CN111778333A

CN111778333A - Application of reagent for determining EDAR expression level and kit

Info

Publication number: CN111778333A
Application number: CN202010638801.6A
Authority: CN
Inventors: 梁艳芳; 王斌; 林碧华; 曾今诚; 邱贤秀; 黄卫娟; 陈莎莎; 柴星星
Original assignee: Dongguan Binhai Bay Central Hospital; Guangdong Medical University
Current assignee: Dongguan Binhai Bay Central Hospital; Guangdong Medical University
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2020-10-16
Anticipated expiration: 2040-07-03
Also published as: CN111778333B

Abstract

The invention provides application of a reagent for determining the expression level of EDAR in preparation of a preparation for auxiliary diagnosis of colorectal cancer, prognosis of colorectal cancer and resistance to colorectal cancer. In a second aspect, the invention provides a kit for the aided diagnosis, prognosis and resistance of colorectal cancer, comprising reagents for measuring EDAR expression levels. In a third aspect, the invention provides a medicament for inhibiting colorectal cancer proliferation comprising an agent for determining EDAR expression levels. The inventor finds that the EDAR is highly expressed in the colorectal cancer tissue, the up-regulated expression of the EDAR is related to the colorectal cancer progression and survival prognosis, and meanwhile, the independent silencing of the EDAR can effectively inhibit the colorectal cancer neoplasia, greatly reduce the tumor volume and weight in vivo, and has very high clinical guidance significance.

Description

Application of reagent for determining EDAR expression level and kit

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a reagent for measuring an EDAR (amplified polymorphic DNA) expression level in preparation of a preparation for auxiliary diagnosis of colorectal cancer, prognosis judgment of colorectal cancer and resistance to colorectal cancer, and a kit for auxiliary diagnosis of colorectal cancer, prognosis judgment of colorectal cancer and resistance to colorectal cancer.

Background

Colorectal cancer (CRC), including Colon Cancer (CC) and Rectal Cancer (RC), is a common lethal malignancy. In the world, 180 ten thousand new cases and 89 ten thousand death cases are newly added in 2018. As the population ages and the incidence of CRC in young adults (under 50 years) rises, the global CRC case data is expected to rise further. Surgery is the primary treatment for early CRC, and surgical removal of the malignant tumor and any affected lymph nodes nearby helps to reduce the risk of metastasis, greatly improving survival. Recent evidence suggests that early intervention therapy can significantly reduce mortality in CRC patients. However, many CRC patients are diagnosed at a time when surgery is not available, and therefore, early diagnosis and early intervention of CRC are of great clinical significance.

Disclosure of Invention

The invention aims to provide a novel colorectal cancer intervention target EDAR, and further provides application of a reagent for determining the expression level of EDAR in preparation of a preparation for colorectal cancer auxiliary diagnosis, colorectal cancer prognosis judgment and colorectal cancer resistance, and a kit for colorectal cancer auxiliary diagnosis, colorectal cancer prognosis judgment and colorectal cancer resistance.

Ecdysone a receptors (EDARs) play an important role in the development of ectodermal tissues such as skin. There are no reports to suggest that EDAR can be used as a molecular marker for prognosis of CRC survival and clinical staging and as a target for therapeutic intervention. The inventor of the invention confirms that the EDAR is highly expressed in colorectal cancer tissues through RNA sequencing data analysis and clinical data research of a large number of samples, the up-regulated expression of the EDAR is related to the colorectal cancer progression and survival prognosis, and the independent silencing of the EDAR can effectively inhibit colorectal cancer neoplasia and greatly reduce the volume and weight of tumors in vivo, so that the EDAR plays an important role in auxiliary diagnosis of colorectal cancer, prognosis judgment and treatment of colorectal cancer.

In order to achieve the above object, the first aspect of the present invention provides the use of a reagent for determining EDAR expression levels for the preparation of a formulation for use in the aided diagnosis of colorectal cancer, the prognostic judgment of colorectal cancer, and anti-colorectal cancer.

