CN116609528A - Pancreatic cancer early diagnosis marker button protein cingulin and novel anticancer drug target - Google Patents
Pancreatic cancer early diagnosis marker button protein cingulin and novel anticancer drug target Download PDFInfo
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- CN116609528A CN116609528A CN202310422451.3A CN202310422451A CN116609528A CN 116609528 A CN116609528 A CN 116609528A CN 202310422451 A CN202310422451 A CN 202310422451A CN 116609528 A CN116609528 A CN 116609528A
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Abstract
The invention discloses a novel pancreatic cancer early diagnosis marker button protein cingulin and an anticancer drug target. The pancreatic cancer early diagnosis marker comprises an antibody of the button protein CGN and a fluorescent quantitative polymerase chain reaction primer sequence, and accurately and clearly displays the expression mode and the variation of the expression quantity of the button protein CGN in the malignant pancreatic cancer occurrence and development process. The invention discloses the expression mode of the button protein CGN in early pancreatic cancer and the expression mode is used as a novel target point of cell proliferation intervention and cell migration invasion intervention. The pancreatic cancer is used as a diagnosis marker, so that early diagnosis of pancreatic cancer is facilitated; the target can be used for developing medicines for inhibiting the growth and metastasis of tumor cells and enhancing the treatment effect on tumors.
Description
Technical Field
The invention relates to the technical field of biomedicine and the technical field of medicines, in particular to a button protein CGN antibody serving as a marker for early diagnosis of pancreatic cancer of malignant tumor, and a button protein CGN down-regulator serving as a medicine for treating pancreatic cancer of malignant tumor.
Background
Pancreatic cancer is a very invasive malignancy of the digestive system, and is characterized by difficulty in early diagnosis and poor prognosis. Pancreatic Ductal Adenocarcinoma (PDA) is the most common type of pancreatic cancer. In all cases, the 5-year survival rate of PDA is less than 9.3% and in metastatic disease patients is less than 2.9%. PDA is currently the fourth leading cause of cancer-related death in the united states, and is expected to be the second leading cause of cancer-related death by 2030. Many PDA patients have distant metastasis at diagnosis and are not responsive to current surgical treatment and chemotherapy. Thus, new early diagnostic techniques and tumor treatment techniques are in need of further improvement and development.
Button protein CGN is a tightly linked family of proteins found earliest in chicken intestinal epithelial brush border cells. Its subcellular localization and tissue distribution show its localization at cellular tight junctions, as well as various polarized epithelium and endothelium. In physiological and pathological situations, the cingulated protein CGN is involved in maintaining the integrity of certain epithelial and endothelial barriers. CGNKnock-downCan increase the permeability of colon cancer epithelial cells, neurons and Purkinje cells, and reduce the epithelial resistance. However, knockout of CGN in MDCK and embryonic stem cell-derived embryoid bodies did not affect tight junction barrier function, suggesting that button protein CGN is not always maintaining epithelial and endothelial barriers. Likewise, cingulan CGN is also involved in many cellular processes by regulating cell movement, cell proliferation, cell cycle and gene expression.
Disclosure of Invention
In order to overcome the inaccuracy and the non-specificity of the existing diagnosis, the invention aims to provide an early diagnosis marker. In order to overcome the defect of poor effect of the prior treatment technology, another object of the invention is to provide a medicament for treating cancers.
In order to achieve the first object, the invention adopts the following technical scheme:
the invention provides a cancer early diagnosis marker, which comprises an antibody of button protein CGN and a fluorescent quantitative polymerase chain reaction primer sequence.
Further, the button protein CGN antibody is an antibody for blocking amino acid residues 7-356 or 307-412 of button protein CGN.
Further, the primer sequence of the button protein CGN fluorescent quantitative polymerase chain reaction is a reverse complementary sequence of CDS (coding sequence of messenger ribonucleic acid) mRNA (messenger ribonucleic acid) combined with the button protein CGN.
Further, when the transcript level and protein level expression of the button protein CGN is upregulated, the subject is in an early stage of pancreatic carcinogenesis.
Further, the pancreatic cancer early-stage diagnostic reagent includes a reagent for detecting the transcription level and the protein level of the button protein CGN.
In order to achieve the second purpose, the invention adopts the following technical scheme:
the invention also provides a medicine for treating malignant tumors, comprising the down regulator of the button protein CGN.
Further, the method comprises the steps of. The button protein down regulator is designed button protein CGN short interfering RNA.
The small interfering RNA sequence of the button protein is a down-regulating sequence aiming at human button protein CGN: GTCCAGATTCGCTTCATCACA, down-regulator sequence 1 for murine button protein Cgn: GTGAGGAGGAAAGTTAGTTTG; down-regulatory sequence 2 for murine button protein Cgn: TGGAGTTCAAATTCGATTTAT.
The invention provides a medicine capable of treating malignant pancreatic cancer, and can inhibit the expression of button protein CGN, inhibit proliferation, migration and invasion of malignant pancreatic cancer cells and inhibit in-vivo tumor growth.
