CN113293208B

CN113293208B - Molecular marker related to lung cancer proliferation and metastasis and application thereof

Info

Publication number: CN113293208B
Application number: CN202010108165.6A
Authority: CN
Inventors: 邢怡明; 张苏红
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-02-21
Filing date: 2020-02-21
Publication date: 2022-05-03
Anticipated expiration: 2040-02-21
Also published as: CN113293208A

Abstract

The invention provides a molecular marker related to lung cancer proliferation and metastasis and application thereof. The invention adopts a CRISPR/Cas9 system of a gene editing technology, finds that TMEM116 gene has close relation with the transfer and proliferation of lung cancer in a human lung cancer cell line, designs the knockout sgRNA of the TMEM116 gene, and obtains the lung cancer cell line of the knock-down TMEM116 gene by a lipotropy method. On the basis, in vitro and in vivo experiments of mice show that the carcinogenic capacity and the metastatic capacity of the lung cancer cells knocked down by the TMEM116 are obviously reduced, the relationship between the TMEM116 gene and the lung cancer is clarified, and the TMEM116 gene is found to be a molecular marker related to lung cancer proliferation and metastasis, so that the TMEM116 gene can be used for diagnosing the lung cancer and provides a new method for diagnosing and treating the lung cancer.

Description

Molecular marker related to lung cancer proliferation and metastasis and application thereof

Technical Field

The invention relates to the field of medical molecular biology, in particular to the technical field of lung cancer diagnosis, and specifically relates to a molecular marker related to lung cancer proliferation and metastasis and application thereof.

Background

Due to the stimulation of smoking, air pollution and some chemical factors, normal lung cells of a human body are damaged, and gene mutation is easy to occur in the process of repairing the damage of the human body, so that tumors and cancers are caused. Currently, lung cancer is also one of the most common malignant tumors, accounting for 18.4% of cancer deaths worldwide. Lung cancer is a highly complex tumor with a generally severe prognosis with a 5-year survival rate of only 15%. In addition, lung cancer cells are likely to metastasize to other organs through blood vessels and lymphatic vessels to form secondary tumors, which is promoted by metabolic changes and immune evasion. Since malignant proliferation and metastasis of cancer cells are the key to the onset and prognostic survival of patients, finding gene indicators or molecular markers for detecting lung cancer proliferation and metastasis is of great significance.

The tmem (transmembrane protein) family, a protein that spans biological membranes, allows cell-to-cell, cell-to-environment communication, and is a large family. The TMEM family of proteins have a very broad range of biological functions in the human body. In recent years, various clinical studies have found that the expression level of TMEM family members varies in cancer tissues, but the variation of different TMEM family members in different tissues is not the same, and not all TMEM family members are related to the occurrence and development of cancer. The function of the TMEM116 gene and the relation with cancer are not reported at present. If a novel molecular marker related to lung cancer proliferation and metastasis can be discovered through the research on TMEM116, a new method and a new way for developing early diagnosis and treatment drugs for lung cancer occurrence in the future are provided.

Disclosure of Invention

The first purpose of the invention is to provide the application of TMEM16 gene in participating in the proliferation and metastasis of lung cancer cells, and further provide a molecular marker related to the diagnosis of lung cancer.

It is a second object of the present invention to provide a diagnostic kit for lung cancer.

The third purpose of the invention is to provide a potential drug which has the capacity of preventing the proliferation, infiltration and migration of lung cancer cells.

The purpose of the invention is realized by the following technical scheme: and collecting lung cancer tissues and lung cancer cell lines, detecting the expression conditions of related genes, and finding that the expression quantity of the TMEM116 gene in the lung cancer tissues and the lung cancer cells is remarkably improved. TMEM116 was knocked out in lung cancer cell a549 to detect functional changes in lung cancer cells. By the CRISPR/Cas9 technology, a TMEM116 knock-down human lung cancer A549 cell line is successfully constructed. In vitro migration and infiltration experiments show that the deletion of TMEM116 significantly inhibits the migration and infiltration capacity of A549 cells. In vitro proliferation and cancer cell clonogenic experiments show that the deletion of TMEM116 significantly inhibits the proliferation and clonogenic of A549 cells. The results show that the TMEM116 has close relation with the proliferation and migration of the lung cancer, and the detection of the expression change of the TMEM116 gene can be used as a diagnostic index for the occurrence and the development of the lung cancer.

