Target CRTAC1 for inhibiting lung adenocarcinoma and application thereof
阅读说明：本技术 抑制肺腺癌的靶标crtac1及其应用 (Target CRTAC1 for inhibiting lung adenocarcinoma and application thereof ) 是由 黄海山 常园园 李海英 金红蕾 于 2020-06-23 设计创作，主要内容包括：本发明公开了一种CRTAC1作为靶点在制备诊断和治疗肺腺癌的药物中的应用,可作为肺腺癌细胞生长增殖和侵袭迁移的靶点。本发明具有如下作用：过表达CRTAC1抑制肺腺癌细胞增殖；过表达CRTAC1抑制裸鼠皮下成瘤能力；过表达CRTAC1抑制肺腺癌细胞迁移和侵袭；过表达CRTAC1抑制裸鼠肺转移灶的形成。(The invention discloses application of CRTAC1 as a target point in preparation of a medicine for diagnosing and treating lung adenocarcinoma, and the CRTAC1 can be used as a target point for growth, proliferation, invasion and migration of lung adenocarcinoma cells. The invention has the following functions: overexpression of CRTAC1 inhibits lung adenocarcinoma cell proliferation; the capability of over-expressing CRTAC1 for inhibiting subcutaneous tumor formation of nude mice; overexpression of CRTAC1 inhibits migration and invasion of lung adenocarcinoma cells; overexpression of CRTAC1 inhibited the formation of lung metastases in nude mice.)
Application of CRTAC1 as a target point in preparation of medicines for diagnosing and treating lung adenocarcinoma.
2. Use according to claim 1, characterized in that: as a target for the growth, proliferation, invasion and migration of lung adenocarcinoma cells.
3. Use according to claim 1 or 2, characterized in that: detecting mRNA of CRTAC1 or protein expression level of CRTAC 1.
4. Use according to claim 3, characterized in that: the primers used for amplifying the CRTAC1 gene fragment are as follows:
Forward 5'-GGTGTCGCCCTGGCTGACTT-3'
Reverse 5'-GAGAACTTGGGTGAGGCGATGTC-3'。
5. use according to claim 3, characterized in that: including antibodies that specifically bind to CRTAC1 protein.
6. Use according to claim 1, characterized in that: the CRTAC1 gene expression enhancer is used for preparing medicines for treating lung adenocarcinoma.
7. Use according to claim 6, characterized in that: the medicament comprises an enhancer of CRTAC1 gene expression and a pharmaceutically acceptable carrier.
8. Use according to claim 6 or 7, characterized in that: the enhancer is CRTAC1 gene overexpression plasmid.
9. Use according to any one of claims 6 to 8, characterized in that it is at least any one of the following:
overexpression of CRTAC1 inhibits lung adenocarcinoma cell proliferation; the capability of over-expressing CRTAC1 for inhibiting subcutaneous tumor formation of nude mice; overexpression of CRTAC1 inhibits migration and invasion of lung adenocarcinoma cells; overexpression of CRTAC1 inhibited the formation of lung metastases in nude mice.
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a new target for research on growth, proliferation and metastasis of lung adenocarcinoma cells and application thereof, wherein the new target is used for detecting the mRNA or protein expression level of CRTAC1, can be further used as a marker for diagnosis and prognosis of lung adenocarcinoma, and is expected to become a new target for treating lung adenocarcinoma.
Background
Lung cancer is one of The most rapidly growing malignant tumors that threaten human health and life, and The research results of The newly published CONCORD-3 (third round of global cancer survival analysis) in The Lancet show that: the five-year survival rate of most of the national lung cancer patients is between 10 and 19 percent, and the survival rate of Chinese patients is between 19.4 and 20.2 percent. According to the latest statistical data of the cancer center in China, the method comprises the following steps: the lung cancer is the first to occur nationwide, and the lung cancer is about 78.1 ten thousand per year and accounts for 20.55 percent of all types of cancers. Lung cancer is the highest incidence and mortality cancer in china and worldwide.
According to the pathological type and characteristics of lung cancer, the lung cancer can be divided into: small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), of which about 80-85% of lung cancers are non-small cell lung cancer (NSCLC), which can be further divided histologically into four categories: (1) lung adenocarcinoma; (2) squamous cell carcinoma; (3) large cell carcinoma; (4) and others. Lung adenocarcinoma is again a relatively common type. Compared with the characteristics of high malignancy, easy invasion and metastasis, short course of disease, fast progress and short survival time of the small cell lung cancer, the non-small cell lung cancer has the characteristics of slow growth and division, late diffusion and metastasis, relatively low malignancy, long course of disease and high five-year survival rate. Although the research on tumor immunity and molecular targeted therapy has a certain breakthrough in the research on lung cancer in recent years, a large number of research achievements on lung cancer are used for guiding clinical treatment, an effective diagnosis and treatment method is currently lacked due to the influence of factors such as target tolerance, off-target effect, epigenetic tolerance, change of tumor microenvironment and the like, and up to 69% of patients with advanced non-small cell lung cancer possibly have potential and feasible molecular targets according to literature reports, so that the identification of a natural molecule capable of specifically inhibiting the occurrence and development of lung cancer has great significance for the clinical treatment of the disease, and the research on a new target of a new lung cancer mechanism is still repeated and far for a large number of researchers.
