CN113817824A

CN113817824A - Tumor gene diagnostic marker combined with carcinoembryonic antigen and its use

Info

Publication number: CN113817824A
Application number: CN202111144757.4A
Authority: CN
Inventors: 不公告发明人
Original assignee: Nanjing Fanyida Biotechnology Co ltd
Current assignee: Nanjing Fanyida Biotechnology Co ltd
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2021-12-21

Abstract

The invention discloses a tumor gene diagnostic marker combined with carcinoembryonic antigen and its application. The invention improves the screening accuracy of colorectal cancer by combining miR-609 and carcinoembryonic antigen, and overcomes the deficiency of low accuracy of carcinoembryonic antigen alone for screening colorectal cancer. Therefore, miR-609 can be combined with carcinoembryonic antigen to prepare a serum detection kit for early diagnosis of colorectal cancer.

Description

Tumor gene diagnosis marker combined with carcinoembryonic antigen and application thereof

Technical Field

The invention belongs to the field of disease diagnosis, and particularly relates to a tumor gene diagnosis marker combined with a carcinoembryonic antigen and application thereof.

Background

Colorectal cancer (CRC) is one of the common malignant tumors worldwide, and the worldwide CRC age-related morbidity is estimated to be 19.5/10 ten thousand and the mortality is estimated to be 9.0/10 ten thousand in 2020. CRC has a high incidence in developed countries, with an age-normalized incidence of CRC of about 38.7/10 ten thousand in the United states for the last 5 years, and a mortality rate of 13.9/10 ten thousand. Practical experience in many countries has shown that the disease burden of colorectal cancer can be reduced by adopting an effective colorectal cancer screening strategy (current research and thinking of colorectal cancer screening strategy, journal of general medical science in china, 09 months in 2021).

Carcinoembryonic antigen (CEA) as one of CRC screening indexes is a tumor-associated antigen first extracted from colon cancer and embryonic tissue by Gold and Freedman in 1965, is an acid glycoprotein having characteristics of a human embryonic antigen, exists on the surface of cancer cells differentiated from endoderm cells, and is a structural protein of cell membranes. Formed in the cytosol, secreted outside the cell through the cell membrane and then into the surrounding body fluids. Therefore, the reagent can be detected from various body fluids and excretions such as serum, cerebrospinal fluid, milk, gastric juice, hydrothorax, ascites, urine, feces and the like. CEA plays an important clinical value in the aspects of differential diagnosis, disease condition monitoring, curative effect evaluation and the like of colorectal cancer.

Alpha-fetoprotein (AFP) is a tumor marker commonly used for tumor screening, and is widely used for diagnosis of tumors such as liver cancer.

However, CEA or AFP are not accurate for colorectal cancer diagnosis. The invention is especially proposed in order to improve the accuracy of the CEA or AFP for colorectal cancer diagnosis.

Disclosure of Invention

The invention provides a tumor gene diagnosis marker combined with carcinoembryonic antigen and application thereof in order to overcome the defects of the prior art.

The technical scheme of the invention is as follows:

the miR-609 and carcinoembryonic antigen are combined to prepare the serum detection kit for early diagnosis of colorectal cancer.

A serum detection kit for early diagnosis of colorectal cancer contains a reagent for detecting serum miR-609 and carcinoembryonic antigen.

The beneficial technical effects are as follows:

according to the invention, the screening accuracy of the colorectal cancer is improved by combining miR-609 and carcinoembryonic antigen, and the defect of low screening accuracy of the carcinoembryonic antigen when being singly used for the colorectal cancer is overcome. Therefore, miR-609 can be combined with carcinoembryonic antigen to prepare a serum detection kit for early diagnosis of colorectal cancer.

