CN111686106A

CN111686106A - Application of compound for improving Dicer expression in preparation of medicine

Info

Publication number: CN111686106A
Application number: CN202010735192.6A
Authority: CN
Inventors: 唐开福
Original assignee: First Affiliated Hospital of Wenzhou Medical University
Current assignee: First Affiliated Hospital of Wenzhou Medical University
Priority date: 2020-04-05
Filing date: 2020-07-28
Publication date: 2020-09-22
Anticipated expiration: 2040-07-28
Also published as: CN111686106B

Abstract

The invention relates to a new application of a medicine for improving Dicer expression. The application of the medicine in preparing the medicine for preventing and treating colitis or colon cancer. The colitis is selected from inflammatory bowel disease, the inflammatory bowel disease is ulcerative colitis and Crohn's disease, and the colon cancer is colitis related colon cancer. The drug for increasing Dicer expression is selected from anastrozole, salts thereof, metabolites thereof and derivatives thereof.

Description

Application of compound for improving Dicer expression in preparation of medicine

The present invention claims priority from chinese patent application CN202010261825.4, and the contents of the specification, drawings and claims of this priority document are incorporated in their entirety into the present specification and are included as part of the original description of the present specification. Applicants further claim that applicants have the right to amend the description and claims of this invention based on this priority document.

Technical Field

The invention relates to the field of treatment of colitis and colon cancer, in particular to a medicine for improving Dicer expression and application of a medicine composition containing the medicine for improving Dicer expression in prevention and/or treatment of colitis and colon cancer.

Background

Dicer is a key enzyme in the RNAi pathway and is essential for the biosynthesis of micrornas (mirnas) and small interfering rnas (sirnas). Recent studies have shown that Dicer expression is down-regulated in various tumor tissues and that a reduction in Dicer expression promotes tumorigenesis. Somatic mutations in Dicer are found in a variety of tumor clocks, and heterozygote mutations in Dicer increase the risk of disease in a variety of tumors, particularly in lung, kidney, ovarian and thyroid tissues.

Global regulation of protein synthesis is crucial for the development and progression of cancer, as highly proliferating cancer cells require increased protein synthesis. Reduction of Dicer expression may promote protein synthesis by a variety of mechanisms. First, Dicer is critical for miRNA biosynthesis, which inhibits protein synthesis by inhibiting transcription or degrading its target mRNA; thus, a decrease in Dicer expression may result in an impairment of the overall expression of mirnas that mediate an increase in protein synthesis. Secondly, Dicer can process the full-length tRNA into small fragments of small rnas (tsrnas), called trnas, thereby inhibiting protein translation, and therefore, decreased Dicer expression leads to increased full-length tRNA levels and decreased tsRNA levels, which in turn leads to increased protein synthesis. Finally, Dicer can process 7SL RNA into small fragments, thereby impairing the formation of Signal Recognition Particles (SRPs), thereby inhibiting SRP-mediated protein targeting. SRP combines the synthesis of nascent proteins with their correct cellular destination; thus, reduced Dicer expression may promote localization of nascent proteins to their correct cellular destinations. Taken together, these findings indicate that decreased Dicer expression may promote carcinogenesis by increasing protein synthesis.

Dicer is critical for DNA repair, and decreased expression of Dicer reduces the efficiency of DNA repair and leads to the accumulation of DNA damage. DNA damage can promote inflammation by inducing expression of NKG2D ligands. In addition, DNA damage results in DNA and micronuclei accumulation in the cytoplasm; this cytoplasmic DNA triggers the production of proinflammatory cytokines, such as type I interferon and interleukin 6 (IL-6). During tumor formation, inflammation is essential, which can promote the development of cancer and the subsequent proliferation, survival and migration of cancer cells. It has been found that Dicer knock-out in intestinal epithelial cells induces inflammation, whereas Dicer-heterozygous mice are more susceptible to colitis-related tumors. Thus, in addition to its mutational consequences, reduced Dicer expression may promote tumorigenesis through induction of inflammation. It was reported that the level of Dicer mRNA was slightly decreased in colon cancer tissues, but significantly increased in rectal cancer tissues, as compared to normal mucosal tissues. Moreover, Dicer mRNA levels in stage I and II colorectal tumor tissues were lower than in stage III and IV tumor tissues. However, the dynamic pattern of Dicer expression during colitis-related carcinogenesis remains unclear. Thus, in the current study, we investigated whether Dicer is down-regulated in inflamed colon tissue prior to malignant changes. Furthermore, we investigated whether restoring Dicer expression in inflamed colon tissue could reduce colonic inflammation and colitis-related tumorigenesis.

Based on the above, the invention provides an improved scheme for treating rectal cancer by using a Dicer expression-improving drug or a combination of the Dicer expression-improving drug and chemotherapy/targeted drug, and the improved scheme has an important development and application prospect in prevention and/or treatment of colitis and colon cancer.

Disclosure of Invention

The invention screens drugs that increase Dicer protein expression based on the recognition that Dicer protein expression is reduced in colitis and colon cancer.

Anastrozole, chemical name α, α, α 'α' -tetramethyl-5- (1H-1,2, 4-triazol-1-ylmethyl) -1, 3-diacetoxybenzene. Is a strong and selective non-steroidal aromatase inhibitor. Can inhibit the conversion of androstenedione generated in adrenal gland of postmenopausal female patients into estrone, thereby obviously reducing the level of estrogen in blood plasma and generating the effect of inhibiting the breast retention growth. Has been approved for the treatment of advanced metastatic breast cancer in postmenopausal women. The invention unexpectedly discovers that anastrozole can improve the expression of Dicer protein, and further tests metabolites of anastrozole, namely hydroxyaitrazole, anastrozole-N-glucuronic acid and hydroxyaitrazole glucuronic acid.

Further, on the basis of screening a medicine for improving Dicer protein expression, the therapeutic effect of the medicine on colitis and colon cancer is verified in cell experiments and animal experiments, and the medicine is used as a preventive and/or therapeutic agent for colitis and colon cancer, thereby completing the invention.

Accordingly, in a first aspect, the present invention provides the use of a medicament for increasing Dicer expression in the manufacture of a medicament for the prevention and/or treatment of a disease or condition resulting from low Dicer expression, said disease or condition being selected from colitis or colon cancer.

In certain embodiments, the disease or disorder is colitis. Further, the colitis includes inflammatory bowel disease, which is ulcerative colitis or crohn's disease.

In certain embodiments, the disease or disorder is colon cancer. Further, the colon cancer is colon cancer associated with colitis.

In a second aspect, the present invention provides a Dicer expression-enhancing agent selected from anastrozole, a salt thereof, a metabolite thereof or an analogue thereof.

In certain embodiments, the anastrozole metabolite is selected from the group consisting of hydroxyaanastrozole, anastrozole-N-glucuronide, hydroxyaanastrozole glucuronide.

In a third aspect, the invention provides the use of anastrozole or a salt, metabolite or analogue thereof in the preparation of a medicament for a disease or condition caused by low Dicer expression.

In certain embodiments, the disease caused by low Dicer expression is selected from colitis or colon cancer.

Further, the disease or condition is colitis, including inflammatory bowel disease, which is ulcerative colitis or crohn's disease.

Further, the disorder or condition is colon cancer.

Further, the colon cancer is colon cancer associated with colitis.

In a fourth aspect, the invention provides the use of anastrozole, a salt, metabolite or analogue thereof in the manufacture of a medicament for the treatment of a subject having a reduced level of the colitis or colon cancer biomarker Dicer as indicated by the results of a test which determines the level of Dicer protein expression in an exosome fraction of a sample obtained from the subject.

In certain embodiments, the sample is an enteroscopy specimen, a surgical specimen, of the subject.

In certain embodiments, the colitis comprises inflammatory bowel disease, which is ulcerative colitis or crohn's disease.

In certain embodiments, the disorder or condition is colon cancer. Further, the colon cancer is colon cancer associated with colitis.

In certain embodiments, characterized in that the anastrozole metabolite is selected from the group consisting of hydroxyaanastrozole, anastrozole-N-glucuronide, hydroxyaanastrozole glucuronide.

In a fifth aspect, the medicament for providing Dicer expression of the invention further comprises a second therapeutic agent selected from a chemotherapeutic agent, a targeted therapeutic agent, or radiation therapy.

In certain embodiments, the chemotherapeutic or targeted therapeutic agent is selected from cisplatin, carboplatin, oxaliplatin, dichloromethyldiethylamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, ifosfamide, fluorouracil (5-FU), gemcitabine, methotrexate, cytarabine, fludarabine, paclitaxel, docetaxel, doxorubicin, daunorubicin, valrubicin, doxorubicin, mitomycin, irinotecan, topotecan, amsacrine, etoposide phosphate, or teniposide.

In certain embodiments, the second therapeutic agent is fluorouracil (5-FU).

In certain embodiments, the Dicer expression-increasing agent is selected from anastrozole, a salt thereof, a metabolite thereof, or an analog thereof.

