CN111793687B

CN111793687B - Target CRTAC1 for inhibiting lung adenocarcinoma and application thereof

Info

Publication number: CN111793687B
Application number: CN202010579044.XA
Authority: CN
Inventors: 黄海山; 常园园; 李海英; 金红蕾
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-06-23
Filing date: 2020-06-23
Publication date: 2021-08-24
Anticipated expiration: 2040-06-23
Also published as: CN111793687A

Abstract

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.

Description

Target CRTAC1 for inhibiting lung adenocarcinoma and application thereof

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 cartilage acid protein 1 and is secreted by chondrocytes, and its expression has tissue specificity: expressed in the interzonal matrix of the cartilage in the deep zone of the joint. Research has shown that: (1) the protein can be used as a novel human marker and can be used for distinguishing human chondrocytes from osteoblasts and mesenchymal stem cells in culture. (2) The protein may be involved in cell-to-cell or cell-to-matrix interactions. The data from the NCBI database show that isoform 1 and isoform 2 encoded by this gene are expressed in brain, and isoform 1 is detectable in both lung and chondrocytes. The research on the CRTAC1 gene mainly focuses on genomics analysis at present, and only few reports on cataract treatment and neurofibromatosis type 1 (NF1) pathogenesis exist in the aspect of basic medicine.

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 CRTAC 1.

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, 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 their relativeInjecting the corresponding control cells into the tumor formation condition of the right back of the nude mice;

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 CRTAC 1;

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:

example 1 consistent downregulation of CRTAC1 expression in Lung adenocarcinoma tissues and cell lines

1) T-test results analysis of the expression of CRTAC1 mRNA from lung adenocarcinoma patients 58 in the TCGA database using GraphPad Prism software, P <0.05 indicated significant differences between the two sets of data compared (fig. 1A).

2) Tissue sample

The lung adenocarcinoma clinical sample used in the invention is provided for cooperative hospitals, and the tissue is diagnosed as lung adenocarcinoma by a pathologist. During specimen collection, a position about 3cm around lung adenocarcinoma tissue is selected as a para-carcinoma tissue contrast, a part of the tissue is fixed by 4% PFA, and the rest is frozen at-80 ℃ for later use. All patients had the details of name, pathological information, etc. Experimental procedures were approved by ethical review.

3) Tissue/cell total RNA extraction

1. Taking out the collected lung cancer tissue sample from a refrigerator at the temperature of minus 80 ℃, cutting 30-50g of the sample, adding the sample into an enzyme-removed EP tube, and placing the enzyme-removed EP tube on ice; cells 2X 10⁶Within one. After 1mL of Trizol was added, the tissue was homogenized with a tissue homogenizer to grind as completely as possible or blow the cells until completely dissolved.

2. After shaking with a vortex oscillator, equilibrate for 5-10min at room temperature.

3. Adding 200 μ l chloroform (chloroform), shaking vigorously for 1min, and standing at room temperature for 2-3 min.

4. 12000g of sample is centrifuged for 15min at 4 ℃, and after centrifugation, the supernatant is sucked up to a new enzyme-removed EP tube as much as possible.

5. Adding equal volume of isopropanol, mixing several times by turning upside down, and standing on ice for 10 min. 12000g, centrifuging for 10min at 4 ℃, and then discarding the supernatant.

6, adding 1mL of 75% absolute ethyl alcohol into an EP tube to gently blow up the precipitate, 7500g, centrifuging at 4 ℃ for 5min, and then discarding the supernatant.

7. Repeat step 6 once.

8. Drying RNA precipitate in air for 5-10min, and dissolving RNA with 10-20 μ L enzyme-free water.

9. The concentration and purity of the samples were measured using a BioDrop instrument.

4) Reverse transcription of mRNA

After the desired tissue and cells were treated and RNA was extracted according to the method described above, SuperScript was purchased from Invitrogen corporation, USA^TMIV, reverse transcriptase is carried out, and an RT reaction system is prepared as follows:

after oscillating and mixing uniformly, quickly centrifuging the PCR tube, placing the PCR tube in a PCR instrument, setting the PCR instrument to react for 5min at 65 ℃, and then placing the PCR tube on ice for 3 min; the RT reaction system was added as follows:

namely, after the system is uniformly mixed, the mixture is quickly centrifuged and then added into a PCR tube which is subjected to the first step, and after the mixture is uniformly mixed, the mixture is put into a PCR instrument, and the program is set: obtaining cDNA at 56 ℃ for 13min → 80 ℃ for 10min, sealing and storing at-80 ℃.

