CN112029866B - Application of WWP1 in pancreatic cancer - Google Patents
Application of WWP1 in pancreatic cancer Download PDFInfo
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- CN112029866B CN112029866B CN202010997886.7A CN202010997886A CN112029866B CN 112029866 B CN112029866 B CN 112029866B CN 202010997886 A CN202010997886 A CN 202010997886A CN 112029866 B CN112029866 B CN 112029866B
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Abstract
The invention relates to a reagent for detecting and judging prognosis of pancreatic cancer based on WWP1 and application of a WWP1 blocker in treating pancreatic cancer. Specifically, the invention provides a detection reagent for detecting pancreatic cancer by detecting WWP1 gene and a kit containing the reagent. In addition, the invention also provides the application of the WWP1 blocker, preferably WWP1ShRNA, in preparing medicines for treating and/or preventing and/or relieving related diseases caused by pancreatic cancer.
Description
Technical Field
The invention relates to the technical field of medical biology, in particular to a pancreatic cancer detection and prognosis reagent based on WWP1 and application of a WWP1 blocker in treating pancreatic cancer
Background
Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most malignant malignancies at present, and also one of the major lethal cancers worldwide, estimated to cause 227,000 patient deaths each year. Usually, diagnosis has advanced, and only less than 20% of pancreatic cancers can be surgically resected; 40% exhibit local infiltration, failing to surgically resect the lesion; lesion metastasis has occurred in the diagnosis of the remaining patients. And pancreatic cancer is insensitive to conventional chemotherapy, radiation therapy (1, 2). The current treatment regimen does not significantly extend patient life and clinical treatment results have not improved significantly over the last 35 years, with overall 5-year survival rates of around 5% and average survival times of most advanced patients of less than one year (2, 3). Early metastasis of pancreatic cancer is its main fatal cause. There is evidence that this process may be achieved by tumor stem cells CSCs, and that such cells have the characteristics of epithelial-to-mesenchymal transition (EMT) (4).
The E3 ubiquitin ligase of WWP1 belonging to NEDD 4-like family has wide distribution, and the protein which is participated in the WWP1 is widely existed in the biochemical processes of a plurality of cells such as RNA transcription and processing, protein transportation and stabilization, cytoskeleton regulation and the like. Studies have shown that WWP1 plays an important role in the development, progression, and prognosis of many tumors, including liver cancer (17), oral cancer (18), prostate cancer (19), and breast cancer (20), among others.
The study found that the mRNA and protein expression levels of WWP1 in gastric cancer were both significantly increased relative to normal stomach tissue, and this was closely related to the degree of tumor differentiation, TNM staging, wetting depth and lymphatic metastasis in gastric cancer patients (21).
A study from liver cancer shows that the expression level of WWP1 protein in liver cancer is significantly higher than that of non-cancer tissues, and simultaneously, the expression level of mRNA is also significantly increased in 7 liver cancer tissues studied (17).
LinJH et al (18) studies showed that WWP1 expression levels were up-regulated in both oral cancer specimens and 6 oral cancer cell lines.
mRNA and protein expression of WWP1 is also frequently upregulated in prostate and breast cancers (19, 20). Gene mutations of ubiquitin ligase E3 have been found in human breast cancer tissues, such as Md2 (22), EPF (23), skp2 (24) and beta-TrCP (22), etc., in previous studies. Ubiquitin ligase E3 has been reported to have carcinogenesis in human breast cancer. WWP1, another ubiquitin ligase E3, whose genome is frequently occurring and is expressed in large scale, plays an important role in the pathogenesis of breast cancer and is considered a potential oncogene (25, 26, 27).
During the above-described tumorigenesis, WWP1 regulates a number of important proteins, which are the transforming regulator signaling pathways that regulate the TI3R1 (28) pathway through TGF- β receptors, as well as ubiquitin-mediated reverse protein degradation of Smad2 (29) and Smad4 (30). In addition, WWP1 can regulate expression in epithelial cells in sodium ion channels (ENaC) (31), runx2 (32), notch (33), KLF5 (34) and KLF21 (35). Among them, KLF5 is a tumor suppressor gene, and down-regulation of KLF2 can increase transcription level in mammary gland carcinogen c-myc, playing an important role in growth of breast cancer and transfer of KLF2 and KLF5 expression.
Studies have shown that NEDD4 acts as a specific E3 ligase for PTEN, degrading PTEN levels by polyubiquitination (36, 37) and promoting activation of Akt signaling pathway (38), and furthermore studies have demonstrated that PTEN-Akt signaling pathway is a key regulator of protooncogene signaling pathway (39), potentially becoming a molecular target for many tumor interventions (40). As a member of the NEDD 4-like family, WWP1 plays a very important role in the development of tumors, and thus may become a new molecular target for tumor gene therapy, and no application of WWP1 in pancreatic tumors has been reported at present.
Thus, in the field of pancreatic tumor treatment, there is an urgent need to develop a reagent or kit based on detection and prognosis of WWP1 pancreatic tumors, and to investigate the use of WWP1 blockers in the treatment of pancreatic tumors.
Disclosure of Invention
The invention aims to provide a reagent or a kit for detecting and/or judging the prognosis situation of pancreatic cancer patients based on detection of WWP1 gene; and the use of WWP1 blockers in the manufacture of a medicament for the treatment of pancreatic cancer was investigated.