CRTAC1 gene, located on chromosome 10 of human body, which encodes glycosylated extracellular matrix protein
Disclosure of Invention
The technical problem to be solved by the invention is to provide a target CRTAC1 for regulating and controlling the progress of lung adenocarcinoma and application thereof, namely, CRTAC1 is provided as a target for growth, proliferation, invasion and migration of lung adenocarcinoma cells and application thereof.
In order to solve the technical problems, the invention provides application of CRTAC1 as a target (specific marker) in preparing a medicament for diagnosing and treating lung adenocarcinoma.
As an improvement of the application of the invention: as a target for the growth, proliferation, invasion and migration of lung adenocarcinoma cells.
As a further improvement of the application of the invention: detecting mRNA of CRTAC1 or protein expression level of
As a further improvement of the application of the invention: the primers used to amplify the CRTAC1 gene fragment (i.e., the primers that specifically amplify CRTAC1 mRNA) were:
Forward 5'-GGTGTCGCCCTGGCTGACTT-3'
Reverse 5'-GAGAACTTGGGTGAGGCGATGTC-3'。
as a further improvement of the application of the invention: including antibodies that specifically bind to CRTAC1 protein.
Description of the drawings: antibodies that specifically bind CRTAC1 protein were used in fig. 1C, 1E, 3A, 3D, 5A, 5D, 7D.
As a further improvement of the application of the invention: the CRTAC1 gene expression enhancer is used for preparing medicines for treating lung adenocarcinoma.
As a further improvement of the application of the invention: the medicament comprises an enhancer of CRTAC1 gene expression and a pharmaceutically acceptable carrier.
As a further improvement of the application of the invention: the enhancer is CRTAC1 gene overexpression plasmid, and specifically comprises the following components: preparing a medicament overexpressing CRTAC1 for treating lung adenocarcinoma.
As a further improvement of the application of the invention, at least one of the following is provided:
overexpression of CRTAC1 inhibits lung adenocarcinoma cell proliferation; the capability of over-expressing CRTAC1 for inhibiting subcutaneous tumor formation of nude mice; overexpression of CRTAC1 inhibits migration and invasion of lung adenocarcinoma cells; overexpression of CRTAC1 inhibited the formation of lung metastases in nude mice.
The invention discloses the effect of CRTAC1 in lung adenocarcinoma for the first time, and the CRTAC1 can promote the proliferation and metastasis of lung adenocarcinoma cells in vitro and in vivo. Meanwhile, the consistency of TCGA and clinical specimens indicates that the expression of CRTAC1 is increased in lung adenocarcinoma and lung squamous carcinoma, but the expression of CRTAC1 is only up-regulated in lung adenocarcinoma to be positively correlated with the survival period of a patient, and further functional experiment results also find that CRTAC1 only plays the biological function in lung adenocarcinoma. Thus, CRTAC1 can be used as a specific prognosis and treatment target of lung adenocarcinoma.
The technical scheme adopted by the invention is as follows: the lung adenocarcinoma data in a TCGA database (Cancer genome atlas) are analyzed through bioinformatics, the expression of CRTAC1 in lung adenocarcinoma is screened to be up-regulated, and the expression of CRTAC1 in human lung adenocarcinoma tissues and paracarcinoma tissues is further detected by adopting Real-time PCR and immunohistochemical staining (IHC) technologies. An in vitro soft agar colony forming experiment (soft agar assay) detects the influence of CRTAC1 on the growth and proliferation capacity of lung adenocarcinoma cells, and an in vitro Transwell experiment proves that CRTAC1 can inhibit the migration and infiltration of lung adenocarcinoma cells in vitro.
Further, by over-expressing CRTAC1, the proliferation, migration and invasion capacity of lung adenocarcinoma cells can be remarkably inhibited in vitro, and lung metastasis of the lung adenocarcinoma cells can be remarkably inhibited in vivo.
The invention has the following beneficial effects: the invention takes lung adenocarcinoma H1299, A549 and HCC827 cells as models, and the over-expression CRTAC1 can inhibit the growth, proliferation, invasion and migration of lung adenocarcinoma cells in vitro and in vivo. It can be seen that lung adenocarcinoma can be treated by increasing exogenous CRTAC1 levels. The method for detecting CRTAC1 expression in most lung adenocarcinoma tissues in clinic by using real-time fluorescence quantitative PCR technology, bioinformatics analysis TCGA database and other methods shows a remarkable down-regulation trend. Therefore, the detection of CRTAC1 expression can help to diagnose lung adenocarcinoma.