Drawings

FIG. 1 is a comparison of the content of CEA, AFP, miR-609 and miR-3934-5p in the serum of colorectal cancer and healthy human; wherein miR-609 and miR-3934-5p are determined by agarose gel electrophoresis, the internal parameter is beta-Actin, 40 rectal cancer samples or healthy population samples in a training set are randomly divided into four parts, 10 samples in each part are mixed into one part, A, C, E, G in an electrophoresis result respectively corresponds to the mixed samples of the healthy population, and B, D, F, H respectively corresponds to the mixed samples of the rectal cancer;

FIG. 2 is a ROC curve for CEA single diagnosis in training set to differentiate colorectal cancer from healthy persons;

FIG. 3 is a ROC curve for AFP alone diagnosis in training sets to differentiate colorectal cancer from healthy persons;

FIG. 4 is a ROC curve for CEA combined miR-609 diagnosis in training set to distinguish colorectal cancer from healthy people;

FIG. 5 is a ROC curve for CEA combined miR-3934-5p diagnosis in training set to distinguish colorectal cancer from healthy people;

FIG. 6 is a ROC curve for AFP in combination with miR-609 diagnosis in a training set to distinguish colorectal cancer from healthy people;

FIG. 7 is a ROC curve for AFP in combination with miR-3934-5p diagnosis in training sets to distinguish colorectal cancer from healthy people.

Detailed Description

The following examples are intended to illustrate the essence of the present invention, but should not be construed as limiting the scope of the present invention.

First, test materials

Total RNA extraction kits were purchased from Invitrogen, USA. Reverse transcription kits were purchased from Thermo Fisher Scientific. RT-PCR measuring instruments were purchased from Qiagen, Germany. RT-PCR primers were synthesized by Chiman Biotech (Shanghai) Ltd.

Second, test method

1. General data

Colorectal cancer sample: 120 patients with colorectal cancer diagnosed in 2018-2020 of the first subsidiary hospital of Nanjing medical university are selected; healthy population samples: 120 healthy people are selected from first subsidiary hospitals 2018-2020 of Nanjing medical university. The age distributions of the two groups of samples have high similarity, P is more than 0.05, and the statistical significance is avoided.

Grouping standard: firstly, the examination is verified by imaging examination or pathological examination such as Computed Tomography (CT); ② before detection, radiotherapy, chemotherapy and other anti-tumor treatments are not carried out; age > 18 years. Exclusion criteria: combining malignant tumors of other parts; ② women in gestation or lactation; and thirdly, diseases of the immune system, diseases of the blood system and the like are combined.

2. Sample grouping

Training set: respectively randomly selecting 40 samples from colorectal cancer samples and healthy population samples to form a training set, wherein the total number of the samples is 80; and (4) verification set: the remaining colorectal cancer samples and healthy population samples constitute a validation set, and the total number of samples is 160.

3. Serum CEA, AFP assay

Extracting 5mL venous blood under fasting condition, centrifuging at 2500r/min for 5min, separating serum, and determining CEA and AFP concentration strictly according to the instruction by using a full-automatic biochemical analyzer and a matched original reagent.

4. Determination of expression levels of miR-609 and miR-3934-5p in serum

5mL venous blood is extracted under the condition of fasting, centrifugation treatment is carried out for 5min at the rotating speed of 2500r/min, serum is separated, and total RNA is extracted by using a total RNA extraction kit. cDNA was synthesized in reverse transcription kit from 2. mu.g of total RNA. Putting cDNA into PCR amplification instrument for amplification, with the reaction condition of 94 ℃ for 15min for pre-denaturation, 60 ℃ for 30s for annealing, 72 ℃ for 30s for extension, and 40 cycles. The Ct value of the PCR product of the target gene and GAPDH was determined by relative quantitation method, and the Ct value was calculated as 2^-ΔΔCtCalculating the relative expression quantity of the target gene, setting 3 multiple wells respectively, and repeating the experiment for 3 times.

miR-609 forward primer sequence: 5'-CGACGTGCACTGCACCAGC-3', respectively;

miR-609 reverse primer sequence: 5'-AGTCACTGCACAGTCAC-3', respectively;

miR-3934-5p forward primer sequence: 5'-GCCAGCTCCTACATCTCAGC-3', respectively;

miR-3934-5p reverse primer sequence: 5'-TGACTACCAGGTTTGAAGA-3', respectively;

GAPDH forward primer sequence: 5'-TGATGACATCAAGAAGGTGGTGAAG-3', respectively;

GAPDH reverse primer sequence: 5'-TCCTTGGAGGCCCAGTGGGCCAT-3' are provided.

