CN112877431A

CN112877431A - Use of snoRNA-U41 in the detection and treatment of pancreatic cancer

Info

Publication number: CN112877431A
Application number: CN202110169452.2A
Authority: CN
Inventors: 李晨蔚; 周慧斌; 曹靖晨; 邓婷婷; 张蕾
Original assignee: Shanghai Sunstem Biotechnology Co ltd
Current assignee: Shanghai Sunstem Biotechnology Co ltd
Priority date: 2021-02-07
Filing date: 2021-02-07
Publication date: 2021-06-01
Anticipated expiration: 2041-02-07
Also published as: CN112877431B

Abstract

The invention relates to the technical field of medical biology, and particularly provides application of a snoRNA-U41 gene, cDNA or a detection reagent thereof, namely the application of the snoRNA-U41 gene, cDNA or the detection reagent thereof in preparing a diagnostic product for detecting whether a subject has pancreatic cancer. In addition, the invention also discovers that the agonism of the snoRNA-U41 overexpression can inhibit the growth and/or proliferation of pancreatic cancer tumor cells, and can be used for preparing a) an inhibitor for inhibiting the growth and/or proliferation of pancreatic cancer cells; and/or b) a medicament for the treatment and/or prevention and/or amelioration of the associated diseases caused by pancreatic cancer.

Description

Use of snoRNA-U41 in the detection and treatment of pancreatic cancer

Technical Field

The invention relates to the technical field of medical biology, in particular to application of snoRNA-U41 in detecting and treating pancreatic cancer.

Background

Pancreatic cancer (PAAD) is one of the seven most lethal cancers, accounting for 4.5% of all cancers. Because of the poor prognosis, it is called "cancer king". Among them, Pancreatic Ductal Adenocarcinoma (PDAC) is the most predominant type of Pancreatic cancer, accounting for over 85% of the cases. The main reason for this is the low diagnosis rate of pancreatic cancer patients in early clinical stage, usually found in advanced stages of pancreatic cancer. Moreover, only less than 20% of pancreatic cancers can be surgically resected, and the remainder of the patients diagnosed with focal metastasis. By 2030, mortality of pancreatic cancer in the united states is expected to exceed that of liver cancer, breast cancer, prostate cancer and large intestine cancer, and is the second leading cause of cancer-related death.

Early metastasis of pancreatic cancer is its major cause of mortality. There is evidence that this process may be achieved by Cancer Stem Cells (CSCs), and that such Cells are characterized by epithelial-mesenchymal transition (EMT). Current conventional cancer treatments such as radiation and chemotherapy do kill most of the rapidly growing malignant cells, but do have little effect on the rare number of tumor stem cells present in the tumor, and the proliferation of residual tumor stem cells after treatment is sufficient to promote cancer recurrence and metastasis. Effective tumor therapy should include treatment regimens directed against tumor stem cell populations. Tumor stem cells are capable of self-renewal indefinitely and have relatively quiescent characteristics that, in combination with epithelial-mesenchymal transition EMT, enable them to avoid cell death induced by conventional chemotherapy methods. Leading to metastasis of the tumor and death of the tumor patient.

Nucleolar small-molecule RNA (snorRNA) is a kind of small-molecule non-coding RNA with a specific structure, widely exists in nucleoli of eukaryote and can form a complex with ribonucleoprotein in nucleoli, namely snorNPs. The gene encoding snoRNA is distributed mainly in the intron region of a protein-coding gene or a non-protein-coding gene, and is processed after transcription to form mature snoRNA.

The snornas are closely related to cancer development, are not only involved in proliferation and migration of cancer cells, but also are expected to play an important role in early diagnosis of cancer.

Therefore, there is an urgent need in the art to develop snornas associated with pancreatic cancer and to study the use of such snornas in diagnosis and treatment of pancreatic cancer.

Disclosure of Invention

The main purpose of the present invention is to develop snornas associated with pancreatic cancer and to study the use of such snornas in the diagnosis and treatment of pancreatic cancer. In particular, the invention provides a snoRNA, snoRNA-U41, down-regulated in pancreatic cancer and the use of snoRNA-U41 in the diagnosis and treatment of pancreatic cancer.

In a first aspect of the present invention, there is provided a use of a snoRNA-U41 gene, cDNA or a detection reagent thereof for the preparation of a diagnostic product for detecting whether a subject has pancreatic cancer.