In a first aspect of the invention, there is provided the use of a WWP1 gene, cDNA, protein or detection reagent thereof for the preparation of a diagnostic product for detecting whether a subject has pancreatic cancer; and/or predicting pancreatic cancer prognosis in a subject.
In another preferred embodiment, the diagnosis comprises an early diagnosis, an auxiliary diagnosis, or a combination thereof.
In another preferred embodiment, the WWP1 gene, cDNA, protein is of human origin.
In another preferred embodiment, the detection is for an ex vivo sample.
In another preferred embodiment, the ex vivo sample is selected from the group consisting of: serum samples, tissue samples, or combinations thereof.
In another preferred embodiment, the detection reagent comprises:
a) WWP 1-specific antibodies, WWP 1-specific binding molecules; and/or
b) A primer pair, a probe, or a combination thereof that specifically amplifies the WWP1 gene.
In another preferred embodiment, the detection reagent contains a primer pair shown in SEQ ID No. 1 and 2 for specifically amplifying the WWP1 gene.
In a second aspect of the present invention, there is provided a kit comprising a first detection reagent for detecting WWP1 gene, cDNA, protein.
In another preferred embodiment, the first detection reagent contains a primer pair shown in SEQ ID No. 1 and 2 for specifically amplifying the WWP1 gene.
In another preferred embodiment, the kit further comprises a label or instructions stating that the kit is for detecting whether the subject has pancreatic cancer; and/or predicting pancreatic cancer prognosis in a subject.
In another preferred embodiment, the subject is a pancreatic cancer patient.
In a third aspect of the present invention, there is provided the use of a WWP1 blocker or a formulation comprising said WWP1 blocker for the manufacture of a medicament for the treatment and/or prevention and/or alleviation of related diseases caused by pancreatic cancer.
In another preferred embodiment, the WWP1 blocker comprises ShRNA of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, selected from the group of sequences blocking WWP1 expression.
In another preferred embodiment, the formulation further comprises an additional tumor therapeutic agent, including gemcitabine.
In a fourth aspect of the invention, there is provided a pharmaceutical composition comprising:
a1 WWP1 blockers;
a2 Other tumor treatment drugs.
In another preferred embodiment, the WWP1 blocker comprises ShRNA of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, selected from the group of sequences blocking WWP1 expression.
In another preferred embodiment, the other tumor therapeutic agent comprises gemcitabine.
In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating and/or preventing and/or alleviating related diseases caused by pancreatic cancer.
In a fifth aspect of the invention, there is provided an in vitro inhibition of pancreatic cancer tumor stem cell replication; and/or a method of inhibiting Snail-mediated EMT, comprising the steps of: contacting the pancreatic cancer tumor stem cells with a pharmaceutically effective amount of a WWP1 blocker, thereby inhibiting expression of WWP1 and inhibiting replication of pancreatic cancer tumor stem cells; and/or inhibit Snail-mediated EMT.
In another preferred embodiment, the WWP1 blocker comprises ShRNA of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, selected from the group of sequences blocking WWP1 expression.
In a sixth aspect of the present invention, there is provided a method for preventing and/or treating and/or alleviating pancreatic cancer, comprising the steps of: administering to a subject in need thereof a pharmaceutically effective amount of a WWP1 blocker, thereby inhibiting pancreatic cancer stem cell replication.
In another preferred embodiment, the subject is a pancreatic cancer patient.
In another preferred embodiment, the WWP1 blocker comprises ShRNA of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, selected from the group of sequences blocking WWP1 expression.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a graph showing qRT-PCR versus changes in the expression level of WWP1 in normal pancreatic tissue, pancreatitis, pancreatic tumor tissue and pancreatic tumor stem cells.
FIG. 2 is a graph showing the relationship between WWP1 expression levels and prognosis of 21 pancreatic cancer patients.
FIG. 3 is an in vitro experimental test to examine the effect of blocking WWP1 on pancreatic cancer cell viability.
Fig. 4 is an experimental animal test to examine the effect of inhibiting WWP1 on pancreatic cancer growth.
Fig. 5 is an in vitro experimental test to examine the effect of blocking WWP1 on pancreatic cancer cell EMT.
Detailed Description
The inventors of the present invention have found for the first time through extensive and intensive studies that WWP1 is highly expressed in pancreatic tumor cells, pancreatic liver metastasis tumor cells and tumor stem cells, and can be used as a detection marker for diagnosis of pancreatic cancer and prognosis judgment, and have completed the present invention on the basis of this.
In particular, the invention provides a reagent or a kit for detecting pancreatic cancer and/or judging pancreatic cancer prognosis based on detection of WWP1 gene. Moreover, the examples of the present invention demonstrate that WWP1 blockers treat and/or prevent and/or alleviate related diseases caused by pancreatic cancer by inhibiting pancreatic tumor stem cells and inhibiting Snai l-mediated EMT. Based on the above, the invention also provides an application of the WWP1 blocker, preferably WWP1shRNA, in preparing medicaments for preventing and relieving pancreatic cancer related diseases.
