阅读说明：本技术 肝癌的肿瘤标志物热休克因子2结合蛋白及其应用 (Tumor marker heat shock factor 2binding protein of liver cancer and application thereof ) 是由 吴荣谦 杜肇清 吕毅 于 2019-12-09 设计创作，主要内容包括：本发明为热休克因子2结合蛋白(HSF2BP)提供了一种新的生物医药用途,即HSF2BP作为一种肝癌肿瘤标志物及应用。本发明公开了HSF2BP mRNA和蛋白的定量检测在制备作为肝癌肿瘤诊断或预后监测试剂中的应用,特别是指HSF2BP mRNA或蛋白表达增高作为肝癌肿瘤的预测指标或预后不良的指标。本发明通过实时定量PCR、Western Blot法及免疫组织化学法鉴定出血清中增高的HSF2BP mRNA或蛋白的表达可作为肝癌的诊断标志物及预后评估指标,具有重要的临床应用价值。(The invention provides a new biological medical application of heat shock factor 2binding protein (HSF2BP), namely HSF2BP as a liver cancer tumor marker and an application thereof. The invention discloses application of quantitative detection of HSF2BP mRNA and protein in preparation of a reagent for diagnosing or monitoring prognosis of liver cancer tumor, in particular to application of HSF2BP mRNA or protein expression increase as a prediction index or an index of poor prognosis of liver cancer tumor. The method identifies the expression of HSF2BP mRNA or protein increased in serum by a real-time quantitative PCR, a Western Blot method and an immunohistochemistry method, can be used as a diagnosis marker and a prognosis evaluation index of liver cancer, and has important clinical application value.)

1. The tumor marker of liver cancer is heat shock factor 2binding protein (HSF2 BP).

2. The use of the detection reagent for liver cancer tumor markers of claim 1 for the preparation of an early liver cancer diagnostic reagent or a prognosis evaluation reagent.

3. The use according to claim 2, wherein the test sample is identified as HSF2BP mRNA using a fluorescent quantitative PCR reaction and/or the test sample is identified as marker HSF2BP protein using the Western Blot method using HSF2BP antibody.

4. The use of claim 3, wherein the tumor marker quantification reagent is used as a quantification reagent for real-time quantitative PCR, PCR sequencing, mass spectrometric detection, and enzyme-linked immunosorbent assay.

5. The use according to claim 4, wherein as a PCR detection reagent, a transcript probe or primer that specifically binds to HSF2BP mRNA; the detection reagent as the HSF2BP protein can specifically recognize the HSF2BP protein.

6. The use of claim 5, wherein the transcript probe or primer carries a molecular marker to facilitate detection.

7. The use of claim 5 or 6, wherein the expression level detection of HSF2BP mRNA is based on PCR primer acquisition, and the upstream primer and the downstream primer are as follows in the sequence table:

the primer sequence of the GAPDH cDNA was:

5'-ACCCAGAAGACTGTGGATGG-3' (upstream);

5'-TCTAGACGGCAGGTCAGGTC-3' (downstream);

the primer sequence of the hsf2bp cDNA was:

5'-GGTGCTCTTGGACACCATATT-3' (upstream);

5'-CTGGACTCTCGCTGATGTATTT-3' (downstream).

8. The use according to claim 5, wherein a kit for detecting HSF2BP mRNA levels is prepared based on the sequence of the primers, the kit comprising the corresponding reverse transcription reagents, internal reference primers, and substances and buffers for RNA amplification including qPCR reaction buffer, dNTPs, DNA polymerase, fluorescent label for DNA synthesis amount, enzyme-free water.

9. The use of the liver cancer tumor marker of claim 1 for the preparation of a medicament for inhibiting or resisting liver cancer tumor.

10. The use of claim 9, wherein the anti-hepatoma tumor and the anti-hepatoma tumor drugs are prepared by constructing a vector for stably transferring and knocking down the HSF2BP gene by using HSF2BP as a target regulatory gene, and preparing a reagent or a drug for in vitro or in vivo use.

11. The use of claim 9, wherein the vector for constructing the stable transgenic knockdown HSF2BP gene is a viral vector or a gene silencing vector.

12. The use of claim 9, wherein the viral vector comprises an adenoviral vector, an adeno-associated vector, and a retroviral vector; the gene silencing vector comprises a CRISPR editing system gene silencing vector and a plasmid low expression vector.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a tumor marker heat shock factor 2binding protein of liver cancer and application thereof.