5. Statistical treatment

The data is processed by adopting SPSS 19.0, all the measurement data are subjected to the homogeneity test of the normal and the variance, the data conforming to the normal distribution are represented by the deviation of the mean value +/-s, the measurement data not conforming to the homogeneity test of the normal and the variance are subjected to the nonparametric test, and the measurement data are subjected to the t test in pairwise comparison among groups. And drawing a Receiver Operating Characteristic (ROC) curve of the diagnosis and the distinction of the colorectal cancer and the healthy human by adopting SPSS 19.0 and serum CEA or AFP alone and combined serum miR-609 or miR-3934-5p, calculating the area under the curve (AUC), and evaluating the diagnosis and the distinction value of the CEA or AFP alone and combined serum miR-609 or miR-3934-5p to the colorectal cancer and the healthy human by using the AUC. AUC is less than or equal to 0.5, the diagnosis accuracy is lower when the AUC is more than 0.5 and less than or equal to 0.7, the diagnosis accuracy is better when the AUC is more than 0.7 and less than or equal to 0.9, the diagnosis accuracy is the highest when the AUC is more than or equal to 0.9, and the difference is statistically significant when P is less than 0.05.

Third, test results

1. Content comparison of CEA, AFP, miR-609 and miR-3934-5p in serum of colorectal cancer and healthy human

In training set, the content of CEA, AFP, miR-609 and miR-3934-5p in the serum of colorectal cancer and healthy human is obviously up-regulated, and the up-regulation times are shown in Table 1 and figure 1.

TABLE 1 fold-upregulation of target markers in colorectal cancer serum relative to healthy humans

	CEA	AFP	miR-609	miR-3934-5p
					Colorectal cancer/healthy person	4.59±1.82	5.17±2.35	6.25±2.18	5.87±1.94

2. ROC curve analysis in training set

In the training set, taking the CEA content of each sample as an independent variable X, taking colorectal cancer and healthy person as dependent variables, and performing binary logistic regression on the CEA content in the colorectal cancer and healthy person samples by using SPSS software to obtain a binary logistic regression equation: ln^[p/(1-p)]Substituting the CEA content of each sample into the binary logistic regression equation to obtain the p value of each serum sample, calculating the sensitivity and specificity by using the possible p values as diagnosis points, and plotting ROC curves, as shown in fig. 2 and table 2, where AUC is 0.783, the sensitivity is 71.2%, and the specificity is 67.4%.

In the training set, taking the AFP content of each sample as an independent variable X, taking colorectal cancer and healthy people as dependent variables, and performing binary logistic regression on the AFP content in the colorectal cancer and healthy people samples by using SPSS software to obtain a binary logistic regression equation: ln^[p/(1-p)]Substituting the AFP content in each sample into the binary logistic regression equation to obtain the p value of each serum sample, calculating the sensitivity and specificity by using the possible p value as a diagnosis point, drawing an ROC curve, as shown in fig. 3 and table 2, wherein AUC is 0.749, the sensitivity is 77.5%, and the specificity is 64.1%.

In the training set, the CEA content of each sample is taken as an independent variable X₁The relative content of miR-609 is an independent variable X₂And carrying out binary logistic regression on the contents of CEA and miR-609 in the colorectal cancer and healthy human samples by using SPSS software according to the group of colorectal cancer and healthy human dependent variables to obtain a binary logistic regression equation: ln^[p/(1-p)]＝3.02X₁+2.77X₂-5.93, substituting the contents of CEA and miR-609 in each sample into the binary logistic regression equation to obtain a p value of each serum sample, calculating the sensitivity and specificity by using the possible p value as a diagnosis point, and drawing an ROC curve, as shown in fig. 4 and table 2, where AUC is 0.912, the sensitivity is 83.6%, and the specificity is 80.2%.

In the training set, the CEA content of each sample is taken as an independent variable X₁The relative content of miR-3934-5p is an independent variable X₂According to the colorectal cancer and healthy human response of the group, SPSS software is used for treating CEA and miR-3934-5p in the colorectal cancerPerforming binary logistic regression on the contents in the cancer and healthy human samples to obtain a binary logistic regression equation: ln^[p/(1-p)]＝2.86X₁+2.39X₂-3.70, substituting the contents of CEA and miR-3934-5p in each sample into the binary logistic regression equation to obtain the p value of each serum sample, calculating the sensitivity and specificity by using the possible p value as a diagnosis point, and drawing an ROC curve, as shown in fig. 5 and table 2, wherein AUC is 0.805, the sensitivity is 70.5%, and the specificity is 75.7%.