The progression of colorectal cancer is usually a progression of disease from benign polyps in the colorectal cancer, to precancerous lesions in the colorectal cancer, to colorectal cancer, and thus, assisted diagnosis of colorectal cancer requires determining at what stage the patient is in the progression of the disease. Preferably, the auxiliary diagnosis of colorectal cancer includes at least one of the following situations:

(a) differentiating colorectal cancer from pre-colorectal cancer lesions;

(b) differentiating colorectal cancer from benign polyps of the colorectal;

(c) colorectal cancer precancerous lesions are distinguished from colorectal benign polyps.

Preferably, the colorectal cancer prognostic judgment includes judgment of survival and/or clinical stages including T stage (tumor primary), N stage (lymph node metastasis) and total stage (case stage). Prognostic indicators are all correlated with EDAR expression levels, and thus determination of EDAR expression levels can be used to aid clinical staging and, more importantly, to predict patient survival.

Preferably, said anti-colorectal cancer agent comprises an agent that reduces EDAR expression levels or binds EDAR sites of action.

Preferably, the means for reducing EDAR expression levels comprises siRNA, shRNA, gene knock-out or antisense oligonucleotides.

Preferably, said determining EDAR expression levels comprises determining protein expression levels of EDAR and/or determining mRNA expression levels of EDAR. The means for measuring the expression level of a protein is usually an immunoassay, and the means for measuring the expression level of mRNA is usually a molecular assay.

Preferably, the reagent for determining the protein expression level of EDAR comprises an antibody specifically recognizing the EDAR protein.

Preferably, protein expression level of EDAR is determined by western blotting, cell fractionation kit is used for nucleoplasm separation, and RIPA buffer solution is used for extracting whole cell lysate.

Preferably, the reagent for determining EDAR mRNA expression level comprises a primer and/or a probe that specifically binds EDAR mRNA.

Preferably, the reagent for determining the mRNA expression level of EDAR is a PCR specific primer with the nucleotide sequence shown in SEQ ID NO. 1 and SEQ ID NO. 2.

5'-GCACCGTCAAGGCTGAGAAC-3'，SEQ ID NO:1

5'-TGGTGAAGACGCCAGTGGA-3'，SEQ ID NO:2

In a second aspect of the invention, a kit for the aided diagnosis, prognosis and resistance of colorectal cancer is provided, comprising reagents for measuring EDAR expression levels.

In a third aspect the invention provides a medicament for inhibiting colorectal cancer proliferation comprising an agent for determining the level of EDAR expression.

The inventor finds that EDAR is highly expressed in colorectal cancer tissues, the up-regulated expression of EDAR is related to colorectal cancer progression and survival prognosis, and meanwhile, the independent silencing of EDAR can effectively inhibit colorectal cancer neoplasia and greatly reduce the tumor volume and weight in vivo. Based on the results, the invention provides the application of the reagent for determining the expression level of EDAR in the preparation of preparations for auxiliary diagnosis of colorectal cancer, prognosis judgment of colorectal cancer and anti-colorectal cancer and a kit for auxiliary diagnosis of colorectal cancer, prognosis judgment of colorectal cancer and anti-colorectal cancer on the one hand, and provides the application of the reagent for determining the expression level of EDAR in the medicines for inhibiting the proliferation of colorectal cancer on the other hand, which has high clinical guidance significance.

Drawings

FIG. 1 shows EDAR expression in paired colon (CC) and Rectal (RC) cancer tissues, where panel A shows mRNA levels; panel B shows protein levels, in which ANT is paracancerous normal tissue; tumor is Tumor tissue.

FIG. 2 is the EDAR expression level in relation to colorectal cancer development, wherein Panel A is an EDAR immunohistochemistry profile at different stages of development; graph B shows EDAR score statistics at different stages of development; graph C is the correlation of N staging with EDAR expression levels; panel D is the correlation of T staging with EDAR expression levels; panel E is the correlation of stage staging with EDAR expression levels; panel F is the overall survival curve for patients with different EDAR expression levels, where L is low expression and H is high expression.