In certain embodiments, the anastrozole, salt thereof, metabolite thereof or analogue thereof increases the therapeutic effect of 5-FU on colon cancer, and the increase in Dicer-expressing drug enhances the inhibitory effect of 5-FU on colon cancer compared to administration of 5-FU alone.

In a sixth aspect, the medicament of the present invention further comprises a pharmaceutically acceptable carrier. The active ingredients and pharmaceutically acceptable carriers are prepared into a pharmaceutical preparation together, and the dosage form of the pharmaceutical preparation is oral preparation, parenteral administration dosage form or transdermal absorption dosage form.

Further, the dosage form is tablet, capsule, solution, suspension, syrup, intramuscular injection, intravenous injection, transdermal patch, suppository.

In a seventh aspect, the anastrozole, its analogue or derivative is used in an amount of 5-20mg/kg, preferably 20mg/kg, more preferably 5 mg/kg.

In an eighth aspect, the invention provides an application of anastrozole, a salt thereof, a metabolite thereof or an analogue thereof in preparing a medicine for improving Dicer expression.

In a ninth aspect, the present invention provides the use of anastrozole, a salt, metabolite or analogue thereof in the preparation of an agent for activating Dicer protein expression in cells of colitis or colon cancer in vitro.

As used herein, "anastrozole" has the chemical name α, α, α 'α' -tetramethyl-5- (1H-1,2, 4-triazol-1-ylmethyl) -1, 3-diacetoxybenzene. Is a strong and selective non-steroidal aromatase inhibitor. The anastrozole has the structure of

As used herein, "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt of anastrozole. Pharmaceutically acceptable salts include ammonium salts, alkali metal salts such as potassium and sodium (including mono-, di-, tri-sodium) salts, alkaline earth metal salts such as calcium and magnesium salts, organic base salts such as dicyclohexylamine salts, N-methyl-D-glucamine, and amino acid salts such as arginine, lysine, and the like.

"metabolite" as used herein refers to any intermediate or product of metabolism. Some examples of anastrozole metabolites include, but are not limited to, hydroxyaanastrozole, anastrozole-N-glucuronide, hydroxyaanastrozole glucuronide. In a particular example, the metabolite is hydroxyastrozole.

As used herein, "patient" refers to any animal, such as a primate, e.g., human, simian, macaque. Any animal can be treated with the methods and compositions of the present invention.

The term "drug" refers to a compound or composition that has a pharmacological activity or effect on a patient. The terms "active ingredient", "compound" and "drug" are used interchangeably herein.

The term "administering" or "administering" includes the route by which a compound of the invention is introduced into a subject to perform its intended function. Examples of routes of administration that can be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal injection), oral, inhalation, rectal and transdermal. The pharmaceutical preparation may be administered in a form suitable for each route of administration. For example, these formulations are administered in the form of tablets or capsules, by injection, and rectally by means of suppositories. Oral administration is preferred. The injection may be a bolus injection or may be a continuous infusion. The compounds of the present invention may be administered in combination with a pharmaceutically acceptable carrier. In addition, the compounds of the present invention may also be administered in the form of a prodrug that is converted to its active metabolite or to a more active metabolite in the body.

As used herein, the term "colon cancer" is a malignancy derived from the mucosal epithelial cells of the colon, a common malignancy of the gastrointestinal tract, and may occur in the rectum, ascending colon, transverse colon, descending colon, sigmoid colon, and the like; chronic inflammation caused by ulcerative colitis, Crohn's disease of colon and schistosomiasis of colon, so that the intestinal mucosa is repeatedly destroyed and repaired to generate canceration; the diffusion and transfer routes mainly include direct infiltration, lymphatic transfer, blood circulation transfer and planting transfer. In certain embodiments, patients with colon cancer may be treated with a pharmaceutical composition of the invention. The pharmaceutical compositions of the present invention can be used to enhance efficacy and reduce the amount of drug needed to achieve that efficacy.

The term "therapeutically acceptable" refers to those compounds (or salts, prodrugs, tautomers, zwitterionic forms, etc.) that are suitable for contact with the tissues of a patient without undue toxicity, inflammation, and allergic response, are commensurate with an appropriate benefit/risk ratio, and are effective for their intended use.

An "effective amount" or "therapeutically effective amount" means the amount of a compound, alone or in combination with another treatment regimen, needed to treat a patient suffering from cancer in a clinically relevant manner. The amount will achieve the goal of reducing or eliminating the disease or disorder. The amount of active compound sufficient to therapeutically treat the condition caused by cancer for the practice of the present invention will vary depending upon the mode of administration, age, weight and general health of the patient. Ultimately, the prescribing physician will determine the appropriate dosage and dosage regimen.

The optimal dosage depends on the severity of the colon cancer being treated and whether there are side effects. The optimal dosage can be determined by routine experimentation. For gastrointestinal administration of anastrozole, a dose of between 0.1mg/kg and 100mg/kg, or between 0.5mg/kg and 50mg/kg, or between 1mg/kg and 25mg/kg, or between 10mg/kg and 25mg/kg, or between 5mg/kg and 20mg/kg, or between 2mg/kg and 16mg/kg, or between 4mg/kg and 16mg/kg is administered and may be administered, for example, once per week, once every other week, once every three weeks, or once per month per treatment round.

The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, involved in carrying or transporting one or more active substances from one organ or portion of the body to another organ or portion of the body. Each carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered gum tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cotton seed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other non-toxic compatible materials used in pharmaceutical formulations.

As used herein, "treating cancer," "treating," and "treatment" include, but are not limited to, preventing or reducing cancer development, alleviating a symptom of cancer, arresting or inhibiting the growth of an established cancer, preventing metastasis and/or invasion of cancer cells of an existing cancer, promoting or inducing regression of cancer, inhibiting or arresting proliferation of cancer cells, reducing angiogenesis, killing malignant or cancerous tumor cells, or increasing the amount of cancer cell apoptosis. In some embodiments, a compound of the invention is administered to a subject at risk of developing cancer with the aim of reducing the risk of developing cancer. The phrases "inhibiting growth" or "inhibiting proliferation" of a cancer cell refer to slowing, halting, arresting or stopping its growth and metastasis, and do not necessarily indicate complete elimination of neoplastic growth.

The term "chemotherapeutic agent" may include, for example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, antitumor antibiotics, hormonal agents, antiangiogenic agents, differentiation inducing agents, cell growth arrest inducing agents, apoptosis inducing agents, cytotoxic agents, and other antineoplastic agents. These drugs may affect cell division or DNA synthesis and function in some way. Representative chemotherapeutic agents include, but are not limited to, alkylating agents (such as cisplatin, carboplatin, oxaliplatin, dichloromethyldiethylamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), antimetabolites (such as fluorouracil (5-FU), gemcitabine, methotrexate, cytarabine, fludarabine, and fluorouracil), antimitotic agents (including taxanes (such as paclitaxel and docetaxel) and vinca alkaloids (such as vincristine, vinblastine, vinorelbine, and vindesine), anthracyclines (including doxorubicin, daunorubicin, valrubicin, idarubicin, and epirubicin, and actinomycin D), cytotoxic antibiotics (including mitomycin, procambin, and bleomycin), and topoisomerase inhibitors (including camptothecins (such as irinotecan and topotecan), and epidophyllotoxin Derivatives of podophyllotoxin (e.g., amsacrine, etoposide phosphate, and teniposide)).

The term "miRNA or" microRNA "refers to RNA of about 10-50 nucleotides in length, preferably single-stranded RNA, preferably 15-25 nucleotides in length, more preferably about 17,18,19,20,21,22,23,24 or 25 nucleotides in length, capable of directing or mediating RNA interference. Naturally occurring mirnas are generated from stem-loop precursor RNAs (pre-mirnas) by Dicer.

The term "Dicer" includes Dicer as well as any Dicer ortholog or homolog capable of processing dsRAN structures into siRNA, miRNA, siRNA-like or miRNA-like molecules.

Advantageous effects

The invention unexpectedly discovers that the medicine for improving the Dicer expression can be used for preventing/treating the colitis and the colon cancer, in particular to the colon cancer related to the colitis. The invention also discovers that the anastrozole, the salt, the metabolite and the analogue thereof can improve the Dicer protein expression, and verifies the prevention and treatment effects of the anastrozole, the salt, the metabolite and the analogue thereof on colitis and colon cancer in cell tests and animal tests. Further, the present inventors have surprisingly found that increasing Dicer-expressing drugs can increase the inhibitory effect of 5-FU on colon cancer compared to administration of 5-FU alone, which is a chemotherapeutic agent. The invention provides a new feasible way for preventing or/and treating colon cancer, and has potential great clinical popularization value.

Drawings

Figure 1 decreased Dicer expression in inflamed colon tissue.