Training centralization, using AFP content of each sample as independent variable X₁The relative content of miR-609 is an independent variable X₂Carrying out binary logistic regression on the contents of AFP and miR-609 in colorectal cancer and healthy person samples by using SPSS software according to the group of colorectal cancer and healthy person dependent variables to obtain a binary logistic regression equation: ln^[p/(1-^p)]＝4.13X₁+2.06X₂And (4) substituting the content of AFP and miR-609 in each sample into the binary logistic regression equation to obtain the p value of each serum sample, calculating the sensitivity and specificity by taking the possible p value as a diagnosis point, drawing an ROC curve, and as shown in FIG. 6 and Table 2, the AUC is 0.692, the sensitivity is 66.8%, and the specificity is 67.5%.

Training centralization, using AFP content of each sample as independent variable X₁The relative content of miR-3934-5p is an independent variable X₂Carrying out binary logistic regression on the contents of AFP and miR-3934-5p in colorectal cancer and healthy person samples by using SPSS software according to the group of colorectal cancer and healthy person artificial variables to obtain a binary logistic regression equation: ln^[p/(1-p)]＝3.45X₁+3.18X₂-4.03, substituting the AFP and miR-3934-5p contents in each sample into the binary logistic regression equation to obtain the p value of each serum sample, calculating the sensitivity and specificity by taking the possible p value as a diagnosis point, and drawing an ROC curve, as shown in fig. 7 and table 2, wherein AUC is 0.923, the sensitivity is 87.6%, and the specificity is 82.4%.

TABLE 2 diagnostic efficacy of different markers for colorectal cancer

As known to those skilled in the art, AUC is less than or equal to 0.5, the diagnosis accuracy is lower when AUC is more than 0.5 and less than or equal to 0.7, the diagnosis accuracy is better when AUC is more than 0.7 and less than or equal to 0.9, and the diagnosis accuracy is the highest when AUC is more than or equal to 0.9. The above results show that: the diagnosis efficiency of the CEA or AFP used for diagnosing the colorectal cancer independently is general, the combined use of the miR-609 and the CEA can obviously improve the diagnosis efficiency of the CEA for diagnosing the colorectal cancer, and the combined use of the miR-3934-5p and the AFP can obviously improve the diagnosis efficiency of the AFP for diagnosing the colorectal cancer. miR-609 has no obvious gain effect on the diagnosis efficiency of AFP for diagnosing colorectal cancer, and miR-3934-5p also has no obvious gain effect on the diagnosis efficiency of CEA for diagnosing colorectal cancer.

3. Validating diagnostic accuracy of centrally validated optimal combinations

The verification is centralized, and the CEA content (X) in each sample is determined₁) Relative content of miR-609 (X)₂) Substituting into the above-mentioned binary logistic regression equation Ln^[p/(1-p)]＝3.02X₁+2.77X₂-5.93, obtaining the regression prediction probability p of each serum sample, wherein the prediction of colorectal cancer which is higher than the optimal cut-off value by 0.524 is contrary to the prediction of healthy people, and the result diagnosis accuracy is 89.4% (143/160).

Verifying and centralizing, namely determining the AFP content (X) in each sample₁) miR-3934-5p relative content (X)₂) Substituting into the above-mentioned binary logistic regression equation Ln^[p/(1-p)]＝3.45X₁+3.18X₂4.03, obtaining the regression prediction probability p of each serum sample, wherein the prediction of colorectal cancer which is higher than the optimal cut-off value by 0.561 is the prediction of healthy people, and the result diagnosis accuracy rate is 91.3 percent (146/160).

In conclusion, miR-609 can be combined with CEA to prepare a serum detection kit for early diagnosis of colorectal cancer, and miR-3934-5p can be combined with AFP to prepare a serum detection kit for early diagnosis of colorectal cancer.

Claims

The application of miR-609 combined carcinoembryonic antigen in preparing a serum detection kit for early diagnosis of colorectal cancer.
2. A serum detection kit for early diagnosis of colorectal cancer is characterized in that: contains a reagent for detecting miR-609 and carcinoembryonic antigen in serum.