The downregulation of the button protein CGN inhibits proliferation of malignant tumor cells and downregulates the clonogenic capacity of malignant tumor pancreatic cancer cells; down-regulating the expression level of the malignant pancreatic cancer cell mitosis, cell proliferation-related antigen Ki 67; down-regulating the tumor growth promoting MAPK/ERK signaling pathway; and growth of pancreatic cancer cells in vivo in mice.
The downregulation of the button protein CGN can inhibit the migration and invasion of malignant tumor cells, downregulate the wound healing capacity of the malignant tumor and weaken the migration and invasion capacity of the cells.
Compared with the prior art, the invention has the following advantages:
the discovery provides differential expression of the button protein CGN in the occurrence and development process of pancreatic cancer of malignant tumors for the first time, and can judge whether a subject is in the early stage of pancreatic cancer occurrence or not by detecting the transcription level and the protein level of the button protein CGN, so that a molecular means is provided for early diagnosis of pancreatic cancer of malignant tumors.
The malignant tumor therapeutic drug provided by the invention can inhibit proliferation, migration and invasion of pancreatic cancer malignant tumor cells and growth of in-vivo tumors by reducing the CGN level of the button protein. The target can be used for developing medicines for inhibiting the growth and metastasis of tumor cells and enhancing the treatment effect on tumors.
Drawings
FIG. 1-A is an Immunohistochemical (IHC) test showing the expression level of Cingatin (Cingatin [ Homo sapiens ]) in pancreatic cancer tissue, paracancerous normal tissue, acinar catheter metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN), scale = 50 μm;
FIG. 1-B is a graph showing the statistical result of the expression level of Cingulin (Homo sapiens) in pancreatic cancer tissue, paracancerous normal tissue, ADM and PanIN;
FIG. 1-C shows the transcription expression level of CGN (Homo sapiens Cingulin (CGN), CDS) and the expression level of Cingulin (Cingulin [ homosapiens ]) protein in each of pancreatic cancer cell lines and pancreatic ductal cell lines, as measured by Real-time PCR and Western blotting;
FIG. 1-D is a dataset analysis of GSE62452, GSE32676 and GSE71729, showing the expression levels of CGN (Homo sapiens Cingulin (CGN), CDS) in pancreas and pancreatic cancer;
FIG. 2 up-regulation of Cgn expression level during pancreatic carcinogenesis in mice
FIG. 2-A is an IHC assay showing the expression level of cingulin (cingulin isocord 1[ mus musculus ]) in normal pancreas, ADM and PanIN of C57BL/6 mice, scale = 50 μm;
FIG. 2-B shows the transcription expression levels of Cgn (Mus musculus cingulin (Cgn), transcript variant, CDS), CK19 (Mus musculus keratin (Krt 19), transcript variant1, mRNA) and Amy2a (Mus musculus Amylase a1 (Amy 2a 1), mRNA) and the protein expression levels of cingulin (cingulin isofan 1[ mus musculus ]), sox9 (transcription factor SOX-9[ mus musculus ]), amylase (amyase 2a1 precursor[Mus musculus ]) in murine ADM by Real-time PCR and Western blotting assays;
FIG. 2-C shows the expression level of cingulin (cingulin isocord 1[ mus musculus ]) in murine ADM, as detected by Immunofluorescence (IF);
FIG. 3 Single cell analysis of expression patterns of CGN in human and murine pancreatic cancers
FIG. 3-A is a GSE155698 dataset analysis showing that CGN (Homo sapiens Cingulin (CGN), CDS) is expressed higher in tumor cells and metaplastic cells than acinar cells;
FIG. 3-B is a GSE141017 dataset analysis showing that Cgn (Mus musculus cingulin (Cgn), transcript variant1, CDS) is found in Ptf1a-Creer, LSL-Kras G12D Expression patterns in LSL-tdTomato (KC) pancreatic cancer model are expressed mainly in ductal and metaplastic cells;
FIG. 4-A shows the expression levels of CGN (Homo sapiens Cingulin (CGN), CDS) at various levels in human pancreatic cancer tissue chips and GSE62452 dataset;
fig. 4-B is a kaplan-meyer curve analysis using GSE71729 dataset, showing the relationship of CGN (Homo sapiens Cingulin (CGN), CDS) to patient survival;
FIG. 4-C is a graph of a Kaplan-Meier curve analysis using a human pancreatic cancer tissue chip, showing the relationship between Cingalin (Cingalin [ Homo sapiens ]) protein expression and patient survival;
FIG. 5 is a multi-factor COX regression analysis using the TCGA pancreatic cancer dataset, demonstrating the correlation of expression of CGN (Homo sapiens Cingulin (CGN), CDS) with pancreatic cancer prognosis survival