(A, B) immunochemical staining for Dicer expression in 56 colon inflammatory tissues and 57 normal colon tissues. Representative immunohistochemical images (a) and semi-quantitative assessments (B) of Dicer protein expression. CD, crohn's disease; UC, ulcerative colitis. (C-E) analysis of Dicer expression in 46 colon inflammatory tissues and 34 normal colon tissues. Representative western blot images of Dicer protein levels in three normal colon tissues and three inflamed colon tissues (C). Dicer and GAPDH protein levels were determined by densitometry using ImageJ and expressed as Integrated Optical Density (IOD) (D). Dicer mRNA levels (E) were determined by real-time RT-PCR. (F, G) determining Dicer expression in mouse model tissues derived from control mice, DSS-induced acute or AOM/DSS-induced colitis-associated colorectal cancer in mice by Western blotting (F) and real-time RT-PCR (G). (n-10 mice per group). Data represent mean ± SEM, × P < 0.01. ns, no significant difference.

Figure 2 oxidative stress inhibits Dicer expression in inflammatory colon tissue by inducing miR-215 expression.

(A) With different doses of H₂O₂FHC cells were treated and Dicer protein levels were determined 24 hours after treatment. (B) With 400 μ M H₂O₂And 2mM NAC treated FHC cells; dicer protein levels were determined 24 hours after treatment. (C, D) mouse model (C) of acute colitis induced in DSS or AOM/DSS Induction by Western blottingOf colon cancer associated with colitis mouse model (D) expression of Dicer in colon tissue, with or without NAC treatment. (E) Correlation between 8-OHdG levels and Dicer levels in 46 inflamed colon tissues. (F) With different doses of H₂O₂FHC cells were treated for 24 hours and the level of miR-215 was quantified. (G) FHC cells were transfected with miR-215 mimetics and Dicer protein levels were determined 48 hours post-transfection. (H) FHC cells were transfected with miR-215 inhibitor and 400. mu. M H was added 24 hours after transfection₂O₂Adding to the culture medium, H₂O₂Dicer protein levels were determined 24 hours after treatment. (I) Quantifying miR-215 levels in colon tissue derived from a mouse model of acute colitis or a mouse model of colitis-related colon cancer with or without NAC treatment; -n-8 mice/group. (J) miR-215 levels were quantified in 34 normal control colon tissues and 46 inflamed colon tissues. (K) Correlation between 8-OHdG levels and miR-215 levels in 46 inflamed colon tissues. Correlation between miR-215 levels and Dicer protein levels in (L)46 inflamed colon tissues. Data represent mean ± SEM,. P<0.01; ns, no significant difference.

FIG. 3H₂O₂Treatment dose-dependently inhibited Dicer expression.

(A) With different doses of H₂O₂Treating CCD-18Co and THP-1 cells; dicer protein levels were determined 24 hours after treatment. (B) FHC cell treatment 100. mu. M H₂O₂The expression of Dicer was examined at different time points later. (C) FHC cells treated different concentrations (10-40. mu.M) of H₂O₂After 72 hours, Dicer expression was examined. (D) With different doses of H₂O₂FHC, CCD-18Co and THP-1 cells are treated; dicer mRNA levels were measured after 24 hours. (E) With 400 μ M H₂O₂And 2mM NAC treatment of CCD-18Co and THP-1 cells; dicer protein levels were determined after 24 hours. Data represent mean ± SEM, ns, with no significant difference.

Figure 48-OHdG levels were elevated in inflammatory colon tissue.

(A) Quantitative measurements 8-OHdG levels were measured in 34 normal colon tissues and 46 inflammatory colon tissues. (B) Examining 8-OHdG levels in colon tissue of a control mouse, a mouse model of DSS-induced acute colitis or a mouse model of AOM/DSS-induced colitis-related colon cancer; n-8 mice/group. Data represent mean ± SEM, × P < 0.01.

FIG. 5H₂O₂Dicer expression is inhibited by inducing miR-215 expression.

(A) Predicted miR-215 binding sites in the 3' -UTR of Dicer (upper panel) and pGL 3-control luciferase reporter constructs (lower panel). Luc: (ii) luciferase; dicer UTR: three different fragments of Dicer 3' -UTR; poly A: poly (A) tail. (B) FHC cells were co-transfected with the miR-215 mimic and different reporter plasmids along with the pRL-CMV reporter plasmid, and luciferase activity was measured 24 hours after transfection. (C, D) CCD-18Co and THP-1 cells were transfected with miR-215 mimetics, and then Dicer protein (C) and mRNA (D) levels were determined 48 hours after transfection. (E, F) CCD-18Co and THP-1 cells were transfected with miR-215 inhibitor, and 400. mu. M H was added to the medium 24h after transfection₂O₂。H₂O₂After 24 hours of treatment, the levels of Dicer protein (E) and mRNA (F) were determined. Data represent mean ± SEM,. P<0.01; ns, no significant difference.

FIG. 6 reduction of Dicer expression sensitizes cells to DNA damage induced by oxidative stress.

FHC and CCD-18Co cells were transfected with control or Dicer siRNA and 400. mu. M H were transfected 24 hours after transfection₂O₂Adding into culture medium. (A) At H₂O₂Dicer expression was analyzed by western blot 24 hours after treatment. (B, C) in H₂O₂DNA damage was determined 24 hours after treatment by comet assay (B) or by immunofluorescence with γ -H2AX (C). Data represent mean ± SEM,. P<0.01。

FIG. 7 reduction of Dicer expression sensitizes cells to oxidative stress-induced apoptosis.

(A) FHC and CCD-18Co cells were transfected with control or Dicer siRNA and different concentrations of H were applied 24 hours after transfection₂O₂Adding into culture medium. H₂O₂After 24 hours of treatment, cell viability was determined using the MTT assay. (B) Use pairFHC and CCD-18Co cells were transfected with either Dicer siRNA and 400. mu. M H cells were transfected 24 hours after transfection₂O₂Adding into culture medium. H₂O₂Apoptosis was detected 24 hours after treatment using Annexin V-FITC/PI staining. (C) Representation of TUNEL stained human normal colon tissue sections and inflamed colon tissue (left panel) and percentage of apoptotic cells (right panel). (D) Representative images of TUNEL stained tissue sections (upper panel) and percentage of apoptotic cells (lower panel) in colon tissue of acute colitis mouse model; at least 12 mice per group. (E) Correlation between Dicer protein levels and percentage of apoptotic cells in human inflamed colon tissue. Data represent mean ± SEM,. P<0.01。

FIG. 8 at H₂O₂After treatment, decreased Dicer expression results in increased expression of cytoplasmic DNA and IL-6.

(A, B) FHC cells were transfected with control or Dicer siRNA and 400. mu. M H cells were transfected 24h after transfection₂O₂Adding into culture medium; h₂O₂Cytoplasmic dsDNA levels (A) and IL-6mRNA levels (B) were determined 24 hours after treatment. (C) Immunofluorescence measures the expression levels of dsDNA in 4 normal colon tissues and 46 inflammatory colon tissues. (D) IL-6mRNA levels were quantified in 34 normal colon tissues and 46 inflammatory colon tissues. (E) Correlation between Dicer protein levels and IL-6mRNA levels in 46 inflamed colon tissues. Data represent mean ± SEM,. P<0.01; ns, no significant difference.

FIG. 9 at H₂O₂After treatment, a decrease in Dicer expression results in an increase in cytoplasmic DNA and IL-6 expression.

(A, B) FCCD-18Co and THP-1 cells were transfected with control or Dicer siRNA and 400. mu. M H were transfected 24 hours after transfection₂O₂Adding into culture medium. H₂O₂Cells were assayed for dsDNA levels (A) and IL-6mRNA levels (B) 24 hours after treatment. Data represent mean ± SEM,. P<0.01。

Figure 10 reduction of Dicer expression promotes inflammatory responses in colon tissue in a mouse model of DSS-induced acute colitis.

(A) Schematic diagram of experimental setup: five-week-old male C57BL/6 mice were rectally instilled with lentiviruses expressing Dicer shRNA or shCon. One week later, acute colitis was induced by oral administration of 3% DSS in drinking water for 7 days, followed by 2 days of normal drinking water. All mice were euthanized on day 9. (B) Dicer expression in colon tissue was determined by western blot. (C) Relative body weight curves, (D) colon length, (E) colon/body weight ratio, (F) colon weight/length (w/l) ratio and (G) representative images of mouse large intestine. (H) Representative HE stained colon sections showing inflammatory infiltration (upper panel) and inflammatory score (lower panel). (I) Mouse colon tissue paraffin sections were immunostained with LY-6G. (J) Serum IL-6 levels. Data represent mean ± SEM of at least 12 mice per group,. P < 0.01; ns, no significant difference.

FIG. 11 reduction of Dicer expression enhances AOM/DSS-induced colonic tissue inflammation and promotes colitis-associated carcinogenesis.

(A) Schematic diagram of experimental setup: six-week old male C57BL/6 mice were injected intraperitoneally with 12.5mg/kg AOM, followed by three cycles of 2.5% DSS treatment. To reduce Dicer expression in colon tissue, a lentivirus containing a Dicer shRNA expression cassette was administered intrarectally to mice 3 times. All mice were euthanized 92 days after AOM injection. Mice that received normal drinking water and no lentivirus instillation were used as controls. (B) Dicer expression in colon tissue was determined by western blot. (C) Relative body weight curve, (D) colon length, (E) colon/body weight ratio, (F) colon weight/length (w/l) ratio, (G) representative image of mouse large intestine and (H) number of tumors in mouse colorectal. (I) Representative HE stained colon sections showing inflammatory infiltration (upper panel) and inflammatory score (lower panel). (J) Representative images of TUNEL stained tissue sections (upper panel) and percentage of apoptotic cells in colon tissue (lower panel). (K) Serum IL-6 levels. Data represent mean ± SEM of at least 11 mice per group,^**P<0.01; ns, no significant difference.

