 protein.
In a fourth aspect of the invention there is provided the use of at least one of the following A1-A3 in at least one of the following B1-B4:
a1, PANX1 gene; a2, PANX1 mRNA; a3, PANX1 protein;
b1, as a diagnostic marker for cancer, or preparing a product for cancer diagnosis;
b2, as a prognostic marker for cancer, or preparing a product for prognostic assessment of cancer;
b3, preparing a product for monitoring the curative effect of the cancer;
b4, preparing a medicament for treating cancer;
further, the cancer is pancreatic ductal adenocarcinoma.
Further, the cancer also comprises more than one of diffuse large B cell lymphoma, esophageal cancer, glioblastoma, brain glioma, gastric adenocarcinoma and thymus cancer.
Further, the cancer diagnosis includes early cancer diagnosis, remote cancer metastasis diagnosis, and/or cancer lymph node metastasis diagnosis.
Further, the products for cancer diagnosis, products for prognosis evaluation of cancer, and products for monitoring the efficacy of cancer include primers, probes, or antibodies that amplify PANX 1.
Further, the products for cancer diagnosis, products for prognosis evaluation of cancer and products for monitoring the curative effect of cancer comprise chips and kits.
Further, the product for cancer diagnosis, the product for cancer prognosis evaluation, the product for cancer efficacy monitoring include: products for detecting PANX1 gene expression by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization, chip or high throughput sequencing and the like; wherein the products of RT-PCR and real-time quantitative PCR comprise a primer and a probe for specifically amplifying PANX1 genes and detecting PANX1mRNA levels; products of immunoassays include antibodies that specifically bind to PANX1 protein; products of in situ hybridization include probes that hybridize to the nucleic acid sequence of the PANX1 gene; the chip includes a protein chip including an antibody that specifically binds to the PANX1 protein and a gene chip including a probe that hybridizes to a nucleic acid sequence of the PANX1 gene.
Further, the medicine for treating cancer comprises: compounds, bioactive substances, or gene therapy agents that inhibit PANX1 gene expression, induce post-transcriptional gene silencing, inhibit PANX1 protein activity, or degrade PANX1 genes, mRNA, or proteins, and the like. Wherein the bioactive substances include proteins, polypeptides, lipids, etc. Gene therapy drugs include siRNA, miRNA, shRNA or vectors comprising the small molecules described above, and the like.
The technical scheme of the invention has the following advantages:
1. the cancer marker provided by the invention comprises pancreatic duct adenocarcinoma, and the cancer marker is PANX1 gene and/or an expression product of PANX1 gene. Wherein the expression product of the PANX1 gene comprises PANX1mRNA and/or PANX1 protein.
According to the invention, the expression level of the PANX1 Gene (Gene ID: 24145) in pancreatic duct adenocarcinoma tissues is found to be significantly higher than that in normal tissues, so that the correlation between the expression level of the PANX1 Gene and pancreatic duct adenocarcinoma is obvious. The PANX1 gene and/or the expression product of the PANX1 gene are ideal targets for early molecular diagnosis of pancreatic duct adenocarcinoma, provide important clinical information for early screening, early diagnosis and early treatment of pancreatic duct adenocarcinoma, and are beneficial to improving the survival quality of patients and the survival rate of the patients.
The invention further verifies the correlation of PANX1 genes and pancreatic duct adenocarcinoma cells proliferation, migration and tumorigenesis through cytology experiments, and after the RNAi technology is used for interfering the expression of the PANX1 genes, the proliferation, migration and tumorigenicity of the pancreatic duct adenocarcinoma cells are inhibited, which indicates that the PANX1 genes influence the proliferation, migration and tumorigenicity of the pancreatic duct adenocarcinoma cells and participate in the occurrence and development of the pancreatic duct adenocarcinoma, and the PANX1 genes can be used as molecular treatment targets of the pancreatic duct adenocarcinoma and provide basis for clinical targeted treatment and personalized treatment of the pancreatic duct adenocarcinoma.
