Application of reagent for detecting TUBA1B gene in preparation of reagent for detecting non-small cell lung cancer bone metastasis and stably transfected lung adenocarcinoma cell strain of low-expression and overexpression TUBA1B gene

Abstract

The invention provides application of a reagent for detecting a TUBA1B gene in preparation of a reagent for detecting non-small cell lung cancer bone metastasis and a stably transfected lung adenocarcinoma cell strain for low-expression and over-expression of the TUBA1B gene, and belongs to the technical field of lung cancer bone metastasis research. Single-cell sequencing data analysis is carried out on lung carcinoma in situ (PT), lung cancer bone metastasis primary lesion (MPT) and metastatic lesion (MT); screening differential genes as candidate genes, and then screening genes with significant difference in survival rate in lung adenocarcinoma in combination with a GEPIA2 database to obtain TUBA1B (Log2FC = 1.73, p < 0.05); according to the application, expression of the TUBA1B gene in lung adenocarcinoma cells A549 is interfered, knock-down or overexpression of the TUBA1B gene has remarkable influence on proliferation, invasion and migration capacities of the A549 cells, and an experimental foundation is laid for subsequent specific bone metastasis mechanism research.

Description

Technical Field

[0001] The present invention belongs to the technical field of lung cancer bone metastasis research, and particularly relates to the use of a reagent for detecting the TUBA1B gene in preparing a reagent for detecting non-small cell lung cancer bone metastasis, and a stably transfected lung adenocarcinoma cell line that underexpresses or overexpresses the TUBA1B gene. Background Art

[0002] According to the 2024 Global Cancer Epidemiology Report, lung cancer will be the malignant tumor with the highest morbidity and mortality worldwide in 2022. Based on its pathological morphology, lung cancer can be preliminarily divided into two major categories: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with NSCLC accounting for approximately 85% of cases. Because nearly 80% of lung cancer patients have distant metastases at the time of diagnosis, survival and prognosis are poor, with a 5-year survival rate of 18%. The incidence of bone metastasis in NSCLC ranges from 20% to 60%. With the advent of PET-CT and other technologies, the detection rate of bone metastasis has greatly improved. Tumor cell bone metastasis is often associated with osteoclasts. Consequently, bone metastasis often leads to skeletal-related complications (SREs) in cancer patients, such as hypercalcemia, severe pain, spinal compression, and pathological fractures. The presence of SREs can further impact physical activity and quality of life in cancer patients. Existing clinical practice has shown that patients with bone metastases in NSCLC who receive immunotherapy have a worse prognosis due to the unfavorable bone microenvironment. Therefore, studying bone metastasis of non-small cell lung cancer and even analyzing the immune microenvironment of bone metastases have very important clinical and scientific significance. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide stably transfected lung adenocarcinoma cell lines that underexpress or overexpress the TUBA1B gene and the use of the TUBA1B gene in preparing a reagent for detecting bone metastasis of non-small cell lung cancer.

[0004] The present invention provides the use of a reagent for detecting the TUBA1B gene in preparing a reagent for detecting bone metastasis of non-small cell lung cancer. When the TUBA1B gene is significantly overexpressed, the risk of bone metastasis of non-small cell lung cancer is high; when the TUBA1B gene is significantly underexpressed, the risk of bone metastasis of non-small cell lung cancer is low.

[0005] The present invention provides a stably transfected lung adenocarcinoma cell line with low TUBA1B gene expression.

[0006] Preferably, the protein is obtained by transfecting a lentiviral vector carrying TUBA1B gene siRNA into lung adenocarcinoma cells.

[0007] Preferably, the lung adenocarcinoma cells are PC9 or A549 cells.

[0008] The present invention provides a stably transfected lung adenocarcinoma cell line overexpressing the TUBA1B gene.

[0009] Preferably, the gene is obtained by transfecting a lentiviral vector carrying the TUBA1B gene into lung adenocarcinoma cells.

[0010] Preferably, the lung adenocarcinoma cells are PC9 or A549 cells.

