CN109692332B

CN109692332B - Application of NUDT5 protein as colorectal cancer treatment target

Info

Publication number: CN109692332B
Application number: CN201710999397.3A
Authority: CN
Inventors: 蔡剑平; 李瑾; 田馨园
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-10-23
Filing date: 2017-10-23
Publication date: 2021-07-09
Anticipated expiration: 2037-10-23
Also published as: CN109692332A

Abstract

The invention relates to application of NUDT5 protein as a colorectal cancer treatment target, belonging to the fields of molecular biology and biomedicine. Application of NUDT5 protein as colorectal cancer treatment target. The invention provides a novel therapeutic target for colorectal cancer, which can be effectively used for colorectal cancer development judgment, treatment scheme selection and/or prognosis evaluation, thereby providing a novel colorectal cancer diagnostic agent and/or therapeutic agent for the field and having clinical application prospect.

Description

Application of NUDT5 protein as colorectal cancer treatment target

Technical Field

The invention relates to application of NUDT5 protein as a colorectal cancer treatment target, belonging to the fields of molecular biology and biomedicine.

Background

Colorectal cancer (CRC) is malignant cancer that occurs due to the action of various carcinogenic factors such as environment and heredity of colonic mucosal epithelium, is one of the most common digestive tract malignant tumors, has a third-place morbidity and a fourth-place mortality in worldwide, has about 120 million new cases every year, and seriously threatens human health. Colorectal cancer is high in incidence in asia, and is mainly related to risk factors such as age, family history, unhealthy diet and living habits in China.

Cells produce large amounts of ROS, either during normal metabolic activity or upon exogenous stimuli, which can damage DNA, RNA, and free nucleotides. Guanine, because of its lowest oxidation potential, its oxidation product, 8-oxoG, is the most abundant oxidized base in the cell. Nucleotides containing 8-oxoG can be incorporated into DNA or RNA, and as 8-oxoG pairs with A and C with equal efficiency, mismatches at the replication and transcription levels are caused.

The NUDT5(Nudix hydrosile 5) protein belongs to the mammalian Nudix hydrolase superfamily. The NUDT5 protein has the activity of hydrolyzing 8-oxo-dGDP, prevents oxidized guanine from being incorporated into DNA, reduces replication mismatch, has wider substrate specificity, and can also hydrolyze 8-oxo-dADP, 2-oxo-dADP and 5-CHO-dUDP. In vitro and in vivo experimental studies find that cDNA expressing NUDT5 in mutT-deficient Escherichia coli obviously reduces the spontaneous mutation rate and obviously inhibits the generation of variant protein, exogenous 8-oxo-dGTP is added after the NUDT5 is knocked down in 293T cells to increase the transversion mutation from A: T to C: G, and the results show that the NUDT5 protein can maintain the fidelity of DNA replication and transcription under oxidative stress. However, studies on the expression of NUDT5 in tumor tissues have not been reported.

Disclosure of Invention

One aspect of the invention provides the use of the NUDT5 protein as a target for colorectal cancer therapy.

The present invention is based on the following findings:

we found for the first time that the NUDT5 protein is expressed in increased amounts in colorectal cancer (CRC) tissues and is associated with CRC progression and prognosis. The expression level of the NUDT5 protein is related to colorectal cancer AJCC stage and lymph node metastasis. The overall survival rate of CRC patients in the high expression NUDT5 group was lower after surgical treatment. The research finds that the expression of the NUDT5 is related to the development and prognosis of CRC, and the inhibition of the expression of the NUDT5 protein causes the reduction of cell viability, so that the NUDT5 protein is a potential new target point for treating colorectal cancer. The present invention has been completed based on this finding.

In a second aspect of the invention, there is provided a use of an inhibitor of the NUDT5 protein in the manufacture of a medicament for the prevention and/or treatment of colorectal cancer.

The medicine comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of the NUDT5 protein.

The inhibitor of the NUDT5 protein is selected from an antibody of the NUDT5 protein and/or a binding protein of the NUDT5 protein.