In conclusion, the invention discloses a target CRTAC1 related to growth, proliferation, invasion and migration of lung adenocarcinoma cells and application thereof. Specifically, the CRTAC1 is found to have the effect of inhibiting the proliferation and metastasis of lung adenocarcinoma cells at a cellular level by combining animal models and clinical tissue specimens. The invention discovers that CRTAC1 can play an important role in the aspect of clinical lung cancer prevention and treatment as a target spot for the growth, proliferation, invasion and migration of lung adenocarcinoma cells for the first time.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows that CRTAC1 is significantly highly expressed in human normal lung epithelial cells and lung normal tissues;
in the context of figure 1 of the drawings,
a is a real-time fluorescence quantitative PCR result of CRTAC1 in lung adenocarcinoma tissue to normal lung tissue in a bioinformatics analysis TCGA database;
b, performing real-time PCR analysis on a clinical lung adenocarcinoma specimen by using 36, and comparing the expression level of CRTAC1 in normal lung tissues;
C-D is to detect the protein expression level of CRTAC1 in the lung adenocarcinoma tissue and the normal tissue by using the immunohistochemical technology;
e is the comparison of CRTAC1 protein expression in human normal lung epithelial cells and lung adenocarcinoma cell lines;
normal stands for human lung adenocarcinoma paracancerous tissue and Tumor for human lung adenocarcinoma tissue.
FIG. 2 is a DFS disease-free survival analysis of lung adenocarcinoma patients.
FIG. 3 shows that the CRTAC1 is over-expressed to remarkably inhibit the proliferation capacity of lung adenocarcinoma cells in vitro;
in the context of figure 3, it is shown,
a is a stable transgenic cell line which successfully constructs over-expressed CRTAC1 in H1299 cells; B. c, after CRTAC1 is over-expressed on H1299, performing soft agar clone formation experiments and data analysis;
d is a stable transgenic cell line which successfully constructs over-expressed CRTAC1 in A549 cells; E. f is soft agar clone formation experiment and data analysis after overexpression of CRTAC1 on A549.
FIG. 4 is a graph showing that the capacity of over-expressing CRTAC1 to significantly inhibit subcutaneous tumorigenesis of lung adenocarcinoma cells in nude mice;
in FIG. 4, A-C is the effect of overexpression of CRTAC1 on subcutaneous neoplasia of H1299 lung adenocarcinoma cells in nude mice; D-F is the influence of over-expression of CRTAC1 on subcutaneous tumor formation of a nude mouse with lung adenocarcinoma A549 cells;
the method comprises the following specific steps:
a: mixing 2.5X 106H1299(CRTAC1) tumor cells or corresponding control cells thereof are injected into the right back of the nude mice for tumor formation;
b: sacrifice injection 2.5X 106Mice bearing H1299(CRTAC1) tumor cells or their corresponding control cells, and tumors were removed and a photograph taken;
c: recording the subcutaneous tumor weight of the mouse, and performing statistical analysis;
d: mixing 2.5X 106A tumor formation condition of a549(CRTAC1) tumor cells or corresponding control cells injected into the right back of a nude mouse;
e: sacrifice injection 2.5X 106Mice bearing a549(CRTAC1) tumor cells or their corresponding control cells, and tumors were removed and a photograph taken;
f: the subcutaneous tumor weight of the mice was recorded and statistically analyzed.
FIG. 5 shows that CRTAC1 inhibits migration and invasion of lung adenocarcinoma cells in vitro;
in the context of figure 5, it is shown,
a is a stable cell line which is successfully constructed and over-expressed CRTAC1 on lung adenocarcinoma HCC 827;
b and C are invasion chamber experiments and quantitative data analysis of HCC827 over-expressing CRTAC1 cells compared with control cells;
d is a stable cell line which successfully constructs over-expressed CRTAC1 on lung adenocarcinoma H1299;
e and F are invasion chamber experiments and quantitative data analysis of H1299 overexpressing CRTAC1 cells compared to their control cells.
FIG. 6 shows that the lung metastasis capacity of nude mice with lung adenocarcinoma cells is remarkably inhibited by over-expression of
in the context of figure 6, it is shown,
a is a stable cell line HCC827(Vector) and HCC827(CRTAC1) injected into tail vein to construct a nude mouse lung metastasis model, and nude mouse lung tissues of the lung metastasis model are fixedly embedded to be paraffin sections and subjected to H & E staining analysis.
And B, counting the number of lung metastases of the nude mice.
FIG. 7 shows that CRTAC1 may not be involved in the progression of squamous cell lung carcinoma cells, and its expression level has no significant correlation with the prognosis of squamous cell lung carcinoma patients;
in the context of figure 7 of the drawings,
a, analyzing a real-time fluorescence quantitative PCR result of CRTAC1 in a lung adenocarcinoma tissue to a normal lung tissue in a TCGA database by bioinformatics;
b, detecting the mRNA expression condition of CRTAC1 in 18 pairs of clinical lung squamous carcinoma tissues by using a real-time fluorescent quantitative PCR (polymerase chain reaction) technology;
c is the DFS disease-free survival analysis of the lung adenocarcinoma patients;
d, a CRTAC1 overexpression stable cell line has been successfully constructed in squamous lung carcinoma H226;
E-F after overexpression of CRTAC1 on H226, soft agar colony formation experiments and data analysis were performed.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: