CN107510843B - DEM1 function and use - Google Patents

Abstract

The invention belongs to the technical field of life science, and particularly relates to functions and purposes of DEM 1. According to the invention, through extensive and intensive research, DEM1 can be used as a colorectal cancer treatment target for the first time, and the inhibition of the expression of DEM1 can inhibit the proliferation of colorectal cancer cells, induce the apoptosis of the colorectal cancer cells, inhibit the clonogenic capacity of the colorectal cancer cells, inhibit the tumorigenic capacity of the colorectal cancer cells and slow down the growth of colorectal cancer tumor bodies. Therefore, the invention provides powerful scientific evidence for the pathogenesis of the colorectal cancer and the clinical treatment of the colorectal cancer from the tissue level, the cell function level and the molecular level of a clinical patient sample.

Description

DEM1 function and use

Technical Field

The invention belongs to the technical field of life science, and particularly relates to functions and purposes of DEM 1.

Background

Colorectal cancer is a common malignancy, occurring with high fat low cellulose diets, chronic inflammation of the large intestine, large intestine adenomas, genetic factors and other factors such as: schistosomiasis, pelvic cavity radiation, environmental factors (such as lack of molybdenum in soil), smoking and the like.

Early colorectal cancer is asymptomatic or has no obvious symptoms, and only feels uncomfortable, dyspepsia, stool occult blood and the like. With the development of cancer, symptoms gradually appear, which are manifested as stool habit change, abdominal pain, hematochezia, abdominal mass, intestinal obstruction, etc., with or without anemia, fever, emaciation, etc. The tumor may cause the change of affected organs due to metastasis and infiltration. Colorectal cancer shows different clinical symptoms and signs depending on the site of onset.

(1) Right colon cancer: the main clinical symptoms of right colon are anorexia, nausea, vomiting, anemia, fatigue, abdominal pain. Right half colon cancer causes iron deficiency anemia, and shows symptoms such as fatigue, hypodynamia, shortness of breath and the like. The right colon has a wide intestinal cavity, and abdominal symptoms can not appear until the tumor grows to a certain volume, which is one of the main reasons for late stage when the tumor is diagnosed.

(2) Left hemicolon cancer: the left half colon cavity is narrower than the right half colon cavity, and the left half colon cancer is easier to cause complete or partial intestinal obstruction. Intestinal obstruction causes changes in stool habits, with symptoms of constipation, hematochezia, diarrhea, abdominal pain, abdominal cramps, abdominal distension, and the like. Stool with fresh bleeding indicated that the tumor was located in the end of the left half colon or rectum. The stage of the disease is often diagnosed earlier than right colon cancer.

(3) Rectal cancer: the main clinical symptoms of rectal cancer are hemafecia, changes in defecation habits and obstruction. The lower part of the cancer and the harder part of the fecal mass are easy to bleed by the friction of the fecal mass, which is bright red or dark red, and are not mixed with the formed feces or attached to the surface of the fecal column, so the bleeding caused by hemorrhoid is misdiagnosed. Secondary infection of lesion stimulation and tumor ulcer, which causes continuous defecation reflex, is easily misdiagnosed as enteritis or bacillary dysentery. The ring-shaped growth of cancer causes intestinal constriction, which manifests as deformation and thinning of stool column in the early stage and incomplete obstruction in the late stage.

(4) Tumor infiltration and metastasis: the most common form of infiltration of large bowel cancer is local invasion, tumor invasion and surrounding tissues or organs, causing the corresponding clinical symptoms. Anal incontinence, persistent pain in the lower abdomen and lumbosacral region are the result of rectal cancer invasion and sacral plexus. Tumor cells are planted and transferred to the abdominal cavity and the pelvic cavity to form corresponding symptoms and signs, mass can be touched in a bladder rectum nest or a uterine rectum nest through digital rectal examination, and tumors are widely planted and transferred in the abdominal cavity and the pelvic cavity to form abdominal dropsy. There are two main ways of distant metastasis of large bowel cancer: lymphatic metastasis and blood-borne metastasis. Tumor cells are transferred to lymph nodes through lymphatic vessels, and also to the liver, lung, bone, etc. through blood circulation.

