CN108061803B

CN108061803B - Liver cancer serum marker CCT3

Info

Publication number: CN108061803B
Application number: CN201810133870.4A
Authority: CN
Inventors: 王佳谊; 张骁; 刘雅; 孙奋勇; 陈岩; 潘秋辉
Original assignee: Shanghai Tenth Peoples Hospital
Current assignee: Shanghai Tenth Peoples Hospital
Priority date: 2018-02-09
Filing date: 2018-02-09
Publication date: 2020-07-31
Anticipated expiration: 2038-02-09
Also published as: CN108061803A

Abstract

The invention relates to a new application of CCT3, namely, a liver cancer serum marker, which has the advantages that the expression of CCT3 in liver cancer is regulated and controlled by YAP, the serum CCT3 level of a liver cancer patient is higher, the higher CCT3 concentration is obviously related to the higher tumor stage in the liver cancer, the serum CCT3 level of the liver cancer patient is in positive correlation with alpha fetoprotein AFP, glutamic pyruvic transaminase A L T and glutamic oxalacetic transaminase AST, the invention discovers that the optimal diagnosis threshold value of serum CCT3 for predicting the liver cancer is 79pg/ml, the sensitivity is 94.5 percent, the specificity is 97.6 percent, and the serum CCT3 is a tumor marker superior to AFP in the liver cancer.

Description

Liver cancer serum marker CCT3

Technical Field

The invention relates to the technical field of inspection medicine, in particular to a novel liver cancer serum marker.

Background

Te chaperone protein (CCT), containing TCP1 complex, mediates protein folding in the cytoplasm. Chaperonins are the ATP-dependent protein folding machinery present in all organisms. They consist of two oligo rings stacked back-to-back, with a cavity at each end, in which protein substrate binding and folding occurs. The Te chaperone protein family comprises mitochondrial heat shock protein 60, plastid Rubisco subunit binding protein, archaebacterium group II chaperone protein and the like. CCT3 is a key component in CCT complexes that plays an important role during protein folding or refolding. We analyzed by bioinformatics prediction that CCT3 is likely to be a key target protein of the liver cancer oncogenic protein YAP. However, whether CCT3 can be used as a novel serum tumor marker in liver cancer is still unknown.

Currently, the detection of tumor markers in serum, such as alpha-fetoprotein (AFP), has been used for early diagnosis of tumors, assessment of prognosis, and observation of recurrence and metastasis of therapeutic effects. Chinese patent document CN107604062A discloses a multi-antigen detection method for liver cancer immunotherapy and discloses that CCT3 is a liver cancer tumor antigen gene. However, the CCT3 of the invention is not reported as a serum tumor marker of liver cancer at present.

Disclosure of Invention

The first purpose of the invention is to provide a new application of CCT3 aiming at the defects in the prior art.

The second purpose of the invention is to provide a kit for diagnosing or staging liver cancer.

In order to achieve the first purpose, the invention adopts the technical scheme that: CCT3 protein is used as the diagnosis marker of liver cancer.

CCT3 protein is used as serum marker of liver cancer.

CCT3 protein is used as a marker in the preparation of detection products for diagnosing liver cancer.

CCT3 protein is used as a marker in the preparation of detection products for liver cancer staging.

CCT3 protein as a serum marker.

The detection product detects serum.

The detection product is a diagnostic kit.

The CCT3 protein serves as the only marker.

In order to achieve the second object, the invention adopts the technical scheme that: a kit for diagnosing or staging liver cancer comprises a reagent for specifically detecting serum CCT3 protein.

The invention has the advantages that:

1. the invention discovers that in liver cancer, the expression of CCT3 is regulated and controlled by YAP, the serum CCT3 level of a liver cancer patient is higher, the higher CCT3 concentration is obviously related to the higher tumor stage in the liver cancer, and the serum CCT3 level of the liver cancer patient is in positive correlation with alpha fetoprotein AFP, glutamic-pyruvic transaminase A L T and glutamic-oxalacetic transaminase AST.