2. According to the cancer marker, clinical data analysis of cancer shows that the expression level of the PANX1 gene is obviously related to diffuse large B cell lymphoma, esophageal cancer, glioblastoma, brain glioma, gastric adenocarcinoma and thymus cancer (P is less than 0.01), and important clinical basis can be provided for diagnosis or treatment of the cancer by detecting the expression of the PANX1 gene.
3. The PANX1 gene, the PANX1mRNA and the PANX1 protein provided by the invention have obvious expression difference in cancers with different pathological stages, and the Log-rank detection analysis of pancreatic duct adenocarcinoma patients with high expression of the PANX1 gene has a survival period obviously shorter than that of the patients with low expression of the PANX1 gene, so that the expression level of the PANX1 gene is closely related to the poor prognosis of the pancreatic duct adenocarcinoma patients, and the PANX1 gene and/or an expression product thereof can be used as a prognosis marker of the pancreatic duct adenocarcinoma for predicting the prognosis condition of the patients. The PANX1 gene and the expression product thereof can also be used as a treatment target point of pancreatic duct adenocarcinoma, be used for preparing medicaments for treating pancreatic duct adenocarcinoma, assist in clinical treatment of pancreatic duct adenocarcinoma and improve the treatment effect of the cancer.
4. The PANX1 gene expression inhibitor siRNA provided by the invention has good interference effect when being used for gene inhibition, can effectively inhibit the PANX1 gene expression, and inhibit proliferation, migration and tumorigenesis of tumor cells, and has important application prospects for gene therapy.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of differential expression detection of PANX1mRNA in paracancerous Normal tissue (Normal) and pancreatic ductal adenocarcinoma tissue (Tumor).
FIG. 2 shows the results of the detection of PANX1mRNA expression in pancreatic ductal adenocarcinoma patients at different pathological stages (stage I, II, III, IV).
FIG. 3 is a graph showing the survival analysis of PANX1 gene expression levels on overall survival and disease-free survival of pancreatic cancer patients.
FIG. 4 is a graph showing the results of differential expression detection of PANX1mRNA in Cancer tissue samples (Cancer) and paracancerous Normal tissue samples (Normal) of 33 Cancer types.
A in fig. 5 is a diagram showing immunohistochemical staining results of PANX1 in human pancreatic ductal adenocarcinoma paracented Normal tissue (Normal), early lesions (acinar ductal metaplasia, acinar-ductal metaplasia, abbreviated ADM; intraepithelial neoplasia of the pancreas pancreatic intraepithelial ne oplasia, abbreviated PanINs) and pancreatic ductal adenocarcinoma tissue (pancreatic ductal adenocarcinomas, abbreviated PD AC) tissues; b in fig. 5 is a graph of immunohistochemical staining results of PANX1 in mouse Normal, ADM, panINs and PDAC.
A in fig. 6 is a graph showing the detection result of protein expression of anti-PANX 1 antibody (cat#hpa 016930) on PANX1 gene knockdown/overexpression of human pancreatic ductal adenocarcinoma cell line; b in FIG. 6 is a graph showing the results of protein expression detection of PANX1 antibody (Cat#D9M1C) on PANX1 gene knockdown/overexpression in human and murine pancreatic ductal adenocarcinoma cell lines.
FIG. 7 is a graph showing the effect of PANX1 gene knockdown in human pancreatic ductal adenocarcinoma PANC-1 and SW1990 cells, PANX1 gene overexpression in human pancreatic ductal adenocarcinoma ASPC-1 and PL-45 cells, and PANX1 gene knockdown/overexpression in murine pancreatic ductal adenocarcinoma cell line Panco2 on proliferation of pancreatic ductal adenocarcinoma cell line.
FIG. 8A is a graph showing the effect of PANX1 gene knockdown in PANC-1 cells on pancreatic ductal adenocarcinoma cell line neoplasia; b in FIG. 8 is a graph of the quantitative result of the effect of knockdown of the PANX1 gene on pancreatic ductal adenocarcinoma cell lines in PANC-1 cells.