FIG. 6 is an analysis of the relationship between CGN and clinical pathology parameters of human pancreatic cancer tissue chips, showing that the expression of Cingulin (Cingulin [ Homo sapiens ]) is significantly correlated with N-grade;
FIG. 7-A shows the transcription expression level of CGN (Homo sapiens Cingulin (CGN), CDS) and the expression level of Cingaulin [ homosapiens ]) protein after CGN overexpression and knockdown, as measured by Real-time PCR and Western blotting;
FIG. 7-B is a graph showing the effect of CCK8 assay on pancreatic cancer cell proliferation, over-expression and knock-down of CGN (Homo sapiens Cingulin (CGN), CDS);
FIG. 8 shows the effect of cell fluorescence detection, over-expression and knock-down of CGN (Homo sapiens Cingulin (CGN), CDS) on Ki67 expression;
FIG. 9-A is a graph showing the effect of cell clone detection, overexpression and knockdown of CGN (Homo sapiens Cingulin (CGN), CDS) on the clonogenic capacity of pancreatic cancer cells;
FIG. 9-B is a graph of the GSE32676 dataset, and the differential expression analysis is performed for both the high and low sets of CGN (Homo sapiens Cingulin (CGN), CDS expression, and the major enrichment pathway of the up-regulated differential expression genes;
FIG. 9-C is a WB assay showing the effect of overexpression and knock-down of CGN (Homo sapiens Cingulin (CGN), CDS) on MAPK/ERK signaling pathway;
FIG. 10-A is the effect of CGN (Homo sapiens Cingulin (CGN), CDS) knockdown on pancreatic cancer cell xenograft/allograft tumor size;
FIG. 10-B is tumor volume change over time of mouse pancreatic carcinoma xenograft/allograft tumors;
FIG. 10-C is the effect of CGN (Homo sapiens Cingulin (CGN), CDS) knockdown on pancreatic cancer cell xenograft/allograft tumor weight;
FIG. 10-D is an IHC assay showing the effect of a cgN (Homo sapiens Cingulin (CGN), CDS) knockdown xenograft/allograft tumor on MAPK/ERK signaling pathway activation and Ki67 expression;
fig. 11-a is a cell scratch assay showing the effect of CGN (Homo sapiens Cingulin (CGN), CDS) overexpression and knockdown on the wound healing capacity of human pancreatic cancer cell lines, scale = 100 μm;
FIG. 11-B is a quantitative result of the effect of CGN (Homo sapiens Cingulin (CGN), CDS) overexpression and knock-down on the wound healing capacity of human pancreatic cancer cell lines;
figure 12CGN (Homo sapiens Cingulin (CGN), CDS) promotes pancreatic cancer cell migration and invasion capacity;
fig. 12-a is a Transwell experimental assay showing the effect of CGN (Homo sapiens Cingulin (CGN), CDS) overexpression and knockdown on human pancreatic cancer cell line migration and invasiveness, scale = 100 μm;
FIG. 12-B is a graph of quantitative results of the effects of CGN (Homo sapiens Cingulin (CGN), CDS) overexpression and knockdown on the ability of human pancreatic cancer cell lines to migrate and invade;
FIG. 12-C shows the effect of WB detection CGN (Homo sapiens Cingulin (CGN), CDS) overexpression and knock-down on vimentin (vimentin [ Homo sapiens ]);
FIG. 13-A shows the expression levels of Cgn (Mus musculus cingulin (Cgn), transcript variant, CDS) over-expression and knock-down for Real-time PCR and Western blotting assays; and CCK8 experiments to detect the effect of over-expression and knock-down Cgn (Mus musculus cingulin (Cgn), transcript variant, CDS) on pancreatic cancer cell proliferation;
fig. 13-B is a cell scratch assay showing the results of Cgn (Mus musculus cingulin (Cgn), transcript variant, cds) overexpression and knock-down on the wound healing capacity of murine pancreatic cancer cell lines, scale = 100 μm;
FIG. 13-C is a quantitative result of the effect of Cgn (Mus musculus cingulin (Cgn), transcript variant, CDS) overexpression and knock-down on the wound healing capacity of murine pancreatic cancer cell lines.
Detailed Description
The present invention provides a therapeutic agent for pancreatic cancer early diagnosis marker and malignant tumor, and for the purpose of making the technical and effect of the present invention clearer, the present invention will be described in further detail with reference to the following specific embodiments. 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.
hTERT-HPNE cells, HPDE6-C7 cells, HPAC cells, and PL45 cells were purchased from deep-bloom biotechnology limited, guangzhou; asPC-1 cells, bxPC-3 cells, caPan-2 cells, CFPAC-1 cells, MIA Paca-2 cells, PANC-1 cells, PK-59 cells and SW1990 cells were purchased from Sakuku Biotechnology in GuangzhouA limited company; 266-6 cells were purchased from Beijing Zhongyuan Polymer biotechnology Co. Panc02 cells were purchased from Zhejiang Meissn cell technology Co. All cell lines were placed in medium containing 5% or 10% fetal bovine serum and penicillin/streptomycin and at 37℃and 5% CO 2 And (5) culturing the conditions.
