CN112029866A - Application of WWP1 in pancreatic cancer - Google Patents
Application of WWP1 in pancreatic cancer Download PDFInfo
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- CN112029866A CN112029866A CN202010997886.7A CN202010997886A CN112029866A CN 112029866 A CN112029866 A CN 112029866A CN 202010997886 A CN202010997886 A CN 202010997886A CN 112029866 A CN112029866 A CN 112029866A
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Abstract
The invention relates to a WWP 1-based pancreatic cancer detection and prognosis judgment reagent and application of a WWP1 blocker in treatment of pancreatic cancer. Specifically, the invention provides a detection reagent for detecting pancreatic cancer by detecting WWP1 gene and a kit containing the detection reagent. In addition, the invention also provides an application of a WWP1 blocker, preferably WWP1ShRNA, in preparing a medicament for treating and/or preventing and/or relieving related diseases caused by pancreatic cancer.
Description
Technical Field
The invention relates to the field of medical biotechnology, 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 currently one of the most malignant malignancies and is also one of the leading lethal cancers worldwide, estimated to cause 227,000 deaths annually. Often diagnosed as having progressed to an advanced stage, with less than 20% of pancreatic cancers surgically resectable; 40% of the lesions are locally infiltrated and can not be removed by operation; the remaining patients had already developed metastasis at the time of diagnosis. And pancreatic cancer is not susceptible to conventional chemotherapy, radiotherapy (1, 2). Current treatment regimens do not significantly prolong patient life, and clinical treatment outcomes have not improved significantly over the last 35 years, with overall 5-year survival rates of around 5%, with the average survival of most advanced patients being less than one year (2, 3). Early metastasis of pancreatic cancer is its major cause of mortality. Evidence suggests that this process is likely to be carried out by tumor stem cells CSCs, and that such cells have the property of epithelial-mesenchymal transition (EMT) (4).
WWP1 belongs to E3 ubiquitin ligase of NEDD 4-like family, has wide distribution, and the protein formed by the E3 ubiquitin ligase exists widely in RNA transcription and processing, protein transportation and stabilization, cytoskeleton regulation and other cell biochemical processes. Research has 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 both the mRNA and protein expression levels of WWP1 were significantly increased in gastric cancer relative to normal gastric tissue, and this was closely related to the degree of tumor differentiation, TNM staging, degree of parenting, and lymphatic metastasis in gastric cancer patients (21).
A study from liver cancer showed that the expression level of WWP1 protein in liver cancer was significantly higher than that in non-cancer tissues, and the expression level of mRNA was also significantly increased in 7 liver cancer tissues studied (17).
LinJH et al (18) study showed that WWP1 was up-regulated in oral cancer specimens and 6 oral cancer cell lines.
The mRNA and protein expression of WWP1 is also frequently up-regulated in prostate and breast cancer (19, 20). In previous research reports, the genetic mutation of ubiquitin ligase E3, such as Md2(22), EPF (23), Skp2(24), beta-TrCP (22) and the like, is found in human breast cancer tissues. Ubiquitin ligase E3 has been reported to have carcinogenic effects in human breast cancer. WWP1, another ubiquitin ligase E3, whose genome is frequently expressed and amplified, plays an important role in the pathogenesis of breast cancer and is considered to be a potential oncogene (25, 26, 27).
During the above tumorigenesis, WWP1 regulates many important proteins, a switch regulator signaling pathway that regulates the TI3R1(28) pathway and ubiquitin-mediated reverse protein degradation by Smad2(29) and Smad4(30) through TGF- β receptors. In addition, WWP1 can also regulate expression in epithelial cells in sodium ion channel (ENaC) (31), Runx2(32), Notch (33), KLF5(34) and KLF21 (35). Among them, KLF5 is a tumor suppressor gene, and downregulation of KLF2 increases the transcriptional level of the breast carcinogen c-myc, playing an important role in the growth and metastasis of breast cancer, KLF2 and KLF5 expression.
Research shows that NEDD4 as a special E3 ligase acting as PTEN can degrade PTEN levels (36, 37) and promote activation of Akt signal pathway (38) through polyubiquitination, and research proves that PTEN-Akt signal pathway is a key regulator of protooncogene signal pathway (39) and can be a molecular target for a plurality of tumor interventions (40). As a member of the NEDD 4-like family, WWP1 plays an important role in the development of tumors, and thus may become a new molecular target for tumor gene therapy, and there is no report on the application of WWP1 in pancreatic tumors.
Therefore, in the field of pancreatic tumor treatment, there is an urgent need to develop a WWP 1-based reagent or kit for pancreatic tumor detection and prognosis, and to study the application of WWP1 blocker 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 condition of a pancreatic cancer patient based on the detection of a WWP1 gene; and the application of the WWP1 blocker in preparing medicines for treating pancreatic cancer is researched.
