CN107429249B

CN107429249B - Female reproductive disease target CMKLR1 and antagonist and related application thereof

Info

Publication number: CN107429249B
Application number: CN201580001232.1A
Authority: CN
Inventors: 张键; 任培根; 黄晨; 王苗苗
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2015-12-29
Filing date: 2015-12-29
Publication date: 2021-04-09
Anticipated expiration: 2035-12-29
Also published as: WO2017113101A1; CN107429249A

Abstract

Provides a novel target CMKLR1 of female reproductive diseases and application of an antagonist aiming at CMKLR1 in preparing a medicament for preventing and/or treating the female reproductive diseases. Also provided is a pharmaceutical composition for the prevention and/or treatment of female reproductive diseases comprising an antagonist against CMKLR 1. Also provided is a method of preventing and/or treating female reproductive disorders using an antagonist to CMKLR 1.

Description

Female reproductive disease target CMKLR1 and antagonist and related application thereof

Technical Field

The invention relates to a novel target related to female reproductive diseases, in particular to a female reproductive disease target CMKLR1, an antagonist thereof and related application.

Background

The national statistical agency issues the data of the main data bulletin of the sixth national census, which shows that the total population of the nation is 1,339,724,852. Compared with the fifth national census in 2000, 7390 thousands of people are increased in a decade, the increase is 5.84%, the average annual increase is 0.57%, and the average annual increase rate is reduced by 0.5% from 1.07% in 1990 to 2000. The data indicate that the population growth in our country is in the stage of low fertility level. 10/2015, Communist Party of China eighteenth central committee fifth conference bulletin states: promoting the balanced development of population, adhering to the basic national policy of family planning, improving the development strategy of population, comprehensively implementing the policy that a couple of couples can breed two children, and actively developing actions for coping with the aging of population.

In recent years, however, fast-paced work and lifestyle, high consumer pressure, have all challenged women married to age. Therefore, the health of women, especially the reproductive health of women, is a social issue that is not overlooked.

(1) Polycystic ovary syndrome (PCOS)

Polycystic ovarian syndrome is a common endocrine disorder disease of women in childbearing age, the incidence rate accounts for 6-10% of women in childbearing age, and the polycystic ovarian syndrome mainly shows that the women have infrequent menstruation or amenorrhea, infertility, polycystic ovarian change, obesity, hirsutism, hyperandrogenism and the like. Treatment of patients with PCOS includes surgical treatment, drug treatment, and assisted reproductive techniques.

Surgical treatment of PCOS can reduce a proportion of granulosa cells in the ovary, and the ovarian stroma produces reduced androgen, thereby reducing circulating androgen levels, and in turn reducing GnRH, causing further reductions in serum androgen concentrations, which also indicates that the ovarian stroma is also regulated by the pituitary-ovarian axis. Surgical treatment of PCOS mainly includes bilateral ovariowedge resection (BOWR), Laparoscopic ovariocautery or laser perforation therapy (LOD), and transvaginal hydro-laparoscopy (THL).

Drug therapy of PCOS has now replaced surgical therapy as a first-line treatment, the purpose of which is mainly related to the fertility requirements of the patient, such as drug therapy with hypoandrogenism, ovulation-promoting drug therapy, or insulin-sensitizing drugs (ISD) therapy.

In addition, assisted fertility techniques for PCOS patients, especially for PCOS patients who have ovulation after treatment using a standard ovulation-promoting cycle of more than 6 months but are not yet pregnant, or for many drugs for ovulation-promoting treatment and for patients who have no ovulation and are In need of pregnancy, may be selected from embryo transfer assisted fertility techniques, including In Vitro Fertilization (IVF) and In vitro oocyte maturation (IVM).

However, the above treatments have advantages and disadvantages, and the key is that the pathogenesis of PCOS is still unclear so far, so the currently adopted treatment is only a temporary solution.

A large number of studies have shown that 40-60% of PCOS patients are obese and develop Insulin Resistance (IR) and secondary elevated blood insulin. It is known that adipose tissue in human body is one of the important target organs of insulin action, so some pathological and physiological changes of adipose tissue will also affect the generation and development of PCOS IR.

(2) Ovarian Cancer (Ovarian Cancer, OAC)

Some of the cancers of female reproduction are related to endocrine. Such as Breast Cancer (Breast Cancer), Endometrial Cancer (Endometrial Cancer), and Ovarian Cancer (OAC). Wherein, the ovarian cancer is a malignant tumor which occurs in the ovary, 90 to 95 percent of the ovarian cancer is primary cancer, and the other 5 to 10 percent of the ovarian cancer is primary cancer metastasis of other parts to the ovary. Although the incidence of ovarian cancer in our country is not as high as that in the European and American countries, eradication-based surgical treatment, as well as cytotoxic chemotherapy, lack the effectiveness of effectively reducing the mortality of ovarian cancer. Because ovarian cancer lacks symptoms in the early stage, even if the ovarian cancer has symptoms, the ovarian cancer is not specific, and the screening effect is limited, the early diagnosis is difficult, 60 to 70 percent of cases are in the late stage, and the late stage cases have poor curative effect. Therefore, although the incidence rate of ovarian cancer is second to the cervical cancer and the endometrial cancer and is the third place of gynecological malignant tumors, the mortality rate exceeds the sum of the cervical cancer and the endometrial cancer, and the gynecological cancer is the first place of gynecological cancer, which is the biggest disease seriously threatening the health of women.

In previous clinical studies, ovarian cancer was considered to have no obvious symptomatic manifestation in early stages. However, some studies show that ovarian cancer may show clinical symptoms in early stage, such as abnormal abdominal distension, fullness, abdominal pain or back pain, lassitude, etc., but these symptoms are often ignored by patients, and metastasis often occurs when diagnosis is carried out, so that ovarian cancer is often described as "silent killer", and the prognosis of ovarian cancer is poor.

Numerous studies have shown that ovarian cancer is often transplanted to distant organs by direct spread and disseminated to the pelvic and abdominal cavities. The main pathway of ovarian cancer metastasis is intraperitoneal vegetative metastasis, which is clinically observed to be the omentum majus, with omentum metastasis being associated in 80% of women with ovarian cancer. At this time, cancer cells on the greater omentum grow at a much faster rate than at the primary lesion. The greater omentum is the peritoneum connecting the greater curvature of the stomach to the transverse colon, contains phagocytes, has an important defense function, is adipose tissue containing a large number of lipid droplets, is an important endocrine organ in the body, and participates in homeostasis. Despite the apparent fact that ovarian cancer metastasizes to the omentum majus, the mechanism has remained unclear. Because the greater omentum part is mainly adipose tissue which is the largest endocrine organ in the body and can secrete fat factors, cytokines and the like, the relationship between adipose cells in the adipose tissue and ovarian cancer cells and the mechanism thereof still need more evidence for elucidation.