In another preferred embodiment, the diagnosis includes early diagnosis, assisted diagnosis, or a combination thereof.

In another preferred embodiment, the snoRNA-U41 gene, cDNA, is 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, a paraffin section sample, or a combination thereof.

In another preferred embodiment, the detection reagent comprises: a primer pair, a probe or a combination thereof for specifically amplifying the snoRNA-U41 gene.

In another preferred embodiment, the detection reagent comprises a primer pair shown in SEQ ID Nos. 2 and 3 for specifically amplifying snorRNA-U41 gene.

In a second aspect of the invention, there is provided a kit comprising a first detection reagent for detecting a snoRNA-U41 gene, cDNA.

In another preferred embodiment, the first detection reagent comprises a primer pair shown in SEQ ID Nos. 2 and 3 for specifically amplifying the snorRNA-U41 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.

In another preferred example, the subject is a pancreatic cancer patient, preferably a pancreatic ductal adenocarcinoma patient.

In a third aspect of the invention there is provided the use of a snoRNA-U41 agonist or a formulation comprising said snoRNA-U41 agonist, in the preparation of a) an inhibitor of the growth and/or proliferation of pancreatic cancer cells; and/or b) a medicament for the treatment and/or prevention and/or amelioration of the associated diseases caused by pancreatic cancer.

In another preferred embodiment, the pancreatic tumor cells comprise pancreatic cancer stem cells.

In another preferred embodiment, the formulation further comprises an additional anti-tumor drug selected from the group consisting of: crizotinib, gemcitabine, 5-fluorouracil, folinic acid, oxaliplatin, irinotecan, erlotinib, or combinations thereof.

In another preferred embodiment, the snoRNA-U41 agonist is an shRNA that overexpresses U41.

In another preferred embodiment, the formulation is oral or non-oral.

In another preferred embodiment, the formulation is in a form selected from the group consisting of: tablets, capsules, granules, suspensions, pills, solutions, syrups, or injections.

In a fourth aspect of the invention, there is provided a pharmaceutical composition comprising:

A1) a snoRNA-U41 agonist;

A2) other antineoplastic agents selected from the group consisting of: crizotinib, gemcitabine, 5-fluorouracil, folinic acid, oxaliplatin, irinotecan, erlotinib, or a combination thereof;

B) other pharmaceutically acceptable carriers or excipients.

In another preferred embodiment, the pharmaceutical combination is used for the preparation of a) an inhibitor for inhibiting the growth and/or proliferation of pancreatic cancer cells; and/or b) a medicament for the treatment and/or prevention and/or amelioration of the associated diseases caused by pancreatic cancer.

In a fifth aspect of the invention, there is provided an in vitro non-diagnostic method of inhibiting pancreatic cancer tumor cell growth and/or proliferation, comprising the steps of: contacting the cell with a medically effective amount of a snoRNA-U41 agonist.

In another preferred embodiment, the cells are derived from a mammal, preferably a human, a mouse.

In another preferred embodiment, the cell is a pancreatic cancer cell or a pancreatic cancer stem cell.

In a sixth aspect of the invention there is provided a method of treating pancreatic cancer by administering to a subject in need thereof a medically effective amount of a snoRNA-U41 agonist.

In another preferred example, the patient is a pancreatic cancer patient, preferably a pancreatic ductal adenocarcinoma patient.

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 the results of the chip, the change in the expression level of snorRNA in normal pancreatic tissue, conventional pancreatic cancer cells and pancreatic tumor stem cells.

FIG. 2 shows the results of qRT-PCR chip, the change of snorNA in BXPC-3 cells and normal pancreatic cells.

FIG. 3 is a cell experiment examining the effect of overexpression of snorRNA-U41 on pancreatic cancer cell proliferation.

FIG. 4 is an animal experiment examining the effect of overexpression of snorRNA-U41 on pancreatic cancer neoplasia.

FIG. 5 is a graph of the effect of snorRNA-U41 in combination with crizotinib on pancreatic cancer cell proliferation.

Detailed Description

The present inventors have made extensive and intensive studies and, as a result, have unexpectedly found that snoRNA-U41 is down-regulated in pancreatic cancer cells as well as pancreatic cancer stem cells, thereby completing the present invention.