A in fig. 9 is a diagram of subcutaneous neoplasia in a murine pancreatic ductal adenocarcinoma cell line Panco2 control (NC) C57BL/6J mice; FIG. 9B is a diagram of subcutaneous tumor formation in C57BL/6J mice of the murine pancreatic ductal adenocarcinoma cell line Panco2 PANX1 knock-down group (si 3-mPANX 1); FIG. 9C is a photograph of subcutaneous tumor cells removed from the control group (NC) and PANX1 knock-down group (mSi 3); in FIG. 9, C is a line graph of tumor diameter of subcutaneous tumor cells removed in the control group (NC) and PANX1 knockdown group (mSi 3).
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
PL45 cells were purchased from deep-bloom biotechnology limited, guangzhou; asPC-1 cells, PANC-1 cells and SW1990 cells were purchased from Saku Biotechnology Co., ltd; panco2 cells were purchased from Zhejiang Meisen cell technologies Co. All cell lines were cultured in medium containing 5% or 10% fetal bovine serum and placed under conditions of 37℃and 5% CO 2.
anti-PANX 1 (cat#d9m1c) antibody was purchased from Cell Signaling Technology; anti-PANX 1 (cat#hpa 016930) antibodies were purchased from Simga; anti-beta-action (Cat# 66009-1) antibodies were purchased from Proteintech; anti-GAPDH (Cat#60004-1) antibody was purchased from Proteintech, WB Anti-Rabbit secondary antibody, goat Anti-Rabbit IgG (H+L), HRP conjugate was purchased from Proteintech, cat#SA0002; WB anti-mouse anti-mouse IgG (H+L), HRP conjugate from Proteint ech, cat#SA0001; matrigel (cat# 356234), purchased from BD.
Specifically targeting human PANX1 (NCBI Gene ID: 24145) and murine PANX1 (NCBI Gene ID: 55991) siRNAs were purchased from Gimar Biolabs. The PANX1 siRNA sequences are shown in Table 1.
A, G, C and U in each oligonucleotide represent adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide and uracil ribonucleotide in sequence. The addition of two tails of dTdT, dADC or dGdG at the 3' end directs the siRNA to form RNAi silencing complexes and also avoids degradation by nucleases.
NC is a commercial design of Ji Ma company, is not self-made siRNA, and is a nonspecific random fragment.
Human PANX1 (NCBI Transcript ID: NM-015368.4) and murine PANX1 (NCBI Transcript ID: NM-019482.2) transcript plasmids (overexpressing plasmids) were purchased from Nanjing Jinsri Biotechnology, inc., human PANX1 overexpressing plasmid abbreviation "h pc PANX1", and murine PANX1 overexpressing plasmid abbreviation "m pc PANX1". The human and murine PANX1 ORF were subcloned into the pcDNA3.1 (+) -C-DYK vector, respectively. The specific steps are as follows:
Gene symbol:hPANX1；
Express Cloning Vector:pcDNA3.1+C-DYK；
NCBI Transcript ID:NM_015368.4；
the CDS sequence (1281 nt) of the human PANX1 ORF region is shown in SEQ ID NO.15 from 5 'to 3';
5' -terminal cleavage site: nheI-HF;
3' -terminal cleavage site: apaI.
Gene symbol:mPanx1；
Express Cloning Vector:pcDNA3.1+C-DYK；
NCBI Transcript ID:NM_019482.2；
CDS sequences (1281 nt) of the murine PANX1 ORF region are shown in SEQ ID NO.16 from 5 'to 3';
5' -terminal cleavage site: nheI-HF;
3' -terminal cleavage site: apaI.
C57BL/6J mice and transgenic mice Pdx1-Cre, LSL-Trp53 R172H/+ And LSL-Kras G12D/+ The C57BL/6J mice are derived from Guangzhou Sjia Jingda Biotechnology Co., ltd., pdx1-Cre and LSL-Kras G12D/+ From Shanghai Nannon model biotechnology Co., ltd and LSL-Trp53 R172H/+ From the racing biotechnology company. All experimental mice were housed within the SPF grade barrier. Animal bodyThe development of the experiment was approved by the laboratory animal ethics committee of the university of south China and followed the animal welfare principle in the process.