With the increasing depth of research into tumors, there is increasing evidence that obesity increases the risk of mortality in cancer patients. For example, Leptin (Leptin) secreted by white adipose tissue can promote the development of breast cancer: in one aspect, leptin is able to promote breast cancer growth by activating the JAK/STAT3, MAPK-ERK 1/2, or PI3K pathway; in addition, leptin can promote the generation of tumor-related blood vessels by inducing the expression of angiogenin, and leptin can also induce the transcription of human epidermal growth factor receptor 2(ErbB-2), participate in the response of insulin-like growth receptor 1(IGF-1) in triple-negative breast cancer cells, activate Epidermal Growth Factor Receptor (EGFR) and promote the invasion and metastasis of the cells. The current research also shows that leptin can promote the process of various cancers such as prostate cancer, thyroid cancer and the like, the expression level of the leptin is positively correlated with tumorigenesis, but the level of the leptin in pancreatic cancer is lower, and the relationship between the leptin and the tumorigenesis is not clear. It is also found that adiponectin has an inhibitory effect on the occurrence and development of tumors.

Obesity has also been reported to increase the risk of ovarian cancer in women. Adipose tissue affected ovarian cancer in obese women, and the investigator studied 216 women, 35 obese women and 108 women of normal weight, and found that obese women had lower survival and shorter survival times in patients with ovarian cancer than women of normal weight. Scientists found that in addition to their differences in cancer lethality and cancer recurrence rates, their tumor cells also behave differently, suggesting that hormones or proteins secreted by adipose tissue may cause ovarian cancer cells to proliferate rapidly. It has also been shown that IL-6 and IL-8 secreted by the adipocytes of the omentum majus in the peritoneal cavity can promote the metastasis of ovarian cancer cells thereto.

A review of the relationship of adipose tissue, and particularly the fat of the greater omentum, to ovarian cancer is provided by professor Ernst Lengyel in Nature Medicine, journal of 10.30.2011. chemerin is a recently discovered adipocyte factor, also called chemotactic factor, plays a role in immune response, inflammatory reaction, adipocyte differentiation and maturation, lipid metabolism and the like, and is related to obesity and metabolic syndrome. The chemerin Gene, also known as Tazarotene-Induced Gene 2(Tazarotene Induced Gene2, TIG2), was first cloned in 1997 and subsequently, in 2003, Wittamer et al isolated its active protein by reverse phase high pressure liquid chromatography in ascites secondary to ovarian cancer.

In 2012, the study by Reverchon et al demonstrated that chemerin and its receptor CMKLR1 (chemokine-like receptor-1) is expressed in major human ovarian granule cells (hGCs) and human ovarian granule-like tumor cells (KGN), again mentioning the relationship of chemerin to ovarian tumors.

In 2011, 10 months, the research of natural media, which is published by the Ernst longyel doctor, finds that FABP4 plays a crucial role in the process of transferring ovarian cancer to the omentum majus, and the chemotactic factors of the omentum majus participate in the process of transferring the ovarian cancer to the omentum majus fat, and detects the chemotactic factors adiponectin, cytokines IL-6, IL-8 and the like in the omentum majus fat cells. However, there is no mention of detection of chemerin, which is also a chemokine.

The prior literatures and the prior arts report that no good specific targets for the diseases of polycystic ovarian syndrome and ovarian cancer are found.

Disclosure of Invention

The invention mainly aims to search a new target of female reproductive diseases so as to better prevent and/or treat the female reproductive diseases, relieve polycystic ovarian syndrome and inhibit the proliferation of ovarian cancer.

The invention identifies the chemerin/CMKLR1 system as a novel target for regulating polycystic ovarian syndrome and inhibiting ovarian cancer.

CMKLR1(chemokine receptor-1), one of the receptors for chemokine/adipokine chemerin, belongs to the family of G protein-coupled receptors.

The inventor finds in research that the interference of a gene expression of CMKLR1 by using a gene knockout technology or shRNA mode or the interference of the action of a CMKLR1 receptor by using a specific antagonist can effectively relieve the symptoms of experimental mouse polycystic ovary syndrome and can also effectively inhibit the in vitro and in vivo proliferation of human ovarian cancer cells.

In a specific experiment of the invention, a human epithelial ovarian cancer SKOV3 permanent cell line (Tomato-shCMKLR1SKOV3) which has fluorescein (Luciferase) and Tomoto fluorescent labels and is subjected to interference knocking weak CMKLR1 gene by utilizing shRNA is established by utilizing a shRNA sequence specifically interfering CMKLR1 gene expression, and in-vitro cell colony proliferation experiments show that compared with a control group, the proliferation of SKOV3 cells knocking weak CMKLR1 gene is obviously inhibited. Furthermore, a tumor-bearing model of a nude mouse is established by subcutaneously injecting Tomato-shCMKLR1SKOV3 cells, the body weight and the tumor growth condition are detected every week, and the Tomato-shCMKLR1SKOV3 cells grow obviously and slowly in the animal body compared with a control group. In addition, by utilizing the effect of CMKLR1 specific small molecule antagonist on antagonizing CMKLR1, the growth of tumor can be obviously inhibited in a nude mouse tumor-bearing model by injecting the CMKLR1 specific small molecule antagonist into the abdominal cavity compared with a control group.

In another specific experiment of the invention, symptoms of experimental mouse polycystic ovary syndrome caused by Dihydrotestosterone (DHT) or Dehydroepiandrosterone (DHEA) can be effectively alleviated by interfering with the gene expression of CMKLR1 or antagonizing the action of CMKLR 1.

Thus, in one aspect, the invention provides the use of an antagonist to CMKLR1 in the manufacture of a medicament for the prevention and/or treatment of female reproductive disorders.

In another aspect, the invention also provides a pharmaceutical composition for the prevention and/or treatment of female reproductive diseases, comprising an antagonist against CMKLR 1.