Terminology
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of a protein encoded by RNA or gene portion, and also includes the presence of a detection substance associated with expression. For example, cDNA, binding of a binding ligand (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic portions of the binding ligand are included within the term "expressed". Thus, an increase in half-pel density in immunoblots, such as western blots, is also within the term "expression" based on biological molecules.
WWP1
WW domain E3 ubiquitin ligase 1 (WWP 1) belongs to the E3 ubiquitin ligase of the NEDD 4-like family, a subfamily of HECT, highly conserved in different animals, the human WWP1 gene is located on chromosome 8q21, comprises 26 exons, is 142kb in size, its protein comprises 922 amino acids, has a molecular weight of 110kDa, comprises 1C 2 domain, 4 WW domains and 1 HECT domain. The HECT domain can interact with ubiquitin conjugating enzyme to affect ubiquitin E3 ligase activity. WWP1 is widely distributed, and proteins involved in it widely exist in RNA transcription and processing, protein transportation and stabilization, cytoskeletal regulation and other cell biochemical processes. Ubiquitination is one of the important post-translational modifications of proteins in vivo and is a signal for protein degradation.
Primer(s)
A primer refers to a macromolecule with a specific nucleotide sequence that stimulates synthesis at the initiation of nucleotide polymerization, and is covalently linked to a reactant. Primers are typically two oligonucleotide sequences that are synthesized, one complementary to one strand of the DNA template at one end of the target region and the other complementary to the other strand of the DNA template at the other end of the target region.
The embodiment of the invention shows that WWP1 is highly expressed in pancreatic cancer tumor, pancreatic cancer liver metastasis and pancreatic cancer tumor stem cells, so that the kit can be used as a marker for detecting pancreatic cancer. Based on the above, the invention designs a primer pair using WWP1 gene as a template, and detects pancreatic cancer and judges pancreatic cancer prognosis by detecting WWP 1.
The primer for detecting the WWP1 gene is shown as SEQ ID No. 1 and 2.
Detection method
The invention provides a method for detecting pancreatic cancer based on detection of WWP1 by utilizing high expression of WWP1 in pancreatic tumor cells, pancreatic liver metastasis tumor cells and tumor stem cells.
The detection method for detecting WWP1 includes (but is not limited to) detection of WWP1 gene, cDNA and protein.
In another preferred embodiment, the present invention provides a qRT-PCR method for detecting pancreatic cancer based on detection of WWP1 gene.
In qRT-PCR, required detection primers are shown in SEQ ID No. 1 and 2.
The operation steps of the qRT-PCR method are as follows.
Detection kit
Based on high expression of WWP1 in pancreatic tumor cells, pancreatic liver metastasis cells and tumor stem cells, the invention also provides a detection kit based on detection of WWP 1.
The kit provided by the invention comprises a first detection reagent, wherein the first detection reagent is used for detecting WWP1 genes, cDNA and proteins.
In another preferred embodiment, the first detection reagent contains a primer pair shown in SEQ ID No. 1 and 2 for specifically amplifying the WWP1 gene.
In another preferred embodiment, the kit further comprises a label or instructions stating that the kit is for detecting whether the subject has pancreatic cancer; and/or predicting pancreatic cancer prognosis in a subject.
Antagonists (blockers)
As used herein, the terms "antagonist" and "blocking agent" have the same meaning and refer to the use of the protein of the present invention (WWP 1 protein) to screen for substances, particularly blocking agents, that interact with the WWP1 protein by various conventional screening methods.
Antagonists of the WWP1 proteins of the invention (including antibodies, antisense nucleic acids, small molecule compounds, and other blocking agents), when administered therapeutically, inhibit the expression and/or activity of the WWP1 protein, thereby inhibiting Snail-mediated EMT and thus pancreatic cancer tumor stem cell replication. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes.
Antagonists useful in the present invention include: antibodies to WWP1 (e.g., WWP1 neutralizing antibodies), inhibitory mRNA, antisense RNA, siRNA, shRNA to WWP1 nucleic acid, small molecule compounds, and activity blockers of WWP 1. Typical WWP1 blockers are, among others, inhibitory mirnas, sirnas and WWP1 neutralizing antibodies.
ShRNA
RNA interference (RNAi) is a process that effectively silences or inhibits the expression of a target gene by selectively inactivating the corresponding mRNA of the target gene by double-stranded RNA (dsRNA). Short hairpin RNA (shRNA) can specifically degrade target mRNA through complementary binding sequence with target mRNAh, and is a tool for inhibiting protein expression through gene silencing. The shRNA is composed of a sense strand and an antisense strand separated by a circular sequence, the expression of which is based on a vector, and a plurality of lentiviral and retrovirus plasmids are suitable for the expression of the shRNA.
Pharmaceutical composition
The present invention also provides a pharmaceutical composition comprising the above-mentioned antagonist of WWP1 (content: 0.001-99% by weight, preferably 0.01-90% by weight), and a pharmaceutically acceptable carrier (content: balance). The pharmaceutical composition can be used for weakening the drug resistance of taxane drugs.
In the present invention, the antagonist includes an antisense nucleic acid (e.g., siRNA, shRNA, antisense RNA, antisense DNA), antibody, or a combination thereof, directed against WWP 1. In addition, the antagonists include small molecule compounds that reduce WWP1 expression or activity.