In a first aspect of the present invention, there is provided a use of a WWP1 gene, cDNA, protein or a detection reagent thereof for preparing a diagnostic product for detecting whether a subject has pancreatic cancer; and/or predicting a prognosis of pancreatic cancer in the subject.
In another preferred embodiment, the diagnosis includes early diagnosis, assisted diagnosis, or a combination thereof.
In another preferred example, said WWP1 gene, cDNA, protein are of human origin.
In another preferred example, the test is a test on an ex vivo sample.
In another preferred embodiment, the ex vivo sample is selected from the group consisting of: a serum sample, a tissue sample, or a combination 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 for specifically amplifying the WWP1 gene.
In another preferred embodiment, the detection reagent contains a primer pair shown in SEQ ID Nos. 1 and 2 for specifically amplifying 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 comprises a primer pair shown in SEQ ID Nos. 1 and 2 for specifically amplifying WWP1 gene.
In another preferred embodiment, the kit further comprises a label or instructions for use of the kit for detecting whether a subject has pancreatic cancer; and/or predicting a prognosis of pancreatic cancer in the subject.
In another preferred embodiment, the subject is a pancreatic cancer patient.
In a third aspect of the present invention, there is provided a WWP1 blocker or a use of a preparation containing said WWP1 blocker for the preparation of a medicament for the treatment and/or prevention and/or amelioration of a disease associated with pancreatic cancer.
In another preferred example, said WWP1 blocker comprises ShRNA selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 that blocks expression of WWP 1.
In another preferred embodiment, the formulation further comprises other tumor treatment drugs, including gemcitabine.
In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising:
A1) WWP1 blocker;
A2) other tumor treating medicines.
In another preferred example, said WWP1 blocker comprises ShRNA selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 that blocks expression of WWP 1.
In another preferred embodiment, the other tumor treatment drug 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 method of inhibiting pancreatic cancer tumor stem cell replication; and/or inhibiting Snail-mediated EMT, comprising the steps of: contacting said pancreatic cancer tumor stem cells with a medically effective amount of a WWP1 blocker, thereby inhibiting expression of WWP1 and inhibiting replication of pancreatic cancer tumor stem cells; and/or inhibiting Snail-mediated EMT.
In another preferred example, said WWP1 blocker comprises ShRNA selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 that blocks expression of WWP 1.
In a sixth aspect of the present invention, there is provided a method for preventing and/or treating and/or ameliorating pancreatic cancer, comprising the steps of: administering to a subject in need thereof a medically 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 example, said WWP1 blocker comprises ShRNA selected from the group consisting of SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 that blocks expression of WWP 1.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a graph comparing changes in expression levels of WWP1 in qRT-PCR compared to normal pancreatic tissue, pancreatitis, pancreatic tumor tissue and pancreatic tumor stem cells.
FIG. 2 is a graph showing the relationship between the expression level of WWP1 and the prognosis in 21 patients with pancreatic cancer.
FIG. 3 is an in vitro assay to examine the effect of blocking WWP1 on pancreatic cancer cell survival.
FIG. 4 is an animal experiment examining the effect of inhibition of WWP1 on pancreatic cancer growth.
FIG. 5 is an in vitro experiment to examine the effect of blocking WWP1 on EMT of pancreatic cancer cells.
Detailed Description
The present inventors have made extensive and intensive studies to find, for the first time, 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 and prognosis of pancreatic cancer, and have completed the present invention based on this finding.
Specifically, the invention provides a reagent or a kit for detecting pancreatic cancer and/or judging pancreatic cancer prognosis based on detecting WWP1 gene. Furthermore, the examples of the present invention show that WWP1 blocker can treat and/or prevent and/or alleviate the related diseases caused by pancreatic cancer by inhibiting pancreatic tumor stem cells and inhibiting Snai-mediated EMT. Based on the WWP1 blocker, preferably WWP1shRNA, and the application of the WWP1 blocker in preparing the medicines for preventing and relieving the pancreatic cancer related diseases are also provided.
Term(s) for
As used herein, the term "comprising" or "includes" 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 portion of a gene, and includes the production of protein encoded by an RNA or gene or portion of a gene, as well as the presence of a test substance associated with expression. For example, cDNA, binding of a binding partner (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic moieties of the binding partner are included within the scope of the term "expression". Thus, an increase in the density of half-spots on immunoblots such as western blots is also within the scope of the term "expression" based on biological molecules.
WWP1
WW domain E3 pan-ligase 1(WWP1) E3 ubiquitin ligase, belonging to NEDD 4-like family, is a subfamily of HECT, highly conserved among different animals, and human WWP1 gene is located on chromosome 8q21, contains 26 exons, has a size of 142kb, and its protein contains 922 amino acids, molecular weight of 110kDa, contains 1C 2 domain, 4 WW domains and 1 HECT domain. The HECT domain can interact with ubiquitin-binding enzymes, affecting the activity of ubiquitin E3 ligase. WWP1 is widely distributed, and the proteins formed by the WWP1 are widely existed in various cell biochemical processes such as RNA transcription and processing, protein transportation and stabilization, cytoskeleton regulation and the like. Ubiquitination, one of the important post-translational modifications of proteins in vivo, is a signal for protein degradation.