In a third aspect of the invention, the use of an inhibitor of the NUDT5 gene in the manufacture of a medicament for the prevention and/or treatment of colorectal cancer is provided.

The medicine comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of the NUDT5 gene.

The inhibitor of the NUDT5 gene is selected from one or more of RNAi specific to the NUDT5 gene, microRNA specific to the NUDT5 gene and an inhibitor for inhibiting a promoter of the NUDT5 gene.

In a fourth aspect of the present invention, there is provided a medicament for preventing and/or treating colorectal cancer, comprising a pharmaceutically acceptable carrier and effective amounts of the following active ingredients: an inhibitor of the NUDT5 protein and/or an inhibitor of the NUDT5 gene.

The inhibitor of the NUDT5 protein is selected from an antibody of the NUDT5 protein and/or a binding protein of the NUDT5 protein; the inhibitor of the NUDT5 gene is selected from one or more of RNAi specific to the NUDT5 gene, microRNA specific to the NUDT5 gene and an inhibitor for inhibiting a promoter of the NUDT5 gene.

The term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

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

Such pharmaceutically acceptable carriers include, but are not limited to: water, saline, buffer, glycerol, ethanol, liposomes, lipids, proteins, protein-antibody conjugates, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.

The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition of the invention can be prepared into injections, oral preparations (tablets, capsules, oral liquids), transdermal agents and sustained-release agents. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to, pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc.

In a fifth aspect of the invention, a method for non-therapeutic inhibition of tumor cells in vitro is provided, comprising culturing the tumor cells in the presence of an inhibitor of the NUDT5 protein or an inhibitor of the NUDT5 gene, thereby inhibiting the tumor cells. Wherein said tumor cell is a colorectal cancer cell.

The expression of NUDT5 in colorectal cancer (CRC) cell strains and colorectal cancer (CRC) tissues is detected, the difference between the expression levels of the CRC cells (tissues) and normal cells (tissues) is compared, the correlation between the expression level of NUDT5 and clinical pathological data (such as sex, age, position, tumor size, clinical stage, differentiation degree and the like) is analyzed, and the condition that the proliferation capacity of colorectal cancer cell strains can be obviously reduced by knocking and reducing NUDT5 protein is determined.

Has the advantages that: the invention provides a novel therapeutic target for colorectal cancer, which can be effectively used for colorectal cancer development judgment, treatment scheme selection and/or prognosis evaluation, thereby providing a novel colorectal cancer diagnostic agent and/or therapeutic agent for the field and having clinical application prospect.

The invention is further illustrated with reference to the following figures and detailed description. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Equivalents of the art made in accordance with the present disclosure are intended to be covered by the present invention.

Drawings

FIG. 1 shows the expression of NUDT5 protein in colorectal cancer tissues (cells) and paired paracancerous tissues (cells); FIG. 1A is a relative quantification of NUDT5 mRNA expression in colorectal cancer cell lines, FIG. 1B is a representative Western blotting band of NUDT5 protein expression in colorectal cancer cell lines, FIG. 1C is a relative quantification of NUDT5 protein expression in colorectal cancer cell lines, FIG. 1D is a representative Western blotting band of NUDT5 protein expression in 6 colorectal cancer tissues and paired paracarcinoma tissues, FIG. 1E is a quantification result of NUDT5 protein expression in 20 colorectal cancer tissues and paired paracarcinoma tissues, and the statistical method is Student's t-test.

FIG. 2 is a representation of the immunohistochemistry of NUDT5 protein in colorectal cancer tissues and paired paracarcinoma tissues

FIG. 3 is a Kaplan-Meier survival curve

FIG. 4 shows that knocking down the NUDT5 protein inhibits SW480 and COLO320 cell proliferation; FIG. 4A shows the effect of Western blotting on the knockdown of the NUDT5 protein in SW480 and COLO320 cells, and FIG. 4B shows the proliferation curve of SW480 and COLO320 cells after the NUDT5 protein is knocked down.

Detailed Description

Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Parts and percentages are by weight unless otherwise indicated.

Example 1.