In another preferred embodiment, the pharmaceutical composition further includes, but is not limited to, the following other tumor therapeutic agents: gemcitabine.
The pharmaceutical compositions of the present invention contain a safe and effective amount of the WWP1 antagonists of the invention described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the present invention may also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the WWP1 antagonist of the invention is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 8 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Tumor stem cells
The definition of tumor stem cells by AACR (11) (American Association for Cancer Research) in 2006 is: cells in a tumor that have self-renewing capacity and are capable of producing heterogeneous tumor cells that are capable of differentiating into different cells that make up the tumor. Normal stem cells have similar biological properties as tumor stem cells: self-renewal, unlimited proliferation, multidirectional differentiation and the like, wherein the difference is that the unlimited proliferation capacity of the normal stem cells is regulated by an organism, and the proliferation capacity of the normal stem cells can be out of control through mutation; whereas proliferation of tumor stem cells is unregulated. Stem cells survive for a long period in vivo, have the potential to self-replicate, and are more likely to accumulate mutations, changing from quantitative to qualitative to neoplastic stem cells (12).
Tumor stem cells are currently defined primarily by cell surface markers, each of which has a unique cell surface marker that can be identified. Leukemia tumor stem cells (CD34+/CD 38-) (6) were isolated for the first time by Dick and Bonnet et al, university of Toronto 1997. Li (Li Chenwei), clarke and Simeone et al demonstrated for the first time that pancreatic cancer stem cells exist (CD44+CD24+ESA+), which have high tumorigenic capacity and tumor stem cell characteristics that can self-renew and produce different cell populations (7). Hermmann et al reported that CD133 positive cells were highly tumorigenic and drug resistant (8). Research by rashed et al found that ALDH positive tumor cells had the properties of tumor stem cells and EMT (9). Li (Li Chenwei) and Simerone report that c-Met is a novel pancreatic cancer stem cell surface marker and can be used as an effective target for treatment (5). Rhim et al have demonstrated a 100-fold increase in tumor cells entering the blood circulation as compared to cd44+cd24+ cells as compared to in situ tumor cells (18).
There are 3 common methods for sorting tumor stem cells:
1. pancreatic cancer stem cells were sorted by phenotype: there was no morphological difference between tumor stem cells and non-tumor stem cells. One way to distinguish between tumor stem cells and non-tumor stem cells is by surface markers. Li et al (7) cultured human pancreatic cancer primary cells by a xenograft model, and about 0.2-0.8% of highly tumorigenic cell populations with phenotypes of CD44+CD24+ESA+ were sorted by flow cytometry using CD44, CD24, ESA as surface markers, and demonstrated that the cells had high tumorigenic capacity in an animal tumorigenic model. Huang et al (14) selected CD44+CD24+ highly tumorigenic cells in pancreatic cancer cell line PANC-1 using CD44, CD24 as surface markers, and demonstrated that the tumorigenic capacity of cells of this phenotype was 20-fold stronger than that of CD44-CD 24-cells in vivo in nude mice in a tumorigenic experiment. Hermann et al (8) selected CD133+ cells from human pancreatic cancer tissue and pancreatic cancer cell line L3.6pl by immunomagnetic bead sorting, and confirmed that the cells had high tumorigenicity in vivo tumorigenic experiments in athymic mice, and 500 such cells could be tumorigenized.
2. Side group cell sorting: without an explicit surface marker of tumor stem cells, SP cells can be sorted using the property of side group (SP) pump-out Hoechest dye in combination with flow cytometry. Zhou et al (15) stained PANC-1 cells with Hoechst33342 and PI dye, and then the side population cells were selected by flow cytometry. The presence of light-stained SP cells in the PANC-1 cell line was confirmed by fluorescence microscopy morphological examination.
3. Balloon culture: suspension cell balloon culture is another common stem cell sorting method. Gou et al (16) base DMEM-F12. EGF, insulin and other factors are added, saccule cells are cultured in PANC-1 cell lines, and 98.10% + -1.26% of cells are not colored through hoehst staining. In vivo oncological experiments in nude mice also demonstrated that balloon cells were about 20-fold more potent in oncological capacity than adherent cells.
Other medicaments for treating tumors
Gemcitabine
Gemcitabine (Gemcitabine) is a novel cytosine nucleoside derivative. Like cytarabine, it is activated by deoxycytidine kinase and metabolized by cytosine nucleoside deaminase after entering the human body. The product is pyrimidine antitumor medicine with the same action mechanism as cytarabine, and its main metabolite is doped with DNA in cell and acts mainly on G1/S phase. However, except that difluoro deoxycytidine can inhibit nucleotide reductase in addition to incorporation into DNA, resulting in reduction of intracellular deoxynucleoside triphosphates; the other difference with cytarabine is that it can inhibit deoxycytosine deaminase to reduce the degradation of intracellular metabolites and has self-synergistic effect. Clinically, the product has different antitumor spectrums with cytarabine, and is effective on various solid tumors.
Gemcitabine has been demonstrated to be superior to fluorouracil in pancreatic cancer since 1997, and none of the single agents has been able to surpass it, even though the combination chemotherapy regimen is based on it, the gemcitabine-based single agent or combination chemotherapy regimen remains the first choice for pancreatic cancer chemotherapy.