In another aspect, the present invention also provides a method of preventing and/or treating female reproductive disorders, the method comprising: uses CMKLR1 as a target to reduce the expression level of CMKLR1 and/or antagonize the action of CMKLR1 so as to prevent and/or treat female reproductive diseases. Specifically, it may be possible to reduce the expression level of CMKLR1 (knock-out of the CMKLR1 gene or knock-out of the expression level of the CMKLR1 gene) and/or antagonize the effect of CMKLR1 using an antagonist against CMKLR 1.

According to a particular embodiment of the invention, the antagonist against CMKLR1 is any agent that reduces the expression level of CMKLR1 and/or antagonizes the effect of CMKLR 1. Agents that function in this manner may include, for example, siRNA, shRNA, antisense RNA, antibodies, or combinations thereof. Such agents are available to those skilled in the art based on the prior art, and may be any of the antagonists known in the art that reduce the level of expression of CMKLR1 and/or antagonize the effect of CMKLR1 per se, or agents that have been modified, engineered based on this formula, and still function to reduce the level of expression of CMKLR1 and/or antagonize the effect of CMKLR 1. In a preferred embodiment of the invention, the antagonist against CMKLR1 is a small molecule antagonist, such as shRNA or α -NETA. More preferably, the shRNA has a sequence shown as SEQ ID No. 1.

According to a specific embodiment of the present invention, the prevention and/or treatment of female reproductive diseases comprises: relieving symptoms of polycystic ovarian syndrome, and/or inhibiting proliferation of ovarian cancer cells.

According to a specific embodiment of the present invention, the pharmaceutical composition for preventing and/or treating female reproductive diseases of the present invention may further comprise a pharmaceutically acceptable carrier or excipient, as necessary, in addition to the antagonist against CMKLR 1.

In summary, the invention proves that CMKLR1 is a new target for preventing and treating polycystic ovarian syndrome and ovarian cancer, and any technology is utilized to interfere the expression of CMKLR1 gene or interfere the action of CMKLR1, so that the symptoms of polycystic ovarian syndrome can be effectively relieved, and the proliferation of ovarian cancer is inhibited.

Drawings

FIG. 1: the knocking-out of CMKLR1 gene can inhibit the expression of the translocation related gene in ovarian cancer cell line SKOV3.

FIG. 2: the knocking-out of CMKLR1 gene can inhibit the migration and proliferation of ovarian cancer cell line SKOV3.

FIG. 3: the knocking-out of CMKLR1 gene can inhibit the proliferation of human ovarian cancer cells under the skin of a nude mouse.

FIG. 4: CMKLR1 specific antagonist can inhibit the proliferation of human ovarian cancer cells in nude mouse skin.

Fig. 5A and 5B: effect of deletion of CMKLR1 gene on oestrus cycle changes induced by dihydrotestosterone. Wherein, WT, wild type: wild type mice. Cmklr 1-/-: CMKLR1 gene deletion mice. Control (Ctl): control placebo group. DHT, dihydrotestosterone: experimental groups implanted with dihydrotestosterone. P: proestrous: in the early estrus stage. D: biestrous: estrus interval. E: estrous: in estrus. M: metestrous: in the anaphase of estrus. And (6) days: days. Estrous Cycles: the estrus cycle.

FIG. 6: deletion of the CMKLR1 gene, effects on serum hormone levels in mice induced by dihydrotestosterone.

FIG. 7: deletion of the CMKLR1 gene, effect on mouse ovarian structure induced by dihydrotestosterone.

Fig. 8A and 8B: the deletion of CMKLR1 gene has the effect on mouse follicle cell apoptosis caused by dihydrotestosterone.

FIG. 9: the effect of CMKLR1 gene deletion on hormone synthesis related enzyme gene expression in ovarian tissues caused by dihydrotestosterone.

FIG. 10: the CMKLR1 specific antagonist, alpha-NETA, influences the mouse ovary structure, hormone synthetase and other gene expression caused by dihydrotestosterone.

FIG. 11: effect of CMKLR 1-specific antagonist, α -NETA, on mouse ovarian architecture induced by DHEA.

Fig. 12A and 12B: effect of CMKLR 1-specific antagonist, α -NETA, on DHEA-induced apoptosis of mouse follicular cells.

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Example 1: interfering the gene expression of CMKLR1 or antagonizing the action of CMKLR1 can effectively inhibit the proliferation of human ovarian cancer cells.

This example demonstrates that interfering with the gene expression of CMKLR1, or antagonizing the effects of CMKLR1, can effectively inhibit the proliferation of human ovarian carcinoma cells. Wherein, a shRNA sequence which specifically interferes with CMKLR1 gene expression is designed, and a human epithelial ovarian cancer SKOV3 permanent cell line (Tomato-shCMKLR1SKOV3) which has fluorescein (Luciferase) and Tomoto fluorescent labels and utilizes shRNA interference to knock down CMKLR1 gene is established.

In vitro cell population proliferation experiments show that the proliferation of SKOV3 cells with knocked-down CMKLR1 gene is obviously inhibited compared with a control group. And (3) subcutaneously injecting Tomato-shCMKLR1SKOV3 cells, establishing a nude mouse tumor-bearing model, and detecting the weight and the tumor growth condition every week, wherein the Tomato-shCMKLR1SKOV3 cells grow remarkably and slowly in the animal body compared with a control group. By utilizing the effect of CMKLR1 specific small molecule antagonist on antagonizing CMKLR1, in a nude mouse tumor-bearing model, the growth of tumors can be remarkably inhibited by injecting the CMKLR1 specific small molecule antagonist into the abdominal cavity compared with a control group.

The method comprises the following specific steps:

1. establishing a human ovarian cancer cell line with fluorescein (Luciferase, Fluc) and Tomato fluorescent markers: SKOV3-Luc-Tomato/LRH and SKOV3. ipl-Luc-Tomato/LRH.