The pathogenesis of colorectal cancer is unclear, most of differential genes screened by the method for comparing healthy colorectal cancer tumor tissues at present have no functions, corresponding medicine intervention is not performed, and the applicability is not strong.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide the function and the application of the DEM1 in colorectal cancer.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

the invention provides application of a DEM1 inhibitor in preparing a medicine for treating colorectal cancer.

The colorectal cancer treatment drug has at least one of the following functions: can inhibit proliferation of large intestine cancer cells, induce apoptosis of large intestine cancer cells, inhibit cloning formation of large intestine cancer cells, inhibit tumorigenicity of large intestine cancer cells, and slow down growth of tumor body of large intestine cancer.

The large intestine cancer includes colon cancer and rectal cancer.

Preferably, the DEM1 inhibitor refers to a molecule having an inhibitory effect on DEM 1.

Having a suppressive effect on DEM1 includes, but is not limited to: inhibit DEM1 activity, or inhibit DEM1 gene transcription or expression.

The DEM1 inhibitor can be siRNA, shRNA, antibody and small molecule compound.

As exemplified in the examples herein, the DEM1 inhibitor can be an siRNA or shRNA. The target sequence of the siRNA or shRNA is shown in SEQ ID NO. 1.

The colorectal cancer treatment drug necessarily comprises a DEM1 inhibitor, and the DEM1 inhibitor is used as an effective component of the functions.

In the colorectal cancer treatment drug, the effective components playing the functions can be only the DEM1 inhibitor, and other molecules playing similar functions can also be contained.

The colorectal cancer treatment medicine can be a single-component substance or a multi-component substance.

The form of the colorectal cancer treatment drug is not particularly limited, and the colorectal cancer treatment drug can be in the forms of various substances such as solid, liquid, gel, semifluid, aerosol and the like.

The colon cancer treatment drug mainly aims at mammals such as rodents, primates and the like.

In a second aspect of the invention, there is provided a method of treating colorectal cancer by administering to a subject an inhibitor of DEM 1.

The object is a mammal or a colon cancer cell of the mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. Preferably, the primate is a monkey, ape or homo sapiens. The colorectal cancer cells can be ex vivo colorectal cancer cells, including but not limited to HT-29 cells and SW620 cells.

The subject may be a patient suffering from colorectal cancer or an individual expected to treat colorectal cancer, or isolated colorectal cancer cells of a colorectal cancer patient or an individual expected to treat colorectal cancer.

The DEM1 inhibitor may be administered to a subject before, during or after treatment for colorectal cancer.

In a third aspect of the invention, the invention provides a colorectal cancer treatment drug which comprises an effective amount of DEM1 inhibitor and a medicinal carrier.

In a fourth aspect of the invention, a colorectal cancer combination therapy is provided, which comprises an effective amount of DEM1 inhibitor and at least one other colorectal cancer therapy drug.

The other colorectal cancer treatment drug is a colorectal cancer treatment drug other than the DEM1 inhibitor.

The combination therapy drug combination may be in any one of the following forms:

firstly), the DEM1 inhibitor and other medicines for treating colorectal cancer are respectively prepared into independent preparations, the preparation forms can be the same or different, and the administration routes can be the same or different.

When the other therapeutic agent for colon cancer is an antitumor antibody, a parenteral administration type is generally used. When other colorectal cancer treatment medicines are chemotherapy medicines, the administration forms can be rich, and the gastrointestinal administration or the parenteral administration can be carried out. Known routes of administration for each chemotherapeutic agent are generally recommended.

Secondly), the DEM1 inhibitor and other medicaments for treating colorectal cancer are prepared into a compound preparation. When the DEM1 inhibitor and the other colorectal cancer therapeutic agent are administered by the same administration route and administered simultaneously, they may be formulated as a combined preparation.

In a fifth aspect of the present invention, there is provided a method for treating colon cancer, comprising administering to a subject an effective amount of DEM1 inhibitor, and administering to the subject an effective amount of another colon cancer treatment drug and/or administering to the subject another colon cancer treatment means.

An effective amount of DEM1 inhibitor and an effective amount of at least one other colorectal cancer treatment agent may be administered simultaneously or sequentially.

The DEM1 is the first discovered colorectal cancer treatment target point, and the combined application of the DEM1 and other colorectal cancer treatment medicines except the DEM1 inhibitor can at least play a role in adding curative effects, so that the inhibition on colorectal cancer is further enhanced.