anti-Cingaulin (Cat# 21369-1-AP), GAPDH (Cat#60004-1-Ig) antibodies were purchased from Proteintech; anti-Cingulin (cat#ab 244406), anti-Ki 67 (cat#ab 15580), anti-pERK (cat#ab 47339), anti-ERK (cat#ab 184699) antibodies were purchased from Abcam; anti-Vimentin (Cat#5741S) antibodies were purchased from Cell Signaling Technology; anti-Amylase (Cat#PA 5-117115) antibodies were purchased from Invitrogen; anti-ELA 3B (cat#mab 14788) antibodies were purchased from Abnova; anti-SOX 9 (Cat#AB 5535-25 UG) antibody was purchased from Sigma-Aldrich.
Specific targeting siRNA was purchased from germacra biosystems and synthesized against the down-regulator sequence of human button protein CGN: GTCCAGATTCGCTTCATCACA, down-regulator sequence 1 for murine button protein Cgn: GTGAGGAGGAAAGTTAGTTTG; down-regulatory sequence 2 for murine button protein Cgn: TGGAGTTCAAATTCGATTTAT. The primers for Real-time PCR are detailed in Table 1.
Human CGN transcript plasmids (over-expression plasmids) and CGN shRNA (knockdown plasmids, the sequences of which are identical to siRNA) were purchased from Guangzhou Ai Ji Biotechnology Co., ltd, and the CGN ORF region was subcloned onto the pCDH-CMV-MCS-EF1-puro vector containing a Flag tag; CGN shRNA was constructed on pLKO.1-U6-EF1a-coGFP-T2A-puro plasmid containing Flag tag. The Cgn transcript plasmid (over-expression plasmid) was purchased from south kyo gold sry biotechnology limited and the Cgn ORF region was subcloned into the pcdna3.1 (+) -C-DYK vector.
The C57BL/6J mice and nude mice are purchased and substituted by the university of North China university animal experiment center in a unified way and are sourced from Guangzhou Sjia Jingda biotechnology Co. All experimental mice were housed within the SPF grade barrier. Animal experiments were developed via the university of south China university laboratory animal ethics committee and followed the animal welfare principle in the process.
Example 1: mouse pancreatic cancer transplantation tumor experiment
In xenograft/allograft tumor models, human pancreatic cancer cells (1×10 6 ) Rat pancreasAdenocarcinoma cells (5×10) 5 ) Resuspended in 0.1mL of matrigel (BD, # 356234) in PBS and injected subcutaneously into the right inguinal of nude mice. The length and width of the tumor were measured with a vernier caliper every three/four days. Tumor-bearing mice were euthanized at the indicated time of dying or post-inoculation, tumor excised and weighed. Tumor volume (mm) 3 ) The calculation formula is that the short diameter is 2 times the long diameter/2.
The experimental results are shown in fig. 10, which is a diagram for describing the pancreatic cancer xenograft/allograft tumor model of the mice, and fig. 10-a is a diagram for the tumor sub-morphology of the mice after the subcutaneous tumor-bearing experiment is finished. FIG. 10-B shows the change in tumor-bearing volume under the skin of the mice. FIG. 10-C shows the weight of the transplanted tumor after the end of the subcutaneous tumor-bearing transplantation model in mice. Results from the morphology, volume and weight of transplanted tumors show that CGN down-regulators can inhibit tumor growth in pancreatic cancer cell mice.
Example 2: three mouse acinar catheter metaplasia (ADM) models
Under standardized conditions, using 6-8 weeks of wild type mice, duct ligation surgery was performed to induce a mouse pancreatitis model (i.e., PDL mouse ADM model), and the pancreatic tissue of the mice was taken 1 week after duct ligation. Using 6-8 week old wild mice, an intraperitoneal injection of ranpirin-induced mice pancreatitis model (i.e., CAE mice ADM model) was performed at an injection rate of 80ug/kg, 8 injections per day, 1 injection every 1 hour every other for 8 consecutive hours, two days total injection, and 2 days after injection, the pancreatic tissue of the mice was taken. The pancreatic tissue of KC mice with ADM lesions already at 2 months of age (i.e. KC mouse ADM model) was taken.
The experimental results are shown in FIG. 2-B and FIG. 2-C. FIG. 2-B shows the expression level of Cgn in Real-time PCR and Western blotting assays, in murine ADM/pancreatitis. FIG. 2-C shows the expression level of Cgn in murine ADM, as measured by IF. The results showed that ADM was increased in Cgn expression and increased in ADM apical cavity compared to the normal pancreas of C57BL/6 mice.