In this experiment, human ovarian carcinoma cells SKOV3 and SKOV3.ipl (purchased from Bod. Wuhan) were selected to prepare a lentiviral vector containing the Tomato/Luciferase gene (the vector was prepared by itself according to the references Ren P. -G. (R.Y.), Lee S-W, Biswal S, Goodman SB. systematic transfection of monoclonal induced by bone fragment peptides in nucleic acids. biomaterials, 2008.29: 4760-. The specific operation is as follows:

selecting human ovarian cancer cells SK-OV3 and SK-OV3.ipl which grow vigorously one day before transfection in a ratio of 5 × 10⁵One/well, inoculating in 6-well plate, culturing until the cell fusion degree is 60% after the second day;

secondly, cell infection is carried out on the lentivirus containing the Tomato/Luciferase gene for 6 hours in daily use;

③ after 6 hours, the cells to be transfected are rinsed once with PBS, 2mL of complete culture medium is added into the holes, and the cells are cultured in a carbon dioxide incubator;

fourthly, after 48 hours, selecting a culture medium containing 1 mu G/mL G418 for screening; obtaining the ovarian cancer cell line stably expressing the Tomato/Luciferase gene after the cells are not dead any more: SKOV3-Luc-Tomato/LRH and SKOV3.ipl-Luc-Tomato/LRH, namely can be used for in vitro experiment of tumor cells.

2. Establishing a luminous human ovarian cancer cell line with a knocked-down CMKLR1 gene: SKOV3-Luc-Tomato-shCMKLR1/LRH and SKOV3.ipl-Luc-Tomato-shCMKLR 1/LRH.

The human CMKLR1 gene shRNA used in this experiment was pSM2c-Hu-shCMKLR1 (available from Open Biosystems), the sequence of Hairpin: 5'-TGCTGTTGACAGTGAGCGAGGTGATGAATACCCTGATTATTAGTGAAGCCACAGATGTAATAATCAGGGTATTCATCACCGTGCCTACTGCCTCGGA-3' (SEQ ID No. 1). The control group was pSM2c-Hu-scramble shRNA.

Selecting a luminous human ovarian cancer cell which grows vigorously: SKOV3-Luc-Tomato/LRH and SKOV3.ipl-Luc-Tomato/LRH, at 5X 10 on the day before transfection⁵One/well, inoculating in 6-well plate, culturing until the cell fusion degree is 60% after the second day;

② the second day, with 6 hole plate culture hole as a unit, using 200 u L of opti-MEM medium dilution 3 u g plasmid, another 200 u L opti-MEM medium dilution 6 u L liposome Lipofectamine 2000, after gently mixing, placed at room temperature for 5 minutes;

③ mixing the two tube dilutions gently, standing for 20 minutes at room temperature, and then adding 600 μ L of opti-MEM culture medium gently into the mixed dilutions;

rinsing the cells to be transfected with PBS slightly once, then adding the mixed diluent into the culture holes slightly, and culturing in a carbon dioxide incubator;

fifthly, after culturing for 4-6 hours, abandoning the culture medium used for transfection, and adding 3mL of complete culture medium into the hole;

sixthly, selecting a culture medium containing 1 mu g/mL puromycin (puromycin) for screening after 48 hours; and obtaining the luminous ovarian cancer cell line which stably expresses the CMKLR1shRNA after the cells are not dead any more. Then screening by using a flow cytometer to sort out cells with high expression of red fluorescence,

seventhly, extracting total RNA by using TRIzol, quantifying 2 mug of RNA for reverse transcription (a reverse transcription kit, purchased from Promega corporation), and performing qPCR by using a specific primer sequence.

The sequence of the specific primer is Hu-CMKLR1 primer sequence:

Fw 5’-GAGGCGTGACATAGAATGGA-3’(SEQ ID No.2)

Rv 5’-TGATATGGATTGGGAGGAAGAC-3’(SEQ ID No.3)

(viii) comparing with the transfected pSM2c-Hu-scramble shRNA, the CMKLR1 gene has an expression level of only 40% +/-2.87%, and the gene is used as a luminous human ovarian cancer cell line with weakened CMKLR1 gene and is named as: SKOV3-Luc-Tomato-shCMKLR 1/LRH; and SKOV3.ipl-Luc-Tomato-shCMKLR 1/LRH; can be used for in vitro and in vivo experiments of tumor cells.

3. In vitro proliferation, migration and invasion experiments of a luminescent human ovarian cancer cell line with a knockdown of the CMKLR1 gene.

(1) MTT proliferation assay:

inoculating the ovarian cancer cells with the shRNA and the idle plasmid into 96-hole cell culture plates at 2000 cells per hole, wherein the volume of a culture medium in each hole is 200 mu L, and simultaneously placing different culture plates into a carbon dioxide incubator to culture for 12 hours, 24 hours, 48 hours and 72 hours respectively;

secondly, adding MTT solution (5mg/mL) into the culture plate at different detection time points, wherein each hole has 20 mu L, and continuously putting the MTT solution into a carbon dioxide incubator for culture for 4 hours;

and thirdly, abandoning the supernatant in the culture plate, adding 150 mu L DMSO (dimethyl sulfoxide), shaking for 10 minutes, selecting 490nm wavelength on a microplate reader for detection, and drawing a growth curve of the cells.

(2) Soft Agar colony formation experiment for Soft Agar:

firstly, 1.2 percent and 0.7 percent of agarose are prepared, sterilized under high pressure and then placed in a water bath at 55 ℃ to keep the agarose in a molten state;

preparing a 2 XRPMI 1640 culture medium containing 20% FBS and 2 Xantibiotics, and preheating at 37 ℃;

filling lower layer glue: mixing 1.2% agarose gel with 2 × culture medium at a ratio of 1: 1, adding into 6-well plate (3 ml per well), standing at room temperature for solidification;

waiting for digesting SKOV3-Luc-Tomato-shCMKLR1/LRH, or SKOV3.ipl-Luc-Tomato-shCMKLR1/LRH, or pSM2c-Hu-scramble shRNA stably transformed cells in the lower layer gel fixation process, counting, adjusting the concentration to 5 multiplied by 104/ml by using a serum-free culture medium, using 100 mu l cells for each hole, and arranging 3 parallel holes;

fifthly, pouring the upper glue after the lower glue is solidified: mixing 0.7% agarose gel with 2 × culture medium at 1: 1 (about 40 deg.C), adding 100 μ l cell suspension (5000 cells), mixing, and adding into a well plate with 3ml per well;

sixthly, culturing at 37 ℃ with 5% CO2, observing the clone size by fluorescence after about 2-3 weeks, counting and comparing the formation of two groups of clones.