FIG. 12 overexpression of Dicer reduces H₂O₂Induced DNA damage.

Transfection with Dicer expression plasmid (pDNA) or pcDNA3.1(HC) FHC, CCD-18Co and THP-1 cells, 800. mu. M H cells were transfected 24h later₂O₂Adding into culture medium. H₂O₂Dicer protein levels were determined 24 hours after treatment by western blotting (a) and DNA damage was determined by comet assay (B) or by immunofluorescence with γ -H2AX (C). The data represent the mean + -SEM,^**P<0.01。

FIG. 13 overexpression of Dicer attenuated H₂O₂Induced cytoplasmic DNA accumulation and IL-6 expression.

(A, B) FHC, CCD-18Co and THP-1 cells were transfected with Dicer expression plasmid (pDNA) or pcDNA3.1 and 800. mu. M H were added 24 hours after transfection₂O₂Adding into culture medium. H₂O₂Cells were assayed for dsDNA levels (A) and IL-6mRNA levels (B) 24 hours after treatment. Data represent mean ± SEM.^**P<0.01。

FIG. 14 Anastrozole enhances Dicer expression and decreases H₂O₂Induced IL-6 expression.

(A) Treating FHC cells with different doses of anastrozole; dicer protein levels were determined 24 hours after treatment. (B-F) FHC cells were incubated with 800. mu. M H₂O₂、800μM H₂O₂+5 μ M anastrozole for 24 hours. Dicer protein levels were determined by western blotting (B), DNA damage by comet assay (C), or cytoplasmic dsDNA levels were determined by immunofluorescence with γ -H2AX (D), by anti-dsDNA antibodies and (E) by IL-6 mRNA. Quantification was performed by real-time RT-PCR (F). The data represent the mean + -SEM,^**P<0.01; ns, no significant difference.

FIG. 15 Anastrozole treatment enhanced Dicer expression and decreased H₂O₂Induced DNA damage.

(A) Treating CCD-18Co and THP-1 with different doses of anastrozole; dicer expression levels were determined 24 hours after treatment. (B-D) 800. mu. M H₂O₂、800μM H₂O₂+ 5. mu.M anastrozole treated CCD-18Co and THP-1; 24 hours after treatment, Dicer expression levels were determined by western blotting (B), DNA damage by comet assay (C) or by gamma-H2AX immunostaining (D). The data represent the mean + -SEM,^**P<0.01。

FIG. 16 Anastrozole relieves H₂O₂Induced cytoplasmic DNA accumulation and IL-6 expression.

800 mu M H for (A, B)₂O₂、800μM H₂O₂+ 5. mu.M anastrozole treated CCD-18Co and THP-1. Cells were assayed for dsDNA levels (A) and IL-6mRNA levels (B) 24 hours after treatment. The data represent the mean + -SEM,^**P<0.01。

FIG. 17 Dicer knock-down partially abrogates anastrozole Pair H₂O₂Effects of induced DNA damage.

(A-C) FHC, CCD-18Co and THP-1 cells were transfected with control or Dicer siRNA, then 24 hours after transfection, 400. mu. M H₂O₂、400μM H₂O₂+ 5. mu.M anastrozole or 400. mu.M MH₂O₂+5 μ M pranoprofen added to the medium; at 24 hours after treatment, Dicer expression levels were determined by western blotting (a) and DNA damage was determined by comet assay (B) or immunostaining with γ -H2AX (C). The data represent the mean + -SEM,^**P<0.01。

FIG. 18 Dicer knock-down partially abrogates anastrozole Pair H₂O₂Influence of induced IL-6 expression.

(A, B) FHC, CCD-18Co and THP-1 cells were transfected with control or Dicer siRNA, then 24 hours after transfection, 400. mu. M H2O2, 400. mu. M H2O2+ 5. mu.M anastrozole or 400. mu. M H₂O₂+5 μ M pranoprofen was added to the medium. H₂O₂Cells were assayed for dsDNA levels (A) and IL-6mRNA levels (B) 24 hours after treatment. The data represent the mean + -SEM,^**P<0.01。

figure 19 overexpression of Dicer reduced inflammation of colon tissue in a mouse model of DSS-induced acute colitis.

(A) Schematic diagram of experimental setup: five-week-old male C57BL/6 mice were rectally instilled with Dicer-expressing adenovirus or control adenovirus. One week later, the acute phase was induced by taking 3% DSS orally in drinking water for 7 days, and then taking 2 days of normal drinking waterColitis (colitis). Mice that received normal drinking water and no lentivirus instillation were used as controls. (B) Dicer expression in colon tissue was determined by western blot. (C) Relative body weight curve, (D) colon length, (E) colon/body weight ratio, (F) colon weight/length (w/l) ratio, (G) representative image of mouse colon. (H) Representative HE stained colon sections showing inflammatory infiltration (upper panel) and inflammatory score (lower panel). (I) Mouse colon tissue paraffin sections were immunostained with LY-6G. (J) Representative images of TUNEL stained tissue sections (upper panel) and percentage of apoptotic cells in colon tissue (lower panel). (K) Serum IL-6 levels. Data represent mean ± SEM of at least 8 mice per group,^*P<0.05；^**P<0.01. ns, no significant difference.

FIG. 20 Dicer overexpression reduces AOM/DSS-induced inflammation in colon tissue and reduces colitis-related canceration.

(A) Schematic diagram of experimental setup: six-week old male C57BL/6 mice were injected intraperitoneally with 12.5mg/kg AOM, followed by three cycles of 2.5% DSS treatment. To increase Dicer expression in colon tissue, an adenovirus containing the Dicer expression cassette was administered intrarectally to mice 3 times. All mice were euthanized 92 days after AOM injection. Mice that received normal drinking water and did not receive adenovirus were used as controls. (B) Dicer expression in colon tissue was determined by western blot. (C) Relative body weight curve, (D) colon length, (E) colon/body weight ratio, (F) colon weight/length (w/l) ratio, (G) representative image of mouse large intestine and (H) number of tumors in mouse colorectal. (I) Representative HE stained colon sections showing inflammatory infiltration (upper panel) and inflammatory score (lower panel). (J) Representative images of TUNEL stained tissue sections (upper panel) and percentage of apoptotic cells in colon tissue (lower panel). (K) Serum IL-6 levels. Data represent mean ± SEM of at least 11 mice per group,^**P<0.01; ns, no significant difference.

Figure 21 restoration of Dicer expression by anastrozole reduces inflammation and prevents colitis-associated carcinogenesis.

(A) Schematic diagram of experimental setup: peritoneum of six-week-old male C57BL/6 miceAn internal injection of 12.5mg/kg AOM was followed by three cycles of 2.5% DSS treatment. To restore Dicer expression in inflammatory colon tissue, 0.4mg/kg anastrozole was added to the drinking water. (B) Dicer expression in colon tissue was determined by western blot. (C) Relative body weight curve, (D) colon length, (E) colon/body weight ratio, (F) colon weight/length (w/l) ratio, (G) representative image of mouse large intestine and (H) number of tumors in mouse colorectal. (I) Representative HE stained colon sections showing inflammatory infiltration (upper panel) and inflammatory score (lower panel). (J) Representative images of TUNEL stained tissue sections (upper panel) and percentage of apoptotic cells in colon tissue (lower panel). (K) Serum IL-6 levels. Data represent mean ± SEM of at least 8 mice per group,^**P<0.01。

figure 22 anastrozole enhanced Dicer expression and reduced inflammation in DSS-induced acute colitis.

(A) Schematic diagram of experimental setup: six-week-old male C57BL/6 mice induced acute colitis by oral administration of 3% DSS in drinking water for 7 days, followed by 2 days in normal drinking water. To restore Dicer expression in inflammatory colon tissue, 0.4mg/kg anastrozole was added to the drinking water. All mice were euthanized on day 9. (B) Dicer expression in colon tissue was determined by western blot. (C) Relative body weight curve, (D) colon length, (E) colon/body weight ratio, (F) colon weight/length (w/l) ratio, (G) representative images of mouse large intestine. (H) Representative HE stained colon sections showing inflammatory cell infiltration (upper panel) and inflammatory score (lower panel). (I) Mouse colon tissue paraffin sections were immunostained with LY-6G. (J) Representative images of TUNEL stained tissue sections (upper panel) and percentage of apoptotic cells in colon tissue (lower panel). (K) Serum IL-6 levels. Data represent mean ± SEM of at least 10 mice per group,^*P<0.05；^**P<0.01; ns, no significant difference.

Figure 23 anastrozole dose-dependently reduced DSS-induced acute colitis.

