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Applications of BCKDK as biomarker in preparation of tumor diagnosis kits

A biomarker, tumor diagnosis technology, applied in the preparation of test samples, instruments, measuring devices, etc., can solve the problem of unclear relationship between tumors and so on

Inactive Publication Date: 2017-11-17
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that BCKDK is closely related to Huntington's dance syndrome [30], maple sugar diabetes [23,31], autism with epilepsy [32] and obesity [33], but the relationship with tumors is still unclear

Method used

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  • Applications of BCKDK as biomarker in preparation of tumor diagnosis kits
  • Applications of BCKDK as biomarker in preparation of tumor diagnosis kits
  • Applications of BCKDK as biomarker in preparation of tumor diagnosis kits

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1 Confirmation that BCKDK promotes the occurrence of colon cancer.

[0089] 1) BCKDK is generally highly expressed in colorectal cancer cell lines

[0090] Six colorectal cancer cell lines including DLD1, HCT8, SW480, WiDr, HCT116, HCT15 and one human normal intestinal epithelial cell line were cultured in vitro, the total protein was extracted, and the expression level of BCKDK was detected by Western blotting. The results showed that BCKDK was generally highly expressed in colorectal cancer cell lines. (See figure 1 )

[0091] 2) Establish cell lines overexpressing BCKDK and silencing BCKDK, and observe changes in cell phenotype

[0092] Using simpleFect (purchased from Wuhan Signal Shuguang Biotechnology Co., Ltd.) to transfect pCMV-c-Flag and pCMV-BCKDK-Flag eukaryotic expression plasmids into JB6Cl41 and WiDr cells with low expression of BCKDK, screened with G418 to obtain JB6-Mock , JB6-BCKDK, WiDr-Mock and WiDr-BCKDK stable cell lines.

[0093] And c...

Embodiment 2

[0097] Example 2 It is clear that the high expression of BCKDK in colon cancer is related to the poor prognosis of colorectal cancer patients.

[0098] After strict screening by pathologists, clinical tissue samples from patients with confirmed colon cancer were selected and made into tissue chips. Immunohistochemical methods were used to analyze the expressions of t-BCKDHA, p-BCKDHA, and BCKDK (scored according to the Remmele scoring method [35], If the score is less than 3, it is negative, and if it is greater than or equal to 3, it is positive), and a follow-up investigation is carried out on each group of patients, and the correlation analysis of survival period is carried out.

[0099] The main steps of immunohistochemical staining (IHC):

[0100] 1) Place the prepared paraffin sections in an oven at 60°C for 30-60 minutes. After dewaxing to washing, the slices were dewaxed in xylene I and II in turn for 15 minutes each, then placed in absolute ethanol I and II for 10 mi...

Embodiment 3

[0118] Example 3 Determining that BCKDK activates the MEK-ERK signaling pathway to promote the occurrence of colorectal cancer

[0119] 1) Overexpression of BCKDK activates MEK and ERK.

[0120] BCKDK overexpressed cells were stimulated with EGF (20 ng / mL for normal cells and 80 ng / mL for tumor cells), a common reagent used in the study of tumor transformation-related signaling pathways, and the total protein was extracted immediately, and MEK, p-MEK, ERK and p-MEK were detected by western blot. - Expression level of ERK. see results Figure 8 , in JB6Cl41 and WiDr cells overexpressing BCKDK, no matter whether they were stimulated with EGF, the levels of p-MEK and p-ERK were higher than those of the control group, and the increase was more obvious after EGF stimulation, while the levels of t-MEK and t-ERK Levels did not differ between groups. The results indicated that BCKDK could activate MEK-ERK signaling pathway.

[0121] 2) Silence BCKDK-inhibited phosphorylation of ME...

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Abstract

The invention discloses the downstream direct interaction molecule of BCKDK, the phosphorylation modification site of BCKDK, and applications of BCKDK as a biomarker in preparation of tumor diagnosis kits, and belongs to the field of biology and disease diagnosis. According to the present invention, the new function and the specific mechanism of the BCKDK molecule are provided, the specific phosphorylation modification site and the corresponding substrate are defined, and the existing BCKDK antibody is used to directly detect clinical tissue specimens so as to develop new uses; and currently the clinical application for the BCKDK molecular detection does not exist on the market, and with the applications of the present invention, the solid foundation is established for the early diagnosis and the prognosis prediction and treatment of tumors, especially colorectal cancer and glioma.

Description

technical field [0001] The invention belongs to the field of biology and disease diagnosis, and specifically relates to BCKDK molecular detection and the development and application of anti-BCKDK antibody in a tumor diagnosis kit. Background technique [0002] The incidence of colon cancer ranks third among male cancers and second among female cancers in the world, and there are as many as 1.4 million new patients with colorectal cancer every year [1]. Analysis of colorectal cancer data in the United States in 2017 showed that the incidence of colorectal cancer was highest among Alaska Natives and blacks, and lowest among Asians and Pacific Islanders [2]. In my country, the mortality rate of colorectal cancer ranks fifth in the tumor mortality rate, and the mortality rate of colorectal cancer is the fastest rising in the male age-standardized cancer mortality rate [3]. In addition, although colorectal cancer is the same as other tumors It is considered as a kind of geriatric...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30G01N1/44G01N33/574
CPCG01N1/30G01N1/44G01N33/57446
Inventor 朱峰段秋红薛佩佩王哲晏伟肖娟娟范林妮全纯涛刘琳袁萍柯昌庶路慧田琴张建民
Owner HUAZHONG UNIV OF SCI & TECH
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