Specifically, the invention discovers that snoRNA which is down-regulated in pancreatic cells and pancreatic cancer stem cells is snoRNA-U41, can be used as a target for detecting pancreatic cancer, and therefore can be used for preparing products or kits for early diagnosis of pancreatic cancer. The invention also discovers that the activation of the over-expression of the snoRNA-U4 can inhibit the growth and proliferation of pancreatic cancer cells, particularly the proliferation of pancreatic tumor cells can be obviously inhibited when the snoRNA-U4 is combined with crizotinib, so that the agonist of the snoRNA-U41 can be used for preparing a medicament for treating pancreatic cancer.

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".

Tumor stem cells

Tumor Stem Cells (CSCs) are Cancer Cells with Stem cell properties, and have the ability to "self-replicate" (self-renewal) and "differentiate" (differentiation), and each tumor cell has a unique cell surface marker that can be identified. In 1997, leukemia tumor stem cells (CD34+/CD38-) are separated from Dick, Bonnet and the like in Toronto theory for the first time, and the research of the cancer stem cells enters a new stage. Hermann et al report that CD133 positive cells are highly tumorigenic and resistant. Rasheed et al found that ALDH-positive tumor cells have characteristics of tumor stem cells and characteristics of Epithelial-Mesenchymal Transition (EMT). We further report that c-Met is a novel pancreatic cancer stem cell surface marker and can be an effective target for therapy. c-Met is activated by Hepatocyte Growth Factor (HGF), is a receptor in the tyrosine kinase family, and regulates the mobility, invasiveness and metastasis of tumor cells. Human pancreatic cancer cells are separated through a xenograft model, and research reports that the c-Met down-regulated cell subset has the characteristics of tumor stem cells. c-Met down-regulated cells have higher tumorigenicity than other markers (e.g., CD44, CD24, ESA, CD133, ALDH). The cell subset with both c-Met + and CD44+ markers was most tumorigenic in vivo.

snoRNA

snornas generally consist of 60-400 nucleotides, have specific conserved result units, and can be classified into three major groups according to their structures: C/D box snorRNA, H/ACA box and MRP RNA (rarely studied). The main functions include: 1) direct post-transcriptional modification of other non-coding RNAs (e.g., ribosomal RNA (rrna), small nuclear RNA (snRNA), etc.), specific functions including participation in modifications such as 2' -O-ribomethylation and pseudouracil of ribosomal RNA; 2) affecting the mRNA 3 'end processing, the U/A-rich SNORD50A has competitive inhibition in mRNA 3' end processing, and finally affects the expression of mRNA level.

The invention discovers that snorRNA-U41 is down-regulated in pancreatic cancer, the purpose of early detecting pancreatic cancer can be achieved by detecting snorRNA-U41 through a primer pair shown in SEQ ID Nos. 2 and 3, and the snorRNA-U41 is an effective pancreatic cancer detection target.

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.

Since the snoRNA-U41 is shown to be down-regulated in pancreatic cancer cells as well as pancreatic cancer stem cells in the examples of the present invention, it can be used as a marker for detecting pancreatic cancer. Based on the above, the invention designs a primer pair using snoRNA-U41 gene as a template, and detects snoRNA-U41 to detect pancreatic cancer.

The primers for detecting the snoRNA-U41 gene are shown in SEQ ID Nos. 2 and 3.

Detection kit

Based on the down regulation of snoRNA-U41 in pancreatic cancer cells as well as pancreatic cancer stem cells, the invention also provides a detection kit based on the detection of snoRNA-U41.

The kit provided by the invention comprises a first detection reagent, and the first detection reagent is used for detecting snoRNA-U41 gene and cDNA.

Agonists

As used herein, the term "agonist" refers to a substance, particularly an agonist or the like, which interacts with the SnoRNA-U41 gene by various conventional screening methods using the SnoRNA of the present invention (SnoRNA-U41).

When being applied (dosed) therapeutically, the agonist of the SnoRNA-U41 can stimulate the expression and/or activity of SnoRNA-U41, thereby inhibiting the 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.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an agonist of snoRNA-U41 as defined above (in an amount of 0.001 to 99 wt%, preferably 0.01 to 90 wt%); other antineoplastic agents selected from crizotinib, gemcitabine, 5-fluorouracil, leucovorin, oxaliplatin, irinotecan, erlotinib, or combinations thereof; and a pharmaceutically acceptable carrier (the balance being the content). The pharmaceutical composition can be used for weakening the drug resistance of the taxane drugs.