Six-week old male C57BL/6 mice were induced by oral administration of 3% DSS in drinking water for 7 days, followed by 2 days of normal drinking waterAcute colitis. To restore Dicer expression in inflamed colon tissue, various doses of anastrozole (1X 0.1mg/kg, 4X 0.4mg/kg) were added to the drinking water. All mice were euthanized on day 9. (A) Relative body weight curve. (B) Colon length, colon/body weight ratio and colon weight/length (w/l) ratio. (C) Representative images of mouse large intestine. (D) Inflammatory cell infiltration and percentage of apoptotic cells in colon tissue. (E) Serum IL-6 levels. Data represent mean ± SEM of at least 10 mice per group;^*P<0.05；^**P<0.01; ns, no significant difference.

FIG. 24 anastrozole dose-dependently alleviates AOM/DSS-induced chronic colitis and inhibits colitis-related carcinogenesis.

Six-week old male C57BL/6 mice were injected intraperitoneally with 10mg/kg AOM, followed by three cycles of 2.5% DSS treatment. To rescue Dicer expression in inflamed colon tissue, various doses of anastrozole (1X ═ 0.1mg/kg, 4X ═ 0.4mg/kg) were used in drinking water. (A) Relative body weight curve. (B) Colon length, colon/body weight ratio and colon weight/length (w/l) ratio. (C) Representative images of mouse large intestine. (D) Number of tumors in colorectal in mice. (E) Inflammatory cell infiltration and percentage of apoptotic cells in colon tissue. (F) Serum IL-6 levels. Data represent mean ± SEM of at least 10 mice per group,^*P<0.05；^**P<0.01; ns, no significant difference.

Figure 25 Dicer knockdown partially abrogated the effect of anastrozole on acute inflammation.

Five-week-old male C57BL/6 mice were rectally instilled with lentiviruses expressing Dicer shRNA or shCon. One week later, acute colitis was induced by oral administration of 3% DSS in drinking water for 7 days, followed by 2 days of normal drinking water. To restore Dicer expression in inflamed colon tissue, 0.4mg/kg anastrozole was added to the drinking water. (A) Dicer expression in colon tissue was determined by western blot. (B) Relative body weight curve. (C) Colon length, colon/body weight ratio and colon weight/length (w/l) ratio. (D) Representative images of mouse large intestine. (E) Infiltration of inflammatory cells in colonic tissue andpercentage of apoptotic cells. (F) Serum IL-6 levels. Data represent mean ± SEM of at least 11 mice per group,^*P<0.05；^**P<0.01; ns, no significant difference.

FIG. 26 the Dicer combination knockout partially abolished the effect of anastrozole on colitis-related carcinogenesis.

Six-week old male C57BL/6 mice were injected intraperitoneally with 10mg/kg AOM, followed by three cycles of 2.5% DSS. To reduce Dicer expression in colon tissue, a lentivirus containing a Dicer shRNA expression cassette was administered intrarectally to mice 3 times. To restore Dicer expression in inflamed colon tissue, 0.4mg/kg anastrozole was added to the drinking water. (A) Dicer expression in colon tissue was determined by western blot. (B) Relative body weight curve. (C) Colon length, colon/body weight ratio and colon weight/length (w/l) ratio. (D) Representative images of mouse large intestine. (E) Number of tumors in colorectal in mice. (F) Inflammatory cell infiltration and percentage of apoptotic cells in colon tissue. (G) Serum IL-6 levels. Data represent mean ± SEM of at least 11 mice per group,^*P<0.05；^**P<0.01; ns, no significant difference.

Figure 27 anastrozole increased the efficacy of 5-Fu in the treatment of colon cancer.

A nude mouse graft tumor model was constructed using HCT116 cells, treated with 5-Fu and fed with anastrozole. Transplant tumor picture (a), mean volume (B), weight (C) and mouse body weight (D) analysis. Data represent mean ± SEM of at least 5 mice per group,^*P<0.05；^**P<0.01; ns, no significant difference.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Materials and methods part:

reagent:

anastrozole was purchased from MCE, 5-FU from dingoto, doxorubicin and methotrexate from Sigma, oxaliplatin from sirolimus, cetuximab from merck, bevacizumab from roche, irinotecan from feverine, cyclophosphamide from Baxter Oncology Gmbh, da carbamazine from south kyo pharmaceutical factory, gemcitabine from li, and mitomycin from zhejiang nordstock.

Cell culture

FHC cell lines, CCD-18Co cells, THP-1 cell lines, and colon cancer cell lines SW480, HT29 were all purchased from ATCC. FHC cell line, HT29 cells containing 10% fetal calf serum RPMI-1640 culture medium culture, CCD-18Co cells, SW480 cells containing 10% fetal calf serum DMEM medium culture, THP-1 cells containing 10% fetal calf serum and 50M beta-mercaptoethanol RPMI-1640 culture medium culture. Placing in a constant temperature incubator containing 5% CO2 at 37 ℃.

Collection of samples from patients with inflammatory bowel disease

Samples of 91 normal tissue specimens and 102 inflammatory bowel disease patients were obtained from the first hospital affiliated to the university of medical Wenzhou, and were collected in two ways: one part is frozen rapidly by liquid nitrogen, and the other part is fixed and preserved by formalin solution. The rapidly frozen sample is stored at the temperature of minus 80 ℃ for extracting RNA reverse transcription to be used as the quantification of miR-215 and extracting protein to be used as the western blot experiment of Dicer protein; the formalin-fixed specimen was used for the immunohistochemical experiments of Dicer and TUNEL experiments of apoptosis by paraffin embedding and sectioning. All experiments had obtained informed consent for the study as a clinical sample, which was approved by the scientific ethics committee of the first hospital affiliated with the university of medical science, wenzhou.

Animal research

5 weeks old C57BL/6J male mice and 5 weeks old BALB/C female nude mice, SPF grade, weight 18 + -2 g, purchased from Beijing Wintolite laboratory animals technology Ltd, license number SCXK (Jing) 2016-. The animals are bred in SPF animal laboratories in the school district of the university of Wenzhou medical school. Before experiment, all mice are kept at room temperature of 22-25 ℃, the relative humidity is controlled to be about 50%, the mice are adaptively fed in cages for 1 week under the condition of natural illumination period, and SPF standard animal feed is fed in the period, and water is freely drunk. All animals were housed and processed according to the animal care and use committee at the university of medical science, wenzhou.

DSS-induced acute enteritis mouse model: male C57BL/6 mice (experimental group) at six weeks of age were given 3% Dextran Sodium Sulfate (DSS) for continuous drinking for 7 days, and after 7 days, they were changed to drinking distilled water for 2 days. The change in body weight of the mice was measured daily.

Mouse model of AOM/DSS-induced colitis-associated colorectal cancer: mice were fed with a small dose of Azoxymethane (AOM) (12.5mg/kg) given a single intraperitoneal injection followed by 2.5% DSS for 5 days followed by 16 days of normal drinking, one cycle of 21 days, for 3 cycles (the last cycle of normal drinking was extended to 46 days), and the change in body weight of the mice was measured daily. Fixing the mouse on a foam plate after euthanasia, dissecting the mouse by using an ophthalmic scissors and a small forceps, taking out the colorectal of the mouse in physiological saline, cleaning intestinal contents, placing the colorectal on clean filter paper, measuring the length (cm) and the weight (g) of the colorectal, and taking a picture; colon tissue specimens were removed and placed in formalin for fixation for at least 24 hours.

Acute enteritis Dicer lentivirus (Dicer lentivirus available from Origene) enema treatment: enema with 100. mu.l/50% ethanol/NaCl solution, enema with 100. mu.l/108 titers lentivirus after 3h, 1 time for 3 days, 2 times total.

Colitis-related colorectal cancer enema treatment: enema with 100. mu.l/50% ethanol/NaCl solution, enema with 100. mu.l/108 titers lentivirus after 3h, 1 time a week for 3 times.

And (3) drug treatment: the drug was added to the mouse's drinking water from the beginning of DSS addition until the end of the experiment.

Nude mice xenograft tumor mouse model construction nude mice were injected subcutaneously with HCT116 cells to generate transplantable tumors, tumor size was measured with a vernier caliper, and tumor volume was calculated according to V ═ L × W2 × 0.5236 (L represents LMajor axis, W for minor axis), when the tumor size becomes visible (-100 mm)³) And the molding is successful.

Successfully modeled mice were divided into 4 groups of 6 mice each.

The first group (model control group), i.p. saline (50mg/kg), once daily for two consecutive weeks;

a second group (5-FU administration group) into which 5-fluorouracil (5-FU, 50mg/kg) was intraperitoneally injected once a day for two consecutive weeks (i.p.);

the third group (anastrozole group) was fed with anastrozole (0.4mg/kg) via drinking water once a day for two consecutive weeks.

(iv) group four (combination group), i.p.) 5-fluorouracil (5-FU, 25mg/kg) was injected intraperitoneally once daily for two consecutive weeks; anastrozole (0.2mg/kg, dose halved) was then fed through the drinking water once daily for two consecutive weeks.

At the end of the two week experiment, mice were sacrificed using cervical dislocation and their tumors were weighed.