FIG. 3 is a graph of the effect of EDAR silencing on cell proliferation, wherein, graph A is EDARmRNA levels after EDAR silencing in colorectal cancer cells; panel B is EDAR protein levels following EDAR silencing of colorectal cancer cells; FIGS. C and D show EDAR silencing of different colorectal cancer cells followed by cell proliferation; panel E is colony formation following EDAR silencing of colorectal cancer cells; FIG. F shows the non-anchoring growth capacity of colorectal cancer cells after EDAR silencing, wherein vec represents cells transfected with a control plasmid; sh #1 indicates cells transfected with EDAR shRNA # 1; sh #2 indicates cells transfected with EDAR shRNA # 2.

FIG. 4 is a graph of the effect of silencing EDAR on cell cycle, where vec in the graph represents cells transfected with control plasmid, sh #1 represents cells transfected with EDAR shRNA #1, and sh #2 represents cells transfected with EDAR shRNA # 2.

FIG. 5 is a graph of the effect of EDAR silencing in vivo on mouse neoplasia, where Panel A is a tumor appearance graph; panel B shows tumor volume; panel C is tumor weight; panel D shows the staining of tumor tissue, wherein vec represents cells transfected with control plasmid; sh #1 indicates cells transfected with EDAR shRNA # 1; sh #2 indicates cells transfected with EDAR shRNA # 2.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1 EDAR expression in colorectal cancer tissue

Clinical samples of this example were obtained from the central hospital in the Jiangmen city. Clinical samples included 10 pairs of fresh CRC tissues (5 CC, 5 RC) and paracancerous normal tissues collected surgically. mRNA and protein level measurements were performed on the above clinical samples.

mRNA measurement: total RNA was extracted using Trizol reagent (TaKaRa, Japan) using RNA extraction and RT-qPCR. Reverse transcription of RNA Using PrimeScript^TMRT Master Mix (TaKaRa, Japan). Real-time PCR was performed using a premix containing SYBR Green (TaKaRa, Japan) and analysis was performed using an Applied Biosystems 7500 system (ThermoFisher, USA) with GAPDH as an internal reference. GAPDH is a specific primer with the nucleotide sequence shown in SEQ ID NO. 1 and SEQ ID NO. 2.

5'-GCACCGTCAAGGCTGAGAAC-3'，SEQ ID NO:1

5'-TGGTGAAGACGCCAGTGGA-3'，SEQ ID NO:2

Wherein, EDAR has the nucleotide sequence shown in SEQ ID NO. 3 and SEQ ID NO. 4.

5'-CAGCCCGAGCGGAATACTC-3'，SEQ ID NO:3

5'-CCGTAGCCACAGGACAGGTA-3'，SEQ ID NO:4

Protein level determination: the whole Cell lysate was extracted using western blotting, nuclear mass isolation using Cell fractionation kit (Cell Signaling Technology), and RIPA buffer (Cell Signaling Technology). Protein concentration was determined using standard Bradford assay (Beyotime, beijing), 50 μ g of each sample was separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, usa), and an incubation reaction was performed using a primary antibody and an HRP-conjugated secondary antibody. Protein bands were detected using the ECL system and α -tubulin was used as an internal control. Among them, EDAR antibody (ThermoFisher, PA5-99058), α -tubulin antibody (CST, #3873 s).

The results are shown in figure 1, and clinical samples showed that EDAR mRNA levels and protein levels were higher in tumor tissues than in paracancerous normal tissue (ANT) in patients with colon (CC) or Rectal Cancer (RC).