Embodiment one: a method for detecting the mRNA expression level of the PANX1 gene in a cancer tissue (Tumor) and a normal tissue (Nor mal) by using a bioinformatic analysis; a method for detecting the mRNA expression level of PANX1 gene in different pathological stages of pancreatic duct adenocarcinoma.
GEPIA2 (http:// ualcan. Path. Uab. Edu/c) is an online data mining platform based on TCGA and GTEx databases, which can be used to analyze gene expression in various cancers. In this study, pancreatic ductal adenocarcinoma tissues were from the TCG a database, and the paracancel tissues were from the TCGA and GTEx databases, using one-way ANOVA analysis methods, and log2 (tpm+1) conversion mapped the expression profile box of PANX1 gene in paracancel normal and pancreatic ductal adenocarcinoma tissues. In the study, mRNA expression of the PANX1 gene in different pathological stages of pancreatic duct adenocarcinoma patients is drawn through a one-way ANOVA analysis method by adopting a GEPIA2 database and log2 (TP M+1) conversion, so as to generate a violin graph.
The experimental results are shown in fig. 1 and 2: PANX1 was significantly higher expressed in mRNA levels (< 0.01) in pancreatic ductal adenocarcinoma tissue (Tumor) compared to paracancerous Normal tissue (Normal) (fig. 1). In the pancreatic ductal adenocarcinoma pathological stage, PANX1mRNA expression was significantly elevated in stage IV (pancreatic ductal adenocarcinoma pathological stage four) (p=0.035) (fig. 2). The expression of the PANX1 gene has correlation with pancreatic duct adenocarcinoma, and the PANX1 gene can be used as an effective cancer marker of the pancreatic duct adenocarcinoma for diagnosis and treatment of cancers.
Embodiment two: a method for analyzing the cancer prognosis correlation of PANX1 gene expression and pancreatic ductal adenocarcinoma by using bioinformatic analysis.
In this study, the Kaplan-Meier curve and log-rank test were used to analyze the correlation of PA NX1 gene expression levels with overall survival and disease-free survival of pancreatic ductal adenocarcinoma patients using the GEPIA2 database, resulting in a survival analysis curve as depicted in fig. 3.
The experimental results are shown in fig. 3: the overall survival time and the disease-free survival time of the pancreatic duct adenocarcinoma patients with high expression of the PANX1 gene are obviously lower than those of the pancreatic duct adenocarcinoma patients with low expression of the PANX1 gene (P is less than 0.01), and the expression of the PANX1 gene is closely related to the poor prognosis of the pancreatic duct adenocarcinoma patients. The expression product of the PANX1 gene can be used as a prognosis marker of pancreatic duct adenocarcinoma, and by detecting the PANX1 gene expression, the expression product can be used for evaluating prognosis of pancreatic duct adenocarcinoma patients, prompting survival time of the patients, monitoring cancer treatment effect, and timely adjusting clinical treatment strategies of the cancers, thereby being beneficial to further improving clinical treatment effect and improving survival quality of the patients.
Embodiment III: a method for correlation analysis of PANX1 gene expression and 33 cancers using bioinformatic analysis.
In this study, the GEPIA2 database was used to obtain expression profiles of PANX1 gene in cancer tissues and paired paracancerous normal tissues of 33 different tumors, wherein cancer tissue data were obtained from TCGA database, normal tissue data were obtained from TCGA and GTEx database, by one-way ANOVA analysis method, and log2 (tpm+1) conversion plotted as a lattice plot of PANX1 gene expression in different cancers, and P value <0.01 was set as a threshold for significant differential expression.
The experimental results are shown in fig. 4: PANX1mRNA levels were significantly higher in 33 cancer tissues compared to paracancerous normal tissues (P < 0.01) including diffuse large B-cell lymphoma, esophageal cancer, glioblastoma, brain glioma, gastric adenocarcinoma, thymus cancer, and pancreatic ductal adenocarcinoma. From these results, it is clear that PANX1 expression is correlated with the occurrence of diffuse large B cell lymphoma, esophageal cancer, glioblastoma, brain glioma, gastric adenocarcinoma, thymus cancer and pancreatic ductal adenocarcinoma, and that PANX1 gene and its expression products can be used as molecular markers for diagnosis and treatment of cancers.