[0011] The present invention provides the use of the stably transfected lung adenocarcinoma cell line that underexpresses the TUBA1B gene or the stably transfected lung adenocarcinoma cell line that overexpresses the TUBA1B gene in the study of bone metastasis of non-small cell lung cancer and analysis of the immune microenvironment of bone metastases.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention screens differentially expressed genes as candidate genes by analyzing single-cell sequencing data of lung carcinoma in situ (PT), primary lesions of lung cancer bone metastases (MPT), and metastatic lesions (MT). The genes with significantly different survival rates in lung adenocarcinoma were then screened using the GEPIA2 database to obtain TUBA1B (Log2FC=1.73, p<0.05). The present invention interferes with the expression of the TUBA1B gene in lung adenocarcinoma cells A549, demonstrating that knocking down or overexpressing the TUBA1B gene significantly affects the proliferation, invasion, and migration abilities of A549 cells. The present invention utilizes lentivirus to construct a stable cell line with low TUBA1B expression, so as to utilize the characteristic of high expression in the primary lesions of patients with bone metastasis to test its effect on bone metastasis of non-small cell lung cancer in subsequent animal experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A This is the heat map of differentially expressed genes between the primary lesion (MPT) and the metastatic lesion (MT) of the epithelial cell population;

[0015] Figure 1B This is the heat map of differentially expressed genes in the osteoclast population MT compared with MPT;

[0016] Figure 1C This is a heat map of differentially expressed genes in macrophage MT compared with MPT.

[0017] Figure 2 This is the intersection diagram of differentially expressed genes after comparison of different groups of epithelial cells, osteoclasts, and macrophages;

[0018] Figure 3 This is the OS chart of TUBA1B in lung adenocarcinoma (red represents high expression of TUBA1B, blue represents low expression of TUBA1B);

[0019] Figure 4To identify the transfection effect using the GFP fluorescent tag in the lentivirus, NC is the control group, 955 is the stable transfection strain code, and 1, 2, and 3 are different experimental groups;

[0020] Figure 5 To identify the transfection effect using the GFP fluorescent tag in the lentivirus, NC was the control group and 1229 was the overexpression experimental group;

[0021] Figure 6 Figure 3: Changes in cell behavior after low expression of TUBA1B. A: Cell invasion ability was significantly reduced after low expression of TUBA1B (top); B: Cell migration ability was significantly reduced after low expression of TUBA1B (bottom);

[0022] Figure 7 Figure 3: Changes in cell behavior after overexpression of TUBA1B. A: Overexpression of TUBA1B significantly enhanced the cell invasion ability; B: Overexpression of TUBA1B significantly enhanced the cell migration ability; C: The multiples of TUBA1B overexpression in different types of lung adenocarcinoma cells (PC9 and A549); D: CCK8 curve of cell proliferation ability of A549 cells after overexpression of TUBA1B. DETAILED DESCRIPTION

[0023] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] The lung adenocarcinoma cell line A549 was constructed by lentiviral transfection to interfere with and overexpress the TUBA1B gene.

[0025] The lung adenocarcinoma cell line PC9 overexpressing TUBA1B gene was constructed by lentiviral transfection.

[0026] Lentivirus: purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0027] Human TUBA1B gene interference lentivirus and human TUBA1B gene overexpression lentivirus were purchased from Shanghai GeneCare Gene Medicine Technology Co., Ltd.

[0028] Lung adenocarcinoma cell line A549 was purchased from the Stem Cell Bank of the Chinese Academy of Sciences.

[0029] Example 1

[0030] 1. Target gene screening

[0031] Single-cell sequencing was performed on clinical samples of bone metastases and primary lesions from three cases of primary lung cancer and three cases of non-small cell lung cancer with bone metastasis (all samples were extracted from lesions of relevant patients in Ward 3 of the Department of Respiratory Medicine, Henan Cancer Hospital; see Table 1 for specific information). The relevant sequencing work was entrusted to Xingeyuan (Nanjing) Biotechnology Co., Ltd.

[0032] Table 1 Sample sources

[0033]

[0034]

[0035] The specific operations are as follows:

[0036] Single cell suspension (2×10 5 cells / ml) by Singleron The single cell processing system is loaded onto the microwell chip. The barcoded magnetic beads are collected for reverse transcription of mRNA to obtain cDNA, which is then fragmented and connected to sequencing adapters after PCR amplification. The library was constructed using the Single Cell RNA Library Kit (Singleron), diluted to 4 nM, mixed, and subjected to paired-end 150 bp sequencing using the Illumina NovaSeq 6000 platform.

[0037] After the data was taken off the computer, the dimensionality reduction and clustering were performed on the data, and the cells were grouped and processed to obtain the primary lung cancer group (PT), the primary lesion of non-small cell lung cancer bone metastasis group (MPT), and the non-small cell lung cancer bone metastasis group (MT). The differences of different cell populations in the three groups were analyzed.