Example 3: pancreatic cancer cell line migration and invasion experiments
CGN overexpression employs a lentiviral system: the target plasmid was first virus-packaged by the pSPAX2 packaging plasmid and the pmd2.G coat plasmid. Then, PANC-1 and PL45 cells were re-infected, and 8ug/ml polybrene (Beyotidme, #C0351) was added simultaneously) Stable transgenic cell lines were obtained by screening with puromycin (InvivoGen, # QLL-40-03) and verified by Real-time PCR and WB. One day in advance, 5×10 5 PANC-1 and PL45 cells were seeded on 6-well plates, and 2mL of cell culture medium containing 10% FBS was added, and after 24 hours, cells were pooled to 90%, cell streaks were performed, PBS was washed 3 times, 2mL of cell culture medium containing 1% FBS was added, and after 0 hour after streaking, cells were photographed for 24 hours. The scratch area was calculated by imageJ.
CGN knockdown, CGN overexpression and CGN knockdown employed Lipo3000 liposome transient vehicle lines: day before transfection, 3×10 5 CFPAC-1 and PK-59 cells were seeded onto 6-well plates and 2mL of cell culture medium containing 10% FBS was added to transfect siRNA for 18-24 hours. Two sterile 1.5ml EP tubes (A and B) were taken, 125. Mu.L of serum-free OPTI-MEM medium was added, 5. Mu.L of Lipofectamine3000 (A tube) and 100pmol of siRNA or 2. Mu.g of DNA (B tube) were added, gently mixed, and left at room temperature for 5 minutes; mixing the tube A and the tube B, shaking completely, and standing at room temperature for 15min; the 250. Mu.LA and B tube mixtures were added to 6-well plates and after 6-8 hours the fresh complete medium was changed. Scoring 48 hours after transfection; PBS was washed 3 times, 2mL of 1% FBS-containing cell culture medium was added, and the cells were photographed at 0 hours, 24 hours after streaking. The scratch area was calculated by imageJ.
Mixing Matrigel with serum-free DMEM medium at a ratio of 1:10 on ice box, adding 60 μl into upper chamber of Transwell chamber, placing at 37deg.C, 5% CO 2 Is cultured in a cell culture incubator for 4 hours. Taking cells of a control group and an experimental group, performing pancreatin digestion, and cleaning the cells with a serum-free culture medium once; taking serum-free culture medium, re-suspending, counting, diluting cell density to 1×10 6 /ml. Taking 1×10 4 (migration experiments) or 1X 10 5 (invasive experiments) cells were inoculated into the upper chamber of a Transwell chamber, 600. Mu.l of the corresponding 10% FBS-DMEM complete medium was added to the lower chamber, and left to stand at 37℃with 5% CO 2 The cells were cultured in an incubator for 24-48 hours. Then the upper chamber is clamped out, placed upside down to remove the culture medium, a 24-well plate is prepared, 600 μl of 4% paraformaldehyde is added into the upper chamber, the upper surface of the upper chamber is gently sucked dry by a cotton swab, placed in the 24-well plate, and fixedAnd (5) setting for 20 minutes. The cells were placed in crystal violet stain for 20 minutes, washed twice with PBS for 3 minutes and dried. Observations were made under an inverted microscope and cell counts were made.
The experimental results are shown in FIG. 11-A, FIG. 11-B, FIG. 12-A, FIG. 12-B, FIG. 13-B and FIG. 13-C. FIG. 11-A is a graph showing the overexpression and knock-down of CGN in human pancreatic cancer cell lines, and the wound healing ability of pancreatic cancer cells was observed by a scratch test. FIG. 11-B is a quantitative result of migration of wound healing experiments calculated by imageJ. FIG. 12-A shows the overexpression and knock-down of CGN in human pancreatic cancer cell lines, and the migration and invasion ability of cells was observed by Transwell experiments. FIG. 12-B is a quantitative result of cell count, as observed under an inverted microscope. FIG. 13-B is an illustration of overexpression and knock-down of Cgn in murine pancreatic cancer cell lines, and the ability of cells to migrate was observed by wound healing experiments. FIG. 13-C is a quantitative result of migration of wound healing experiments calculated by imageJ. All experimental results show that after the expression of the CGN is knocked down by siRNA, the migration and invasion capacities of pancreatic cancer cells CFPAC-1 and PK59 with high expression of the CGN are weakened; pancreatic cancer cells PANC-1 and PL-45, which underexpress CGN, have enhanced migration and invasion ability by overexpressing CGN.
Example 4: cell growth experiments
CGN overexpression Using a lentiviral System, the embodiment is the same as example two, 3000 cells of PANC-1 and PL45 overexpressing CGN were inoculated into 96-well plates one day in advance, cultured in a 1% FBS-containing cell culture medium for 5 days, and the relative growth of the cells was measured on each day by adding CCK8 (Dojindo, #CK04) and measuring the absorbance value under an enzyme-labeled instrument.
CGN knockdown employs Lipo3000 liposome transient system, 3000 CFPAC-1 and PK-59 cells were inoculated into 96-well plates one day in advance, and the embodiment was the same as example two, cultured under the condition of containing 1% FBS cell culture medium for 5 days after transfection, and the relative growth of the cells was measured by adding CCK8 on each day and measuring absorbance value under an enzyme-labeled instrument.