The main advantages of the invention include:
a) Reagents for detecting pancreatic cancer based on detection of WWP1 were first developed.
B) The detection reagent or the kit containing the reagent not only can detect pancreatic cancer, but also can judge the prognosis of pancreatic cancer in advance.
C) The use of WWP1 blockers, preferably WWP1ShRNA, for the preparation of a medicament for the treatment and/or prevention and/or alleviation of pancreatic cancer was first developed.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: the conditions described in the laboratory Manual (NewYork: cold spring harbor laboratory Press, 1989) or according to the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.
The method for establishing and testing the curative effect of the animal models of the immunohistochemical experiments, qRT-PCR, MTT and pancreatic cancer transplantation tumor and the method for sorting pancreatic cancer stem cells and the statistical analysis method comprise the following operation steps of:
1. immunohistochemical procedure
1. Dewaxing and hydration: the tissue chips should be left at room temperature for 60 minutes or baked in an incubator at 60℃for 20 minutes before deparaffinization.
a) Soaking the tissue chip in xylene for 10min, and soaking for 10min after changing xylene
b) Soaking in absolute ethanol for five minutes
c) Soaking in 95% ethanol for five minutes
d) Soaking in 75% ethanol for five minutes
2. Antigen thermal remediation:
heating in a microwave oven, heating 0.01 sodium citrate buffer (ph 6.0) to boiling, placing the tissue chip into the microwave oven, switching off, repeating for 1-2 times at intervals of 5-10 minutes. Paraffin embedded tissue chip for formalin fixation
3. The specific operation steps of the immunohistochemical staining SP method are as follows:
(1) Dewaxing and hydration
(2) PBS was washed 2-3 times for 5min each
(3) 3% H2O2 (80% methanol) was added dropwise to TMA and allowed to stand at room temperature for 10 minutes
(4) PBS was washed 2-3 times for 5min each
(5) Antigen retrieval
(6) PBS was washed 2-3 times for 5min each
(7) Dripping normal goat serum blocking solution, and throwing off excessive liquid at room temperature for 20min
( 8) Drop-in I anti 50 μl, standing at room temperature for 1-2 hr (antibody: WWP1 antibody (N-20) SantaCruzBiotech )
(9) After overnight at 4℃it was necessary to rewire at 37℃for 45 minutes
(10) PBS wash 3 times for 2 minutes each
(11) Dripping 45-50 μl of II antibody, standing at room temperature or 37deg.C for 1 hr
(12) The second antibody can be added with 0.05% of tween-20.
(13) PBS was washed 3 times for 5 minutes each
(14) DAB color development is carried out for 5-10 minutes, and the dyeing degree is mastered under a microscope
(15) PBS or tap water rinse for 10min
(16) Hematoxylin counterstain for 2 min, hydrochloric acid alcohol differentiation
(17) Flushing with tap water for 10-15 min
(18) Dehydrating, transparency, sealing and microscopic examination.
4. Result evaluation
The cancer tissue and the normal tissue of the tissue specimen are respectively evaluated, and the brown yellow particles of the cells are used as positive expression markers. The following expression judgment standards are formulated according to the references, wherein the dyeing area is less than 10% -0 minutes; 11% <25% -1 min; 26% <50% -2 min; 51% -3 min. Negative-0 score of dyeing intensity; weak-1 score; medium-2 minutes; strong-3 minutes.
2. qRT-PCR procedure
First) Total RNA extraction
1) Washing a cell sample in a cell culture dish twice by using PBS, sucking the PBS to be clean by using a 1ml gun, adding 1mlTrizol (invitrogen) solution, blowing and mixing uniformly, sucking the mixture into a 1.5ml RNasedreEP tube to fully lyse the cells, and standing the mixture for 5min at room temperature; fully grinding the tissue sample by liquid nitrogen, adding 1mlTrizol (Invitrogen) solution, uniformly mixing, and standing at room temperature for 5min to fully crack;
2) Adding 200 μl chloroform, shaking vigorously, mixing for 30s, allowing the water phase and the organic phase to fully contact, and standing at room temperature for 3-5min; (the centrifuge tubes are sequentially discharged during centrifugation, and the centrifuge tubes are sequentially arranged after centrifugation, and the sequence of the centrifuge tubes is consistent with that of the first step)
3) Centrifuging at 4deg.C for 15min at 14,000g to obtain three layers, and transferring RNA into another new RNasedreEP tube in the upper water phase;
4) Precipitating RNA: adding equal volume of isopropanol, gently and fully mixing (reversing for 6-8 times), and standing at room temperature for 10min;
5) Centrifuging at 4deg.C for 10min at 14,000g, collecting RNA precipitate, and removing supernatant;
6) Washing twice with 75% ethanol (centrifuging at 12,000g for 5min, and air drying in a super clean bench;
7) An appropriate amount of DEPC water (at least 15 ul) was added depending on the amount of precipitate to dissolve the precipitate.
Two) genome removal procedure:
1) Adding equal volume of phenol/chloroform, mixing, standing at room temperature for 5min, centrifuging at 14,000rpm for 15min, and collecting supernatant.
2) Adding equal volume of chloroform, mixing, standing for layering, centrifuging at 14,000rpm for 15min, and collecting supernatant.