[0038] To study the mechanism of promoting bone metastasis in primary lung cancer, in epithelial cell populations, the differential genes of MPT vs. MT group were analyzed and the TOP20 up-regulated differential genes were screened; in osteoclast and macrophage populations, the differential genes of MT vs. MPT group were analyzed and the TOP20 up-regulated differential genes were screened. In macrophage populations, the TOP20 up-regulated differential genes of MT vs. MPT were screened ( Figure 1A 、 Figure 1B and Figure 1C Then, the veen graph was used to intersect the up-regulated expression parts of the three groups of differentially expressed genes, and the differentially expressed gene TUBA1B ( Figure 2 ).

[0039] After querying the survival difference of TUBA1B in lung adenocarcinoma (LUAD) using the GEPIA2 database, it was found that high expression of TUBA1B can significantly reduce the survival of LUAD (Log2FC=1.73, p<0.05) ( Figure 3 ).

[0040] TUBA1B affects cell behavior

[0041] After confirming that TUAB1B may be a key gene influencing bone metastasis in NSCLC, we conducted experiments in non-small cell lung cancer cell lines, including PC9 and A549, to examine whether knocking down TUBA1B expression alters cell proliferation, invasion, and migration. The siRNA used to knock down TUBA1B is: genOFF™ st-h-TUBA1B_001; target sequence: AGGAGATCATTGACCTTGT; siRNA order source: Guangzhou Ruibo Biotechnology Co., Ltd.

[0042] The results showed that after knocking down TUBA1B using siRNA, the invasion and migration ability of cells was significantly reduced.

[0043] Specific steps:

[0044] (1) Cell preparation

[0045] A549 cells in good condition were plated into 6-well plates according to experimental requirements and cultured in a constant temperature incubator for 6 to 24 hours. When the cells adhered to the wall, the cell density reached 50 to 60% (about 2 × 10 cells per well) at the time of transfection. 5 indivual).

[0046] (2) Viral infection:

[0047] Before infection, take out the virus from the -80℃ freezer and slowly thaw it on ice for later use.

[0048] Remove the A549 cells to be transfected from the incubator and observe the cell status and cell density under a microscope. If the cells meet the requirements, transfer them to a biosafety cabinet. Discard the original culture medium and wash three times with sterile PBS. Add 1 ml of fresh RPMI-1640 complete medium (containing 10% FBS, 1% penicillin-streptomycin, 4 μg / ml Polybrene, and 2 μg / ml puromycin) to each well.

[0049] According to the MOI value of A549 (recommended as 10), add the corresponding virus to the corresponding wells (3 wells for the interference expression group and 3 wells for the control group). The amount of virus added to each well (μl) = MOI × number of cells / virus titer (TU / ml) × 1000, where the titer / concentration of human TUBA1B gene interference lentivirus -1 / 2 / 3 is 5×10 8 TU / ml, i.e., 4 μl was added to each well (the interference group virus was 3 groups); the overexpression lentivirus NC titer / concentration was 1×10 8 TU / ml, that is, add 20 μl to each well (the overexpression group virus is 1 group). Then put it in the incubator for incubation.

[0050] 4 h after lentiviral infection, the culture medium was replenished to 2 ml and the medium was changed 24 h after infection.

[0051] Change the medium or passage the cells according to the cell status and density, monitor GFP, and adjust the puromycin concentration to 1μg / ml after the status stabilizes. The transfection effect was monitored 72 hours after transfection according to the instructions. Specifically, the transfection effect was identified by using the GFP fluorescent tag in the lentivirus. NC is the control group, 955 is the code for the stable transfected strain with low TUBA1B expression, and 1, 2, and 3 are different experimental groups. The results are shown in the figure below. Figure 4 The results of the stable strains overexpressing TUBA1B are shown in Figure 5 shown.

[0052] Table 2 Transfection ratio of stable transfectants with low expression and over expression of TUBA1B

[0053] Experimental groups Cell type GFP+% GFP+% cell concentration Total cell number DENC A549 36.88 4.48E+05 1677 DE9551TUBA1B A549 96.76 8.86E+0.5 1267 DE9552TUBA1B A549 95.85 8.17E+05 1180 DE9553TUBA1B A549 96.29 8.04E+05 1155 OENC A549 36.97 9.71E+05 1813 OE1229TUBA1B A549 95.81 9.52E+05 1375

[0054] Cell proliferation, invasion and migration ability detection

[0055] Proliferation Assay Method: 1) Transfected cells were divided into Ctrl and OE groups. Cell suspensions were prepared by trypsinization and seeded into 96-well plates at 1000 viable cells per well. Five replicates were set up for each group. 0-hour, 24-hour, 48-hour, and 72-hour assays were also established. 2) After cells adhered (approximately 4-6 hours), the culture medium from the 0-hour group was removed. Cell culture medium (RPMI1640 + 1% streptomycin / penicillin + 10% FBS + 1% puromycin) was mixed with CCK-8 assay reagent at a ratio of 10:1. 110 μL of this mixture was added to each well. Five blank wells were also added with the same medium as a blank control and incubated for an additional 2 hours. 3) After 2 hours, the absorbance was read at 450 nm using a microplate reader. The OD450 value for the Ctrl and OE groups was deducted from the average of the blank wells to record as the 0-hour OD450 value for each group. 4) Thereafter, the detection and calculation methods for 24h, 48h, and 72h are the same as above.