The experimental results are shown in FIG. 7-B and FIG. 13-A. FIG. 7-B shows that CGN overexpression promotes proliferation of PANC-1 and PL45 pancreatic cancer cells, and CGN knockdown inhibits proliferation of CFPAC-1 and PK-59 pancreatic cancer cells. FIG. 13-A is that Cgn overexpression promotes proliferation of Panc02 murine pancreatic cancer cells; cgn knockdown inhibited Panc 02C 57BL/6 murine pancreatic cancer cell proliferation. All experimental results show that the CGN over-expression promotes the proliferation of pancreatic cancer cells in vitro, and the CGN knockdown inhibits the proliferation of pancreatic cancer cells in vitro.
Example 5: immunofluorescence (IF) and Immunohistochemistry (IHC)
Paraffin sections or pancreatic cancer tissue chips were de-waxed by xylene and reconstituted by gradient alcohol, then antigen retrieval was performed with sodium citrate buffer or EDTA buffer. Slice at 3% H 2 O 2 Treatment in methanol for 10 min (immunofluorescence skipped this step), blocking with 5% goat serum at room temperature for 1 hour, then incubation with anti-Cingulin antibody overnight at 4 ℃. The level of expression of these antigens was then detected with HRP-conjugated DAB, or with a fluorescent secondary antibody.
The experimental results are shown in FIG. 1-A, FIG. 2-C and FIG. 10-D. FIG. 1-A shows the expression of pancreatic cancer tissue chip CGN at paracancerous normal, ADM, panIN and pancreatic cancer. FIG. 2-A shows the expression of CGN in normal mice (a and e) KC mice (b, c, d, f, g and h). FIG. 2-C shows the expression of CGN in ranpirin-induced ADM. FIG. 10-D shows the expression of CGN, ki67 and pERK in mouse pancreatic cancer xenografts. The results show that CGN is low-expressed in normal acinus and ducts beside the human pancreatic cancer tissue chip, and the expression level of ADM, panIN and pancreatic cancer is gradually increased; acinar, ductal, and islet underexpression in normal C57BL/6 mouse pancreas, with progressively increased ADM and PanIN expression in KC; in ranpirin-induced ADM, luminal expression increases at the top of ADM; in contrast, in CGN knockdown pancreatic cancer cell xenografts, the expression levels of Ki67 and pERK were reduced.
Example 6: cell fluorescence
CGN overexpression and knock-down employ a lentiviral system, and the implementation is the same as in example two. Cells were inoculated one day in advance into 12-well plates with adherent slides and 2mL of cell culture medium containing 10% FBS was added and placed at 37℃in 5% CO 2 Is cultured in a cell culture incubator for 48 hours. Then fixed with 4% paraformaldehyde at room temperature, and permeabilized with 1% Triton X-100 permeabilized solution at room temperature and 2% BSA roomThe incubation was performed for 1 hour, followed by incubation with anti-Ki 67 antibody at 4 ℃ overnight, and then detection of antigen expression levels with a fluorescent secondary antibody.
The experimental results are shown in FIG. 8, which shows Ki67 increase and Ki67 down-regulation after pancreatic cancer cells over-express and knock-down CGN.
Example 7: western blotting
CGN was overexpressed and knocked down in pancreatic cancer cells using the transfection protocol in example two. The protein was recovered, the medium was discarded, washed 3 times with PBS pre-chilled at 4℃and cells were lysed by addition of an appropriate amount of protease inhibitor-SDS lysate (Bootidme, #P0013G). The protein concentration was determined by sonication for 10 seconds with a sonicator 40HZ and using a Biyundin BSA protein concentration determination kit (Beyotime, # ST 025), followed by addition of 5 XSDS-PAGE and boiling for 10 minutes. The loading was adjusted to 10ug and standard Western blotting procedures were performed to detect the expression levels of the corresponding antigens using Cingulin, elastase, amylase, SOX, pERK, ERK, vimentin and GAPDH antibodies. Protein bands were detected using a high-sensitivity chemiluminescent substrate (Millipore, # wbkls0500). Quantifying the Western blot result by using imageJ software; GAPDH expression level was normalized. Under a single blot, its value from the internal reference appears as a fold change in its expression.
The experimental results are shown in FIG. 1-C, FIG. 2-B, FIG. 7-A, FIG. 9-C, FIG. 12-C and FIG. 13-A. FIG. 1-C shows the expression level of CGN in each of the pancreatic cancer cell lines and pancreatic ductal cell lines, and CGN was expressed at higher levels in pancreatic cancer cells than in ductal cells. FIG. 2-B shows the expression level of CGN in the murine normal pancreas, duct ligation, ranpirin model, and the results show that the expression level of CGN in the three ADM models is increased compared with the normal pancreas. FIGS. 7-A and 13-A show that the over-expressed and knocked down CGN/Cgn both achieve an increase and decrease in the amount of CGN/Cgn as measured by their transfection efficiency. FIG. 9-C shows activation of MAPK/ERK signaling pathway after CGN overexpression and knockdown, and shows promotion of MAPK/ERK signaling pathway after CGN overexpression. FIG. 12-C shows the effect on Vimentin expression after over-expression and knock-down of CGN, showing that over-expression of CGN promotes increased expression.