In the present invention, the agonist also includes a small molecule compound which can reduce the expression or activity of SnoRNA-U41.

The pharmaceutical composition of the invention contains a safe and effective amount of the SnoRNA-U41 agonist 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 the case of pharmaceutical compositions, a safe and effective amount of the SnoRNA-U41 agonist of the invention is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed 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 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.

Application method

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity (i.e., anti-aging function) in a human and/or animal and is acceptable to the human and/or animal.

As used herein, an ingredient of the term "pharmaceutically acceptable" is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical composition of the invention can be prepared into injections, oral preparations (tablets, capsules, oral liquids), transdermal agents and sustained-release agents. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the active ingredient of the invention is administered at a daily dose of about 0.00001mg to 50mg per kg of animal body weight (preferably 0.0001mg to 10mg per kg of animal body weight). For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

Typically, when the SnoRNA-U41 agonist is administered orally, the daily average dose in a subject (human) of 60kg body weight is usually 10-500mg, preferably 20-300mg, more preferably 50-250 mg. The daily dose may be administered in one, two or more divided doses.

The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.

The main advantages of the invention include:

(a) the invention discovers a snorRNA-U41 capable of being used as a target for detecting pancreatic cancer for the first time.

(b) The snorRNA-U41 discovered by the invention can be used as a target for early diagnosis of pancreatic cancer, so that the patient can be helped to treat the pancreatic cancer in early stage of the disease, the disease is not delayed, and the recovery probability is improved.

(c) The invention discovers that the activation of snoRNA-U41 overexpression can inhibit the growth and proliferation of pancreatic cancer, and particularly, the combination of the activation of snoRNA-U41 and crizotinib can more obviously inhibit the growth and proliferation of pancreatic cancer tumor cells.

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: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

First, experiment method related in embodiment of the invention

The operation steps of the RNA chip technology, the qRT-PCR, the CCK-8, the establishment of the pancreatic cancer transplantation tumor animal model and the curative effect test method, the pancreatic cancer stem cell sorting method and the statistical analysis method are respectively as follows.

1.1 RNA chip technology

1. Tumor tissues of pancreatic cancer patients are digested into single-cell suspensions, and after the single-cell suspensions are incubated with specific antibodies, the single-cell suspensions are sorted by a flow cytometer to obtain pancreatic cancer stem cells of CD44+ CD24+ ESA + and pancreatic cancer cells of CD44-CD 24-ESA-.

2. And extracting RNA of pancreatic cancer stem cells of CD44+ CD24+ ESA +, CD44-CD 24-ESA-pancreatic cancer cells and cancer paracancerous cells.

3. The RNA sample is sent to a chip for detection.

1.2 qRT-PCR procedure

One) Total RNA extraction

1) Washing the cell sample in the cell culture dish with PBS twice, then sucking the PBS clean with a 1ml gun, adding 1ml Trizol (invitrogen) solution, blowing and mixing uniformly, sucking the mixture into a 1.5ml RNase free EP 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, wherein RNA in the upper water phase is transferred to another new RNase free EP 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: taking 1 mul RNA sample to dilute 50 times, measuring OD value on nucleic acid protein detector, the ratio of OD260/OD280 is more than 1.8, which shows that the prepared RNA is purer and has no protein pollution.

2) Total RNA integrity test: mu.l of the RNA sample was subjected to 1% agarose gel electrophoresis for 80 V.times.20 min, EB staining for 10min, and bands of 5s rRNA, 18s rRNA and 28s rRNA of the total RNA were observed and photographed using a gel imaging system.

Four) RNA reverse transcription operation steps:

1) to the PCR tube of RNase free, 1.0. mu.g of Total RNA 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 specific reaction system and the reaction conditions such as formal experiments need to be tested by qPCR before the formal experiments of the primers designed according to RNA, and each pair of primers needs to be used as template water control. .

2. Preparing a system:

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.

1.3 Experimental procedure of CCK-8 method

1. Subculturing with DMEM (Hyclone) containing 10% fetal calf serum (Thermo) at 5% CO2 and 37 ℃ to prepare single cell suspension, and inoculating 1000-10000 cells per well to a 96-well plate with each well volume of 100 ul.;

2. the same culture conditions, 5% CO2, 37 ℃, were applied until the cell monolayer was plated to the bottom of the well (96 well flat bottom plate).