5) Fluorescent quantitative PCR detection of mRNA

The expression of mRNA was detected by fluorescent quantitative PCR, and the desired cDNA obtained from the above tissues and cells was subjected to PCR using Kit available from Qiagen, USA, as follows: the PCR reaction system was prepared as follows, and the reaction was carried out on ice.

The reaction system was mixed well, added to 384-well plates with 3 replicates per sample, centrifuged briefly to mix the reagents well, and placed in a Q6 fluorescent quantitative PCR instrument for detection. q-PCR reaction, pre-denaturation 95 ℃, 30s, cycling conditions as follows: amplification was performed for 40 cycles, 95 ℃, 5 seconds for denaturation, 58 ℃, 30 seconds for annealing, 72 ℃ for extension, 30 seconds. As shown in fig. 1B, Real-time PCR analysis of 36 clinical lung adenocarcinoma specimens revealed a significant reduction in the expression level of CRTAC1 in lung adenocarcinoma tissue compared to normal lung tissue.

The primer sequences are as follows:

CRTAC1 q-PCR detection primer

Forward 5'-GGTGTCGCCCTGGCTGACTT-3'

Reverse 5'-GAGAACTTGGGTGAGGCGATGTC-3'

GAPDH q-PCR detection primer

Forward 5'-GACTCATGACCACAGTCCATGC-3'

Reverse 5'-CAGGTCAGGTCCACCACTGA-3'

6) Immunohistochemistry (Immunochemistry) was used to explore the expression of CRTAC1 in lung adenocarcinoma tissue. Immunohistochemistry experiments were performed on 24 clinical lung adenocarcinoma tissue specimens and photographed using a DS-Ri2 Eclipse Ni microsystem software (FIG. 1C). Statistical analysis was then performed using Image-Pro Plus software and the results of the statistics were analyzed using GraphPad Prism software (fig. 1D), results table CRTAC1 for down-regulation of expression in lung adenocarcinoma tissue.

7) Protein immunoblotting experiments were used to explore the protein expression of CRTAC1 in lung normal bronchial epithelial cells and lung adenocarcinoma cell lines, and it was found that the protein expression of CRTAC1 in lung adenocarcinoma cell lines H1299, a549 and HCC827 was significantly down-regulated (fig. 1E).

Examples 2,

By using GraphPad Prism software to perform Kaplan-Meier survival analysis on the survival information of 401 cases of lung adenocarcinoma patients, namely CRTAC1 in the TCGA database, the overall survival rate results of lung adenocarcinoma samples classified by high-expression CRTAC1(> media; n ═ 109) and low-expression CRTAC1(< media; n ═ 292) show that P is less than 0.05, which indicates that the two groups of data compared have significant difference, and the survival period of the lung adenocarcinoma patients is prolonged along with the increase of the expression level of CRTAC1 in the cancer tissues of the lung adenocarcinoma patients (fig. 2A). That is, the CRTAC1 expression level was significantly and positively correlated with survival in lung adenocarcinoma patients (fig. 2A).

Example 3 overexpression of CRTAC1 significantly inhibits the proliferative capacity of lung adenocarcinoma cells

1) H1299 and A549 cells are selected as research objects, GFP-CRTAC1 plasmid and GFP no-load plasmid virus supernatant are infected in the H1299 and A549 cells, Puromycin drugs are used for screening, and stable cell strains H1299(CRTAC1), A549(CRTAC1) and control cell strains H1299(Vector) and A549(Vector) are established. The over-expression effect was detected by Western Blot using beta-Actin as an internal control, as shown in FIGS. 3A and 3D.