Example 2 EDAR upregulation expression correlates with colorectal cancer development

Clinical samples of this example were obtained from the central hospital in the Jiangmen city. The clinical specimens were paraffin-embedded tissues, including those of colorectal cancer patients and large intestinal polyps, and were classified into three groups, 294 cases of malignant (malignant), 70 cases of Benign (non-cancerous), and 35 cases of precancerous polyps (Precancerosis), respectively. CRC is usually caused by adenomatous polyps characterized by non-cancerous growth of the inner surface of the colon or rectum, which may become cancerous, progress to precancerous and further to carcinoma. EDAR expression was determined by immunohistochemistry on the above different types of tissues.

The immunohistochemical method comprises the following steps: using EnVision^TMThe paraffin-embedded sections were cut with the kit (danish DAKO). Baked at 60 ℃ for 1h, dewaxed in xylene and hydrated in a series of graded alcohols. EDAR antibody (ThermoFisher) was then added, the secondary antibody was incubated at 37 ℃ for 1h, then treated with hematoxylin at room temperature for 30s, and EDAR positive was calculatedThe EDAR protein expression level is comprehensively scored according to the immunohistochemical tumor cell positive rate and the staining depth, two pathologists respectively and independently judge under the double-blind condition, and the average value of the two is taken;<10% of the tumor cells were positive: 1 minute; 10% -35% of the tumor cells are positive: 2 min; 35-70% of the tumor cells are positive: 3 min;>70% of the tumor cells were positive: and 4, dividing. Scoring is then performed according to the overall depth of staining of positive tumor cells: no staining signal was 0 min; the light yellow is 1 minute; the deep yellow is 2 points; the brown-yellow color is 3 points. Finally, the positive rate score was multiplied by the Staining intensity score to obtain 9 levels of immunohistochemical Staining Score (SI) of 0, 1, 2, 3, 4, 6, 8, 9 or 12, wherein SI ═ 4 was calculated as the median of EDAR expression levels in 294 cases of malignancy (i.e., colorectal cancer patients), and the samples with SI ≤ 4 were set as EDAR low-expression groups, and SI was calculated as the median of EDAR low-expression groups>6 was set as EDAR high expression group for dividing colorectal cancer tissue into 2 groups and survival curves were plotted.

EDAR immunohistochemistry maps for different types of tissues are shown in FIG. 2A, immunohistochemical scores for different types of tissues are statistically analyzed, and the results are shown in FIG. 2B, EDAR expression levels are correlated with colorectal cancer development, and malignant tissue samples show higher scores, wherein tissues with SI > 6 are all developed into colorectal cancer or can be used to distinguish colorectal cancer from non-colorectal cancer (including benign colorectal polyps or precancerous lesions of colorectal cancer), and subjects are identified as non-cancerous (including benign colorectal polyps or precancerous lesions of colorectal cancer) when SI ≦ 1. As can be seen from the above results, EDAR can be used to determine the progression of colorectal cancer, the stages of progression including from colorectal polyps, to colorectal precancerous lesions, to the progression of colorectal cancer, and in particular, can distinguish colorectal cancer from colorectal precancerous lesions, colorectal precancerous lesions from colorectal benign polyps, colorectal cancer from colorectal benign polyps, and the like.

Further clinical stage correlation evaluation was performed on colorectal cancer samples classified into EDAR low-and EDAR high-expressing groups, and as a result, as shown in fig. 2C-E, EDAR high-expression was associated with tumor size (T stage), lymph node metastasis (N stage), and case stage (stage) of colorectal cancer, and as can be seen by survival curve analysis, EDAR high-expression predicts worse survival rate (fig. 2F). As can be seen from the above results, EDAR can be used to predict prognosis of survival, including survival prediction and clinical staging. The overall survival rate of the patient can be determined from fig. 2F, providing guidance for improving the course of the patient.

Example 3 silencing EDAR inhibits colorectal cancer cell proliferation

Human colorectal cancer cell lines SW620 and T84 were selected for testing and were purchased from the Shanghai cell institute of Chinese academy of sciences. The cells were cultured in DMEM medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, penicillin G (100U/ml) and streptomycin (100mg/ml) at 37 ℃ with 5% CO₂。

In the above cells, a cell model for silencing EDAR was constructed, plasmid construction and transfection of EDAR shRNA (1# or 2#), and the control vector was designed by GenePharma (suzhou, china).