3) Adding equal volume of isopropanol, gently and fully mixing (reversing for 6-8 times), standing at 20deg.C for 15min;
4) Centrifuging at 4deg.C for 15min at 14,000g, collecting RNA precipitate, and removing supernatant;
5) Washing twice with 75% ethanol (centrifuging at 12,000g for 5 min), and air drying in a super clean bench;
6) An appropriate amount of DEPC water (at least 15 ul) was added to dissolve the precipitate.
Third) Total RNA purity and integrity detection
1) And (3) purity detection: taking 1 mu l of RNA sample for 50-time dilution, and measuring the OD value on a nucleic acid protein detector, wherein the ratio of OD260/OD280 is more than 1.8, which indicates that the prepared RNA is purer and has no protein pollution.
2) Total RNA integrity detection: RNA samples were taken 1. Mu.l, 1% agarose gel electrophoresed for 80 V.times.20 min, EB stained for 10min, and the 5srRNA,18srRNA and 28srRNA bands of total RNA were observed and photographed using a gel imaging system.
Fourth), mRNA reverse transcription procedure:
1) A total volume of 12. Mu.l of solution was prepared by adding TotalRNA1.0. Mu.g and H2O to the PCR tube of RNasetree.
2) Blowing the solution uniformly, and preserving the temperature at 85 ℃ for 5min to denature RNA. Immediately thereafter ice-cooling to prevent RNA renaturation;
3) Promega reagent was added to the PCR tube
4) Incubating the 20. Mu.l of the reaction solution at 30℃for 10min;
5) Preserving heat at 42 ℃ for 50min;
6) Preserving heat at 85 ℃ for 10min;
7) Preserving at-20 ℃.
Fifth) quantitative PCR detection
1. Primer test:
qPCR is required to test the specificity and amplification efficiency of the primers designed according to mRNA before the formal experiments, and a template control is required to be carried out on each pair of primers according to a specific reaction system and reaction conditions such as the formal experiments.
2. Preparing a system:
H2O4ul
SYBRGreenPCRMastermix10ul (TOYOBO) (uniform oscillation before use)
Upstream primer 0.5ul (10 uM)
0.5ul of downstream primer (10 uM)
Total volume of 15ul
After the total system is prepared, the system is uniformly vibrated in an oscillator or uniformly sucked by a gun, and then 15ul of each tube is split into 8 connecting tubes.
3. The cDNA is diluted with sterilized pure water to a proper concentration, usually 1:20, and the cDNA is added to the reaction system just prepared after being discharged in a certain order. After the sample addition, the eight-connecting tube cover is covered, and the sequence of 1-12 is marked on the uppermost edge of the eight-connecting tube cover.
4. The eight rows of tubes were placed on a palm centrifuge and centrifuged for several seconds.
5. Opening the sample frame, putting the eight connecting pipes, closing the sample frame, selecting the hole sites of the reaction pipes on the software, and eliminating the hole sites without the reaction pipes.
6. The sample name of each reaction well and the name of the detection gene are marked on 7500 software, and the result file is saved in a classified mode.
After the reaction is completed, the eight-way tube is filled into a sealing bag, the file name is marked on the bag, and the name of a customer is marked on the bag. (three repeated experiments of the same customer only need to store one repeated reaction tube, and the rest can be discarded)
3. MTT method Experimental procedure
1. Inoculating cells: subculturing with DMEM medium (Hyclone) containing 10% fetal calf serum (Thermo) 5% CO2 at 37deg.C, panc-1 from ATCC, WWP1ShRNA (TL 301750) as OriGene product, preparing single cell suspension, inoculating into 96-well plate with 1000-10000 cells per well, and 200ul per well volume;
2. culturing the cells: 5% CO2, 37℃to confluence of cell monolayers to the bottom of wells (96 well flat bottom plate).
3. Color development: after 3-5 days of incubation, 20ul of MTT (thiazole blue) solution (5 mg/ml in PBS, pH=7.4) was added to each well, incubation was continued for 4h, incubation was terminated, the in-well culture supernatant was carefully aspirated, and centrifugation was required for suspension cells before the in-well culture supernatant was aspirated. 150ul DMSO was added to each well and the mixture was shaken for 10min to allow sufficient melting of the crystals.
4. Colorimetric: the light absorption value of each hole is measured on an ELISA monitor by selecting 490nm wavelength, and the result is recorded.
4. Method for establishing pancreatic cancer transplantation tumor animal model and testing curative effect
1. Human colon cancer tissue was determined and the cancer tissue was obtained by surgery, or cultured cell lines such as Panc-1, wwp1shrna (TL 301750) were collected as OriGene products.
2. The residence time of the tumor tissue or cells in vitro is shortened as much as possible so as to maintain the freshness of the tumor tissue to the maximum extent.
3. After skin disinfection with 70% alcohol, 3 tumor tissues were transplanted to each side and disinfected, or pancreatic tumor cells were directly injected. The immunodeficient mice may be BALB/C or NOD/SCID mice.
4. Each group takes 6. When the transplanted tumor of each immunodeficient mouse grows to 150mm on average 3 Dosing was started at that time. Dosing regimens include a negative control and different treatment regimens and concentrations.