Example 8: real-time PCR
Total RNA of the tissue or pancreatic cancer cell lines was extracted using TRIzol reagent (Invitrogen, # 15596018) and the concentrations were measured spectrophotometrically. First strand cDNA (Takara, # RR047A) was synthesized using reverse Tra acetic acid reverse transcriptase according to the manufacturer's instructions and used as a template in the following Real-time PCR. The content of CGN, CK19, amylase, 18s and GAPDH genes was measured using TB Green Premix Ex Taq (Takara, # RR420A) reagent.
The experimental results are shown in FIG. 1-C, FIG. 2-B, FIG. 7-A and FIG. 13-A. FIG. 1-C shows the transcriptional expression of CGN in pancreatic cancer cell lines and pancreatic ductal cell lines, with higher levels of CGN expressed in pancreatic cancer cells than ductal cells HPNE and HPDE. FIG. 2-B shows the expression level of CGN in the C57BL/6 mice normal pancreas, duct ligation, ranpirin and KC models, and shows that the expression level of CGN in the three ADM models is increased compared with the normal pancreas. FIGS. 7-A and 13-A show that the over-expression and knock-down CGN/Cgn both achieve the increase and decrease of the transcription expression level of CGN/Cgn, as a result of detecting the transfection efficiency.
Example 9: cell clone formation
CGN was overexpressed and knocked down in pancreatic cancer cells using the stable transfection protocol in example two. 500 PANC-1 and PL45 cells, 1000 CFPAC-1 and PK-59 cells were inoculated into a 6-well plate, cultured in a 10% FBS-containing cell culture medium for 2 weeks, fixed with methanol for 20 minutes, stained with crystal violet (Beyotime, # C0121) for 30 minutes, washed three times with PBS, and the number of clone formations was observed and counted, and the 6-well plate was photographed on a white background plate.
The experimental results are shown in FIG. 9-A, and FIG. 9-A shows that the clonogenic capacity of pancreatic cancer cells is enhanced and reduced after the pancreatic cancer cells overexpress and knock down CGN.
Example 10: credit analysis
Single cell transcriptome sequencing data:
the data used in this experiment were single cell transcriptome sequencing data in the Gene Expression Omnibus (GEO) dataset: data set one: GSE141017 uses single cell RNA-seq to analyze the development and progression of pancreatic cancer from pre-invasive stage to pre-invasive stage in a mouse model by KC mouse model pre-tumorigenic seven stage data. Data set two: GSE155698 contains 16 PDA tissue samples and 3 adjacent normal pancreas samples, which were minced mechanically, digested with collagenase P to give single cells, and sequenced using a 10x genomic platform to give data. Data set three: GSE180212 pancreatic tissue was collected at different time points before and after acute inflammation (WT Day1, day7 and Day 28) in C57BL/6J wild-type mice, single cell suspensions were obtained after digestion and libraries were prepared using 10X single cell 3' v3 chemistry.
Data quality control and preprocessing
The quality control of cells for the downloaded dataset using the setup package of R was performed according to the following filter criteria: screening the original transcription count of the genetic cell matrix according to the cells to remove cells with transcription number less than 200 and cells higher than 6000; and cells containing more than 5% or 10% mitochondrial genes. In addition, genes expressed in less than 3 cells were removed from the analysis. Thereafter, normalization was performed according to library size using a normazedata function, and normalization of data was performed with a ScaleData function according to the median of all cell library sizes.
Data dimension reduction and clustering
The highly variable genes were screened using the semat package for calculation of principal components (principal components, PC) using the highest variance gene for linear dimensionality reduction (principal component analysis), and the number of principal components used in downstream analysis was selected taking into account the pchetmap and Elbowplot of semat. Cell clustering and UMAP dimension reduction visualization were performed with FindClusters and RunTSNE functions, respectively, we annotated each cell cluster with a uniquely expressed gene for each cluster, and cell type annotation for each cell cluster was performed using a known Marker.
Sub-group analysis forest map:
transcriptome sequencing data and clinical information data for the TCGA pancreatic cancer dataset were downloaded via the UCSC XENA database. All samples were divided into two groups, CGN expression high and low, according to the mean CGN expression values. The "survivinal" package in R was used for both sub-set and Survival analysis of CGN high and low expression data sets. And a forest map is visualized via the R package "forest plot".