Based on the findings of the present invention, in a first aspect, the present invention provides the use of the TMEM116 gene for metastasis, infiltration and/or proliferation of lung cancer cells.

The invention provides application of an expression inhibitor of a TMEM116 gene in preparation of a medicament for inhibiting lung cancer cell metastasis, infiltration and/or proliferation.

The expression inhibitor is a biological agent or a chemical agent for inhibiting or reducing the expression quantity of the TMEM116 gene in the cell, and the biological agent is RNAi, a transcription inhibitor, a post-transcriptional processing inhibitor, a function inhibitor after transcriptional processing maturation, or a DNA fragment and sgRNA for inhibiting or reducing the expression of the TMEM116 gene in the cell by a gene knockout or knock-down method.

Preferably, the sgRNA sequence has a DNA sequence of ACGGGTGCGCTTCTACCCAG, which is shown in SEQ ID No. 1.

In a second aspect, the invention provides an application of TMEM116 gene as a diagnostic marker in preparing a lung cancer diagnostic kit, a diagnostic reagent or a chip.

The invention provides application of a reagent for detecting TMEM116 gene expression level in preparation of a lung cancer diagnosis kit, a diagnosis reagent or a chip.

The invention provides application of a reagent for detecting TMEM116 gene expression level in preparation of a lung cancer cure evaluation system.

The reagent contains a primer and/or a probe for detecting the TMEM116 gene expression level.

The application is judged only by detecting the expression level of the TMEM116 gene in the genome of the sample to be detected, and when the expression level of the TMEM116 gene in the genome of the sample to be detected is obviously different from a negative control and is not obviously different from a positive control, the tissue to be detected is a lung cancer tissue or a tissue infiltrated or transferred by lung cancer cells; the negative control is a normal tissue or a tissue beside the cancer, and the positive control is a lung cancer tissue.

In a third aspect, the invention provides a lung cancer diagnostic kit or a lung cancer cell infiltration or metastasis detection kit, which contains a detection reagent for detecting the expression level of TMEM116 protein in tumor tissues or tumor extracellular matrix.

In a fourth aspect, the invention provides sgRNA specifically targeting TMEM116 gene, whose DNA sequence is ACGGGTGCGCTTCTACCCAG, as shown in SEQ ID No. 1.

The CRISPR/Cas9 targeting vector containing the sgRNA belongs to the protection scope of the invention.

The CRISPR/Cas9 targeting vector contains a drug screening marker G418.

The invention provides application of the CRISPR/Cas9 targeting vector in specific recognition and targeted modification of TMEM 116.

The invention provides application of the CRISPR/Cas9 targeting vector in a TMEM116 gene knockdown cell line or a model animal.

The sequence of the TMEM116 gene in the embodiment of the invention is shown in SEQ ID NO. 4.

The invention has the beneficial effects that: compared with the expression level of TMEM116 protein secreted by normal cells, the expression level of the TMEM116 protein of lung cancer cells is obviously improved for the first time, the lung cancer cell protein has the diagnosis capability of indicating lung cancer, and can assist the traditional lung cancer detection method to determine whether a pathological sample is from lung cancer tissues. Further, according to the invention, through a CRISPR/Cas9 system of a gene editing technology, in a human lung cancer cell line, a close relation between the TMEM116 gene and the transfer and proliferation of lung cancer is found, sgRNA is knocked out by the TMEM116 gene, and the lung cancer cell line with the TMEM116 gene knocked down is obtained through a lipotropy method. On the basis, the carcinogenic capacity and the metastatic capacity of the TMEM116 knocked-down lung cancer cells are obviously reduced through in vitro and in vivo experiments of mice. The discovery provides a new idea for the research and development of a therapeutic drug for lung cancer, and the TMEM116 expression inhibitor can be developed and used for preparing a drug for inhibiting the proliferation, infiltration or metastasis of lung cancer cells so as to treat the lung cancer.