Embodiment four: immunohistochemistry was performed to examine protein expression levels of the PANX1 gene in pancreatic ductal adenocarcinoma bypass Normal tissue (Normal), early lesion (acinar ductal metaplasia, acinar-ductal metaplasia, ADM for short; intraepithelial neoplasia of the pancreas pancreat ic intraepithelial neoplasia, panINs for short) and pancreatic ductal adenocarcinoma tissue (pancreatic ductal aden ocarcinomas, PDAC for short).
1. Sample source
Clinical test samples for human pancreatic ductal adenocarcinoma were from clinical patients affiliated with the hospitals at the university of south China. Murine pancreatic ductal adenocarcinoma samples from LSL-Trp53 R172H/+ ；LSL-Kras G12D/+ The method comprises the steps of carrying out a first treatment on the surface of the Pdx1-Cre (KPC for short) mouse model. The KPC mouse model construction scheme is as follows: LSL-Kras is first prepared G12D/+ Mice and LSL-Trp53 R172H/+ Mice were hybridized to produce LSL-Kras G12D/+ ；LSL-Trp53 R172H/+ Mice, i.e., KP mice. KP mice were then crossed with Pdx1-Cre mice to generate mice with LSL-Trp53 at the same time R172H/+ ；LSL-Kras G12D/+ The method comprises the steps of carrying out a first treatment on the surface of the Mice of three genotypes of Pdx1-Cre, i.e., KPC mice. The survival in KPC mice was about 5 months, with 100% mortality at 12 months. KPC mice developed acinar metaplasia (ADM) or intraepithelial neoplasia of the pancreas (PanINs) within the pancreas at 8 to 10 weeks of age; at 16 weeks of age, locally invasive Pancreatic Ductal Adenocarcinoma (PDAC) developed with a dense fibrotic response. Tumors typically have a moderately differentiated ductal morphology with extensive connective tissue hyperplasia, similar to the most common morphology observed in humans. Samples of KPC mice pancreatic tissue were harvested at the corresponding age groups. First, an ice physiological saline heart is required to perfuse, then the pancreatic tissue is gently and rapidly peeled off completely, the blood clot is washed away in the ice physiological saline to remove the effect of blood cells on immunohistochemical staining, and then immersed in fresh 10% neutral formalin. In this example, a conventional paraffin tissue fixation, dehydration, transparency, and embedding procedure was used. The slice thickness was 4um.
2. Immunohistochemical staining experiments
The detection concentration was 1 using an anti-PANX 1 (cat#hpa 016930) antibody: 100, detecting a human pancreatic ductal adenocarcinoma sample; anti-PANX 1 (D9M 1C) antibody, detected at a concentration of 1:100, detecting a murine pancreatic ductal adenocarcinoma sample. By adopting a conventional immunohistochemical staining experiment scheme, it is noted that the microwave antigen retrieval method is adopted in the embodiment, ED TA and PH9.0 antigen retrieval liquid are slightly boiled in 6 to 7 minutes of high fire, and the temperature is changed to low fire for 15 minutes to maintain a slightly boiling state; the 5% goat serum was blocked for 30 min to 1 hour at room temperature, the primary antibody was incubated overnight at 4 degrees, the secondary antibody was incubated for 1 hour at room temperature, developed using conventional DAB, and the nuclei were counterstained with hematoxylin.
From example four (see fig. 5 for experimental results): staining results of PANX1 protein in normal, ADM, panINs and PDAC of human (a) and murine (B) (40X magnification). In normal tissues, PANX1 protein staining was a negative result, while staining in ADM, panINs, and PDAC was a positive result. The expression level of the PANX1 gene is obviously different from that of normal tissues and early lesion tissues, and the PANX1 gene has obvious correlation with the occurrence and development of pancreatic duct adenocarcinoma, so that the PANX1 gene is expected to be used as a cancer marker of the pancreatic duct adenocarcinoma for early diagnosis of cancer.