Background

The liver cancer (HCC) is a malignant disease with fifth morbidity and second mortality, and the liver cancer is most common in patients with chronic alcoholic cirrhosis, non-alcoholic fatty liver and chronic viral hepatitis, the continuous inflammation and repeated healing of liver cells are considered as the root causes of the development of liver cancer, the coexistence of liver inflammation and cirrhosis complicates the early diagnosis of liver cancer, and the tumor-related biomarkers can effectively screen the risk of early liver cancer from inflammation and cirrhosis patients, and obviously improve the prognosis of such patients, and meanwhile, the lack of characteristic symptoms in the early stage is also an important cause of poor prognosis of liver cancer, more than 60% of patients have metastasis when first diagnosed, resulting in 5-year survival of 16%, on the contrary, if early diagnosis is confirmed, the prognosis of disease can be obviously improved, more than 70% of patients can survive in 5 years, and if the Grading of BCG in 0 and 0, the early stage of liver cancer, the early stage of survival of 5-year survival of liver cancer is not reached, even if the diagnosis is carried out by other significant diagnostic methods, the biological markers can still effectively screen the early stage diagnosis of liver cancer, even if the AFP is found, the early stage diagnosis is carried out, and the diagnosis is carried out, even if the biological markers are not reached more than the early stage diagnosis of liver cancer, the early stage diagnosis is found, the early stage of liver cancer, the diagnosis is more than the diagnosis is found, the clinical diagnosis is more than the clinical diagnosis is found, the clinical diagnosis is more than the clinical diagnosis is found, the more than the.

Heat Shock Response (HSR) is a protective stress response, and when a body is subjected to harmful stimuli such as hypoxia, ischemia, overheating and inflammation, heat shock transcription factors (HSFs) are activated to promote the expression of heat shock proteins and protect the body from being subjected to protein toxicity stress to progress a pathological state, thereby alleviating damage and increasing the survival ability of cells. Members of the human HSFs family include HSFl, HSF2, HSF3, and HSF4, of which HSF2 is an important regulator of the regulation of the cellular heat shock response. Heat shock factor 2binding protein (HSF2BP) is associated with HSF2, and the interaction occurs between the trimerization domain of HSF2 and the amino-terminal hydrophilic region of HSF2BP that contains two leucine zipper sequences. HSF2BP was first discovered in human testis cDNA library, and it is a binding partner of HSF2, involved in regulating the activation of HSF2, and plays a potential role in human spermatogenesis. Meanwhile, the protein can be used as a ubiquitination substrate and plays a role in the protein modification process. Currently, there are few studies on HSF2BP, and no studies have been published on malignant tumors in all systems. The invention firstly provides the expression characteristics of HSF2BP in human liver cancer and the relation between the expression characteristics and the biological behavior of liver cancer, aims to provide a new diagnostic marker for early detection and risk assessment of liver cancer, and has important clinical significance.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a tumor marker heat shock factor 2binding protein of liver cancer and application thereof, provides a new biological medical application of the heat shock factor 2binding protein (HSF2BP), and can be applied to the preparation of early liver cancer diagnosis reagents, liver cancer tumor inhibition drugs, liver cancer tumor resistance drugs or prognosis evaluation reagents.

In order to achieve the purpose, the invention adopts the technical scheme that:

the tumor marker of liver cancer is heat shock factor 2binding protein (HSF2 BP).

The detection reagent of the liver cancer tumor marker can be used for preparing an early liver cancer diagnosis reagent or a prognosis evaluation reagent.

The early liver cancer diagnosis, namely the liver cancer detection, refers to the quantitative detection of mRNA and total protein extracted from the obtained liver cancer tissues and corresponding tissues beside the liver cancer of clinical liver cancer patients.

Wherein, when preparing early liver cancer diagnosis reagent or prognosis evaluation reagent, the tumor marker is identified and applied in the form of HSF2BPmRNA or protein. Specifically, a marker HSF2BPmRNA in a sample to be detected can be obtained by fluorescent quantitative PCR reaction identification, and a marker HSF2BP protein in the sample to be detected is obtained by Western blot method identification based on an HSF2BP antibody. The expression of HSF2BP mRNA or protein is obviously increased in clinical liver cancer tissues, and the expression is used as a pointer for poor prognosis of liver cancer.

Through comparative analysis with tissues beside the cancer of a patient, the liver cancer shows that the specific high-expression HSF2BP mRNA or protein has obvious correlation with clinical pathological parameters, and the obvious difference of prognosis is caused, so that the liver cancer has important clinical application prospect.

The tumor marker can be used as a quantitative reagent for real-time quantitative PCR, PCR sequencing, biological mass spectrometry detection and enzyme-linked immunosorbent assay.

The detection reagent of HSF2BP mRNA specifically binds to the corresponding transcript probe or primer, and the transcript probe or primer may carry a molecular marker to facilitate detection. The detection reagent of the HSF2BP protein is an antibody and/or an antibody fragment which can specifically recognize the HSF2BP protein.

The expression level detection of the HSF2BPmRNA is based on PCR primer acquisition, and an upstream primer and a downstream primer are as follows in a sequence table:

the primer sequence of the GAPDH cDNA was:

5'-ACCCAGAAGACTGTGGATGG-3' (upstream);

5'-TCTAGACGGCAGGTCAGGTC-3' (downstream);

the primer sequence of the hsf2bp cDNA was:

5'-GGTGCTCTTGGACACCATATT-3' (upstream);

5'-CTGGACTCTCGCTGATGTATTT-3' (downstream).

Based on the sequences of the primers, a kit for detecting HSF2BP mRNA levels can be further prepared, which should include the corresponding reverse transcription reagents, internal reference primers, and materials and buffers for RNA amplification. Wherein the substances and buffers for RNA amplification may comprise qPCR reaction buffer, dNTPs, DNA polymerase, fluorescent label for DNA synthesis amount, and enzyme-free water.