5. Tumor size changes in each immunodeficient mouse after administration require measurement every 3-5 days.
6. The calculation formula of the drug effect comprises the following steps: T/C (treatment/control) X100% to evaluate the efficacy of all regimens.
5. Pancreatic cancer stem cell sorting method
1. Taking a human pancreatic cancer specimen or a tumor specimen obtained from a nude mouse transplanted tumor model, preparing a cell suspension by using a collagen enzyme digestion method, digesting tumor tissues for 2-3 hours at 37 ℃, and filtering by using a 40uM filter screen after digestion to prepare a single cell suspension.
2. With 2% FCSRPMI 1640HBSS adjusts the cell concentration to 1-5×10 6 /ml. Cells were labeled with antibodies CD44 and c-Met (BD) (20 min at 4 ℃) and washed 2 times with washing solution, about 4ml of each wash solution was added, and centrifuged at 1000rpm X5 min.
Removal of non-pancreatic cancer cells (nude mouse cells) by H2-Kd, removal of dead cells by 4, 6-diamidino-2-phenylindole (DAPI)
4. Flow cytometry (BDAria) sorted human primary pancreatic cancer cd44+c-met+ cells, cells were sorted 2 times, guaranteeing sorted cell purity >90%.
6. Statistical analysis method
Data are shown as mean ± SE. Statistically significant differences were measured using the student's t test and X under different conditions 2 Analytically determine, and define as P<0.05。
The sequences of all primers used in the examples are shown in Table 1 below
TABLE 1 primer sequence listing
Example 1 detection of WWP1 expression level in pancreatic tumor cells, pancreatic hepatic metastasis cells and tumor Stem cells
Clinical specimen selection standard, namely receiving surgical excision cases, and diagnosing postoperative pathology as pancreatic duct adenocarcinoma; the complete histological specimen is obtained by puncture biopsy, and the pathological diagnosis is pancreatic duct adenocarcinoma. Patients who participated in this study did not receive chemotherapy, radiation therapy or immunotherapy prior to surgery or biopsy.
The method comprises the following steps:
normal pancreatic tissue, pancreatitis, pancreatic tumor tissue was obtained post-operatively and biopsied, and pancreatic tumor stem cells were isolated by flow sorting. After RNA extraction, qRT-PCR was performed, and the procedure was as above. qRT-PCR compares changes in the expression level of WWP1 in normal pancreatic tissue, pancreatitis, pancreatic tumor tissue, and pancreatic tumor stem cells.
Results:
as shown in fig. 1, quantitative PCR data found that WWP1 expression levels were lower in normal pancreatic tissue, with a slight increase in pancreatitis WWP1 relative to normal tissue, and 6.3-fold in tumor tissue elevation, and approximately 11-fold in pancreatic tumor stem cells.
Quantitative PCR 10 samples were analyzed per group. As shown in Table 1, immunohistochemical examination revealed that only 2.7% of normal pancreatic patients were WWP1 positive, pancreatitis was 7.1%, pancreatic tumor tissue was 75%, and pancreatic tumor liver metastasis tissue was 87%. Immunohistochemical examination was performed on 16 specimens per group. It can thus be inferred that WWP1 is a significant tumor detection target, and that WWP1 quantitative PCR and immunohistochemical examination can be used to diagnose pancreatic cancer.
TABLE 1 immunohistochemical detection of WWP1 Positive Rate
| Normal tissue | Inflammation | Cancer of the human body | Metastatic cancer |
| 6.3％ | 12.5％ | 75％ | 87.5％ |
Example 2 detection of WWP1 in relation to pancreatic tumor cell Positive level and prognosis
2.1 method:
21 pancreatic cancer patients with complete follow-up records are selected, pathological sections are obtained for WWP1 immunohistochemical examination, and the steps of the immunohistochemical operation are as above. All selected patients were post-operatively treated with conventional gemcitabine chemotherapy alone, with no other treatment record.
The WWP1 immunohistochemical tests of 21 pancreatic cancer patients were divided into three groups (strong positive 3, positive 2, weak positive 1) and 7 for each group, followed by prognosis and patient survival time.
2.2 results:
as shown in fig. 2, the prognosis follow-up data shows that pancreatic tumor cell positive levels and prognosis survival are inversely related, the prognosis for strongly positive patients is poor, survival is short, and the prognosis for weakly positive patients is better, survival is longer.
It can thus be inferred that WWP1 immunohistochemical examination levels can be used to predict prognosis and can be used as an index for predicting therapeutic effects.
Example 3 detection of the growth relationship of blocking WWP1 expression and pancreatic tumor cells
3.1 blocking WWP1 inhibits pancreatic tumor cell survival
The method comprises the following steps:
in vitro experiments MTT experiments were performed using the Panc-1 cell line, and the effect of blocking WWP1 on pancreatic cancer cell viability was analyzed. Gemcitabine served as a control.
Results:
as shown in fig. 3, gemcitabine has little effect on pancreatic cancer cell survival in vitro, whereas blocking WWP1 in vitro significantly reduces pancreatic cancer cell survival.
It can therefore be inferred that blocking WWP1 in vitro experiments is effective in inhibiting the survival of pancreatic tumor cells.
3.2 animal experiments prove that blocking WWP1 can inhibit pancreatic tumor growth.