Fifth embodiment: and (3) identifying the knockdown/over-expression effect of the siRNA/over-expression plasmid on the PANX1 gene.
1. Cell transient transfection assay
Cells were seeded in 6-well plates, 50 to 70 ten thousand cells per well (cell confluence at about 70% after cell attachment, transfection) and after 12-16h plating, transfection was performed. The transfection method comprises the following steps: 100pmol PANX1 siRNA (or 2. Mu.g of plasmid DNA and 5. Mu. l P3000 reagent) was added to 125. Mu.l of reduced serum medium (Opti-MEM) and gently mixed; mix lipofectamine 3000 reagent, dilute 5 μl lipofectamine 3000 with 125 μl Opti-MEM, mix gently, and leave at room temperature for 5 minutes; mixing the diluted siRNA/DNA with diluted lipofectamine 3000; gently mixed and left at room temperature for 15 minutes to form siRNA/lipofectamine complexes (or DNA/lipofectamine). Mu.l of siRNA/lipofectamine (or DNA/lipofectamine) complex was added to the wells of the corresponding plates containing cells and 1ml of medium, and after 6h the cell medium was replaced or replenished.
2. Detection of gene knockdown/over-expression effect by western blotting
Protein samples can be harvested 48 to 72 hours after cell transfection.
A. Sample lysis: before lysis with RIPA lysate, cells were washed 3 times with pre-chilled PBS to remove residual serum, RIPA lysate was added, cells were scraped off, resting on ice for 5 minutes, broken for 20 seconds with an ultrasonic breaker at 60W on ice, the supernatant was centrifuged at 4 ℃, and the cell lysate was assayed using Bradford method.
B. Protein denaturation: protein supernatants were mixed with 5 XSDS-PAGE running buffer and heated at 95℃for 5 minutes and samples were added to 8 or 10% polyacrylamide gel wells.
C. The protein gel electrophoresis and transfer method is a routine experimental procedure. In this experimental example, a 0.22um PVDF membrane 5% skimmed milk powder shaker was used to block for 2 hours at room temperature, a primary antibody (Cat#HPA 016930, dilution concentration 1:1000; anti-human, murine PANX1 antibody (Cat#D9M1C, dilution concentration 1:1000)) was incubated overnight at 4 degrees shaker, a secondary antibody (Goat Anti-Rabbit IgG (H+L), HRP conjugate, proteintech, cat#SA0002, dilution concentration 1:5000) was incubated at shaker room temperature for 1 hour, a PANX1 protein band was obtained by conventional chemiluminescence method, an internal primary antibody (Anti-. Beta. -action (Cat# 66009-1); anti-GAPDH (Cat#60004-1), dilution concentration 1:5000), and incubation of a secondary antibody (Goat i-moIgG (H+L), HRP conjugate, cat#SA0001, dilution concentration 1:5000) as PA 1 was developed.
From example five (see fig. 6 for experimental results): the Human si2 and si3 have obvious knockdown effects on SW1990 and PANC-1 cell line PAN X1 genes; the Human PANX1 over-expression plasmid has obvious over-expression effect on PL-45 and ASPC-1 cell line PANX1 genes. Also, it was demonstrated that the anti-PANX 1 antibody (Cat#HPA 016930) had a test effect on human samples. The Mouse si1, si3 has obvious knockdown effect on PANX1 gene in the rat pancreatic duct adenocarcinoma cell line Panco 2; the Mo use PANX1 over-expression plasmid has obvious over-expression effect on PANX1 genes in a rat pancreatic duct adenocarcinoma cell line Panco 2; the Human PANX1 over-expression plasmid has obvious over-expression effect on the PL-45 cell line PANX1 gene. Meanwhile, the anti-PANX 1 antibody (Cat#D9M1C) has detection effect on human and mouse samples.
Example six: the effect of PANX1 gene knockdown/overexpression on cancer cell proliferation, invasion and neoplasia was examined.