Materials and methods

1. Experimental Material

(1) Human embryonic intestinal mucosal cell CCC-HIE-2 (basic medical cell center of institute of basic medical science of Chinese academy of medical sciences), 6 colorectal cancer cell strains SW480, SW620, COLO320, T84(AJCC, USA), LoVo and HCT116 (basic medical cell center of institute of basic medical sciences of Chinese academy of medical sciences).

(2)44 colorectal cancer tissue and corresponding paracancerous normal tissue (provided by the first Hospital affiliated with the university of Jia Musi)

(3) Colon cancer tissue chip-87 cancer tissues and corresponding paracancerous normal tissues (Shanghai core Biotechnology Co., Ltd., before radical treatment of colorectal cancer, patients were treated with radiotherapy and chemotherapy

(4) anti-NUDT 5 antibody (Abcam)

2. Experimental methods

(1) The expression of NUDT5 mRNA and protein in 6 human colorectal cancer cell strains HCT116, SW480, SW620, LoVo, COLO320, T84 and human normal intestinal mucosa epithelial cells CCC-HIE-2 is respectively detected by qRT-PCR and Western Blotting. The specific steps of qRT-PCR are as follows:

(ii) extraction of total RNA from the cell line using TRIzol (Thermo Fisher, USA).

② 2ug of RNA was reverse transcribed into cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Kyoto Seiki Kagaku Co., Ltd.).

Using KAPA

Quantitative PCR was performed with FAST Universal qPCR Kits (KAPA, USA), with NUDT5 primer: forward 5'-GTTCTCCAGCGGTCTGTATG-3' (SEQ ID NO:1) and reverse 5'-CTTCGGCCTTGCGTTTTCG-3' (SEQ ID NO:2), GAPDH primer: forward 5'-CCTCTCCAGAACATCATCC-3' (SEQ ID NO:3) and reverse 5'-GTGTCGCTGTTGAAGTCAG-3' (SEQ ID NO: 4).

Western Blotting comprises the following specific steps:

adding RIPA lysate (Beijing Solebao science and technology Co., Ltd.) containing 1X PMSF (Solebao), 1X protease and phosphatase inhibitor (CST, USA), standing on ice for 30min, centrifuging at 4 deg.C 12000g for 20min, and transferring the supernatant to a new 1.5ml EP tube.

② the BCA method (Thermo fisher, USA) to determine the total protein concentration.

③ 12 percent SDS-PAGE electrophoresis, the sample loading quantity for detecting the expression of GAPDH is 10ug, and the sample loading quantity for detecting the expression of NUDT5 is 40 ug.

(iv) transferring the membrane, and electrotransfering the protein in the PAGE to a PVDF membrane (Millipore, USA).

Fifthly, sealing the mixture by immunity, and sealing the mixture for 2 hours by using 5 percent of skimmed milk.

Sixthly, primary anti-incubation, wherein the dilution ratio of the anti-NUDT 5 antibody is 1: 1000, anti-GAPDH antibody dilution ratio 1:2000, 4 ℃ overnight, 5 washes in TBST, 5min each.

And seventhly, incubating the goat anti-rabbit IgG-HRP (Biyuntian biotechnology, Co., Ltd., 1:2000), incubating for 2 hours at room temperature, and washing for 5 times (5 min each time) by TBST.

Exposure, and a developing solution (Millipore, USA) 1: 1, uniformly dripping the mixture on a strip after mixing, and carrying out exposure and color development.

(2) The expression level of NUDT5 in colorectal cancer tissues and corresponding paracancerous normal tissues is detected 44 by Western Blotting, and the correlation between the expression level of NUDT5 protein and clinical pathological data (such as sex, age, position, tumor size, clinical stage, differentiation degree and the like) is analyzed. The specific experimental procedures are as in reference (1).

(3) The expression level of NUDT5 in 87 cases of cancer tissues and corresponding paracancer normal tissues (tissue chips) is detected by immunohistochemistry, the cancer tissues are divided into a high expression group and a low expression group according to the degree of staining and the staining area, and the correlation between the expression level of NUDT5 and clinical pathological data (such as sex, age, position, tumor size, clinical stage, differentiation degree and the like) is analyzed. The method comprises the following specific steps:

the tissue chip is dewaxed by xylene for 2d, and is subjected to gradient hydration for 2min by 100%, 95%, 85% and 75% ethanol and is hydrated for 10min by PBS.