Such other colorectal cancer treatment drugs include, but are not limited to: antitumor antibodies, chemotherapeutic drugs or targeted drugs, etc.

The DEM1 inhibitor may be administered parenterally or parenterally. The other colorectal cancer treatment drugs may be administered gastrointestinal or parenteral. For antitumor antibodies or chemotherapeutic drugs, parenteral administration is generally employed.

In a sixth aspect of the invention, there is provided a further novel use of DEM1, said DEM1 inhibitor having at least one of the following functions: can inhibit proliferation of large intestine cancer cells, induce apoptosis of large intestine cancer cells, inhibit cloning formation of large intestine cancer cells, inhibit tumorigenicity of large intestine cancer cells, and slow down growth of tumor body of large intestine cancer.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, through extensive and intensive research, DEM1 can be used as a colorectal cancer treatment target for the first time, and the inhibition of the expression of DEM1 can inhibit the proliferation of colorectal cancer cells, induce the apoptosis of the colorectal cancer cells, inhibit the clonogenic capacity of the colorectal cancer cells, inhibit the tumorigenic capacity of the colorectal cancer cells and slow down the growth of colorectal cancer tumor bodies. Therefore, the invention provides powerful scientific evidence for the pathogenesis of the colorectal cancer and the clinical treatment of the colorectal cancer from the tissue level, the cell function level and the molecular level of a clinical patient sample.

Drawings

FIG. 1: DEM1 protein is expressed differently in the cytoplasm of cancer and paracancer.

FIG. 2A: after 3 days of shRNA lentivirus infection, the expression level of the DEM1 gene at the mRNA level in HT-29 cells in the experimental group was inhibited.

FIG. 2B: after 3 days of shRNA lentivirus infection, the expression level of the DEM1 gene at the mRNA level in HT-29 cells in the experimental group was inhibited.

FIG. 2C: exogenous expression of DEM1 gene in HT-29 showed significant knock-down at the protein level.

FIG. 2D: after shRNA lentivirus infection, the proliferation rate of HT-29 cells in an experimental group is obviously inhibited.

FIG. 2E: after shRNA lentivirus infection, the proliferation rate of HT-29 cells in an experimental group is obviously inhibited.

FIG. 2F: the number of HT-29 cell colonies in the experimental group decreased after shRNA lentiviral infection.

FIG. 2G: the experimental group showed a significant increase in apoptotic HT-29 cells 6 days after shRNA lentivirus infection.

FIG. 3A: after 3 days of shRNA lentivirus infection, the expression level of DEM1 gene in SW620 cells in the experimental group was inhibited at the mRNA level.

FIG. 3B: after 3 days of shRNA lentivirus infection, the expression level of DEM1 gene in SW620 cells in the experimental group was inhibited at the mRNA level.

FIG. 3C: exogenous expression of DEM1 gene in SW620 cells in experimental group had significant knock-down at the protein level.

FIG. 3D: after shRNA lentivirus infection, the proliferation rate of SW620 cells in the experimental group is obviously inhibited.

FIG. 3E: after shRNA lentivirus infection, the proliferation rate of SW620 cells in the experimental group is obviously inhibited.

FIG. 3F: after shRNA lentivirus infection, the number of colonies of SW620 cells in the experimental group decreased.

FIG. 3G: after 6 days of shRNA lentivirus infection, the experimental group showed a significant increase in apoptotic SW620 cells.

FIG. 4A: after DEM1 gene knockdown, nude mice developed significantly less tumor volume.

FIG. 4B: after DEM1 gene knockdown, nude mice developed significantly less tumor volume.

FIG. 4C: compared with the NC group, the DEM1 gene-knocked-down KD group has reduced tumor body growth (P < 0.05).

FIG. 4D: compared with the NC group, the DEM1 gene-knocked-down KD group has reduced tumor body growth (P < 0.05).

Detailed Description

The research discovers that DEM1 can be used as a colorectal cancer treatment target, and the inhibition of the expression of DEM1 can inhibit the proliferation of colorectal cancer cells, induce the apoptosis of colorectal cancer cells, inhibit the clonogenic capacity of colorectal cancer cells, inhibit the tumorigenic capacity of colorectal cancer cells and slow down the growth of colorectal cancer tumor bodies.