(3) Transwell invasion experiments:

the effect of interfering with CMKLR1 gene expression on migration and invasion of ovarian cancer cells was examined using Transwell cells as well as those precoated with Metrigel. The specific operation is as follows:

starvation culturing ovarian cancer cells with shRNA and no-load plasmid for 24 hours, digesting, centrifuging, discarding supernatant, resuspending with serum-free medium, counting, and adjusting cell density to 5 × 10⁵Per mL;

600. mu.L of a medium containing 10% FBS was placed in the lower chamber of a Transwell chamber (Pore size 8.0 μm, available from Corning) and a BD Biocoat chamber (Metrigel Invasion chamber, Pore size 8.0 μm, available from BD), and 200. mu.L of the prepared cell suspension was placed in the upper chamber and then cultured in a carbon dioxide incubator for 6 hours;

③ using PBS to rinse the Transwell chamber lightly, using cotton swab to wipe off the cells in the upper chamber, using methanol to fix for 30 minutes, then drying the chamber fully, using DAPI solution containing 1 microgram/mL to dye for 2 minutes in the dark, using distilled water to rinse lightly, then observing under a fluorescence microscope, randomly selecting 5 visual fields to shoot, counting the cell nucleus and counting the number of the migrated cells.

FIG. 1 shows that knocking down CMKLR1 gene can suppress the expression of the transgene in ovarian cancer cell line SKOV3.

In an ovarian cancer cell line SKOV3, an RNA (shCMKLRA) technology interfering CMKLR1 gene is transfected by lentivirus, the expression of endogenous CMKLR1 gene in weak cells (about 50% of the expression of CMKLR1 gene is weakened), and then the influence on the expression levels of tumor metastasis gene mucin family-16 (MUC16) and matrix metalloproteinase-2 (MMP2) in SKOV3 after the CMKLR1 gene is weakened is detected, and the result shows that the expression levels of the MUC16 gene and the MMP2 gene are obviously reduced. P < 0.05, P < 0.01, P < 0.001.

FIG. 2 shows that knocking down the CMKLR1 gene inhibited migration and proliferation of the ovarian cancer cell line SKOV3.

The effect on migration (migration) and invasion (invasion) of ovarian cancer cell line SKOV3 after knocking down CMKLR1 was examined. shCMKLR1 is a cell line transfected with shCMKLR1 interfering RNA; SKOV3.psm2c is SKOV3 cell line transfected with blank vector only.

In the migration experiment (panel A in FIG. 2), as a control cell line, SKOV3.pSM2c, the number of migrated cells increased gradually with the increase in the concentration of chemerin after the chemerin treatment at various concentrations (from 0nM to 0.5nM), reached the maximum number of migrated cells at the concentration of 0.02nM, and started to decrease at the concentration of 0.5nM for chemerin.

SKOV3 cells showed little migration after treatment with different concentrations of chemerin when expression of CMKLR1 was knocked down.

In the invasion experiment (panel B in fig. 2), chemerin treatment increased the number of control SKOV3 cell lines (SKOV3.psm2c) invaded, but after attenuation of CMKLR1, the number of invaded cells was significantly reduced.

Meanwhile, a research group establishes Tomato fluorescence labeled cell lines of SKOV3-pSM2c and SKOV3-shCMKLR1 cell lines. The proliferation of cancer cells was detected by soft agar cloning experiments (panel C in FIG. 2), and the results showed that SKOV3-shCMKLR1 cell line with attenuated CMKLR1 gene resulted in significantly lower numbers and sizes of clones than the control cell line without attenuated CMKLR1 gene (SKOV3-pSM 2C).

4. In vivo proliferation, migration and invasion experiments of a luminescent human ovarian cancer cell line with a knockdown of the CMKLR1 gene.

In this experiment, a nude mouse subcutaneous tumor-bearing experiment was performed to verify the effects of in vivo proliferation, migration and invasion of the luminescable human ovarian cancer cell line in which the CMKLR1 gene was knocked down. The specific operation is as follows:

selecting 6-8 weeks old nude mice at random, preparing luminous human ovarian cancer cells with attenuated CMKLR1 gene into cell suspension, and preparing 1 × 10^-70.2 ml of cell suspension (about 2X 10 cells) per ml concentration^-6One) of the cells were injected into the cell suspension to inoculate the dorsal skin of nude mice, and pSM2 c-Hu-scarmble shRNA stably transformed cells with the same cell number were inoculated to serve as a control group;

secondly, the living animal imager is used for dynamically tracking and observing the growth condition of the two groups of tumor cell models and the transfer condition of the tumors in vivo.

FIG. 3 shows that knocking down the CMKLR1 gene can inhibit the proliferation of human ovarian cancer cells under the skin of nude mice.

SKOV3 cells are injected into a nude mouse subcutaneous tissue, a tumor-bearing tumor model in SKOV ovarian cancer cell bodies is established, and the condition that a cell line with a weakened CMKLR1 gene forms tumors in a mouse body is detected. Shmmklr r1 transfected light emitting cell line with shmaio-skov 3.shCMKLR 1; Tomato-SKOV3.sramble is SKOV3 light-emitting cell line transfected with non-specific interfering RNA (sramble RNA) alone, as a control.

SKOV3 ipl-sramble RNA-Tomato cells and SKOV3 ipl-shCMKLR1-Tomato cells were injected subcutaneously into mice, and the intensity of Fluc bioluminescence was measured on day 7 (D7), day 14 (D14), day 28 (D28) and day 35 (D35), to infer the extent of tumor growth under the transplanted skin. The stronger the luminescence signal, the larger the area of the luminescence signal, indicating the formation of a larger tumor. With time, SKOV3 ipl-sramble RNA-Tomato cells grew significantly in mice, while shCMKLR1 cells grew little.

FIG. 4 shows that CMKLR 1-specific antagonists inhibit the proliferation of human ovarian cancer cells subcutaneously in nude mice.

The Tomato-labeled SKOV3-Fluc-Tomato cell line was injected subcutaneously into mice, while the mice were injected intraperitoneally with different doses of the drug a-NETA (a-NETA is a CMKLR1 specific Small Molecule Antagonist, which was self-prepared as Graham KL, Zhang JV (Zhang-Key), Lew é n S, Burke TM, Dang T, Zoudilova M, Sobel RA, Butcher EC, Zabel BA.A Novel CMKLR1 Small Mobile antibody absorption monitor PLANE, NET Dec 1; 9(12) eye mouse Influmorm analysis plan. PLOS ONE; NET Dec 1; 9 (ou) 01125. eye mouse antibody degradation; 10.1371/112jnnal. 01125. ecol), 0.1mg per kg per day per body weight per day (a-0.1 mg/kg/day), 0.1mg/kg per day per injection A-1 mg/kg per day per mouse. The intensity of Fluc bioluminescence was measured at day 7 (D7), day 14 (D14), day 28 (D28) and day 35 (D35) after cell injection, and the extent of growth of the transplanted subcutaneous tumor was inferred. The stronger the luminescence signal, the larger the area of the luminescence signal, indicating the formation of a larger tumor.