Primer and method for producing the same
A primer is a macromolecule with a specific nucleotide sequence, which is stimulated to be synthesized at the beginning of nucleotide polymerization and is linked with a reactant in a covalent bond mode. The primers are typically two oligonucleotide sequences synthesized by man, one primer complementary to one DNA template strand at one end of the target region and the other primer complementary to the other DNA template strand at the other end of the target region.
The WWP1 is shown to be highly expressed in stem cells of pancreatic cancer tumors, liver metastases of the pancreatic cancer and pancreatic cancer tumors in the embodiment of the invention, so the WWP1 can be used as a marker for detecting pancreatic cancer. Based on the above, the present invention designs a primer pair using the WWP1 gene as a template, detects WWP1 to detect pancreatic cancer, and determines the prognosis of pancreatic cancer.
The primers for detecting the WWP1 gene are shown as SEQ ID Nos. 1 and 2.
Detection method
By utilizing high expression of WWP1 in pancreatic tumor cells, pancreatic liver metastasis tumor cells and tumor stem cells, the invention provides a method for detecting pancreatic cancer based on WWP 1.
The detection method for detecting WWP1 includes (but is not limited to) detecting WWP1 gene, cDNA and protein.
In another preferred embodiment, the present invention provides a qRT-PCR method based on the detection of WWP1 gene to detect pancreatic cancer.
In the qRT-PCR, the required detection primers are shown as SEQ ID Nos. 1 and 2.
The operational procedure of the qRT-PCR method is described below.
Detection kit
Based on high expression of WWP1 in pancreatic tumor cells, pancreatic liver metastasis tumor cells and tumor stem cells, the invention also provides a detection kit based on WWP 1.
The kit provided by the invention comprises a first detection reagent, and the first detection reagent is used for detecting WWP1 gene, cDNA and protein.
In another preferred embodiment, the first detection reagent comprises a primer pair shown in SEQ ID Nos. 1 and 2 for specifically amplifying WWP1 gene.
In another preferred embodiment, the kit further comprises a label or instructions for use of the kit for detecting whether a subject has pancreatic cancer; and/or predicting a prognosis of pancreatic cancer in the subject.
Antagonists (blockers)
As used herein, the terms "antagonist" and "blocker" have the same meaning and refer to substances, particularly blockers and the like, which interact with the WWP1 protein, can be screened by various conventional screening methods using the protein of the present invention (WWP1 protein).
The antagonist of WWP1 protein (including antibody, antisense nucleic acid, small molecule compound and other blocker) can inhibit expression and/or activity of WWP1 protein when applied (dosed) in therapy, thereby inhibiting Snail-mediated EMT and further inhibiting replication of pancreatic cancer tumor stem cells. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical composition 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 to WWP1 nucleic acids, siRNA, shRNA, small molecule compounds, and blockers of the activity of WWP 1. Among the typical WWP1 blockers are inhibitory mirnas, sirnas and WWP1 neutralizing antibodies.
ShRNA
RNA interference (RNAi) is a process of effectively silencing or inhibiting the expression of a target gene by selective inactivation of the corresponding mRNA of the target gene by double-stranded RNA (dsrna). Short hairpin RNAs (shrnas) can specifically achieve degradation of target mrnas by complementary binding sequences to target mRNAh, and are tools for inhibiting protein expression by gene silencing. shRNA consists of sense and antisense strands separated by a circular sequence, and its expression is based on a vector, and there are many lentivirus and retrovirus plasmids suitable for the expression of shRNA.
Pharmaceutical composition
The present invention also provides a pharmaceutical composition comprising the above-mentioned antagonist of WWP1 (in an amount of 0.001 to 99 wt%, preferably 0.01 to 90 wt%), and a pharmaceutically acceptable carrier (in the balance). The pharmaceutical composition can be used for weakening the drug resistance of the taxane drugs.
In the present invention, the antagonist includes an antisense nucleic acid (e.g., siRNA, shRNA, antisense RNA, antisense DNA), an antibody, or a combination thereof against WWP 1. In addition, the antagonist also comprises a small molecule compound which can reduce the expression or activity of WWP 1.
In another preferred embodiment, the pharmaceutical composition further comprises, but is not limited to, the following other tumor treatment drugs: gemcitabine.
The pharmaceutical composition of the present invention contains a safe and effective amount of the WWP1 antagonist of the present invention 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 preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. 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.