Western blot analysis

1. Extraction of total protein of cell and tissue

Cell: to the cell samples, 100ul of pre-chilled RIPA strong cell lysate was added, and protease inhibitors were added to lyse the cells on ice, during which the cells were blown 2 times. The cells were centrifuged at 12000g for 5 minutes (4 ℃ C.), and the supernatant was extracted as total cell protein.

Organizing: placing a soybean-sized tissue into a centrifuge tube, adding 200ul of precooled RIPA strong lysate, adding protease inhibitor, homogenizing by a homogenizer, fully crushing the tissue, beating for 30 minutes on ice, blowing for 2-3 times, centrifuging for 15 minutes at 12000g and 4 ℃, and sucking the supernatant to obtain the total protein of the tissue.

2. BCA method for determining protein concentration

The method for measuring the total protein concentration of cells/tissues by using the BCA protein concentration measuring kit comprises the following specific steps:

a. preparation of protein standard: melting a tube of BSA protein standard in 1.2ml of protein standard preparation solution to prepare a standard with the concentration of 25mg/ml, and taking out an appropriate amount of sample to prepare 0.5mg/ml to be used as a standard for measuring the protein concentration;

b. 0, 1, 4, 8, 12, 16, 20ul of standard was added to a 96-well plate, and a standard dilution was added to give a final volume of 20ul, corresponding to standard concentrations: 0. 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/ml;

c. add 20ul protein sample to 96 well plate

d. 200ul of BCA working solution is added into the air where the sample and the standard are added, and the air is incubated for 30 minutes at 37 ℃, wherein the formula of the working solution is as follows: adding BCA reagents A and B according to the proportion of 50:1 to prepare a proper amount of BCA working solution;

e. the absorbance at a562 was measured using a multifunctional microplate reader.

f. And drawing a protein standard curve according to the standard substance, and calculating the concentration of the protein in the sample according to the absorbance of the protein standard curve, wherein the protein standard curve is shown in figure 1.

SDS-PAGE gel electrophoresis

a. Preparation of protein samples:

the total protein extracted from the cells/tissues was added to a suitable volume of 5 x protein buffer, mixed well with shaking, the protein samples were boiled for 10 minutes at 95 ℃ in a metal cooker for denaturation, and immediately cooled on ice. The loading of each protein was taken as 30. mu.g depending on the protein concentration determined and the volume of all samples was made up to 20. mu.l with 1 Xprotein buffer.

b. Protein electrophoresis

Preparing 6% of concentrated gel and 6% of separation gel, adding a sample into a sample loading hole, adding 1X electrophoresis liquid, running the concentrated gel by using 80V constant voltage electrophoresis, adding voltage to 120V constant voltage electrophoresis to run the separation gel after a protein marker generates a strip in the separation gel, stopping electrophoresis after bromophenol blue in protein reaches the bottom of the gel, removing a glass plate, taking out the gel containing the protein sample, and preparing for membrane transfer.

c. Rotary film

The PVDF membrane was activated in methanol for 10 minutes before being transferred. Putting a film transferring tool on the film transferring clamp in the following sequence: the method comprises the following steps of (1) putting a white board, a wet sponge mat, 1 layer of thick filter paper, 2 layers of thin filter paper, PVDF (polyvinylidene fluoride) membrane, gel containing a protein sample, 2 layers of thin filter paper, 1 layer of thick filter paper, the wet sponge mat, a blackboard, paying attention to discharge bubbles among all layers, locking a membrane rotating clamp, enabling the blackboard surface of the membrane rotating clamp to face the black surface of a red-black box, enabling a transparent plate of the membrane rotating clamp to face the red surface of the red-black box, putting the red-black box into a membrane rotating groove, adding a precooled 1x electric rotating buffer solution into the membrane rotating groove, putting a precooled ice core into the membrane rotating groove, and rotating the membrane overnight at a constant voltage of 25V.

d. Sealing of

And after the membrane is transferred, taking out the PVDF membrane, tearing open and shearing the PVDF membrane according to the size of the required protein and the size of a protein Marker, putting the PVDF membrane into TBST (tert-butyl-tert-butyl) solution for cleaning, putting the torn and sheared membrane into 5% of pre-prepared skim milk, slightly shaking and sealing the membrane on a shaking table for 90 minutes, and washing the membrane with TBST for 3 times and 6 minutes each time.

e. Incubation primary antibody

The washed membrane was placed in 5ml of a prepared primary antibody in the following proportions: dicer (Abcam)1: 1000; anti-GAPDH (2118S) (Cell Signaling Technology)1:3000, incubated on a shaker at room temperature for 3-4 hours, the antibody can be used repeatedly, washed 3 times with TBST for 6 minutes each.

f. Incubation secondary antibody

Selecting a secondary antibody of each primary antibody corresponding to the species marked by horseradish peroxidase, preparing the secondary antibody by using 5% of milk prepared by TBST according to the proportion of 1:10000, adding a PVDF membrane into the prepared secondary antibody, incubating for 1 hour at normal temperature on a shaking table, and washing for 3 times by using TBST for 6 minutes each time.

g. Development

Preparing an ECL developing substrate in advance according to the proportion of ECLA to B1:1, taking out a washed PVDF film, sucking the liquid on the surface by using filter paper, placing the film on a preservative film, paying attention to the requirement that the front side of the film is upward, dripping 500ul of ECL developing substrate on each film, uniformly coating the substrate on the film by using a liquid transfer gun, incubating for 2 minutes, taking out the film, slightly sucking the liquid on the film, placing the film into a developing clamp, bringing the clamp and a developing film into a dark room, fixing the film on the film, covering the clamp, regulating the developing time according to the strength of a target protein strip, taking out the film, immediately placing the film into the developing solution, developing and fixing in a fixing solution.

h. Experimental data processing

Scanning the developed film, and analyzing the target strip by using Image J software

Paraffin tissue section

1. Material taking and fixing:

the tissue specimens were placed in formalin solution for fixation for at least 24 hours.

2. And (3) dehydrating:

since the water in the fixed tissue cannot be mixed with the tissue embedding fluid, the water in the tissue needs to be removed before embedding the tissue. The tissues were dehydrated as follows:

3. and (3) transparency:

the method for replacing alcohol in the tissue specimen by using dimethylbenzene to keep the tissue transparent comprises the following steps: the dehydrated tissue specimen was placed in xylene for 30 minutes and then in fresh xylene for 30 minutes to make the tissue specimen transparent.

4. Soaking in paraffin wax

And (3) soaking the tissue specimen which is transparent by using the dimethylbenzene in the paraffin for 1 hour, then soaking another fresh paraffin for 1 hour, and replacing the dimethylbenzene in the tissue specimen by using the paraffin.

5. Embedding in paraffin wax

And (3) putting the tissue specimen soaked by the paraffin into a metal embedding frame, adding the paraffin, covering a plastic embedding frame after the paraffin begins to cool, adding the paraffin to fully embed the tissue specimen, placing the tissue specimen on a cooling plate at the temperature of minus 20 ℃ until the paraffin is completely cooled, and detaching the metal embedding frame to obtain a well-embedded paraffin tissue block.

6. Paraffin section

Fixing paraffin block on a slicer, adjusting the thickness of the slice to 5 μm, placing the tissue slice in a 50 deg.C water bath with forceps to completely unfold the tissue slice, slowly taking out the unfolded tissue slice with an adhesive glass slide, air drying at room temperature, and oven drying at 65 deg.C overnight to obtain paraffin tissue slice.

TUNEL experiment

1. Deparaffinization and hydration of tissue slices

2. Adding 20 mu g/ml protease K without DNase into the tissue slices, incubating for 30 minutes at 37 ℃, removing endogenous protein in the tissues, and fully washing the slices for 3 times by using PBS solution;

3. preparing 3% H2O2 with PBS, adding 100ul of the solution into each tissue slice, incubating for 20 minutes at room temperature, removing peroxide in the tissue specimen, and washing for 3 times with PBS;

4. adding 50 μ l of biotin labeling solution to the sample, and incubating at 37 ℃ for 60 minutes, wherein the biotin labeling solution comprises the following components:

PBS washing 3 times;

5. adding 100ul of labeled reaction stop solution, incubating for 10 minutes at room temperature, and washing for 3 times with PBS;

6. adding a Streptavidin-HRP biotin labeling solution to the sample, wherein the Streptavidin-HRP comprises the following components:

PBS washing 3 times;

DAB coloration

At 850ul ddH₂Adding A, B, C reagent into O, preparing DAB solution at a ratio of 50ul, adding 200ul DAB solution into each tissue section for staining, incubating for 15 minutes at normal temperature, and washing for 3 times with ddH 2O;

8. dyeing process

Staining the tissue sections with hematoxylin for 10 seconds, and turning blue with PBS;

9. dehydrated transparent

Dehydrating and transparentizing according to the following steps:

10. sealing sheet

A drop of neutral resin is dropped on the tissue section, and then a cover glass is covered, so that the result can be observed under a microscope or can be kept for a long time.