DEM1 inhibitors

Refers to molecules having inhibitory effects on DEM 1. Having a suppressive effect on DEM1 includes, but is not limited to: inhibit DEM1 activity, or inhibit DEM1 gene transcription or expression. The DEM1 inhibitor comprises siRNA, shRNA, antibody and small molecule compound.

By inhibiting DEM1 activity is meant reducing DEM1 activity. Preferably, DEM1 viability is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, and most preferably by at least 90% compared to that prior to inhibition.

The DEM1 gene transcription or expression inhibition refers to: the gene of DEM1 is not transcribed, or the transcription activity of the gene of DEM1 is reduced, or the gene of DEM1 is not expressed, or the expression activity of the gene of DEM1 is reduced.

The skilled person can use conventional methods to modulate the transcription or expression of DEM1 gene, such as gene knock-out, homologous recombination, interfering RNA, etc.

The inhibition of DEM1 gene transcription or expression was confirmed by PCR and Western Blot.

Preferably, DEM1 gene transcription or expression is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, still more preferably by at least 90%, most preferably by no expression of DEM1 gene as compared to wild type.

Small molecule compounds

The invention refers to a compound which is composed of several or dozens of atoms and has the molecular mass of less than 1000.

DEM1 inhibitor preparation medicine

The DEM1 inhibitor is used as main active ingredient or one of the main active ingredients for preparing the medicine. Generally, the medicament may comprise one or more pharmaceutically acceptable carriers or excipients in addition to the active ingredient, according to the requirements of different dosage forms.

By "pharmaceutically acceptable" is meant that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

A "pharmaceutically acceptable carrier or adjuvant" should be compatible with, i.e., capable of being blended with, the DEM1 inhibitor without substantially diminishing the effectiveness of the pharmaceutical composition as is often the case. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as glycerol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and may be prepared into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by a conventional method. The choice of the pharmaceutical dosage form should be matched to the mode of administration.

Combination therapeutic drug combinations and methods of administration

The combination therapy drug combination may be in any one of the following forms:

firstly), the DEM1 inhibitor and other antitumor drugs are respectively prepared into independent preparations, the dosage forms of the preparations can be the same or different, and the administration routes can be the same or different. When in use, several medicines can be used simultaneously or sequentially. When administered sequentially, the other drugs should be administered to the body during the period that the first drug is still effective in the body.

Secondly), the DEM1 inhibitor and other antitumor drugs are prepared into compound preparations. When the DEM1 inhibitor and the other antitumor agent are administered by the same route of administration and simultaneously administered, they may be formulated as a combined preparation.

The antibody is usually administered by intravenous injection, intravenous drip or arterial infusion. The usage and the dosage can refer to the prior art.

The small molecule compounds are usually administered by either gastrointestinal or parenteral administration. The siRNA, shRNA and antibody are generally administered parenterally. Can be administered locally or systemically.

An effective amount of DEM1 inhibitor and an effective amount of other colorectal cancer drugs may be administered simultaneously or sequentially. When in use, an effective amount of the DEM1 inhibitor and an effective amount of other colorectal cancer drugs can be used simultaneously, or an effective amount of the DEM1 inhibitor and an effective amount of other colorectal cancer drugs can be used successively. When administered sequentially, the other drug should be administered to the organism during the period that the first drug is still effective for the organism.

Chemotherapeutic agents include alkylating agents (e.g., nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, and glyphosate), antimetabolites (e.g., nucleotide analogs such as doxifluridine, doxycycline, fluorouracil, mercaptopurine, methotrexate), antitumor antibiotics (e.g., antibiotics such as actinomycin D, doxorubicin, and daunorubicin), antitumor animal and plant components (e.g., vinorelbine, taxol, cephalotaxine, irinotecan, taxotere, and vinblastine), antitumor hormonal agents (e.g., atalmentane, anastrozole, aminoglutethimide, letrozole, formestane, and tamoxifen), and conventional chemotherapeutic agents such as cisplatin, dacarbazine, oxaliplatin, lesonidine, carboplatin, mitoxantrone, and procarbazine.