2) The soft agar clone formation experiment is adopted, under the room temperature environment, medium is prepared in an ultra-clean workbench, and 12.6ml (1.8ml multiplied by 7) is taken to be preheated for more than 10min at the temperature of 45-46 ℃. Heating and boiling the agar by microwave for 3 times, cooling to 45-46 ℃, quickly and uniformly mixing 8.4ml (1.2ml multiplied by 7) of agar in a medium preheating tube, sucking 3 ml/hole, paving lower-layer agar glue, and naturally solidifying for more than 3 hours. At room temperature, the old culture medium where the cells are located is removed by aspiration in a super clean bench, a proper amount of PBS is added for gentle washing and removal by aspiration, a proper amount of pancreatin is added, the digestion of the cells is observed under a microscope until the outline becomes round (generally 1-2min), and then the pancreatin is immediately removed by aspiration. The pancreatin digestion was then stopped by adding the appropriate amount of cell culture medium, the cells were gently blown down with a 1ml pipette gun, and the cell suspension was pipetted into the corresponding centrifuge tube or EP tube. The cell suspension was centrifuged at 1500r/min for 5 minutes at room temperature. The old medium was discarded and the corresponding fresh medium was added and mixed gently. Preparing a clean and pollution-free abalone counting plate in advance, sucking 10ul of cell suspension, filling the pool and counting. Counting the amount of cells added per well. After the medium and the cells are mixed evenly, the agar is quickly added and mixed gently, no air bubbles are generated, and the mixture is quickly added into a six-hole plate and is statically solidified. Naturally cooling and solidifying for more than 3h, sealing with sealing filmStanding at 37 deg.C and 5% CO₂Culturing in an incubator. And observing the formation of the clone after 1-2w, and observing the formation of the clone in real time. The number of cells of each clone in the counted clones is generally greater than or equal to 32, and the appropriate time is selected for clone shooting. As shown in FIGS. 3B-C, 3E-F, the differences were statistically significant.

From FIGS. 3B-C, 3E-F, it can be seen that: after the CRTAC1 is over-expressed, the proliferation capacity of lung adenocarcinoma cells is obviously weakened.

Example 4 ability of overexpression of CRTAC1 to significantly inhibit subcutaneous neoplasia in nude mice

Subcutaneous tumor formation experiment of nude mice

1) The experimental animals used in the invention are BALB/C-nu female nude mice, the age of 3-4 weeks, the weight of 15 +/-0.5 g, the weight is supplied by Jiangsu Jiejiaokang Biotechnology Limited, the experimental animals are raised in SPF-level experimental area of experimental animal center of Wenzhou medical university, and all animal experiments are carried out according to the experimental scheme approved by the ethical committee of experimental animals of Wenzhou medical university.

2) Subcutaneous tumor formation experiment of nude mice

Culturing lung adenocarcinoma cells, and when the required cell amount is reached and the cells are in a logarithmic growth phase, removing a culture medium, and washing with PBS once; 1min after 0.25% pancreatin digestion, the medium was terminated, centrifuged and resuspended in PBS, cell viability was determined and the total number of cells was counted, the cell concentration was adjusted to 2.5X 10⁷The stably transfected cell lines A549/H1299(Vector), A549/H1299(CRTAC1) were inoculated subcutaneously with 0.1mL of 4-week-old nude mice, 5 per group.

About 45 days after inoculation, analysis by statistical software revealed that: the overexpression of CRTAC1 can obviously inhibit the growth of ectopic transplantation tumor in nude mice of lung adenocarcinoma cells.

Example 5 ability of overexpression of CRTAC1 to significantly inhibit migration and invasion of lung adenocarcinoma cells

Stable cell lines overexpressing CRTAC1 have been successfully constructed on lung adenocarcinoma HCC827 and H1299 (fig. 5A and 5D); GFP-CRTAC1 plasmid and GFP-unloaded plasmid virus are infected in lung adenocarcinoma cell HCC827, and Puromycin drug screening is carried out to establish a stable cell strain HCC827(CRTAC1) and a control cell strain HCC827 (Vector).