HE staining (i.e. hematoxylin-eosin staining method)

1. Deparaffinization and hydration of tissue slices

2. Staining sappan wood semen for 5min

3. Slightly washing with running water for 1-3s

4.1% ethanol hydrochloride for 1-3s

5. Slightly washing for 10-30s

6. Washing with distilled water for 1-2s

Dyeing with 7.0.5% red liquor for 1-3min

8. Slightly washing with distilled water for 1-2s

9. Dehydrated transparent

10. Neutral gum encapsulation

The scoring criteria for HE were: inflammatory cell infiltration was rated 0, 0%;

grade

1, 1% -20%;

grade

2, 21% -40%; grade 3, 41% -61%; 4 grade, 61-80 percent and 5 grade, 81-100 percent.

Comet assay (Comet assay)

1. Preparation of primer

Immersing the clean glass slide in 0.2% normal-melting-point agarose for 1min, and then baking the glass slide in an incubator at 37 ℃ for overnight; before the experiment, the cell suspension and low-melting point agarose (42 ℃) are mixed uniformly in a centrifuge tube according to the proportion of 1:1, glue is poured immediately (firstly, a cover glass is used for pouring glue after a small groove with the size of 22mm multiplied by 10mm multiplied by 0.17mm is arranged on a coated slide glass), the cover glass is taken down after the mixture is kept stand for 15min at the temperature of 4 ℃, and a glue layer containing cells is prepared.

2. Cracking at 4 ℃ for 1 h.

3. Washing with water at 4 deg.C ddH2O for 3 times (5 min/time).

4. Unwinding at 4 deg.C for 20 min.

5. Electrophoresis was carried out at 4 ℃ for 20min at 0.8V/cm.

PBS neutralization 3 times, 5min each time.

7. Gradient alcohol dehydration of 50%, 70%, 80%, 90%, 100%, 5min each time at room temperature. 8. Staining (EB, 20. mu.g/ml, 20. mu.l) was followed by microscopic examination and analysis using CASP analysis software.

Comet assay lysate (100 ml):

pH＝13

comet assay electrophoretic fluid (1L):

immunofluorescence assay

1. When the cells grow and fuse to 80% -90% on the cover glass, taking out the cells from the incubator

2. Washing with 4 deg.C pre-cooled 1 XPBS for 3 times, each for 10 min

3. 4% formaldehyde is fixed for 20-30 minutes at room temperature

4.1 XPBS wash 3 times for 10 minutes each

5. 0.1% Triton X-100 for 10 min

6. 1 XPBS wash 3 times for 10 minutes each

7. Blocking with 1% BSA for 30 min at RT

8. Incubate anti-dsDNA/ssDNA (Millipore 1: 500), γ -H2AX (Cell Signaling technology 1: 800) (configured with 1% BSA) in a wet box at 4 degrees overnight

9. 1 XPBS wash 3 times for 10 minutes each

10. Incubation Secondary antibody (goat anti-mouse/goat anti-rabbit Bostor 1: 500) (formulated with 1% BSA) incubated for 60 min at ambient temperature, light was blocked

11. 1 XPBS wash 3 times for 10 minutes each

12. 20ul of DAPI (Biyunyan day) was added to each slide, incubated at room temperature for 5min, and washed 1 times with 1 XPBS and ddH2O each for 5min

13. Mounting with anti-fluorescence decay mounting tablet (Bostor) (4 degree storage)

Note that 0.1% Triton and 1% BSA were diluted with 1 XPBS

Quantitative RT-PCR

1. 1ug of RNA was added to a centrifugal tube without RNase, and DNA contained in the RNA was digested in the following manner

The DNA digestion system was as follows:

2. 1ul of stop solution was added to each system, the DNse I digestion was terminated, and the reaction was carried out at 65 ℃ for 10 minutes

3.1 ul of random primer (500ug/ml) was added to each system, 5min at 70 ℃ and immediately ice-cooled for 2 min;

4. adding 13ul of reverse transcription system into each system for reverse transcription rate;

the reverse transcription system is as follows:

5. quantitative PCR is carried out on cDNA obtained by reverse transcription by using SYBR Premix Ex Taq (Perfect read-Time) quantitative kit instructions,

the quantitative system is as follows:

volume of ingredients (μ l)

The primers are as follows: dicer (human),5'-TCCACGAGTCACAATCAACACGG-3' and 5'-GGGTTCTGCATTTAGGAGCTAGATGAG-3';

IL-6(human),5'-CAATCTGGATTCAATGAGGAGAC-3'and 5'-CTCTGGCTTGTTCCTCACTACTC-3'；

GAPDH(human),5'-ATGACATCAAGAAGGTGGTG-3'and 5'-CATACCAGGAAATGAGCTTG-3'；

Dicer(mouse),5'-GCCAAGAAAATACCAGGTTGAGC-3'and 5'-GCGATGAACGTCTTCCCTGAG-3'；

GAPDH(mouse),5'-ACGGCCGCATCTTCTTGTGCA-3'and 5'-ACGGCCAAATCCGTTCACACC-3'.

serum IL-6 detection

1. Samples or standards of different concentrations were added to the corresponding wells at 100. mu.l/well, the reaction wells were sealed with a sealing plate (clear), and incubated at room temperature for 120 minutes.

2. The plate was washed 5 times and the last time was patted dry on thick absorbent paper.

3. Biotinylated antibody was added at 100. mu.l/well. The reaction wells were sealed with a plate-sealing membrane (clear) and incubated at room temperature for 60 minutes.

4. The plate was washed 5 times and the last time was patted dry on thick absorbent paper.

5. 100. mu.l/well of Streptavidin was labeled with horseradish peroxidase. The reaction wells were sealed with a sealing plate (white) and incubated at room temperature in the dark for 20 min.

6. The plate was washed 5 times and the last time was patted dry on thick absorbent paper.

7. Add color reagent TMB solution 100u l/hole, seal the reaction hole with sealing plate film (white), room temperature and light-proof incubation for 20 minutes.

8. Add stop solution 50. mu.l/well and measure A450 value immediately after mixing.

Example 1: discussion of relationship of Dicer enzyme to colitis and colon cancer

1. Correlation and mechanism of Dicer expression and colitis and colon cancer pathogenesis

In order to study Dicer expression in pre-malignant colitis, paraffin-embedded colon samples from 56 patients with inflammatory bowel disease (27 patients with crohn's disease and 29 patients with ulcerative colitis) and 57 normal human paraffin-embedded tissue samples were examined, immunohistochemical analysis showed reduced Dicer expression in inflammatory colon tissue relative to normal tissue samples (fig. 1A-B), and further analysis using an additional 46 patients with inflammatory bowel disease frozen inflammatory colon tissue and 34 normal human tissue samples showed reduced Dicer protein levels but not reduced mRNA levels (fig. 1C-E); also, Dicer protein levels, but not mRNA levels, were reduced in the DSS-induced acute colitis mouse model or AOM/DSS-induced colitis-associated colorectal cancer mouse model (fig. 1F-G), and it is clear from the above results that Dicer protein levels have been down-regulated before colitis became cancerous.

2. Inhibition of Dicer expression in inflammatory colonic tissue by oxidative stress

To investigate whether oxidative stress could inhibit Dicer expression in inflammatory colon tissue, H was used₂O₂The results of FHC cells, CCD-18Co cells and THP-1 cells treated show that H₂O₂Dicer protein expression was inhibited in a dose-dependent manner, but had no effect on mRNA (fig. 2A, fig. 3A-D). Treatment of H-treated with the antioxidant NAC₂O₂Treated cells as described above, and as a result, NAC was found to partially restore H₂O₂Induced Dicer down-regulation (fig. 2B, fig. 3E). Hydrogen peroxide can cause the reduction of the Dicer protein level, and NAC can relieve the reduction of the Dicer protein level caused by hydrogen peroxide; also in the mouse model of inflammatory bowel disease, oxidative stress and antioxidant NAC treatment achieved the same effect (fig. 2C-D). 8-OHdG, a biomarker of oxidative stress and oxidative loss of DNA, was found to be increased in the human and mouse inflammatory colon tissues compared to normal tissues (FIGS. 4A-B), and in the human inflammatory colon tissues, a negative correlation between Dicer protein levels and 8-OHdG was detected (FIG. 2E). From the above results, it is understood that oxidative stress causes a decrease in the expression level of Dicer protein in inflammatory colon tissue.

3. Oxidative stress inhibits Dicer expression in inflammatory colon tissue by inducing miR-215 expression.

The target gene prediction software predicted 3 potential miR-215 binding sites in the 3' -UTR in Dicer (fig. 5A). To verify whether miR-215 modulates Dicer expression, we performed a dual luciferase assay using three reporter vectors consisting of a luciferase coding sequence andthe subsequent different fragment compositions of Dicer 3' -UTR (fig. 5A). As shown in fig. 5B, miR-215 inhibited luciferase activity of all three reporter genes. Transient transfection of miR-215 results in Dicer protein expression, but does not affect Dicer mRNA expression (FIGS. 2F-G and 5C-D). Warp H₂O₂After treatment, miR-215 inhibitors partially restored Dicer protein, but failed to restore mRNA expression (fig. 2H and fig. 5E-F). Overall, these findings indicate H₂O₂Treatment inhibited Dicer expression by inducing miR-215 expression.