2. The serum CCT3 is found to have the optimal diagnosis threshold value of 79pg/ml for liver cancer prediction, the sensitivity of 94.5% and the specificity of 97.6%, which indicates that the serum CCT3 is a tumor marker superior to AFP in liver cancer.

Drawings

FIG. 1: CCT3 is regulated by liver cancer promoting protein YAP.

FIG. 2 is a drawing: CCT3 expression specificity is increased in liver cancer. In fig. 2B, p is 0.000 compared to the liver cancer group.

FIG. 3 shows that serum CCT3 is positively correlated with AFP, A L T and AST.

FIG. 4 is a drawing: serum CCT3 liver cancer diagnostic performance is better than AFP.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Specimen collection

All specimens were collected from 3 months to 3 months of 2017 in Renjin Hospital 2015, mean age of 55.27 + -7.73 years for liver cancer patients, mean age of 2.34:1 for men and women, mean age of 37.46 + -6.43 years for hepatitis B patients, mean age of 1.35:1 for men and women, mean age of 57.37 + -10.93 years for hepatitis C patients, mean age of 1.31:1 for men and women, mean age of 57.64 + -9.69 years for liver cirrhosis patients, mean age of 2.39:1 for men and women, mean age of 55.45 + -13.58 years for colon cancer patients, mean age of 3.41:1 for men and women, mean age of 51.52 + -2.54 years for lung cancer patients, mean age of 4.4:1 for men and women, mean age of 57.12 + -10.44 years for stomach cancer patients, mean age of 3.8:1 for men and mean age of 37.66 + -15.61 for breast cancer patients, all female patients were informed about to obtain informed scan and magnetic resonance imaging results to confirm that liver cancer patients had more than liver cancer and liver cancer patients were diagnosed by clinical diagnosis by clinical analysis, and liver cancer diagnosis by clinical analysis³Copied hepatitis b or hepatitis c virus DNA (kowa, shanghai, china) while 200 healthy volunteers (average age 54.42 ± 14.08 years, male-female ratio 1.31:1) were recruited as controls in the shanghai ruijin hospital from 2015 to 2017, metastatic liver tumor patients were not included in the study, liver function indices glutamic-oxalacetic transaminase AST and glutamic-pyruvic transaminase a L t were determined using an automated biochemical analyzer, the study protocol was performed according to the ethical guidelines announced by helsinki 1975.

Cell purchase

Both hepatoma cell lines and normal hepatocytes were purchased from the cell bank of the Chinese academy of sciences.

Immunoblotting experiments

The cell protein lysate was separated in an SDS polyacrylamide gel and transferred onto nitrocellulose membrane. Membranes were blocked in blocking buffer (5% milk in Tris buffered saline with 0.5% tween) and then incubated with specific antibodies. The primary antibodies used were anti-GAPDH [ Cell Signaling Technology (CST), Boston, MA, #5174], anti-TEAD (Abcam, # ab197589), anti-CCT 3(Abcam, # ab225878), anti-YAP (Abcam, # ab52771), anti-TFCP 2(Abcam, # ab 80445).

Enzyme-linked immunosorbent assay (E L ISA)

An enzyme-linked immunosorbent assay was performed to assess the concentration of serum AFP and CCT3 serum samples were diluted in dilution buffer (1: 4) and 50 μ Ι of the diluted samples/well were added to a 96-well microtiter plate for AFP and CCT3 concentration analysis the E L ISA kit was purchased from L ichen Biotech ltd (shanghai, china).

Statistical analysis

Continuous variable differences between groups were compared using independent sample t-test, analysis of variance, Kruskal-Wallis test. Spearman rank correlation tests were performed to calculate the correlation coefficients between the rank variables. Area under the ROC curve (AUC-ROC) was used to assess CCT3, AFP and the combination of CCT3 and AFP predicted the diagnostic value of liver cancer. We also determined the diagnostic threshold by ROC curve. P <0.05 was considered statistically significant.