Drawings

FIG. 1 shows the expression of TMEM116 gene in lung tissue of a patient with lung cancer. The upper panel is lung adenocarcinoma tissue, the lower panel is lung squamous carcinoma tissue, and TMEM116 is expressed in airway epithelium, stroma and tumor region of lung adenocarcinoma and lung squamous carcinoma tissue. CC10, beta-tubulin, is a marker protein for epithelial Clara cells and ciliated cells, and alpha-SMA is a marker protein for mesenchymal cells.

FIG. 2 shows TMEM116 expression in normal human lung cell lines and lung cancer cell lines. 16HBE is a human lung airway epithelial cell line, and A549, H1299 and H441 are human lung cancer cell lines.

FIG. 3 is a graph of the alignment peaks after transfection of cells against different target sites designed for the TMEM116 gene. The first row is a peak image obtained after the target point 1 transfects cells and extracts DNA, the second row is a peak image obtained after the target point 2 transfects cells and extracts DNA, the peak image obtained after the PCR sequencing is performed, and the third row is a peak image obtained after the target point 3 transfects cells and extracts DNA, and the peak image obtained after the PCR sequencing is performed.

FIG. 4 is a graph of TMEM116 knockdown of TMEM116 gene and detection of translation levels in human lung cancer cells. Panel A shows the detection of protein expression levels of TMEM116 in two knock-out cell lines and grey scale analysis. And B, detecting the TMEM116 gene sequences of two cell lines, wherein the first row is the TMEM116 gene sequence in a normal cell line, the second row is the gene sequence in a knockout cell line, and the TMEM116 gene sequence can be found to be a single gene knockout cell line and is named as a knock-down cell line.

FIG. 5 shows the function of TMEM116 in vitro to detect knockdown cell lines. Panel a is a measure of the ability of knockdown cells to migrate and infiltrate, with more cells migrating into the lower layer indicating greater ability. And B, detecting the proliferation capacity of the knockdown cells, wherein the higher the absorbance value is, the stronger the proliferation capacity is. The C picture is a clonality test, and the more and the larger the clonality, the stronger the clonality is indicated.

Figure 6 is the function of TMEM116 to detect knockdown cell lines in vivo. The A picture is the lung tissue photograph of tail vein injection mouse, white lung cancer nodule can be seen, and the B picture is the number of lung cancer metastasis nodule in the statistical control group and the knock-down group. Panel C is the size of subcutaneous tumors in mice injected subcutaneously with cells, and panel D is the statistical control and knockdown subcutaneous tumor weight.

Detailed Description

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified; the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, reagents and materials used in the following examples are all commercially available.

Example 1 detection of TMEM116 expression in Lung cancer tissues and Lung cancer cell lines

By immunohistochemical techniques, TMEM116 expression was first detected in lung tissue sections from lung cancer patients. The results show that TMEM116 is significantly more expressed in cancer tissues than in paracancerous tissues (fig. 1). Firstly, the paraffin-embedded lung tissue is subjected to xylene dewaxing and gradient ethanol hydration, the tissue antigen is repaired by a microwave high-pressure method, the tissue section is subjected to antigen sealing by serum diluent, and TMEM116, CC10, beta-tubulin and alpha-SMA antibodies are incubated overnight at 4 ℃. The next day, a 546 red fluorescent secondary antibody against TMEM116, CC10, β -tubulin, and a 488 green fluorescent secondary antibody against SMA were used, and incubated at room temperature for 1 hour, followed by observation and photographing through an Olympus microscope. The experimental results showed that in normal human lung tissue, TMEM116 was expressed in airway epithelial cells (including Club cells and ciliated cells) and hardly expressed in lung interstitial cells. In the cancerous tissue region, the expression level of TMEM116 was significantly increased. This result shows that TMEM116 has a close relationship with lung cancer development.