In using the pharmaceutical compositions, a safe and effective amount of the WWP1 antagonist of the invention is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is 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 the ability to self-renew and can give rise to heterogeneous tumor cells, which can differentiate 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, and the difference is that the unlimited proliferation capacity of normal stem cells is regulated and controlled by an organism, and the proliferation capacity of the normal stem cells can be out of control through mutation; while the proliferation of tumor stem cells is unregulated. Stem cells survive long in vivo, have the potential to self-replicate, and are more likely to accumulate mutations, thereby changing from quantitative to qualitative to tumor stem cells (12).
Tumor stem cells are currently defined primarily by cell surface markers, each tumor cell having a unique cell surface marker that can be identified. Leukemia tumor stem cells (CD34+/CD38-) (6) were first isolated in 1997 by Durongian Dick and Bonnet et al. Li (Li Chen Wei), Clarke and Simeone and the like firstly confirm the existence of pancreatic cancer stem cells (CD44+ CD24+ ESA +) in 2007, and the cells have high tumorigenic capacity and the characteristics of tumor stem cells which can self-renew and generate different cell populations (7). Hermann et al reported that CD133 positive cells are highly tumorigenic and resistant (8). Rasheed et al found that ALDH positive tumor cells have the characteristics of tumor stem cells and EMT (9). Li (Li Chen-W.E.) and Simeon reported that c-Met is a new surface marker for pancreatic cancer stem cells and can be an effective target for therapy (5). Rhim et al have demonstrated a 100-fold increase in tumor cells entering the blood circulation compared to CD44+ CD24+ cells in situ (18).
There are 3 methods commonly used for sorting tumor stem cells:
1. sorting pancreatic cancer stem cells by phenotype: there was no difference in morphology between tumor stem cells and non-tumor stem cells. One way to distinguish tumor stem cells from non-tumor stem cells is by surface markers. Li et al (7) cultured primary human pancreatic cancer cells in a xenograft model, separated approximately 0.2% to 0.8% of a highly tumorigenic cell population with the phenotype of CD44+ CD24+ ESA + by flow cytometry using CD44, CD24, ESA as surface markers, and confirmed that the cells have high tumorigenic capacity in an animal tumorigenic model. Huang et al (14) used CD44 and CD24 as surface markers to sort out highly tumorigenic cells of CD44+ CD24+ from pancreatic cancer cell line PANC-1, and confirmed that the tumorigenic capacity of the cells of this phenotype is 20 times stronger than that of cells of CD 44-CD 24-in vivo tumor formation experiments of nude mice. Hermann et al (8) have found that cells of CD133+ are sorted by immunomagnetic bead sorting method in human pancreatic cancer tissues and pancreatic cancer cell lines L3.6pl, and that the cells have high tumorigenic ability as demonstrated in vivo tumorigenic experiments in athymic mice, and 500 such cells can be tumorigenic.
2. Side population cell sorting: in the absence of a clear surface marker for tumor stem cells, SP cells can be sorted using the property of side population cells (SP) to 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 was sorted by flow cytometry. Morphological examination with a fluorescence microscope confirmed the presence of lightly stained SP cells in the PANC-1 cell line.
3. Balloon culture: suspension cell balloon culture is another common stem cell sorting method. Gou et al (16) used DMEM-F12 as the basal medium. Adding factors such as EGF, insulin and the like, culturing saccule cells in PANC-1 cell strain, and finding that 98.10% +/-1.26% of cells are not stained by Hoeehst staining. In vivo tumorigenesis experiments of nude mice also confirmed that balloon cells have about 20 times stronger tumorigenic capacity than adherent cells.
Other drugs 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 a pyrimidine antitumor drug, has the same action mechanism as cytarabine, and the main metabolite of the product is doped with DNA in cells and mainly acts on G1/S phase. But differs in that, in addition to incorporation into DNA, difluorodeoxycytidine inhibits ribonucleotide reductase, resulting in a decrease in intracellular deoxynucleoside triphosphate; the other difference from cytarabine is that it can inhibit deoxycytidine deaminase to reduce the degradation of intracellular metabolites and has the function of self-synergism. Clinically, the product has different anti-tumor spectrum from cytarabine and is effective on various solid tumors.
Gemcitabine has not outperformed fluorouracil since its efficacy in pancreatic cancer demonstrated in 1997, neither of the single drugs is superior, even though combination chemotherapy regimens are mostly based, and gemcitabine-based single or combination chemotherapy regimens remain the first choice for pancreatic cancer chemotherapy.
The main advantages of the invention include:
A) for the first time, a reagent for detecting pancreatic cancer based on the detection of WWP1 was developed.
B) The detection reagent or the kit containing the reagent can detect pancreatic cancer and can also judge the prognosis condition of the pancreatic cancer in advance.