3. After 3-5 days of culture, 10ul of CCK-8 solution is added to each well, and the incubation is continued for 1-2h, and the culture is stopped.

4. Selecting the wavelength of 430nm and 460nm, measuring the light absorption value of each hole on a microplate reader, and recording the result.

1.4 pancreatic cancer Stem cell sorting method

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 concentrations were adjusted to 1 million to 5 million/ml with 2% FCS RPMI 1640/HBSS. The cells were washed 2 times with washing solutions, about 4ml each time, with antibodies CD24, CD44 and ESA markers (incubation for 20min at 4 ℃), and centrifuged at 1000 rpm. times.5 min.

H2-Kd ablation of non-pancreatic cancer cells (nude mouse cells), 4, 6-diamidino-2-phenylindole (DAPI) ablation of dead cells

4. Flow cytometry (BD Aria) was used to sort human primary pancreatic cancer CD44+ CD24+ ESA + and CD44-CD 24-ESA-cells 2 times, ensuring that the purity of the sorted cells was > 90%.

1.5 statistical analysis method

Data are shown as mean ± SE. Statistically significant differences were determined in different cases using Student's t-test and X2 analysis and were defined as P < 0.05.

1.6 primers

The gene sequences in this example are shown in table 1 below:

TABLE 1 Gene sequences in this example

Second, example

Example 2.1 RNA chip technology to detect the expression level of snorRNA-U41 in normal pancreatic islet duct cells, conventional pancreatic cancer cells and pancreatic cancer stem cells; qRT-PCR verification of chip results

The clinical specimen of the embodiment has the selection standard that the patient is subjected to surgical excision and the pathological diagnosis after the surgery is pancreatic ductal 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 pancreas tissue and pancreas tumor tissue after operation and biopsy are obtained, pancreas tumor cells are separated through flow type sorting, and snoRNA chip detection is carried out after RNA is extracted. The qRT-PCR chip is adopted to verify the result, total RNA of BXPC-3 cells (the proportion of CD24+ CD44+ ESA + of the BXPC cells is about 70, the BXPC cells can be used as approximate stem cells, published articles use the BXPC cells as pancreas cancer stem cells to study) and normal pancreatic cells is extracted, and the subsequent operation steps are as above.

As a result: as shown in fig. 1, snoRNA chip data found that expression levels of snoRNA-U41 were down-regulated in pancreatic cancer stem cells relative to normal pancreatic cells and conventional pancreatic cancer cells. The obtained result is basically consistent with the result of chip calculation and analysis.

Example 2.2 Up-regulation of snorRNA-U41 expression in relation to growth of pancreatic tumor cells

1) Up-regulation of snorRNA-U41 expression inhibits the growth of BXPC-3 cells

The method comprises the following steps: in vitro experiments, overexpression snoRNA-U41 vector pCDNA3.1(-) is used for transfecting a cell line BXPC-3, so that the overexpression can be realized, a CCK-8 experiment is carried out on BXPC-3 cell strains, and the influence of up-regulation of snoRNA-U41 on the survival rate of pancreatic cancer cells is analyzed.

As a result: as shown in FIG. 3, in vitro experiments, upregulation of snorRNA-U41 significantly reduced pancreatic cancer cell survival, relative to the empty plasmid transfected group, by only about 40% of the control group. Upregulated snoRNA-U41 in combination with crizotinib the proliferation rate of pancreatic tumor cells was slower compared to the upregulated snoRNA-U41 group alone.

And (4) conclusion: up-regulation of snoRNA-U41 in vitro experiments was effective in inhibiting the survival of pancreatic tumor cells.

2) Animal experiments prove that up-regulation of snoRNA-U41 can inhibit pancreatic tumor growth.

The method comprises the following steps: BXPC-3 pancreatic cancer cells over expressing snorRNA-U41 and control group pancreatic cancer cells are inoculated to immunodeficient mice to establish a human pancreatic cancer animal model for research, and the over-expression of snorRNA-U41 is detected

Effect on pancreatic cancer growth in animal models.

As a result: as shown in FIG. 4, overexpression of snorRNA-U41 inhibited pancreatic tumor growth in animal models. Compared with the common BXPC-3 cell, the tumor growth of the nude mice injected subcutaneously after the snorRNA-U41 is over-expressed is inhibited.