There was no significant change in fluorescence intensity in the three treatment groups (controls, NETA-0.1mg/kg/D and 1mg/kg/D) at D7, and there was a significant increase in the control tumor tissue and a gradual increase in fluorescence intensity at D14, D28 and D35. Whereas in the α -NETA-treated group, the α -NETA treatment slowed the growth of SKOV cell tumors compared to the control group, and the fluorescence intensity was significantly reduced, particularly in D28 and D35.

Example 2 interfering with the gene expression of CMKLR1, or antagonizing the effects of CMKLR1, may be effective in alleviating the symptoms of experimental mouse polycystic ovary syndrome caused by DHT or DHEA.

In the example, it is verified that the interference of the gene expression of CMKLR1 or the antagonism of CMKLR1 can effectively relieve the symptoms of experimental mouse polycystic ovary syndrome caused by DHT or DHEA. Wherein, an experimental mouse polycystic ovary syndrome model caused by high androgen DHT or DHEA is established. However, compared with wild type mice, the CMKLR1 gene knock-out mice have obviously relieved the symptoms of polycystic ovary syndrome, including: the physiological cycle is maintained, a certain level of progesterone is contained in serum, and the corpus luteum is still generated on the ovary, so that the apoptosis of ovarian stroma and follicular membranous cells caused by DHT is reduced. In an experimental mouse polycystic ovarian syndrome model caused by DHT or DHEA, the symptom of the polycystic ovarian syndrome can be relieved by intraperitoneal administration of a CMKLR1 specific small molecule antagonist alpha-NETA, and the experimental mouse polycystic ovarian syndrome model comprises the following components: some level of progesterone is maintained in the serum and the corpus luteum is still produced in the ovary.

The method comprises the following specific steps:

(1) DHT experimental mouse polycystic ovary syndrome model

CMKLR1 knockout mice on a C57BL/6J background were purchased from Deltagen, Inc. and supplied by the university of Stanford, doctor Zable laboratory. C57BL/6J wild-type female mice were obtained from the center of medical laboratory animals in Guangdong province.

Animals were kept in a light-dark cycle rearing room at constant temperature and humidity for 12 hours. The feed and water can be taken freely. Procedures for all animals were performed following procedures approved by the animal welfare ethics committee.

Wild-type mice: female mice were randomized on postnatal day 19 (D19) into: placebo, DHT, CMKLR1 antagonist, DHT + CMKLR1 antagonist, at least 10 mice per group).

CMKLR1 gene-deleted mice: female mice were randomized on postnatal day 19 (D19) into: placebo, DHT, groups of at least 10 mice per group.

DHT group: DHT sustained release particles were implanted subcutaneously (american, sarasotan, innovative research institute, florida). These granules contained 7.5 mg of DHT for 90 consecutive days at a daily release dose of 83.3 microgram).

Placebo group: mice were implanted subcutaneously with placebo granules for 90 days.

CMKLR1 antagonist group: a1 mg CMKLR1 antagonist alpha-NETA slow release pump was implanted subcutaneously.

DHT + CMKLR1 antagonist group: the DHT sustained release particles were implanted subcutaneously and the granules contained 7.5 mg of DHT for 90 consecutive days at a daily release dose of 83.3 micrograms. A1 mg CMKLR1 antagonist alpha-NETA slow release pump was implanted subcutaneously.

Body weights were weighed at the beginning and end of treatment, and mice were sacrificed at the end of the 90 day treatment period. In addition, at the end of the 90-D treatment period, blood samples and tissues were collected. Mice were anesthetized with isoflurane and blood samples were collected by retroorbital puncture. Ovary and uterus, gonadal fat were isolated, weighed, and fixed overnight in 4% paraformaldehyde fluid. In addition, ex vivo tissues were snap frozen in liquid nitrogen until further processing.

Fig. 5A and 5B show the effect of deletion of the CMKLR1 gene on the oestrus cycle changes induced by dihydrotestosterone.

As shown in FIG. 5A, wild type mice (WT-control) using placebo typically have 2-3 estrus cycles (each cycle from pre-estrus (P), estrus (E), post-estrus (M), to inter-estrus (D)) within 12 days; whereas wild type mice treated with Dihydrotestosterone (DHT) for 90 days (WT-DHT) had physiological cycles in the estrus (D) phase with no complete estrus cycle. Dihydrotestosterone severely affected the normal physiological cycle of the mice.

In FIG. 5B, a is a comparison of quantification between the control and treated groups of wild-type mice, statistically significant (aP < 0.01).

CMKLR1 gene-deleted mice (CMKLR1-/-control) using placebo also had 2-3 oestrus cycles within 12 days. In FIG. 1B, B is a quantitative comparison between two groups of CMKLR1 gene deletion groups and has a statistically significant meaning (bP < 0.05), D is a comparison between two groups of wild-type mice treated with dihydrotestosterone and CMKLR1 gene deletion mice, and CMKL 1 gene deletion obviously improves the influence of dihydrotestosterone on the physiological cycle of the mice and has a statistically significant meaning (dP < 0.001).

WT, wild type: wild type mice. Cmklr 1-/-: CMKLR1 gene deletion mice. Control (Ctl): control placebo group. DHT, dihydrotestosterone: experimental groups implanted with dihydrotestosterone. P: proestrous: in the early estrus stage. D: biestrous: estrus interval. E: estrous: in estrus. M: metestrous: in the anaphase of estrus. And (6) days: days. Estrous Cycles: the estrus cycle.

FIG. 6 shows the effect of the deletion of the CMKLR1 gene on the level of hormones in the serum of mice induced by dihydrotestosterone.