The tumor marker of the liver cancer can also be used for preparing a medicament for inhibiting the liver cancer tumor or a medicament for resisting the liver cancer tumor.

In the liver cancer tumor inhibiting medicine and the liver cancer tumor resisting medicine, HSF2BP is used as a target regulation gene, a vector for stably transferring and knocking down the HSF2BP gene is constructed, and a reagent or a medicine used in vitro or in vivo is prepared. The stable transfer of the low expression HSF2BP gene comprises the inhibition or deletion of a coding gene of the expression HSF2BP, and the reduction or inhibition of the copy, transcription and translation of the HSF2BP gene, thereby inhibiting the exertion of the function of the HSF2BP protein.

Wherein, the vector for constructing the stable transfer knockdown HSF2BP gene is a viral vector or a gene silencing vector. The viral vectors include adenoviral vectors, adeno-associated vectors and retroviral vectors; the gene silencing vector comprises a CRISPR editing system gene silencing vector and a plasmid low expression vector.

By constructing an HSF2BP knock-down stable hepatoma cell strain and transplanting the strain to a nude mouse subcutaneous tumor, the HSF2BP expression is proved to be down-regulated to reduce the capacity of the nude mouse subcutaneous tumor, which shows that the HSF2BP inhibition can be used as a novel anti-tumor scheme for liver cancer.

Compared with the prior art, the invention provides a new biological medical application of the heat shock factor 2binding protein (HSF2BP), namely HSF2BP is used as a liver cancer tumor marker and the application. The invention discloses application of quantitative detection of HSF2BP mRNA and protein in preparation of a reagent for diagnosing or monitoring prognosis of liver cancer tumor, in particular to application of HSF2BP mRNA or protein expression increase as a prediction index or an index of poor prognosis of liver cancer tumor. The method identifies the expression of HSF2BP mRNA or protein increased in serum by a real-time quantitative PCR, a Western Blot method and an immunohistochemistry method, can be used as a diagnosis marker and a prognosis evaluation index of liver cancer, and has important clinical application value. Meanwhile, the compound can be used for preparing medicines for inhibiting liver cancer tumors and medicines for resisting the liver cancer tumors.

Drawings

In order to more intuitively explain embodiments of the present invention or technical solutions in the prior art, the drawings required in the embodiments or the technical solutions are briefly described below, meanwhile, the following drawings only represent embodiments of the present invention, and other drawings can be obtained by those skilled in the art according to the following drawings without creative efforts.

FIG. 1: 45 examples the expression level of HSF2BP mRNA in cancer tissues and paracancerous normal tissues of patients with liver cancer.

FIG. 2 shows the expression of HSF2BP protein in cancer tissue and paracancer normal tissue of liver cancer patient analyzed by Western Blot method, (left side) the expression of liver cancer tissue and paracancer normal tissue in 4 examples, (right side) the expression of 99 clinical liver cancer and corresponding paracancer normal tissue obtained from 99 examples, β -actin is used as reference for HSF2BP protein expression.

FIG. 3: the immunohistochemical method analyzes the expression of the HSF2BP protein in the cancer tissue and the paracancer normal liver tissue of a liver cancer patient in 3 clinical liver cancer cases.

FIG. 4: constructing a cell strain for stably transforming low expression HSF2BP in human liver cancer cell Bel-7404: (left side) infecting the liver cancer cell strain Bel-7404 cells with HSF2BP interference and no-load control group lentivirus vectors, and after 72 hours, performing transfection efficiency under a fluorescence microscope; (right) two groups of transfected cell total RNA were extracted and the expression level of HSF2BPmRNA was determined by real-time quantitative PCR.

FIG. 5: by constructing an HSF2BP knock-down stable hepatoma cell strain, transplanting the strain to form tumor under the skin of a nude mouse, and discussing the influence of the HSF2BP expression down-regulation on the capability of transplanting the strain to form tumor under the skin of the nude mouse: and observing the tumorigenicity of two groups of liver cancer cells after the two groups of liver cancer cells are transplanted to the tumor under the skin of the nude mouse by using an in-vitro small animal imaging system.

FIG. 6: by constructing an HSF2BP knock-down stable hepatoma cell strain, transplanting the strain to form tumor under the skin of a nude mouse, and discussing the influence of the HSF2BP expression down-regulation on the capability of transplanting the strain to form tumor under the skin of the nude mouse: the tumor volume of two groups of cells after subcutaneous tumor formation of nude mice is increased along with the change of time

FIG. 7: by constructing an HSF2BP knock-down stable hepatoma cell strain, transplanting the strain to form tumor under the skin of a nude mouse, and discussing the influence of the HSF2BP expression down-regulation on the capability of transplanting the strain to form tumor under the skin of the nude mouse: comparative analysis of tumor tumors after subcutaneous tumorigenesis of two groups of cells in nude mice.

Detailed Description

The present invention will be further described with reference to the following examples for the purpose of illustrating the invention in more detail, but the examples are not intended to limit the invention in any way. Unless otherwise indicated, all reagents, methods and equipment used in the following description are conventional in the art.