In addition, the RT-PCR detection result shows that the expression level of TMEM116 in the human lung cancer cells is obviously higher than that of the normal human lung cells (figure 2). Human lung trachea epithelial cell line 16HBE and human lung cancer cell lines A549, H1299 and H441 are cultured, RNA is extracted, and reverse transcription is carried out. Relative expression level of TMEM116 in cells was determined by RT-PCR using GAPDH as an internal control. As can be seen, the expression level of TMEM116 in the lung cancer cell lines A549 and H1299 is significantly higher than that of the normal lung bronchial epithelial cells 16 HBE.

Example 2 construction of TMEM116 knockdown human Lung cancer cell line

The TMEM116 gene was knocked down in the human a549 lung cancer cell line by using CRISPR/Cas9 technology.

By utilizing a tool website, a plurality of target spots aiming at the TMEM116 are designed, and 3 candidate target spots are determined after further optimization and screening, namely the target spot 1: acgggtgcgcttctacccag, target 2: gtatttcccagacagaatac and target 3: cataaagctgactaagccac are provided. Subsequently, the target was ligated to a W2 vector containing the G418 drug screening gene, and 3 vectors were individually transferred into A549 lung cancer cells by lipofection. 48 hours after transfection, G418 drug selection was performed, and 1 week later the selected cells were harvested and DNA was extracted for sequencing detection. Sequencing results show that the sequence of the target 1 is more obvious and has more miscellaneous peaks (figure 3), so that the best beating efficiency of the target 1 can be known, and the target 1 is selected for later-stage experiments. A549 cells transfected with target 1 were subjected to dilution culture, and 500 cells were added per 10cm dish. After 2 weeks, cell monoclonals gradually formed. And when the monoclonal cells grow to be of enough size, randomly cloning and selecting by using a microscope, extracting DNA of the selected cloned cells for sequencing detection, and finally obtaining the ideal TMEM116 gene knock-down A549 cell line.

TMEM116

knockdown cell lines

1 and 2 were finally obtained by sequencing comparison and screening, and further analysis was performed on 2 knockdown cell lines. TA clone sequencing comparison shows that one chain of TMEM116 gene double chains of the knock-down

cell lines

1 and 2 is subjected to gene knockout, and the other chain is a normal gene sequence, so that the two cell lines are single-chain knockout cell lines, and the TMEM116 is not completely knocked out, so that the two cell lines are named as the knock-down cell lines 1 and 2 (B in figure 4). Subsequently, the amount of TMEM116 protein synthesis in knockdown cells was examined at the translational level. By using western blotting techniques, the amount of TMEM116 protein synthesis in

knockdown cell lines

1 and 2 was found to be significantly reduced. The gray scale analysis is used for quantification, and the result shows that the reduction amount of the TMEM116 protein in the

knockdown cell lines

1 and 2 is nearly 70%, which indicates that the construction of the TMEM116 knockdown cell line is successful and can be used for later-stage experiments. Finally obtaining a TMEM116 knockdown cell line by designing a target spot, connecting a vector, transfecting an A549 cell line, screening a G418 drug and selecting a monoclonal cell; and verified at the gene transcription and translation level (a of fig. 4), it was confirmed that TMEM116 achieved successful knockdown on the a549 cell line, with about 70% reduction in TMEM116 expression.

Example 3 functional validation of TMEM116 knockout cells

The experiments of detecting proliferation, clone formation, migration and infiltration of lung cancer cells show that the functions of TMEM116 for knocking down human lung cancer cells are obviously reduced, as shown in figure 5.

Cell migration detection: adding equal amount of control cells and knockdown cells on an upper layer polycarbonate membrane with the pore size of 8 microns by using a transwell technology, adding a culture medium containing fetal calf serum into a lower layer culture plate, culturing for 48 hours in a constant temperature incubator, removing cell culture solution, migrating the cells into the lower layer culture plate by using a crystal violet staining marker, and taking pictures and counting by using a microscope.