The effect of overexpression of CRTAC1 on the migration and invasiveness of human lung adenocarcinoma H1299 and HCC827 cells was examined by invasive laboratory experiments. Using BD BioCoat^TM Matrigel^TMInvasion Chamber measures the migration and Invasion capabilities of cells overexpressing CRTAC1 as well as control cells. Statistical analysis indicated that there was a significant difference between H1299 and HCC827 cells by asterisk (P)<0.05). FIGS. 5B-C and 5E-F show that the migration and invasion abilities of cells overexpressing CRTAC1 are significantly inhibited compared to their control cells.

Example 6 overexpression of CRTAC1 significantly inhibited lung metastatic capacity in nude mice with lung adenocarcinoma cells

Nude mouse tail intravenous injection experiment:

the tail of the injection part of the nude mouse is disinfected by 75 percent alcohol. Cell suspensions (2X 10 each) were mixed well before inoculation⁷/mL HCC827(CRTAC1) and its control cells), 100 μ L of cell suspension was aspirated with a 1mL sterile syringe, and the cell suspension was injected into the tail vein of mice, 5 nude mice per cell. After 5 weeks the nude mice were sacrificed by photographing, the tumors were taken out and weighed, and the size was recorded by photographing. As shown in fig. 6A-B, the experimental group overexpressing CRTAC1 had a smaller number of lung metastases and significantly smaller volume of lung metastases compared to the control group; thus, from fig. 6, it can be seen that: overexpression of CRTAC1 can significantly inhibit lung metastasis ability of lung adenocarcinoma cells in vivo.

Example 7, CRTAC1 expression was upregulated in squamous cell lung carcinoma tissue without affecting the function of squamous cell lung carcinoma cells

1) T-test results analysis of the expression of CRTAC1 mRNA from 4 versus squamous cell lung carcinoma patients in the TCGA database using GraphPad Prism software, P <0.05 indicated significant differences between the two sets of data compared (fig. 7A).

In addition, mRNA levels of CRTAC1 were also detected in 18 pairs of clinical lung squamous carcinoma specimens, with significantly reduced CRTAC1 expression in lung squamous carcinoma tissues compared to normal lung tissues (fig. 7B). Survival information of 374 cases of lung adenocarcinoma patients CRTAC1 in the TCGA database was subjected to Kaplan-Meier survival analysis by using GraphPad Prism software, and it was found that the expression of CRTAC1 in squamous cell lung carcinoma was not significantly correlated with the survival time of the patients (FIG. 7C).

2) Stable transgenic cell lines overexpressing CRTAC1 were constructed in lung squamous carcinoma cells H226 and identified by western immunoblotting (fig. 7D). The soft agar colony formation experiment was performed, and the cells were plated on soft agar, and characteristic images were taken and analyzed at 37 ℃ in a 5% CO 2 incubator for 3-4 weeks. It was found by cell clone counting that overexpression of CRTAC1 did not affect the proliferative capacity of lung squamous carcinoma cells (fig. 7E-F).

Thus, it can be demonstrated that: CRTAC1 may exert a different specific regulation effect in the proliferation direction of lung adenocarcinoma than lung squamous carcinoma, indicating that CRTAC1 specifically inhibits the biological properties of lung adenocarcinoma.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

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Claims

The application of an enhancer of CRTAC1 gene expression in preparing a medicament for treating lung adenocarcinoma is characterized in that: the enhancer is CRTAC1 gene overexpression plasmid.
2. Use according to claim 1, characterized in that: the medicament comprises an enhancer of CRTAC1 gene expression and a pharmaceutically acceptable carrier.
3. Use according to claim 1 or 2, characterized in that it is any of the following: overexpression of CRTAC1 inhibits lung adenocarcinoma cell proliferation, overexpression of CRTAC1 inhibits subcutaneous tumorigenicity capacity of nude mice, overexpression of CRTAC1 inhibits migration and invasion of lung adenocarcinoma cells, and overexpression of CRTAC1 inhibits formation of lung metastases of nude mice.