CCK-8 method for detecting influence of PANX1 gene knockdown/overexpression on cancer cell proliferation
PANX1 Gene knockdown/overexpression in cancer cells As shown in example five, it should be noted that in this experimental example, the siRNA was Human si3 and Mouse si1, 96-well plates were used, the number of cell plating was 3000-5000 cells per well, and the final transfection system was 100ul medium and 10ul siRNA/plasmid DNA+lipofectamine complex/well. Relative cell growth was measured daily using the CCK-8 method (expressed as "OD" value). The P-value is determined by one-way analysis of variance.
The experimental results are shown in fig. 7: knocking down the expression of the PANX1 gene in a human pancreatic ductal adenocarcinoma cell line SW1990, PANC-1 and a murine pancreatic ductal adenocarcinoma cell line Panco2 can inhibit proliferation of cancer cells; the human/mouse PANX1 gene is overexpressed in human pancreatic duct adenocarcinoma cell lines PL-45, ASPC-1 and mouse pancreatic duct adenocarcinoma cell lines Panco2, so that the proliferation of cancer cells can be promoted.
Detection of the influence of PANX1 Gene knockdown on cancer cell migration by the Wound health method
PANX1 Gene knockdown protocol in cancer cells As shown in example five, after 48 hours of siRNA Human si3 transfection, cell streaking was performed using a 200ul pipette tip after starvation treatment for 24 hours with a medium containing 0.5-2% fetal bovine serum, and the cells were washed 3 times with PBS, supplementing 2ml of cell medium containing 0.5-2% fetal bovine serum; cell culture conditions were 37 ℃,5% carbon dioxide, and after 0, 12, 24 hours of cell culture, photographs were taken. The scratch location blank area was counted using ImageJ. The P-value is determined by one-way analysis of variance.
The experimental results are shown in fig. 8: knocking down the expression of PANX1 gene in human pancreatic ductal adenocarcinoma cell line PANC-1 can inhibit the migration of cancer cells (12 h x p < 0.001;24h x p < 0.0001).
3. Mouse pancreatic ductal adenocarcinoma (pancreatic ductal adenocarcinoma) subcutaneous transplantation tumor experiment to detect effects of PANX1 gene knockdown on cancer cell neoplasia
PANX1 Gene knockdown protocol in cancer cells As shown in example five, the siRNA was Mouse si1. In an allograft tumor model, pancreatin digested cells were centrifuged at 1000rpm for 5 min, the supernatant was discarded, resuspended in 2ml PBS, counted using trypan blue (cytometer available from Invitrogen), centrifuged, the supernatant discarded, resuspended in PBS, and a PBS/matrigel (Cat #356234, available from BD) suspension was placed on ice at a cell concentration of 1X 10≡6100ul, injected subcutaneously into the flank of C57BL/6 mice. After observing that the subcutaneous tumor-bearing volume is no longer decreasing, the length and width of the tumor is measured with calipers every three days. Tumor-bearing mice were euthanized at the indicated time points upon dying or post-inoculation, and tumors were resected. Tumor volume (mm) 3 ) The calculation formula is that the short diameter 2 X long diameter/2. The P-value is determined by one-way analysis of variance.
The experimental results are shown in fig. 9: measurements from subcutaneous tumor-bearing morphology and tumor volume showed that PANX1 gene knockdown was able to inhibit cancer cell neoplasia (DAY 25, P < 0.001).
From the implementation of six (experimental results, shown in figures 7-9), the expression of the PANX1 gene promotes proliferation, migration and tumorigenesis of pancreatic duct adenocarcinoma cells, and the PANX1 gene participates in the progress of pancreatic duct adenocarcinoma, so that effective information is provided for targeted treatment of pancreatic duct adenocarcinoma, and the survival rate and the survival quality of patients are improved.
The above examples are only preferred embodiments of the present invention, and are merely for illustrating the present invention, not for limiting the present invention, and those skilled in the art should not be able to make any changes, substitutions, modifications and the like without departing from the spirit of the present invention.

Claims (10)

  1. Use of panx1 inhibitors for the preparation of a medicament for the prevention and/or treatment of cancer.
  2. 2. The use according to claim 1, wherein the PANX1 inhibitor is selected from siRNA targeting PANX 1.