C) The application of a WWP1 blocker, preferably WWP1ShRNA, in the preparation of medicines for treating and/or preventing and/or relieving pancreatic cancer is developed for the first time.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: ColdSpringHarbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The operational steps of the immunohistochemical experiment, the establishment of the qRT-PCR, MTT and pancreatic cancer transplantation tumor animal model and the curative effect testing method, the pancreatic cancer stem cell sorting method and the statistical analysis method related in the invention are respectively as follows:
first, immunohistochemical operation step
1. Dewaxing and hydrating: the tissue chips should be left to bake for 60 minutes at room temperature or 20 minutes in a 60 ℃ incubator prior to deparaffinization.
a) Soaking the tissue chip in xylene for 10min, replacing xylene, and soaking for 10min
b) Soaking in anhydrous ethanol for five minutes
c) Soaking in 95% ethanol for five minutes
d) Soaking in 75% ethanol for five minutes
2. Antigen heat repair:
heating in microwave oven, heating 0.01 sodium citrate buffer solution (ph6.0) in microwave oven to boil, placing the tissue chip in the microwave oven, powering off, separating for 5-10 min, and repeating for 1-2 times. Paraffin embedded tissue chip for formalin fixation
3. The immunohistochemical staining SP method comprises the following specific operation steps:
(1) dewaxing and hydrating
(2) PBS wash 2-3 times for 5 minutes each
(3) 3% H2O2 (80% methanol) was added dropwise to TMA, and the mixture was allowed to stand at room temperature for 10 minutes
(4) PBS wash 2-3 times for 5 minutes each
(5) Antigen retrieval
(6) PBS wash 2-3 times for 5 minutes each
(7) Adding normal goat serum confining liquid dropwise, standing at room temperature for 20min, and removing excessive liquid
(8) Dropwise adding I to resist 50 mu l, standing at room temperature for 1-2 hours (antibody: WWP1 antibody (N-20) Santa Cruz Biotech)
(9) The temperature of the mixture is required to be rewarming for 45 minutes at 37 ℃ after the mixture is kept overnight at 4 DEG C
(10) PBS wash 3 times for 2 min each
(11) Adding II antibody 45-50 μ l dropwise, standing at room temperature or 37 deg.C for 1 hr
(12) 0.05% tween-20 was added to the antibody II.
(13) PBS wash 3 times for 5 minutes each
(14) DAB color development for 5-10 min, and control the staining degree under microscope
(15) Rinsing with PBS or tap water for 10min
(16) Counterstaining with hematoxylin for 2 min, and differentiation with hydrochloric acid and ethanol
(17) Washing with tap water for 10-15 min
(18) Dehydrating, transparentizing, sealing and microscopic examination.
4. Result evaluation
Cancer tissues and normal tissues of the tissue specimen were evaluated separately, and the appearance of brown yellow particles in the cells was used as a positive expression marker. Making the following expression evaluation standard according to the reference, wherein the dyeing area is less than 10-0 min; score > 11% < 25% -1; 26% < 50% -2 min; score > 51% -3. The staining intensity was negative-0 min; weak-1 point; medium-2 points; strong-3 points.
II, qRT-PCR operation steps
One) Total RNA extraction
1) Washing the cell sample in the cell culture dish twice by using PBS, then sucking the PBS by using a 1ml gun, adding 1ml of Trizol (invitrogen) solution, blowing, uniformly mixing, sucking the mixture to a 1.5ml of RNasereeEP tube to fully crack the cells, and standing the mixture for 5min at room temperature; fully grinding the tissue sample by using liquid nitrogen, adding 1ml of Trizol (Invitrogen) solution, uniformly mixing, and standing at room temperature for 5min to fully crack;
2) adding 200 μ l chloroform, shaking vigorously and mixing for 30s to make the water phase and organic phase contact sufficiently, standing at room temperature for 3-5 min; (centrifuge tubes are arranged in order during centrifugation, and after centrifugation, the centrifuge tubes are also arranged in order, which is the same as the order of the first step)
3) Centrifuging at 14,000g for 15min at 4 deg.C to obtain three layers, transferring RNA to the upper water phase, and transferring to another new RNasereeEP tube;
4) and (3) RNA precipitation: adding equal volume of isopropanol, gently mixing well (reversing for 6-8 times), standing at room temperature for 10 min;
5) centrifuging at 4 deg.C for 10min at 14,000g, collecting RNA precipitate, and removing supernatant;
6) washed twice with 75% ethanol (12,000g centrifuged for 5min, air dried on a clean bench;
7) depending on the amount of precipitate, an appropriate amount of DEPC water (at least 15ul) was added to dissolve the precipitate.
II) genome removing step operation:
1) adding equal volume of phenol/chloroform, mixing by turning upside down, standing at room temperature for 5min, centrifuging at 14,000rpm for 15min, and collecting supernatant.
2) Adding equal volume of chloroform, mixing by turning upside down, standing for layering, centrifuging at 14,000rpm for 15min, and collecting supernatant.