② the antigen repair (pH6.0 citric acid repair liquid) microwave repair for 30min, and cooling to room temperature.

③ sealing the goat serum (Beijing Zhongshirt Jinqiao Biotech Co., Ltd.) for 30 min.

Fourthly, primary antibody incubation, wherein the dilution ratio of the anti-NUDT 5 antibody is 1: 1000, 4 ℃ overnight, PBS wash 5min X3 times.

Fifthly, secondary antibody incubation, using PV-6001 goat anti-rabbit IgG/HRP polymer, incubation for 20 minutes at room temperature, washing 5min X3 times with PBS.

DAB (Chinese shirt and gold bridge) coloration

Seventhly, counterstaining hematoxylin, gradient dewatering and transparent sealing.

(4) Firstly, mRNA expression levels in SW480 and COLO320 cell strains are respectively knocked down by using siNUDT5 (with a target sequence of 5'-CATGGATCCTACTGGTAAA-3' (SEQ ID NO:5)) screened by a laboratory, then Western Blotting is carried out to verify knocking-down effects, and finally, the influence of transient knocking-down of NUDT5 in the SW480 and COLO320 cell strains on cell viability is detected by a CCK-8 experiment. The method comprises the following specific steps:

SW480 cells and COLO320 cells were plated in 96-well plates at 1X104 cells per well.

12h later, 50nM siNUDT5 and siControl were transfected using RNAImax (Thermo Fisher, USA), 6 wells per group.

③ at 24h, 48h and 72h after transfection, the absorbance at 450nm is detected by using a CCK-8 kit (full-scale gold).

3. The expression high-low scoring standard of NUDT5 protein in the tissue chip is as follows:

(1) staining intensity 0 (none); 1 (weak); 2 (middle); 3 (Strong)

(2) Dyeing area: 0 (0%); 1 (1-25%); 2 (26-50%); 3 (51-75%); 4 (76-100%);

(3) the final score is the staining intensity X stained area: low expression (0-6); high expression (7-12).

4. Statistical method

Software SPSS statistics version 19.

(1) Student's t-test compares the two sets of means;

(2) pearson 'X2 or Fisher's exact test compares the relevance of the NUDT5 protein to CRC clinical pathological data;

(3) Kaplan-Meier analysis calculated the Overall Survival (OS) and the log-rank test compared the two groups of OS.

Second, experimental results

1. Increased expression level of colorectal cancer cell line NUDT5

(1) The qRT-PCR result shows that the expression level of NUDT5 mRNA in colorectal cancer cell strains HCT116, SW480, SW620, LoVo, COLO320 and T84 is obviously higher than that of CCC-HIE-2 (Student's T-test, P is less than 0.05, and figure 1A).

(2) Western blotting results show that the expression level of the NUDT5 protein in colorectal cancer cell lines is also remarkably higher than that of CCC-HIE-2 (Student's t-test, P is less than 0.05, and figures 1B-1C).

2. Increased expression of NUDT5 in colorectal cancer tissue

(1) We firstly carried out Western blotting detection 20 to detect the expression of NUDT5 protein in human colorectal cancer tissues and matched paracancer normal tissues, and the result shows that the expression of NUDT5 in the cancer tissues is obviously higher than that in the normal tissues (Student's t-test, P < 0.001, figure 1D-figure 1E).

(2) We then performed immunohistochemical detection of the expression of the NUDT5 protein in the tissue chip (87 human colorectal cancer tissue and paired paracancerous tissue), again demonstrating upregulation of the protein expression in cancer tissue (fig. 2). The staining degree of cancer tissues is different, weak, medium and strong. According to the intensity and the range of immunostaining of the cancer tissues, 42 NUDT5 proteins were highly expressed in 87 cancer tissues.