Wherein shRNA1# is a specific primer with the nucleotide sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6.

5'-CCGGCCTCATCATCATGTTCTACATCTCGAGATGTAGAACATGATGATGAGGTTTTTG-3'，SEQID NO:5

5'-AATTCAAAAACCTCATCATCATGTTCTACATCTCGAGATGTAGAACATGATGATGAGGT-3'，SEQID NO:6

shRNA2# is a specific primer with the nucleotide sequence shown in SEQ ID NO. 7 and SEQ ID NO. 8.

5'-CCGGGCCATTTGATTGCCTCGAGAACTCGAGTTCTCGAGGCAATCAAATGGCTTTTTTG-3'，SEQID NO:7

5'-AATTCAAAAAGCCATTTGATTGCCTCGAGAACTCGAGTTCTCGAGGCAATCAAATGGCT-3'，SEQID NO:8

A plasmid containing the shRNA-encoding gene or a control plasmid was transfected into cells using Lipofectamine 3000(Thermo Fisher, USA). Silencing efficiency was determined by qRT-PCR and Western blotting (same procedure as in example 1). The results of cell model construction for silencing EDAR are shown in fig. 3A and 3B, and 2 shrnas can effectively down-regulate EDAR expression.

Testing cell models for proliferative capacity: cell proliferation capacity was assessed by Cell Counting Kit-8(CCK-8) analysis. Briefly, cells were seeded at a density of 1000 cells per well in 96-well plates containing complete medium. The assay was performed every 24h, 10. mu.L of CCK-8 solution was added to each well and incubated at 37 ℃ for 4 h. CCK-8 absorbance was measured at 450nm and the results are shown in FIGS. 3C and 3D, where the absorbance was lower for 2 shRNAs, indicating that EDAR silencing inhibits the proliferative capacity of colorectal cancer cells.

The cell models were tested for colony forming ability: cells were seeded at a density of 500 cells per well in 6-well plates containing complete medium and cultured at 37 ℃ for 2 weeks in 5% CO2, colonies were washed with PBS and fixed with methanol for 30min, then stained with 0.1% crystal violet, allowed to stand for 30min, photographed and counted by a microscope, and the results are shown in FIG. 3E, where the number of colonies of 2 shRNAs is less, indicating that silencing EDAR inhibits the colony forming ability of colorectal cancer cells.

Testing the non-anchoring growth capacity: 3X 103 cells were seeded into 2mL of complete media containing 0.3% agar, and agar cell cultures were plated on complete media containing 0.6% agar, media was changed every 3 days, photographed and counted after 14 days, and the results are shown in FIG. 3F, where 2 shRNA groups grew smaller, indicating that silencing EDAR inhibits the non-anchoring growth ability of colorectal cancer cells.

Example 4 silencing EDAR induces G1 cycle arrest in colorectal cancer cells

Cell cycle analysis method: cells were washed twice with pre-chilled PBS and fixed with pre-chilled 75% ethanol overnight, then cells were gently resuspended in pre-chilled PBS and ribonuclease was added to degrade RNA and incubated at 37 ℃ for 30min, followed by staining with propidium iodide (Dojindo, japan) at room temperature for 20 min. Cell cycle analysis was performed using a flow cytometer.

The results are shown in FIG. 4, where EDAR silenced cells had an increased subpopulation of G0/G1 cells with a corresponding decrease in S phase, indicating that silencing EDAR induces cell cycle arrest at the G1 stage.

Example 5 silencing EDAR inhibits mouse tumor growth in vivo

Tumor transplantation experiment: female BALB/c nude mice (4-5 weeks old, n ═ 18) were randomly selected and inoculated with 5 × 106 SW620 cells of different models (EDAR silent or control plasmid). Body weight and tumor size of mice were measured 3 times per week and tumor volume was calculated using the following method: volume (width) 2 × length/2. On day 35, after sacrifice of the mice by cervical dislocation, tumor weight was measured and HE staining of tumor tissue was performed.