The method comprises the following steps:
pancreatic cancer cells transfected with WWP1ShRNA and control pancreatic cancer cells were inoculated to immunodeficiency
Mice establish an animal model of human pancreatic cancer for research, and the influence of blocking WWP1 on the growth of pancreatic cancer in the animal model is examined. Gemcitabine (100 mg/kg twice weekly) was dosed for 4 weeks and tumor volumes were measured for 8 weeks.
Results:
as shown in fig. 4, blocking WWP1 inhibited pancreatic tumor growth in animal models. Compared with the common Panc-1 cells, the tumor growth of nude mice injected subcutaneously after the Panc-1 cells are transfected with WWP1ShRNA is inhibited.
And the blocking WWP1 has better treatment effect in combination with Gemcitabine (Gemcitabine, gem), the first choice drug for pancreatic cancer. The experimental results using the primary human pancreatic cancer animal model (Patient-developed Xenograft-PDX) were consistent with those using Panc-1.
It was therefore concluded that blocking WWP1 significantly inhibited pancreatic cancer cell growth in nude mice.
3.3 blocking WWP1 inhibits pancreatic tumor stem cells and EMT of tumors.
The method comprises the following steps:
and (3) digesting the tumor tissue obtained by the animal experiment by collagenase to generate single cells, and carrying out flow separation on CD44 and c-Met antibodies to obtain CD44+c-Met+pancreatic cancer stem cells, so as to study and block the influence of WWP1 on the tumor stem cells. Quantitative PCR analysis of RNA extracted from cells was performed to investigate the effect of blocking WWP1 on the EMT pathway.
Results:
blocking WWP1 with WWP1ShRNA significantly reduced the number of pancreatic tumor stem cells, and blocking WWP1 combined with Gemcitabine (Gem) resulted in better therapeutic effect (table two).
As shown in fig. 5, quantitative PCR assays with digested tumor cells were performed, and blocking WWP1 was found to inhibit Snail-mediated EMT.
Blocking WWP1 Effect on pancreatic tumor Stem cells
| Control | sShRNA | GEM | sShRNA+GEM |
| 2.0％ | 0.64％ | 4.3％ | 0.52％ |
Conclusion:
blocking WWP1 significantly reduced pancreatic tumor stem cell numbers and inhibited Snail-mediated EMT.
In conclusion, the research proves that WWP1 is highly expressed in pancreatic tumor cells and tumor stem cells, and the WWP1 can be applied to diagnosis of pancreatic cancer and can be used as an index for predicting treatment effect to predict prognosis; the target WWP1 can inhibit the growth of pancreatic tumors and inhibit pancreatic tumor stem cells, and can be used as a novel drug for treating pancreatic cancer and a novel target for biological immunotherapy.
Discussion of the invention
There are two theories about the formation of tumors. Stochastic considers that every tumor cell is homogeneous and every tumor cell has the potential to form a new tumor. But its proliferative division cycle into cells is controlled by some small probability of random events. The Hierarchy theory states that there is a subset of cells in tumor cells: tumor initiating cells, only cells in this subset have the ability to form new tumors, while such cells have the ability to self-renew and differentiate in multiple directions. Tumor initiating cells, i.e. tumor stem cells, lead to the development and progression of tumors (13).
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (10)
1. Use of a WWP1 gene, cDNA, protein or detection reagent thereof for the preparation of a diagnostic product for detecting whether a subject has pancreatic cancer; and/or predicting pancreatic cancer prognosis in a subject.
2. The use according to claim 1, wherein the detection reagent comprises a primer pair of SEQ ID nos. 1 and 2 for specific amplification of the WWP1 gene.
3. A kit, comprising a first detection reagent for detecting WWP1 gene, cDNA, protein.
4. The kit according to claim 3, wherein the first detection reagent contains a primer pair for specifically amplifying the WWP1 gene shown in SEQ id nos. 1 and 2.
5. Use of a WWP1 blocker or a formulation comprising said WWP1 blocker, for the preparation of a medicament for the treatment and/or prevention and/or alleviation of pancreatic cancer;
wherein the WWP1 blocker consists of ShRNA sequences of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 for blocking WWP1 expression.
6. The use of claim 5, wherein the formulation further comprises an additional tumor therapeutic agent, the additional tumor therapeutic agent comprising gemcitabine.
7. A pharmaceutical composition, comprising:
a1 WWP1 blocking agent, wherein said WWP1 blocking agent consists of the following group of ShRNA sequences blocking WWP1 expression: SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6;
a2 Other tumor treatment drugs.
8. The pharmaceutical composition of claim 7, wherein the additional tumor therapeutic agent is gemcitabine.
9. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is used for the preparation of a medicament for the treatment and/or prevention and/or alleviation of pancreatic cancer.
10. A method of inhibiting pancreatic cancer tumor stem cell replication and/or inhibiting Snail-mediated EMT in vitro comprising the steps of: contacting the pancreatic cancer tumor stem cells with a pharmaceutically effective amount of a WWP1 blocker, thereby inhibiting expression of WWP1 and inhibiting replication of pancreatic cancer tumor stem cells; and/or inhibit Snail-mediated EMT;
wherein, the WWP1 blocker consists of the following ShRNA sequences for blocking WWP1 expression: SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6.
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