Comparing the content of DHT (DHT) in blood serum of four groups of mouse treatment groups (WT-Control, WT-DHT, CMKLR1-/-Control and CMKLR 1-/-DHT), wherein the content of DHT in the WT-Control group and the CMKLR1-/-Control group is about 100pg/mL, and the content of DHT in the blood serum of the WT-DHT treatment group and the CMKLR1-/-DHT treatment group is respectively increased to about 200pg/mL, thus proving that the molding of the DHT treatment group is successful.

The levels of estrogen (estradiol) and progesterone (progestin) in the serum were then measured separately: estrogen was maintained at around 5pg/mL in WT-Control and CMKLR1-/-Control groups, whereas DHT treatment reduced estrogen levels in WT mice; in CMKLR 1-/-mice, DHT treatment did not significantly affect the level of estrogen in the serum. Also, DHT treatment reduced progesterone levels in the serum of WT mice, whereas DHT did not affect the expression of normal levels of progesterone in the serum of CMKLR 1-/-mice. In FIG. 6, a is a quantitative comparison between the control group of wild type mice and the two groups of DHT-treated groups; b is a quantitative comparison between the CMKLR1 gene deletion control group and the DHT treated group; d is a comparison between two groups of DHT dihydrotestosterone treated wild-type mice and CMKLR1 gene-deleted mice. P < 0.05 represents statistically significant differences.

FIG. 7 shows the effect of deletion of the CMKLR1 gene on the ovarian structure of mice induced by dihydrotestosterone.

Comparing the morphology of ovaries of four groups of mice treated (Wild type-placebo, Wild type-DHT, CMKLR 1-/-placebo, CMKLR 1-/-DHT), the ovaries of WT-Control and CMKLR1-/-Control mice had follicles and corpus luteum at various developmental stages. Most of the follicles of DHT-treated WT mice (WT-DHT) had atrophic and the ovary had no corpus luteum. While the development of follicles was more normal in DHT-treated CMKLR1-/- - (CMKLR1-/- -DHT) mice, and an intact corpus luteum was also observed.

Fig. 8A and 8B show the effect of deletion of the CMKLR1 gene on dihydrotestosterone induced apoptosis of mouse follicle cells.

The apoptotic signal (Tunnel staining-green) and the blue signal are nuclear-staining DAPI signals in the ovaries of the four groups of mouse treated groups (WT-Control, WT-DHT, CMKLR1-/-Control, CMKLR 1-/-DHT). No significant apoptotic staining (green) was found in all of the ovarian follicular granulosa cells (granulosa cells), theca cells (theca cells), and interstitial follicular cells (interstitial cells) of the control group of wild mice (WT) and CMKLR1 gene-deleted mice (CMKLR1-/-), whereas in all of the ovaries of the Dihydrotestosterone (DHT) -treated group of wild mice (WT) and CMKLR1 gene-deleted mice (CMKLR1-/-), follicular granulosa cells (granulosa cells), theca cells (theca cells), and interstitial cells (interstitial cells) (as shown in FIG. 8A). Where the numbers of apoptotic cells in the follicular granulosa cells (granulosa cells), theca cells (theca cells), and interstitial follicular cells (interstitial cells), respectively, were calculated for the four groups of mouse treated groups in fig. 8B, statistical analysis showed that DHT treatment increased the number of apoptotic cells in the three tissues, where DHT treatment significantly increased apoptotic cells in the CMKLR 1-/-mice among the granulosa cells (granulosa). DHT treatment resulted in increased apoptosis signals in membrane (theca) and mesenchymal (mesenchymal cells) cells in wild type mice, and correspondingly DHT treatment resulted in significantly weaker apoptosis signals in CMKLR1 gene-deleted mice (CMKLR1-/-) membrane and mesenchymal cells than in wild type mice. In the figure, a is a quantitative comparison between the control group of wild type mice and the two groups of DHT-treated groups; b is a quantitative comparison between the CMKLR1 gene deletion control group and the DHT treated group; d is a comparison between two groups of DHT dihydrotestosterone treated wild-type mice and CMKLR1 gene-deleted mice. P < 0.05 represents statistically significant differences.

FIG. 9 shows the effect of CMKLR1 gene deletion on dihydrotestosterone induced gene expression of hormone synthesis-related enzymes in ovarian tissue.

In four groups of mouse treatment groups (WT-Control, WT-DHT, CMKLR1-/-Control, CMKLR 1-/-DHT), expression of mRNA levels of steroid hormone synthesis rapid regulatory protein (StAR), cholesterol side chain lyase (cholesterol-side-chain cleavage enzyme, P450scc), 3 β -hydroxysteroid dehydrogenase (3bHSD), bone morphogenetic protein 2(BMP2) and bone morphogenetic protein 4(BMP4) in the ovary was examined.

Expression of StAR, P450scc, 3bHSD did not differ significantly in the ovaries of the wild mouse (WT) and the CMKLR1 gene-deleted mouse (CMKLR1-/-) control-treated group. When Dihydrotestosterone (DHT) was treated, the expression of three genes was significantly reduced in WT mice, while in CMKLR 1-/-group, the expression of three genes was significantly increased compared to the WT group.

BMP2 and BMP4 are signaling molecules in the BMP signaling pathway. BMP2 was significantly elevated in the CMKLR 1-/-mouse ovary compared to the wild mouse ovary. While DHT treatment did not have a significant effect on the expression of polybmp 2.

Whereas BMP4 was expressed indifferently in the mouse ovaries of WT and CMKLR1-/-control groups, DHT treatment increased BMP4 expression in the wild mouse ovaries, but not significantly in the CMKLR 1-/-group.

In FIG. 9, a is a comparison of quantification between the control group of wild type mice and the two groups of DHT-treated groups; b is a quantitative comparison between the CMKLR1 gene deletion control group and the DHT treated group; d is a comparison between two groups of DHT dihydrotestosterone treated wild-type mice and CMKLR1 gene-deleted mice. P < 0.05 represents statistically significant differences.

FIG. 10 shows the effect of a CMKLR 1-specific antagonist, alpha-NETA, on dihydrotestosterone-induced gene expression of mouse ovarian structure, hormone synthase, etc.

The morphology of the ovaries of four groups of wild mice (control, NETA, DHT + NETA) was observed. Control (solvent Control group), α -NETA (α -NETA single treatment group), DHT (dihydrotestosterone single treatment group), DHT + NETA (α -NETA and dihydrotestosterone synergistic treatment group).