3) Adding isovolumetric isopropanol, gently mixing well (reversing for 6-8 times), standing at 20 deg.C for 15 min;
4) centrifuging at 4 deg.C for 15min at 14,000g, collecting RNA precipitate, and removing supernatant;
5) washing twice with 75% ethanol (12,000g, centrifuging for 5min), and air drying on a super clean bench;
6) the precipitate was dissolved by adding appropriate amount of DEPC water (at least 15 ul).
Three) Total RNA purity and integrity assays
1) And (3) purity detection: and (3) taking 1 mu l of RNA sample for 50-fold dilution, and determining 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 relatively pure and has no protein pollution.
2) Total RNA integrity test: mu.l of RNA sample was subjected to 1% agarose gel electrophoresis for 80 V.times.20 min, EB staining for 10min, and 5s rRNA, 18s rRNA and 28s rRNA bands of total RNA were observed and photographed using a gel imaging system.
Four) mRNA reverse transcription operation steps:
1) to a PCR tube of RNasefree, TotalRNA1.0. mu.g and H2O were added to prepare a total volume of 12. mu.l solution.
2) The solution is blown and beaten evenly and is kept at 85 ℃ for 5min to denature RNA. Immediately followed by ice cooling to prevent RNA renaturation;
3) promega reagent was added to the PCR tube
4) Keeping the temperature of the 20 mu l reaction solution at 30 ℃ for 10 min;
5) keeping the temperature at 42 ℃ for 50 min;
6) keeping the temperature at 85 ℃ for 10 min;
7) storing at-20 deg.C.
Five) quantitative PCR detection
1. And (3) primer testing:
the specificity and the amplification efficiency of the primers designed according to mRNA need to be tested by qPCR before formal experiments, specific reaction systems and reaction conditions such as formal experiments need to be compared with templates for each pair of primers.
2. Preparing a system:
H2O4ul
SYBRGreenPCRMastermix10ul (TOYOBO) (uniform oscillation is required before use)
Upstream primer 0.5ul (10uM)
Downstream primer 0.5ul (10uM)
Total volume 15ul
After the total system is prepared, the mixture is evenly oscillated in an oscillator or evenly sucked and beaten by a gun, and then 15ul of each tube is subpackaged into 8 tubes.
3. The cDNA is diluted with sterile purified water to a suitable concentration, typically 1:20, and the cDNA is added to the freshly prepared reaction system after being sequenced in a certain order. After the sample is added, the eight-tube-connected cover is covered, and the 1-12 sequence is marked on the edge of the uppermost edge of the eight-tube-connected cover.
4. Each row of eight tubes was placed on a palm centrifuge and centrifuged for several seconds.
5. Opening the sample holder, putting the eight-connection tube, closing the sample holder, selecting the hole site of the placed reaction tube on the software, and removing the hole site of the non-reaction tube.
6. The sample name and the name of the detection gene of each reaction well are marked on 7500 software, and result files are stored in a classified mode.
After the reaction is finished, the eight connecting pipes are arranged in a sealing bag, and the file name and the name of a customer are marked on the bag. (three repeated experiments of the same customer only need to store one of the repeated reaction tubes, and the rest can be discarded)
Third, MTT method experiment step
1. Inoculating cells: subculturing at 37 ℃ by using 10% fetal calf serum (Thermo) containing DMEM (Hyclone) 5% CO2, wherein Panc-1 is from ATCC, WWP1ShRNA (TL301750) is an OriGene product, preparing a single cell suspension, and inoculating 1000-10000 cells per hole to a 96-hole plate, wherein each hole volume is 200-200 ul.;
2. culturing the cells: 5% CO2, 37 ℃ until cell monolayers have plated to the bottom of the wells (96 well flat bottom plate).
3. Color generation: after 3-5 days of incubation, 20ul. MTT (thiazole blue) solution (5mg/ml in PBS, pH 7.4) was added to each well for further incubation for 4h, the incubation was terminated, the culture supernatant in the wells was carefully aspirated, and the culture supernatant in the wells was again aspirated after centrifugation for suspension cells. 150ul DMSO was added to each well, and the mixture was shaken for 10min to completely melt the crystals.
4. Color comparison: the 490nm wavelength is selected, the light absorption value of each pore is measured on an enzyme linked immunosorbent instrument, and the result is recorded.
Establishment and curative effect test method of pancreatic cancer transplantation tumor animal model
1. The human body is determined to be cancer-bearing tissue and the cancer tissue is obtained by surgery, or cultured cell lines, such as Panc-1, WWP1ShRNA (TL301750) are OriGene products.
2. The retention time of the tumor tissues or cells in vitro is shortened as much as possible so as to keep the freshness of the tumor tissues to the maximum extent.
3. After skin sterilization with 70% alcohol, 3 tumor tissues were transplanted to each side of both sides and sterilized, or pancreatic tumor cells were directly injected. The immunodeficient mouse can be a BALB/C or NOD/SCID mouse.
4. Each group was 6. When the transplanted tumor of each immunodeficient mouse grows to 150mm on average3Administration is started. The dosing regimen included a negative control and a differenceTreatment regimen and concentrations.