And (4) conclusion: the overexpression of the snorRNA-U41 can obviously inhibit the growth of pancreatic cancer cells in a nude mouse.

Taken together, studies demonstrated that snoRNA-U41 expression is down-regulated in tumor stem cells; the over-expression of snorRNA-U41 can inhibit the growth of pancreatic tumor and the proliferation of stem cells of the pancreatic tumor, and can be used as a new drug for treating pancreatic cancer and a new target point of biological immunotherapy.

Discussion of the related Art

For example, snoRNA U50 can be used as a marker for the progression of B-cell lymphoma, breast and prostate cancer, and the like. During the development of non-small cell lung cancer (NSCLC), levels of SNORD33, SNORD66, and SNORD76 were significantly elevated in plasma, while activation of SNORA42 was significantly carcinogenic. Silencing SNORA55 inhibits the proliferation and migration of prostate cancer cells. SNORD113-1 has strong ability in inhibiting growth and proliferation of liver cancer cells, thereby playing a good role in inhibiting cancer in the development process of liver cancer. The research finds that SNORD50A/B is the switch of the strongest oncogene K-Ras, and SNORD50A/B can bind to K-Ras, thereby reducing the activity thereof. In addition, in the patients with 12 common cancers such as lung cancer, liver cancer, breast cancer, ovarian cancer and the like, the SNORD50A/B is 10-40% of the patients with the common cancers, which can cause the reduction of the survival rate. In pancreatic ductal adenocarcinoma, the levels of nucleolar small RNA molecules U91 and SNORA23 were significantly increased, and in nude mice in the CDX model, it was found that an increase in SNORA23 resulted in an increase in the expression level of SYNE2, thereby promoting growth metabolism of tumor tissues.

The invention discovers that snoRNA-U41 is down-regulated in pancreatic cancer cells, particularly pancreatic cancer stem cells, and therefore can be used as a target for detecting and treating pancreatic cancer. In vitro and in vivo experiments prove that the over-expression of snoRNA-U41, especially when used together with crizotinib, can obviously inhibit the proliferation and growth of pancreatic cancer cells and help to treat pancreatic cancer.

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.

Sequence listing

<110> Shanghai Shang Tai Biotechnology Ltd

<120> snoRNA-U41 for use in detecting and treating pancreatic cancer

<130> P2021-0191

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 70

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 1

tgggaagtga tgacacctgt gactgttgat gtggaactga tttatcgcgt attcgtactg 60

gctgatcctg 70

<210> 2

<211> 25

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

gcggcgggtg ggaagtgatg acacc 25

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

atccagtgca gggtccgagg 20

Claims

1. Use of the snoRNA-U41 gene, cDNA or a detection reagent therefor in the preparation of a diagnostic product for detecting whether a subject has pancreatic cancer.

2. The use according to claim, wherein the detection reagent comprises a primer pair shown in SEQ ID Nos. 2 and 3 which specifically amplifies the snorRNA-U41 gene.

3. A kit comprising a first detection reagent for detecting snoRNA-U41 gene, cDNA.

4. The kit of claim 3, wherein the first detection reagent comprises a primer pair shown in SEQ ID Nos. 2 and 3 that specifically amplifies the snorRNA-U41 gene.

5. The kit of claim 3, further comprising a label or instructions indicating that the kit is for detecting whether a subject has pancreatic cancer.

6. Use of a snoRNA-U41 agonist or a formulation comprising said snoRNA-U41 agonist for the preparation of a) an inhibitor of the growth and/or proliferation of pancreatic cancer cells; and/or b) a medicament for the treatment and/or prevention and/or amelioration of the associated diseases caused by pancreatic cancer.

7. The use of claim 6, wherein the formulation further comprises an additional anti-neoplastic agent selected from the group consisting of: crizotinib, gemcitabine, 5-fluorouracil, folinic acid, oxaliplatin, irinotecan, erlotinib, or combinations thereof.

8. A pharmaceutical composition, comprising:

A1) a snoRNA-U41 agonist;

B) other pharmaceutically acceptable carriers or excipients.

9. A method for non-diagnostically inhibiting the growth and/or proliferation of pancreatic cancer tumor cells in vitro, comprising the steps of: contacting the cell with a medically effective amount of a snoRNA-U41 agonist.

10. A method of treating pancreatic cancer comprising administering to a subject in need thereof a medically effective amount of a snoRNA-U41 agonist.