  3. 3. The use according to claim 2, wherein the PANX1 targeting siRNA is an oligomeric nucleic acid Human si2, human si3, mouse si1, mouse si2 and/or Mouse si3;
    the Human si2 consists of a single-stranded nucleic acid molecule 1 and a single-stranded nucleic acid molecule 2, wherein the single-stranded nucleic acid molecule 1 consists of a single-stranded RNA molecule shown in SEQ ID NO.1 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 2 consists of a single-stranded RNA molecule shown in SEQ ID NO.2 and 2 dGs, wherein the 2 dGs are positioned at the 3' end;
    the Human si3 consists of a single-stranded nucleic acid molecule 3 and a single-stranded nucleic acid molecule 4, wherein the single-stranded nucleic acid molecule 3 consists of a single-stranded RNA molecule shown in SEQ ID NO.3 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 4 consists of a single-stranded RNA molecule shown in SEQ ID NO.4 and dAdC, wherein the dAdC is positioned at the 3' -end;
    the Mouse si1 consists of a single-stranded nucleic acid molecule 5 and a single-stranded nucleic acid molecule 6, wherein the single-stranded nucleic acid molecule 5 consists of a single-stranded RNA molecule shown in SEQ ID NO.5 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 6 consists of a single-stranded RNA molecule shown in SEQ ID NO.6 and 2 dT, wherein the 2 dT is positioned at the 3' end;
    the Mouse si2 consists of a single-stranded nucleic acid molecule 7 and a single-stranded nucleic acid molecule 8, wherein the single-stranded nucleic acid molecule 7 consists of a single-stranded RNA molecule shown in SEQ ID NO.7 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 8 consists of a single-stranded RNA molecule shown in SEQ ID NO.8 and 2 dT, wherein the 2 dT is positioned at the 3' end;
    the Mouse si3 consists of a single-stranded nucleic acid molecule 9 and a single-stranded nucleic acid molecule 10, wherein the single-stranded nucleic acid molecule 9 consists of a single-stranded RNA molecule shown in SEQ ID NO.9 and 2 dT, and the 2 dT is positioned at the 3' end; the single-stranded nucleic acid molecule 10 consists of a single-stranded RNA molecule shown in SEQ ID NO.10 and 2 dT, wherein the 2 dT is positioned at the 3' end.
  4. 4. A pharmaceutical composition for preventing and/or treating cancer, comprising a PANX1 inhibitor.
  5. 5. The pharmaceutical composition of claim 4, wherein the PANX1 inhibitor is selected from siRNA targeting PAN X1.
  6. 6. A cancer marker, wherein the cancer marker is one or more than two of PANX1 gene, PANX1mRNA and PANX1 protein.
  7. 7. Use of at least one of the following A1-A3 in at least one of the following B1-B4:
    a1, PANX1 gene; a2, PANX1 mRNA; a3, PANX1 protein;
    b1, as a diagnostic marker for cancer, or preparing a product for cancer diagnosis;
    b2, as a prognostic marker for cancer, or preparing a product for prognostic assessment of cancer;
    b3, preparing a product for monitoring the curative effect of the cancer;
    b4, preparing a medicament for treating cancer;
    the cancer is pancreatic duct cancer.
  8. 8. The use of claim 7, wherein the cancer further comprises one or more of diffuse large B-cell lymphoma, esophageal cancer, glioblastoma, glioma, gastric adenocarcinoma, and thymus cancer.
  9. 9. The use according to claim 7, wherein the cancer diagnosis comprises early cancer diagnosis, remote cancer metastasis diagnosis and/or cancer lymph node metastasis diagnosis.
  10. 10. The use according to claim 7, wherein the products for cancer diagnosis, for cancer prognosis evaluation and for cancer efficacy monitoring comprise primers, probes or antibodies for amplification of PANX 1; the forms of the product for cancer diagnosis, the product for cancer prognosis evaluation and the product for cancer curative effect monitoring comprise a chip and a kit.
CN202311285902.XA 2023-10-07 2023-10-07 Cancer marker PANX1 and application thereof Pending CN117599078A (en)

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