5. After administration, the change in tumor size of each immunodeficient mouse was measured every 3-5 days.
6. The calculation formula of the drug effect is as follows: T/C (treatment/control) X100% to assess the efficacy of all regimens.
Method for sorting pancreatic cancer stem cells
1. Taking a human pancreatic cancer specimen or obtaining a tumor specimen from a nude mouse transplanted tumor model, preparing a cell suspension by utilizing a collagen enzyme digestion method, digesting a tumor tissue at 37 ℃ for 2-3h, and filtering the digested tissue by using a 40uM filter screen to prepare a single cell suspension.
2. Cell concentration was adjusted to 1-5X 10 using 2% FCSRPMI 1640/HBSS6And/ml. The cells were labeled with antibodies CD44 and c-Met (BD) (binding time 20min at 4 ℃) and washed 2 times with washing solution, approximately 4ml of washing solution each time, and centrifuged at 1000rpm × 5 min.
H2-Kd ablation of non-pancreatic cancer cells (nude mouse cells), 4, 6-diamidino-2-phenylindole (DAPI) ablation of dead cells
4. The flow cytometer (BDAria) sorts human primary pancreatic cancer CD44+ c-Met + cells for 2 times, ensuring that the purity of the sorted cells is more than 90%.
Sixthly, statistical analysis method
Data are shown as mean ± SE. Statistically significant differences in different cases the student test and X were used2Analytically determined and defined as P<0.05。
The sequences of all primers used in the examples are given in Table 1 below
TABLE 1 primer sequence Listing
Example 1 detection of expression level of WWP1 in pancreatic tumor cells, pancreatic hepatoma metastatic cells and tumor stem cells
The clinical specimen selection standard is that the patient is subjected to surgical excision and the pathological diagnosis after surgery is pancreatic duct adenocarcinoma; needle biopsy yielded a complete histological specimen and was pathologically diagnosed as ductal adenocarcinoma of the pancreas. Patients participating 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 were obtained post-operatively and biopsied and pancreatic tumor stem cells were isolated by flow sorting. After RNA extraction, qRT-PCR was performed, the procedure of qRT-PCR was as above. qRT-PCR contrasts changes in expression levels of WWP1 in normal pancreatic tissue, pancreatitis, pancreatic tumor tissue, and pancreatic tumor stem cells.
As a result:
as shown in FIG. 1, the expression level of WWP1 was found to be low in normal pancreatic tissue, and WWP1 was slightly increased in pancreatitis relative to normal tissue, while WWP was 6.3-fold increased in tumor tissue and nearly 11-fold increased in pancreatic tumor stem cells by quantitative PCR data.
TABLE 1 immunohistochemical detection of WWP1 Positive Rate
| Normal tissue | Inflammation(s) | Cancer treatment | Metastatic cancer |
| 6.3％ | 12.5％ | 75％ | 87.5％ |
Example 2 detection of the relationship between WWP1 Positive level of pancreatic tumor cells and prognosis
2.1, method:
21 pancreatic cancer patients with complete follow-up records were selected, and pathological sections were obtained for WWP1 immunohistochemical examination, the immunohistochemical procedures were as above. Selected patients were post-operatively treated with conventional gemcitabine chemotherapy alone, with no other treatment record.
WWP1 immunohistochemical examination of 21 pancreatic cancer patients was divided into three groups (strong positive of 3, positive of 2, weak positive of 1), 7 of each group, and prognostic follow-up was performed to follow-up the survival time of the patients.
2.2 results:
as shown in FIG. 2, the prognosis follow-up data shows that the positive level of pancreatic tumor cells and the prognosis survival are in inverse proportion, and the prognosis of the strong positive patient is poor and the survival is short, while the prognosis of the weak positive patient is better and the survival is longer.
It can therefore be concluded that the level of immunohistochemical examination of WWP1 can be used to predict prognosis and as an indicator for predicting therapeutic efficacy.
Example 3 detection of the relationship between blocking expression of WWP1 and growth of 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 Panc-1 cell lines to analyze the effect of blocking WWP1 on pancreatic cancer cell survival. Gemcitabine was used as a control.
As a result:
as shown in FIG. 3, gemcitabine has little effect on pancreatic cancer cell survival in vitro, while blocking inhibition of WWP1 in vitro can significantly reduce pancreatic cancer cell survival.
Therefore, it can be concluded that blocking WWP1 in vitro experiments is effective in inhibiting the survival rate of pancreatic tumor cells.
3.2 animal experiments demonstrated that blocking WWP1 inhibits pancreatic tumor growth.
The method comprises the following steps:
pancreatic cancer cells transfected with WWP1ShRNA and control group pancreatic cancer cells were inoculated to the immunodeficiency
The mouse establishes a human pancreatic cancer animal model for research, and the influence of blocking WWP1 on the growth of pancreatic cancer in the animal model is tested. Gemcitabine (100mg/kg twice weekly) was discontinued after 4 weeks of continuous dosing and tumor volume was measured for 8 weeks.