There was no significant difference in the amount of Corpus Luteum (CL) in the ovaries of the solvent control group and the NETA-treated group; no corpus luteum was observed in the mouse ovaries after DHT treatment, while corpus luteum still appeared in the ovaries of mice co-treated with NETA and DHT.

The contents of DHT, estrogen (Estradiol) and progesterone (progasterone) in the sera of the four groups of mice are respectively detected, wherein the content of DHT in the sera of the mice is increased by the independent treatment of DHT and the synergistic treatment of DHT and NETA (proved that DHT modeling is successful), the content of progesterone in the sera is reduced by the independent treatment of DHT, the content of DHT in the sera is not influenced by the synergistic treatment of NETA and DHT, but the level of progesterone in the sera caused by the DHT treatment is increased. The estrogen content of the serum was not affected.

The results of detecting the expression of the mRNA level of hormone key synthetase 3 beta-hydroxysteroid dehydrogenase (3bHSD) in the ovary show that DHT treatment reduces the mRNA level of 3b-HSD, while NETA alone does not affect the mRNA level of 3b-HSD, but NETA and DHT jointly increase the mRNA level of 3 b-HSD.

Increased levels of Caspase-3 (Caspase-3) may indicate increased apoptotic signals in the ovaries. The expression of an ovarian apoptosis-related enzyme Caspase3 can be remarkably increased by singly treating DHT, and after the DHT and NETA are subjected to synergistic treatment, NETA can reduce the stimulation effect of DHT on Caspase3 expression. P < 0.05, P < 0.01, P < 0.001.

(2) DHEA experimental mouse polycystic ovary syndrome model

Wild-type mice: female mice were randomized on postnatal day 19 (D19) into: control group, DHEA group, CMKLR1 antagonist group, DHEA + CMKLR1 antagonist group, at least 10 mice per group).

CMKLR1 gene-deleted mice: female mice were randomized on postnatal day 19 (D19) into: control, DHEA group, at least 10 mice per group).

Control group: sesame oil was injected subcutaneously at 6mg/100g, and physiological saline was injected intraperitoneally at 1 mg/kg.

DHEA group: DHEA was injected subcutaneously at 6mg/100g, and normal saline was injected intraperitoneally at 1 mg/kg.

CMKLR1 antagonist group: sesame oil was injected subcutaneously at 6mg/100g, and CMKLR1 antagonist α -NETA was injected intraperitoneally at 1 mg/kg.

DHEA + CMKLR1 antagonist group: DHEA was injected subcutaneously at 6mg/100g and the CMKLR1 antagonist α -NETA was injected intraperitoneally at 1 mg/kg.

Body weights were weighed at the beginning and end of treatment, and mice were sacrificed at the end of the 21 day treatment period. In addition, at the end of the 21 day treatment period, blood samples and tissues were collected. Mice were anesthetized with isoflurane and blood samples were collected by retroorbital puncture. Ovary and uterus, gonadal fat were isolated, weighed, and fixed overnight in 4% paraformaldehyde fluid. In addition, ex vivo tissues were snap frozen in liquid nitrogen until further processing.

FIG. 11 shows the effect of the CMKLR1 specific antagonist alpha-NETA on DHEA induced ovarian architecture in mice.

Comparison of follicle morphology in ovaries in four groups of mouse treatment (cnotrol, α -NETA, DHEA, DHEA + α -NETA). Control (solvent Control group), α -NETA (α -NETA single treatment group), DHEA (dehydroepiandrosterone single treatment group), DHEA + NETA (α -NETA and dehydroepiandrosterone co-treatment group). In both control and NETA treated groups, morphologically intact secondary follicles were observed, indicating that the development of solvent control and NETA treated multiple follicles was not significantly affected. DHEA treatment significantly altered the development of the follicles, and no secondary and preovulatory mature follicles were found in the ovaries. In the DHEA + NETA treatment group, the development of the secondary follicle is obviously improved, and mature follicles exist, which indicates the protective effect of NETA on the development of the follicles.

FIGS. 12A and 12B show the effect of the CMKLR 1-specific antagonist, alpha-NETA, on DHEA-induced apoptosis of mouse follicular cells.

Comparison of apoptotic signals in ovaries in four wild mouse (WT) treated groups (cnotrol, NETA, DHEA + NETA). Apoptotic signal (Tunnel staining-green), blue signal is nuclear-staining DAPI signal. Control (solvent Control group), α -NETA (α -NETA single treatment group), DHEA (dehydroepiandrosterone single treatment group), DHEA + NETA (α -NETA and dehydroepiandrosterone co-treatment group).

FIG. 12A shows that there were more apoptotic cells in Granulosa Cells (GC), membrane cells (Theca cells, TC) and Interstitial Cells (IC) of the follicle in the wild mouse dehydroepiandrosterone-treated group (WT-DHEA); after the synergistic treatment with NETA (WT-NETA-DHEA), apoptotic cells in membrane cells (TC) are obviously reduced, and apoptotic cells in Interstitial Cells (IC) are also obviously reduced. Fig. 12B comparatively calculates the number of apoptotic cells in GC, TC and IC in Ctrl, NETA, DHEA + NETA treated groups, respectively, of WT mice. P < 0.05, P < 0.01, P < 0.001.

Claims

1. Use of an antagonist to CMKLR1 in the manufacture of a medicament for the prevention and/or treatment of female reproductive disease; the female reproductive disease is polycystic ovarian syndrome and/or ovarian cancer.

2. The use of claim 1 wherein the antagonist to CMKLR1 is an agent that reduces the expression level of CMKLR1 and/or antagonizes the effect of CMKLR 1.

3. The use of claim 2, wherein the antagonist to CMKLR1 is an siRNA, shRNA, antisense RNA, antibody, or a combination thereof.

4. The use of claim 1, wherein the antagonist to CMKLR1 is a combination of α -NETA or shRNA and α -NETA.

5. The use according to claim 3 or 4, wherein the sequence of the shRNA is as shown in SEQ ID No. 1.

6. The use of claim 1, wherein the treatment of female reproductive disorders is: relieving symptoms of polycystic ovarian syndrome, and/or inhibiting proliferation of ovarian cancer cells.

7. A pharmaceutical composition for the prevention and/or treatment of female reproductive diseases comprising an antagonist against CMKLR 1; the antagonist against CMKLR1 is a shRNA.

8. The pharmaceutical composition according to claim 7, wherein the sequence of the shRNA is as shown in SEQ ID No. 1.