The experimental results are shown in FIG. 1-D, FIG. 3-A, FIG. 3-B, FIG. 4-A, FIG. 4-B, FIG. 5, and FIG. 9-B. FIG. 1-D shows the expression of CGN in pancreatic and pancreatic cancers in GSE62452, GSE32676 and GSE71729 data sets, and shows that the expression of CGN in tumors is significantly increased compared with normal. FIG. 3-A is a GSE155698 single cell transcriptome sequencing dataset analysis, showing that CGN is more highly expressed than normal acinar cells in tumor cells and metaplastic cells in epithelial cells. FIG. 3-B is a GSE141017 single cell transcriptome sequencing dataset analysis, showing that the KC mouse model data at various stages before and after the tumorigenesis show that CGN is mainly highly expressed in metaplastic cells and duct cells and is low expressed in normal acinar cells. Fig. 4-a shows the GSE62452 dataset analysis, showing that CGN was more expressed in the G2 and G3 phases than in the normal group, and significantly higher than in the normal group. Fig. 4-B is a survival analysis of GSE71729 dataset, showing that CGN high expression is significantly correlated with short patient survival. FIG. 5 shows the correlation of CGN expression with pancreatic cancer prognosis survival using a multi-factor COX regression analysis with the TCGA pancreatic cancer dataset. FIG. 9-B shows that CGN was positively regulated in MAPK signaling pathways and cell migration by differential expression analysis of two groups of CGN expression levels in GSE32676 dataset, and then up-regulated differential expression genes were mainly enriched in these pathways.
TABLE I primers and peptide fragments
| Primer name | Sequence (5 '-3') |
| CGN primer forward | TGGAGTCCAGATTCGCTTCAT |
| CGN primer reverse | CCCGTAGGTACTGGCTCTTG |
| GAPDH primer forward | TCCATGACAACTTTGGTATCG |
| GAPDH primer reverse | TGTAGCCAAATTCGTTGTCA |
| Cgn primer forward | CAGGCTGAGCTTACCCGAAA |
| Cgn primer reverse | GTGGCACTCTTCAGCCTTCT |
| Gapdh primer forward | AAGGTGGTGAAGCAGGCATCTGAG |
| Gapdh primer reverse | GGAAGAGTGGGAGTTGCTGTTGAAGTC |
| 18s primer forward | ATAAACGATGCCGACTGGCGA |
| 18s primer reverse | AAATTAAGCCGCAGGCCCAC |
| C19primer forward | ATTGGGTCAGGGGGTGTTTT |
| CK19primer reverse | TGTCCAAGTAGGAGGCGAGA |
| Amy2a primer forward | TGCCAAGGAATGTGAGCGAT |
| Amy2a primer reverse | TCCACAGGTACTGCTTGTTCC |
Claims (10)
1. The pancreatic cancer early diagnosis marker is characterized by comprising an antibody of the button protein CGN and a fluorescent quantitative polymerase chain reaction primer sequence, and accurately and clearly displays the expression mode and the variation of the expression quantity of the button protein CGN in the malignant pancreatic cancer occurrence and development process.
2. The marker for early diagnosis of pancreatic cancer according to claim 1, wherein said antibody against calmerin is an antibody blocking amino acid residues 7 to 356 or 307 to 412 of calmerin CGN.
3. The marker for early diagnosis of pancreatic cancer according to claim 1, wherein said primer sequence for fluorescence quantitative polymerase chain reaction of the button protein CGN is a reverse complement of CDS binding to the coding sequence of the button protein CGN mRNA.
4. The pancreatic cancer early-diagnosis marker according to claim 1, wherein said button protein CGN antibody is an antibody blocking the head or the screw stem of button protein.
5. The pancreatic cancer early-diagnosis marker according to claim 1, wherein the subject is at an early stage of pancreatic carcinogenesis when the transcript level and protein level expression of said button protein CGN is up-regulated.
6. The use of the pancreatic cancer early-diagnosis marker according to any one of claims 1 to 5, characterized in that a button protein CGN antibody is used as a pancreatic cancer early-diagnosis marker and a down-regulator of button protein CGN is used as a therapeutic agent for malignant tumor.
7. The use according to claim 6, wherein the down-regulator of the button protein CGN is a designed button protein CGN short interfering RNA.
8. Use according to claim 7, characterized in that it is directed against the down-regulator sequence of human button protein CGN: GTCCAGATTCGCTTCATCACA, down-regulator sequence 1 for murine button protein Cgn: GTGAGGAGGAAAGTTAGTTTG; down-regulatory sequence 2 for murine button protein Cgn: TGGAGTTCAAATTCGATTTAT; inhibit proliferation, migration and invasion of pancreatic cancer cells.
9. The use according to claim 8, wherein the inhibition of proliferation of malignant pancreatic cancer cells is a downregulation of clonogenic capacity of malignant pancreatic cancer cells; down-regulating the expression level of the malignant pancreatic cancer cell mitosis, cell proliferation-related antigen Ki 67; down-regulating the tumor growth promoting MAPK/ERK signaling pathway; and growth of pancreatic cancer cells in vivo in mice.
10. The use according to claim 8, wherein inhibiting malignant pancreatic cancer cell migration and invasion is down-regulating malignant cell wound healing capacity and attenuating pancreatic cancer cell migration and invasion capacity.
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