As a result:
as shown in fig. 4, blocking WWP1 inhibited pancreatic tumor growth in animal models. Compared with the ordinary Panc-1 cells, the Panc-1 cells transfect WWP1ShRNA and then subcutaneously inject nude mice to form tumors which are inhibited from growing.
And the WWP1 blocking effect is better when combined with the first choice medicament Gemcitabine (Gemcitabine, Gem) for treating pancreatic cancer. The experimental results using the primary human pancreatic cancer animal model (Patient-derived xenogenic model-PDX) are consistent with those using Panc-1.
Therefore, it is concluded that blocking WWP1 can significantly inhibit pancreatic cancer cell growth in nude mice.
3.3 blocking WWP1 inhibits the EMT of pancreatic tumor stem cells and tumors.
The method comprises the following steps:
the tumor tissues obtained by the animal experiments are digested by collagenase to generate single cells, CD44 and c-Met antibodies are used for flow separation to obtain CD44+ c-Met + pancreatic cancer stem cells, and the influence of WWP1 on the tumor stem cells is blocked. Quantitative PCR analysis of RNA extracted from cells was performed to investigate the effect of blocking WWP1 on the EMT pathway.
As a result:
blocking WWP1 with WWP1ShRNA significantly reduced the number of pancreatic tumor stem cells, and blocking WWP1 combined with Gemcitabine (Gemcitabine, Gem) was more effective (table two).
As shown in fig. 5, blocking WWP1 was found to inhibit Snail-mediated EMT by quantitative PCR detection using digested tumor cells.
TABLE II blocking the effect of WWP1 on pancreatic tumor stem cells
| Control | sShRNA | GEM | sShRNA+GEM |
| 2.0％ | 0.64％ | 4.3％ | 0.52％ |
And (4) conclusion:
blocking WWP1 significantly reduced the number of pancreatic tumor stem cells and inhibited Snail-mediated EMT.
In conclusion, the research proves that WWP1 is highly expressed in pancreatic tumor cells and tumor stem cells, WWP1 can be applied to diagnosis of pancreatic cancer and used as an index for predicting treatment effect to predict prognosis; the targeted WWP1 can inhibit the growth of pancreatic tumor and stem cell of pancreatic tumor, and can be used as new medicine for treating pancreatic cancer and new target of biological immunotherapy.
Discussion of the related Art
There are two theories of tumor formation. The Stochastic theory states that every tumor cell is homogeneous and that every tumor cell has the potential to form a new tumor. But its entry into the cell's proliferative division cycle is controlled by some small probability of random events. The Hierarchy theory states that a subpopulation of cells exists 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 divergently. Tumor initiating cells, i.e., tumor stem cells, contribute to the development and progression of tumors (13).
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. Use of a WWP1 gene, cDNA, protein or a detection reagent thereof for the preparation of a diagnostic product for detecting whether a subject has pancreatic cancer; and/or predicting a prognosis of pancreatic cancer in the subject.
2. The use according to claim 1, wherein the detection reagent comprises a primer pair shown in SEQ ID Nos. 1 and 2 for specifically amplifying the WWP1 gene.
3. A kit, comprising a first detection reagent for detecting WWP1 gene, cDNA, protein.
4. The kit of claim 3, wherein the first detection reagent comprises a primer pair shown in SEQ ID Nos. 1 and 2 for specifically amplifying the WWP1 gene.
5. The application of a WWP1 blocker or a preparation containing the WWP1 blocker is characterized by being used for preparing a medicine for treating and/or preventing and/or relieving related diseases caused by pancreatic cancer.
6. The use according to claim 5, wherein said WWP1 blocker comprises a ShRNA that blocks expression of WWP1 selected from SEQ ID No 3, SEQ ID No 4, SEQ ID No 5SEQ ID No 6.
7. The use of claim 5, wherein said formulation further comprises an additional oncological agent, said additional oncological agent comprising gemcitabine.
8. A pharmaceutical composition, wherein said pharmaceutical composition comprises:
A1) WWP1 blocker;
A2) other tumor treating medicines.
9. An in vitro method for inhibiting pancreatic cancer tumor stem cell replication; and/or inhibiting Snail-mediated EMT, comprising the steps of: contacting said pancreatic cancer tumor stem cells with a medically effective amount of a WWP1 blocker, thereby inhibiting expression of WWP1 and inhibiting replication of pancreatic cancer tumor stem cells; and/or inhibiting Snail-mediated EMT.
10. A method for preventing and/or treating and/or ameliorating pancreatic cancer, comprising the steps of: administering to a subject in need thereof a medically effective amount of a WWP1 blocker, thereby inhibiting pancreatic cancer stem cell replication.
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