Targeted drugs include EGFR blockers such as Gefitinib (Gefitinib, Iressa and Iressa) and Erlotinib (Erlotinib, Tarceva), monoclonal antibodies to specific cell markers such as Cetuximab (Cetuximab, Erbitux) and anti-HER-2 mabs (Herceptin, Trastuzumab, Herceptin), tyrosine kinase receptor inhibitors such as Crizotinib (Crizotinib, Xalkori), anti-tumor angiogenesis drugs such as Bevacizumab, endostatin and Bevacizumab, etc., Bcr-Abl tyrosine kinase inhibitors such as Imatinib and Dasatinib, anti-CD 20 mabs such as Rituximab, IGFR-1 kinase inhibitors such as NVP-AEW541, mTOR kinase inhibitors such as CCI-779, ubiquitin-proteasome inhibitors such as Bortezomib, etc.

Other tumor treatment modalities may be selected from one or more of surgical resection, radio frequency ablation, argon helium superconducting surgical treatment, laser ablation therapy, high intensity focused ultrasound, and radiation therapy including X-ray, R-ray, 3D-CRT, and IMRT.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise stated, the experimental METHODS, detection METHODS, AND preparation METHODS disclosed herein are based on conventional techniques IN the art, such as molecular biology, biochemistry, Chromatin structure AND analysis, analytical chemistry, cell culture, recombinant DNA techniques, AND related fields, which are well described IN the prior art, see, IN particular, Sambrook et al MO L ECU L AR C L ONING: A L ABORATORATOR MANUA 8652, Second edition, Cold Spring Harbor L analysis Press, 1989AND Third edition, 2001, Ausubel et al, CURRENT PROTOCO L S INMO L ECU L AR BIO L OGY, John Wiley & Sons, New York, 1987AND biological orders, the devices will be used, METHODS of DIDIDISY 2 IN L, Actic, Experimental & Sons, Sandwith et al, sample # Press, AND Press, sample # 1998, sample # Press, AND P, sample # Press et al, AND sample # Press et al.

Example 1 DEM1 Gene function study

Clinical colorectal cancer tissue chip research

The clinical collection of 38 colorectal cancer patients and tissues beside the cancer were subjected to tissue chip analysis of DEM1 on 38 samples, and the results are shown in FIG. 1, Table 1 and Table 2, which revealed that DEM1 protein has differential expression in the cytoplasm of cancer and beside the cancer, and P is less than 0.05.

TABLE 1

TABLE 2

(II) preparation of RNAi lentivirus aiming at DEM1 gene

1. Selecting an effective siRNA target point for the DEM1 gene, wherein the sequence of the target point is shown as SEQ ID NO.1, and specifically comprises the following steps: 5'-GATTGATGAGCTGCACTAT-3' are provided.

shRNA was designed against the siRNA target (SEQ ID No.1), the target gene contained a targeting hairpin, a transcription termination sequence, and a restriction site that allowed insertion of lentiviral vector GV115(GeneChem, Shanghai, China), while the alignment sequence also had a hairpin structure without the target gene synthesized using the same technique. The lentivirus component is composed of a sequencing portion and a production portion. The packaging and purification system of the virus is a lentivirus expression system of GeneChem (Shanghai, China).

Further, the sequence of the shRNA is shown as SEQ ID NO.2, and specifically comprises:

5’-GATTGATGAGCTGCACTATCTCGAGATAGTGCAGCTCATCAATC-3’。

the sequence of the siRNA corresponding to the shRNA is shown as SEQ ID NO.3, and specifically comprises the following steps:

5’-GAUUGAUGAGCUGCACUAU-3’。

the comparison sequence is shown as SEQ ID NO.4, and specifically comprises: 5'-TTCTCCGAACGTGTCACGT-3' are provided.

(II) mRNA level detection by qPCR DEM1 gene reduction efficiency

The shRNA lentivirus is transfected into HT-29 and SW620 colorectal cancer cells respectively, the result is shown in figure 2A and figure 2B, and the expression level of DEM1 gene in the HT-29 cell in an experimental group at the mRNA level is inhibited after 3 days of shRNA lentivirus infection; as shown in fig. 3A and 3B, the expression level of DEM1 gene at the mRNA level was suppressed in SW620 cells of the experimental group 3 days after shRNA lentivirus infection.

(III) reducing DEM1 gene protein level expression by Western Blot detection target

As shown in fig. 2C, exogenous expression of DEM1 gene in experimental group HT-29 had a significant knock-down effect at the protein level. As shown in fig. 3C, exogenous expression of DEM1 gene in experimental SW620 cells had a significant knock-down effect at the protein level.