[0056] Migration: 1) Place the chamber in a 24-well plate. 2) Add 100 μL of serum-free culture medium to the chamber. 3) Add 200 μL of a serum-free culture medium suspension of 5,000 cells to be tested to the chamber. 4) Add 750 μL of complete culture medium containing serum to the corresponding lower chamber. 5) Incubate the above 24-well plate in a cell incubator at 37°C and 5% CO2 for 18 hours. 6) Aspirate the culture medium from the chamber and rinse the chamber twice in PBS solution. 7) Fix the cells with formaldehyde solution for 20 minutes. 8) After removing the formaldehyde solution, rinse again twice in PBS solution. 9) Soak the chamber in crystal violet stain for 30 minutes. 10) Remove the stain and rinse three times with PBS solution. 11) Gently wipe the non-migrated cells in the upper chamber with a cotton swab. 12) Observe and count the cells under an inverted optical microscope.

[0057] Invasion: 1) Place the chamber in a 24-well plate. 2) 2 hours before the experiment, dilute Matrigel with cell culture medium at a ratio of 1:5 and evenly spread 60 μl of Matrigel per well in the upper chamber, avoiding air bubbles. After spreading, place the plate in an incubator to solidify for 1 hour. 3) Subsequent steps are the same as for migration.

[0058] The results are as follows Figure 6 As shown, the cell invasion ability was significantly reduced after low expression of TUBA1B, and the cell migration ability was significantly reduced after low expression of TUBA1B.

[0059] The results are as follows Figure 7 As shown, overexpression of TUBA1B significantly enhanced the cell invasion ability, overexpression of TUBA1B significantly enhanced the cell migration ability, and overexpression of TUBA1B also enhanced the cell proliferation ability.

[0060] As shown in the above examples, the present invention screened for the TUBA1B gene by analyzing single-cell sequencing data from lung carcinoma in situ (PT), primary lung cancer metastasis (MPT), and metastatic lesions (MT) of bone. Knockdown or overexpression of the TUBA1B gene significantly affected the proliferation, invasion, and migration of A549 cells. The stable transgenic strains with low TUBA1B expression provided by the present invention served as a control for subsequent animal experiments, screening multiple generations of low-affinity bone metastatic strains, and laid the experimental foundation for the specific mechanism of bone metastasis.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting the TUBA1B gene in the preparation of a reagent for detecting bone metastasis of non-small cell lung cancer, characterized in that: When the TUBA1B gene is significantly overexpressed, the risk of bone metastasis of non-small cell lung cancer is high, and when the TUBA1B gene is significantly underexpressed, the risk of bone metastasis of non-small cell lung cancer is low.

2. A stably transfected lung adenocarcinoma cell line with low expression of the TUBA1B gene.

3. The stably transfected lung adenocarcinoma cell line with low TUBA1B gene expression according to claim 2, characterized in that: This was achieved by transfecting a lentiviral vector carrying TUBA1B gene siRNA into lung adenocarcinoma cells.

4. The stably transfected lung adenocarcinoma cell line with low TUBA1B gene expression according to claim 3, characterized in that: The lung adenocarcinoma cells are PC9 or A549 cells.

5. A stably transfected lung adenocarcinoma cell line overexpressing the TUBA1B gene.

6. The stably transfected lung adenocarcinoma cell line overexpressing the TUBA1B gene according to claim 5, characterized in that: It was obtained by transfecting a lentiviral vector carrying the TUBA1B gene into lung adenocarcinoma cells.

7. The stably transfected lung adenocarcinoma cell line overexpressing the TUBA1B gene according to claim 6, characterized in that: The lung adenocarcinoma cells are PC9 or A549 cells.

8. Use of the stably transfected lung adenocarcinoma cell line with low TUBA1B gene expression according to any one of claims 2 to 4 or the stably transfected lung adenocarcinoma cell line with overexpression of TUBA1B gene according to any one of claims 5 to 7 in the study of bone metastasis of non-small cell lung cancer and analysis of the immune microenvironment of bone metastases.

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