Detecting the infiltration capacity of the cells: matrigel was added to 8 micron polycarbonate membrane and the other steps were as described above for the migration experiment. From the above experiments, the results showed that both cell migration and infiltration capacity were significantly reduced for

knockdown cell lines

1 and 2 relative to the control group (a of fig. 5).

The proliferation capacity of the knockdown cells was examined. Using the CCK8 technique, equal amounts of cells were added to 96-well cell culture plates, incubated for 0-4 days, CCK8 was added, cells were harvested at appropriate times and absorbance at 450nm was measured. The data obtained from the assays showed a significant decrease in cell proliferation capacity of both TMEM116 knockdown cell lines 1 and 2 (fig. 5B).

Detection of clonogenic Capacity of cells: equal amounts of cells were added to 6-well plates, cultured for 14 days, harvested and subjected to crystal violet staining. The detection results show that the TMEM116 knockdown cell line has obviously reduced clone formation quantity, and the volume of the formed single clone is obviously smaller than that of the clone formed by normal cells. This result indicates that the clonogenic capacity of the cells was reduced after knockdown of TMEM116 (C of fig. 5).

In vivo experiments with mice as experimental animals, cells were injected via tail vein and subcutaneous injection, respectively, and the results showed that both the tumorigenic capacity of TMEM116 for knocking down lung cancer cells and the metastatic capacity of cancer cells were significantly reduced (fig. 6). Selecting a BALB/C female nude mouse with the age of 6 weeks, respectively injecting lung cancer cells or TMEM116 with the same quantity into a tail vein and a subcutaneous way to knock down the lung cancer cells, and detecting the migration and proliferation capacities of the cells. After 7 weeks of injection, the experimental mice were sacrificed and lung tissue and subcutaneous tumor tissue were obtained, respectively. And (3) carrying out body type microscope photographic detection on the lungs of the tail vein injection mice, and calculating and counting the number of lung metastasis nodules. And (3) separating a subcutaneous injection mouse, obtaining subcutaneous tumors, weighing, measuring the volume, and counting the weight and the volume of the subcutaneous tumors. The above experiment results show that the number of lung metastatic nodules of mice with knockdown cells is significantly reduced in tail vein injection experiments (fig. 6, a and B), which indicates that the knockdown of TMEM116 significantly weakens the metastatic capacity of lung cancer cells; in the subcutaneous injection experiment, the weight and volume of the subcutaneous tumor of the mice injected with the knockdown cells are also obviously smaller than those of the control group (C and D in figure 6), which shows that the knockdown of the TMEM116 obviously weakens the proliferation capacity of the lung cancer cells.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain modifications and improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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tgggtgacag ggcgagactc catctcaaaa a 931

Claims

Use of an expression inhibitor of the TMEM116 gene in the preparation of a medicament for inhibiting metastasis, infiltration and/or proliferation of lung cancer cells.
2. The use of claim 1, wherein the expression inhibitor is a biological agent or a chemical agent that inhibits or reduces the expression level of the TMEM116 gene in the cell.
The application of TMEM116 gene as a diagnostic marker in the preparation of lung cancer diagnostic kits, diagnostic reagents or chips.
4. The application of the reagent for detecting the TMEM116 gene expression level in preparing a lung cancer diagnosis kit, a diagnosis reagent or a chip.
5. The application of the reagent for detecting the TMEM116 gene expression level in preparing a lung cancer cure evaluation system.
6. The use of any one of claims 3 to 5, wherein the determination is made only by detecting the expression level of the TMEM116 gene in the genome of the sample to be tested, and when the expression level of the TMEM116 gene in the genome of the sample to be tested is significantly different from that of the negative control and is not significantly different from that of the positive control, the tissue to be tested is lung cancer tissue or tissue infiltrated or transferred by lung cancer cells; the negative control is a normal tissue or a tissue beside the cancer, and the positive control is a lung cancer tissue.