Example 1: expression of CCT3 is regulated by YAP

After overexpression of YAP in the highly carcinogenic hepatoma cell line Bel-7402, we found a significant increase in expression level of CCT3 (FIG. 1A). Furthermore, we found that expression levels of CCT3 were significantly decreased after knocking down YAP in the hepatoma cell lines Bel-7402 and SMMC-7721 (fig. 1B). We also found an interaction between YAP and CCT3 (fig. 1C). It was demonstrated that in liver cancer, CCT3 expression is regulated by YAP.

Example 2: the liver cancer patient has high serum CCT3 level

Serum samples from liver cancer patients and healthy individuals were analyzed for protein expression levels using immunoblotting. anti-CCT 3 antibody a specific band of 61kDa was found in serum samples from liver cancer patients, and no similar band was detected in healthy individuals (fig. 2A). We did not detect YAP and its associated other proteins such as TEAD and TFCP2 in serum samples, demonstrating that although these YAP-associated proteins play a critical role in liver cancer development, they could not be used as liver tumor markers. We also measured the concentration of CCT3 in the serum of healthy people and other liver diseases (including hepatitis b/c and cirrhosis), other types of digestive cancers (including colon and gastric cancers) and breast and lung cancer patients to confirm whether CCT3 is specifically elevated in the serum of liver cancer patients. The results show that the serum CCT3 concentration (705.464 ± 660.223pg/ml, n ═ 204) of the liver cancer patient is significantly higher than that of healthy individuals (38.442 ± 12.041pg/ml, n ═ 200, p ═ 0.000), hepatitis B patients (43.322 ± 19.105pg/ml, n ═ 53, p ═ 0.000), hepatitis c patients (49.409 ± 17.027pg/ml, n ═ 56, p ═ 0.000), cirrhosis patients (35.414 ± 20.112pg/ml, n ═ 53, p ═ 0.000), gastric cancer patients (42.475 ± 21.607pg/ml, n ═ 52, p ═ 0.000), colon cancer patients (43.844 ± 2pg/ml, n ═ 50, p ═ 0.000), breast cancer patients (33.010 ± 17.241pg/ml, n ═ 51 ═ 0.000, p ═ 51 ═ 92 ═ 2/ml, and lung cancer patients (graph B51 ═ 2). In addition, higher CCT3 concentrations were significantly associated with higher tumor stages in liver cancer (fig. 2C).

Example 3: relation between serum CCT3 level of liver cancer patient and liver function index

Serum alpha-fetoprotein (AFP) is a classical marker of liver cancer, the scatter distribution of serum CCT3 and serum AFP demonstrates a positive correlation between CCT3 and AFP (R ═ 0.720, P ═ 0.000) (fig. 3A), and in addition, there is a clear positive correlation between CCT3 and important liver function indices glutamic-pyruvic transaminase a L T (R ═ 0.511, P ═ 0.000) (fig. 3B) and glutamic-oxalacetic transaminase AST (R ═ 0.372, P ═ 0.001) (fig. 3C).

Example 4: serum CCT3 is a tumor marker superior to AFP in liver cancer

AUC-ROC analysis showed that serum CCT3 (AUC: 0.971) was a better diagnostic marker for liver cancer than AFP (AUC: 0.846). The optimal diagnostic threshold for predicting liver cancer was 79pg/ml, sensitivity was 94.5%, and specificity was 97.6% (fig. 4). AUC-ROC analysis also showed that CCT3 combined with AFP diagnosed liver cancer (AUC: 0.984) only slightly better than CCT 3. These results indicate that CCT3 alone is promising for clinical applications as a liver tumor marker.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

The application of CCT3 protein as a marker in the preparation of a detection product for specifically identifying liver cancer and any one of the following diseases, wherein the diseases are selected from the following: hepatitis B, hepatitis C, gastric cancer, colon cancer, breast cancer and lung cancer, and the CCT3 protein is used as a unique marker.
2. The use according to claim 1, characterized in that CCT3 protein is used as serum marker.
3. The use of claim 1, wherein the test product is a test serum.
4. The use of claim 1, wherein the test product is a diagnostic kit.