Results figure 5 shows that silencing EDAR can significantly reduce tumor volume, weight. Wherein the tumor volume decreased by approximately 50% and the tumor weight decreased by approximately 50% at day 35 compared to the control (vector). HE staining results indicated that EDAR silencing decreased the proliferative capacity of cancer cells in mice.

The results show that the EDAR independently silences the colorectal cancer resistance effect. Modulation of EDAR expression levels can prevent or treat colorectal cancer.

Finally, it should be noted that the above examples are only for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred examples, the contents of the preparation raw materials, the preparation process parameters, etc. are not limited to those listed in the examples, and it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> coastal Bay Central Hospital of Dongguan City (Taiping people Hospital of Dongguan city, fifth people Hospital of Dongguan city)

<120> application of reagent for measuring EDAR expression level and kit

<141>2020-07-03

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA/RNA

<213>Artificial Sequence

<400>1

gcaccgtcaa ggctgagaac 20

<210>2

<211>19

<212>DNA/RNA

<213>Artificial Sequence

<400>2

tggtgaagac gccagtgga 19

<210>3

<211>19

<212>DNA/RNA

<213>Artificial Sequence

<400>3

cagcccgagc ggaatactc 19

<210>4

<211>20

<212>DNA/RNA

<213>Artificial Sequence

<400>4

ccgtagccac aggacaggta 20

<210>5

<211>58

<212>DNA/RNA

<213>Artificial Sequence

<400>5

ccggcctcat catcatgttc tacatctcga gatgtagaac atgatgatga ggtttttg 58

<210>6

<211>59

<212>DNA/RNA

<213>Artificial Sequence

<400>6

aattcaaaaa cctcatcatc atgttctaca tctcgagatg tagaacatga tgatgaggt 59

<210>7

<211>59

<212>DNA/RNA

<213>Artificial Sequence

<400>7

ccgggccatt tgattgcctc gagaactcga gttctcgagg caatcaaatg gcttttttg 59

<210>8

<211>59

<212>DNA/RNA

<213>Artificial Sequence

<400>8

aattcaaaaa gccatttgat tgcctcgaga actcgagttc tcgaggcaat caaatggct 59

Claims

1. Use of a reagent for determining the level of EDAR expression for the preparation of a formulation for use in the aided diagnosis, prognosis and anti-colorectal cancer of colorectal cancer.

2. Use according to claim 1, wherein said assisted diagnosis of colorectal cancer comprises at least one of:

(a) differentiating colorectal cancer from pre-colorectal cancer lesions;

(b) differentiating colorectal cancer from benign polyps of the colorectal;

3. Use according to claim 1, wherein said colorectal cancer prognostic assessment comprises a determination of survival and/or clinical staging, including T-staging, N-staging and total stage staging.

4. Use according to claim 1, characterized in that the anti-colorectal cancer agent comprises an agent that reduces EDAR expression levels or an agent that binds EDAR sites of action.

5. The use according to claim 4, wherein the means for reducing the level of EDAR expression comprises siRNA, shRNA, knock-out or antisense oligonucleotides.

6. The use according to claim 1, wherein said determining the EDAR expression level comprises determining the protein expression level of EDAR and/or determining the mRNA expression level of EDAR.

7. The use according to claim 6, wherein the reagent for determining protein expression levels of EDAR comprises an antibody specifically recognizing the EDAR protein.

8. Use according to claim 6, characterized in that the reagent for determining the mRNA expression level of EDAR comprises primers and/or probes that specifically bind to EDAR's mRNA.

9. A kit for the aided diagnosis, prognosis and resistance of colorectal cancer, which is characterized by comprising a reagent for measuring the expression level of EDAR.

10. A medicament for inhibiting the proliferation of colorectal cancer comprising an agent for determining the level of EDAR expression.