Correlation of NUDT5 protein expression with colorectal cancer clinicopathological parameters

(1) According to the result of the immunohistochemical staining of the NUDT5 protein, 87 individual colorectal cancer tissues in the tissue chip are respectively divided into a high expression group and a low expression group, and the correlation of protein expression with age, sex, position, tumor size, AJCC stage, T stage, N stage, M stage, differentiation degree and vascular metastasis is analyzed by using Pearson 'X2 or Fisher's exact test, and the result shows that the expression level of NUDT5 is obviously correlated with the AJCC stage and the N stage (lymph node metastasis) (P is less than 0.05, and Table 1).

TABLE 1 correlation of NUDT5 protein expression with colorectal cancer clinicopathological parameters (immunohistochemistry, n ═ 87)

^aChi-square test

^bFisher’s exact test

*P＜0.05

(2) Western blotting examined the relative expression of the NUDT5 protein in 44 human colorectal tissues, and the Student's T-test was used to analyze the correlation between the protein expression and CRC clinical pathology, and the results showed that the expression level of NUDT5 was significantly correlated with the AJCC stage, T stage (tumor infiltration degree) and N stage (lymph node metastasis) (P < 0.05, Table 2).

TABLE 2 correlation of NUDT5 protein expression with colorectal cancer clinicopathological parameters (Western Blotting, n ═ 44)

Effect of NUDT5 protein expression on prognosis of patients with colorectal cancer

According to the result of the immunohistochemical staining of the NUDT5 protein, 87 individual colorectal cancer tissues in the tissue chip are respectively divided into a high expression group and a low expression group, the Kaplan-Meier analysis is used for calculating the total survival rate (OS) of CRC patients after surgical treatment, and log-rank test is simultaneously used for comparing the OS of the high expression group and the OS of the low expression group, so that the result shows that the survival rate of the NUDT5 high expression group is lower (P is 0.003, figure 3).

5. Knocking-down NUDT5 protein for inhibiting proliferation capacity of colorectal cancer cell line

The CCK-8 experiment result shows that after the NUDT5 protein is knocked down in SW480 and COLO320 cell strains for 72h, the SW480 and COLO320 cells have obviously reduced activity and reduced cell proliferation capacity (figure 4).

Conclusion III

In the present study, the NUDT5 protein is firstly found to be expressed in the CRC tissue in an increased amount and is related to the CRC progression and prognosis.

The expression level of the NUDT5 protein is related to colorectal cancer AJCC stage and lymph node metastasis. The overall survival rate of CRC patients in the high expression NUDT5 group was lower after surgical treatment.

The research finds that the expression of the NUDT5 is related to the development and prognosis of CRC, and the inhibition of the expression of the NUDT5 protein causes the reduction of cell viability, so that the NUDT5 protein is a potential new target for treating colorectal cancer.

Sequence listing

<110> Cai, Jianping

Application of <120> NUDT5 protein as colorectal cancer treatment target

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Claims

Use of an inhibitor of the NUDT5 gene for the manufacture of a medicament for the prevention and/or treatment of colorectal cancer; the inhibitor of the NUDT5 gene is selected from one or two of RNAi with the specificity of the NUDT5 gene and microRNA with the specificity of the NUDT5 gene.
2. Use according to claim 1, characterized in that: the medicine comprises a pharmaceutically acceptable carrier and an effective amount of active ingredients, wherein the active ingredients are inhibitors of the NUDT5 gene.
3. The drug for preventing and/or treating colorectal cancer is characterized by comprising a pharmaceutically acceptable carrier and an effective amount of an inhibitor of the NUDT5 gene, wherein the inhibitor of the NUDT5 gene is selected from one or two of RNAi specific to the NUDT5 gene and microRNA specific to the NUDT5 gene.
4. A method of non-therapeutically inhibiting tumor cells in vitro comprising: culturing a tumor cell in the presence of an inhibitor of the NUDT5 gene, thereby inhibiting the tumor cell, wherein the tumor cell is a colorectal cancer cell; the inhibitor of the NUDT5 gene is selected from one or two of RNAi specific to the NUDT5 gene and microRNA specific to the NUDT5 gene.