(IV) Celigo assay the Effect of DEM1 Gene depletion on cell proliferation

As shown in fig. 2E, 3 days after shRNA lentivirus infection, cells were plated in 96-well plates at 2000 plates. Celigo continued the assay for 5 days and found that the proliferation rate of HT-29 cells was significantly inhibited in the experimental group. The DEM1 gene is obviously related to the proliferation capacity of HT-29.

As shown in fig. 3E, 3 days after shRNA lentivirus infection, cells were plated in 96-well plates at 2000 plates. Celigo continued the assay for 5 days, and found that the proliferation rate of SW620 cells was significantly inhibited in the experimental group. The DEM1 gene was suggested to be significantly related to the proliferative capacity of SW620 cells.

(V) MTT assay for the Effect of DEM1 Gene depletion on cell proliferation

As shown in fig. 2D, cells were plated in 96-well plates at 2000 plates 3 days after shRNA lentivirus infection. After 5 days of continuous detection, the proliferation rate of HT-29 cells in the experimental group is remarkably inhibited. The DEM1 gene is obviously related to the proliferation capacity of HT-29.

As shown in fig. 3D, cells were plated in 96-well plates at 2000 plates 3 days after shRNA lentivirus infection. After 5 days of continuous detection, the proliferation rate of SW620 cells in the experimental group is remarkably inhibited. Indicating that DEM1 gene is significantly related to the proliferative capacity of SW 620.

(six) FACS detection of Effect of DEM1 Gene depletion on apoptosis

As shown in FIG. 2G, the experimental group showed a significant increase in the number of HT-29 cells undergoing apoptosis 6 days after shRNA lentiviral infection, suggesting that the DEM1 gene was significantly associated with the apoptosis of HT-29 cells.

As shown in fig. 3G, SW620 cells that were apoptotic in the experimental group increased significantly after 6 days of shRNA lentiviral infection, suggesting that DEM1 gene was significantly associated with apoptosis of SW620 cells.

(VII) detecting the influence of DEM1 gene reduction on cell clonogenic capacity

As shown in fig. 2F, 3 days after shRNA lentivirus infection, HT-29 cells were plated in 6-well plates at 600, and the number of clones was observed after 15 days, as a result: the number of colonies of HT-29 cells was reduced in the experimental group, suggesting that the DEM1 gene was significantly related to the clonogenic capacity of HT-29 cells.

As shown in fig. 3F, SW620 cells were plated in 6-well plates 3 days after shRNA lentivirus infection, the number of plated cells was 600, and the number of clones was observed 15 days later, as a result: the number of colonies of SW620 cells in the experimental group was reduced, suggesting that DEM1 gene was significantly related to clonogenic capacity of SW620 cells.

In conclusion, the results of the experiments prove that the interference target gene DEM1 has the function of inhibiting the proliferation of the colon cancer cells HT-29 and SW 620.

Example 2 DEM1 animal experiments

The function of DEM1 was further studied in a nude mouse tumorigenesis experiment, and cells infected with DEM1 lentivirus in the previous step were injected into nude mice, and compared with untransfected nude mice three weeks later, as shown in FIGS. 4A and 4B, it was found that after DEM1 gene knockdown, the tumor volume formed by the nude mice was significantly reduced, and 70% of the nude mice did not form significant tumors within three weeks. As shown in fig. 4C and fig. 4D, tumor body growth was reduced (P <0.05) for the DEM1 gene-knocked-down KD group compared to the NC group.

According to the conclusion, DEM1 is a key gene for colorectal cancer cell proliferation, and the down-regulation of DEM1 expression can obviously inhibit colorectal cancer cell proliferation and induce colorectal cancer cell apoptosis, so that the method can provide a direction for the research of drugs for treating colorectal cancer in the future.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

SEQUENCE LISTING

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Claims (2)

1. The application of the DEM1 inhibitor in preparing the colorectal cancer treatment medicine is characterized in that the DEM1 inhibitor is shRNA, and the sequence of the shRNA is shown in SEQ ID NO. 2.
2. The application of the DEM1 inhibitor in preparing the colorectal cancer treatment medicine is characterized in that the DEM1 inhibitor is siRNA, and the sequence of the siRNA is shown in SEQ ID NO. 3.

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