We then investigated whether oxidative stress inhibited Dicer expression by inducing miR-215 expression in inflammatory colon tissue. As shown in figure 2I, miR-215 is up-regulated in inflammatory colon tissue in a mouse model of colitis; in contrast, NAC administration partially inhibited the up-regulation of miR-215. Furthermore, miR-215 expression was significantly up-regulated and positively correlated with 8-OHdG levels in human inflammatory colon tissue (fig. 2J-K). The expression level of Dicer protein was inversely correlated with miR-215 levels in human inflammatory colon tissue (fig. 2L). Taken together, these results indicate that oxidative stress inhibits Dicer expression by inducing miR-215 expression.

4. Reduction of Dicer expression enhances sensitivity of cells to oxidative stress-induced DNA damage and apoptosis

Since oxidative stress induces DNA damage, and Dicer plays an important role in DNA repair, we investigated whether reduced Dicer expression increases the sensitivity of cells to oxidative stress induced DNA damage. Comet assay and immunostaining with gamma-H2 AX antibody showed that H₂O₂More DNA damage was induced in Dicer-knockdown cells than in control cells (FIGS. 6A-C). Cell proliferation and apoptosis assays also showed that Dicer knockdown could be paired with H₂O₂The treatment was sensitive (fig. 7A-B). TUNEL staining showed increased apoptosis in inflammatory colon tissue (fig. 7C), while Dicer knockdown further increased DSS-induced apoptosis in inflammatory colon tissue (fig. 7D). Interestingly, we found that the level of Dicer protein expression in human inflammatory colon tissue was negatively correlated with the level of apoptosis (fig. 7E).

5. Decreased Dicer expression increases H₂O₂After treatmentAnd promoting IL-6 expression

DNA damage leads to accumulation of cytosolic DNA, triggers production of IL-6, and promotes inflammatory diseases and cancer, H₂O₂Treatment increased cytoplasmic DNA in different cells, while Dicer inhibition further increased H₂O₂Induced accumulation of cytoplasmic DNA (FIGS. 8A and 9A). Dicer knockdown of cells in H compared to control cells₂O₂Higher IL-6 levels were expressed after treatment (FIG. 8B and FIG. 9B). Furthermore, we found that IL-6 expression was increased in human inflammatory colon tissue compared to normal healthy colon tissue (FIG. 8C), and that the expression level of Dicer protein was observed to be inversely correlated with the mRNA level of IL-6 in human inflammatory colon tissue (FIGS. 8D-E). Thus, these results indicate that reduced Dicer expression promotes H₂O₂Accumulation of cytoplasmic DNA after treatment, thereby enhancing H₂O₂Induced IL-6 expression.

6. Reduced Dicer expression enhances DSS-induced colonic tissue inflammation and promotes colitis-associated carcinogenesis

Consistent with the finding that heterozygous knockout of Dicer in intestinal epithelial cells increased inflammatory cell infiltration in colon tissue, we found that Dicer knockout in colon tissue in a DSS-induced mouse model of acute colitis promoted inflammation development in the form of weight loss, terminal bowel length, inflammatory cell infiltration and apoptosis in colon tissue, and elevated serum IL-6 levels (fig. 10A-J). In addition, Dicer knockout in colon tissue from AOM/DSS-induced colitis-associated colon cancer mouse models not only increased the severity of inflammation, but also promoted carcinogenesis (fig. 11A-K).

In summary, it can be seen from the present examples that the reduced Dicer protein expression level, whether at the cellular level or the animal model level, is associated with colitis and colon cancer, i.e. the reduced Dicer protein expression level promotes the occurrence and development of colitis and colon cancer, and therefore there is an incentive to investigate whether compounds capable of reversing the reduced Dicer protein expression level can be used for preventing and or treating colon cancer.

Example 2: function of anastrozole in hydrogen peroxide induced cell injury

Consistent with our previous studies on Dicer overexpression promoting DNA repair, Dicer overexpression reduced H₂O₂Induced DNA damage, reduction of H₂O₂Induced increase in cytoplasmic DNA and reduction of H₂O₂Induced expression of IL-6 (FIGS. 12A-C and 13A-B). By screening drug libraries, we found that anastrozole enhanced Dicer expression (fig. 14A and 15A). Increased anastrozole-induced Dicer expression, decreased in H₂O₂Susceptibility to induced DNA damage (FIGS. 14B-D and 15B-D). Thus, treatment with anastrozole reduced H₂O₂Induced increases in cytoplasmic DNA and IL-6 expression (FIGS. 14E-F and 16A-B). Silencing Dicer expression may partially abolish the effects of anastrozole (FIGS. 17A-C and 18A-B). Together, these findings suggest that anastrozole can prevent H by promoting Dicer expression₂O₂Induced DNA damage.

Example 3 Effect of anastrozole in mouse models of colitis and colitis-related Colon cancer

This example discusses whether restoring Dicer protein expression in inflammatory colon tissue can improve colitis and inhibit colitis-associated colon cancer.

Specifically, overexpression of lentiviral-mediated Dicer in colon tissue of a mouse model of acute colitis can improve inflammatory symptoms in terms of mouse body weight, colon length, inflammatory cell infiltration, apoptosis, IL-6 expression level, and the like (fig. 19A-K). Overexpression of Dicer protein in colon tissue of mouse model of chronic colitis not only reduced the inflammatory response but also significantly reduced colon cancer apoptosis (fig. 20A-K).

Administration of anastrozole to a mouse model of colitis restored Dicer expression in inflamed colon tissue (FIGS. 21A-B and 22A-B). Restoration of Dicer expression reduced the degree of inflammation in the mouse model of acute colitis from the final colon length, inflammatory cell infiltration and apoptosis in colon tissues and serum IL-6 levels (fig. 22C-K and fig. 23A-E). Similar to Dicer overexpression, restoration of Dicer expression reduced the severity of inflammation and the formation of colon tumors in a mouse model of colitis-associated colorectal cancer (fig. 21C-K and fig. 24A-F). Silencing of Dicer expression in colon tissue partially abolished the effects of anastrozole on inflammation and inflammation-associated colon cancer (FIGS. 25A-F and 26A-G), which suggests that anastrozole reduces the severity of colitis by upregulating Dicer, as well as preventing the occurrence and progression of colon cancer associated with colitis.

Example 4 study of drug combinations for increasing Dicer expression drug anastrozole in treatment of colon cancer.

In constructing a nude mouse xenograft tumor model from HCT116 cells, treatment of colon cancer cells with 5-FU followed by administration of a Dicer-expressing-enhancing drug was more effective in inhibiting the growth of the transplanted tumor and significantly improved the level of life of the mice, compared to administration of 5-FU alone (fig. 27).

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. Use of anastrozole, or a salt, metabolite or analogue thereof, in the manufacture of a medicament for the treatment of a disease or condition caused by low Dicer expression.

2. Use according to claim 1, wherein said disease or condition caused by low Dicer expression is selected from colitis or colon cancer.

3. Use according to claim 2, characterized in that the disease or condition is colitis, including inflammatory bowel disease, preferably ulcerative colitis or crohn's disease;

or the disorder or condition is colon cancer, preferably colon cancer associated with colitis.

4. Use of anastrozole, a salt, analogue or derivative thereof, in the manufacture of a medicament for the treatment of a subject having a reduced level of the colitis or colon cancer biomarker Dicer as indicated by the results of a test which determines the level of Dicer protein expression in an exosome fraction of a sample obtained from the subject.

5. Use according to claim 4, said sample being selected from an enteroscopy specimen, a surgical specimen, a lymph, saliva, urine or plasma sample of said subject.

6. Use according to claim 5, said colitis comprising inflammatory bowel disease being ulcerative colitis or Crohn's disease.

7. Use according to claim 5, wherein the colon cancer is colitis-related colon cancer.

8. Use according to any one of claims 1 to 7, wherein the disease or condition is colon cancer and the medicament further comprises a second therapeutic agent selected from chemotherapeutic agents.

9. Use according to claim 8, characterized in that said chemotherapeutic agent is selected from cisplatin, carboplatin, oxaliplatin, dichloromethyldiethylamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, ifosfamide, fluorouracil (5-FU), gemcitabine, methotrexate, cytarabine, fludarabine, paclitaxel, docetaxel, doxorubicin, daunorubicin, valrubicin, doxorubicin, mitomycin, irinotecan, topotecan, amsacrine, etoposide phosphate or teniposide, said second therapeutic agent preferably being fluorouracil (5-FU).

10. Use according to any one of claims 1 to 9, characterized in that the medicament further comprises a pharmaceutically acceptable carrier, and the medicament is in the form of an oral preparation, a parenteral administration, or a transdermal absorption formulation, preferably in the form of tablets, capsules, solutions, suspensions, syrups, intramuscular injections, intravenous injections, transdermal patches, suppositories.

11. Use of anastrozole, a salt, metabolite or analogue thereof in the preparation of a reagent for increasing in vitro the expression of a Dicer protein in a cell of colitis or colon cancer, said colon cancer being colitis-related colon cancer.