ARID1A and FAT1 double-mutant human non-small cell lung cancer cell strain and application thereof

Abstract

The application discloses a kind of ARID1A and FAT1 double-mutation human non-small cell lung cancer cell strains and its application, belong to the field of tumor biology.The cell strain is named human non-small cell lung cancer cell strain HC3167, and the preservation number is CCTCC NO:C202369.ARID1A and FAT1 two anticancer genes of the cell strain are all mutated, and cell morphology has epithelial-mesenchymal transition, expresses Pan-Keratin, Vimentin and N-cadherin protein.The cell strain can be used to study the morphological and biological characteristics of human non-small cell lung cancer cells, analyze the sensitivity of antitumor drugs, screen and evaluate antitumor drugs, and study the mechanism of drug resistance, the mechanism of drug resistance reversal, construct human non-small cell lung cancer in vivo and in vitro tumor model, so as to seek more effective tumor treatment for human, and has higher scientific research and production application value.

Description

Technical Field

[0001] This invention belongs to the field of tumor biology and relates to an ARID1A and FAT1 double mutant human non-small cell lung cancer cell line and its application. Background Technology

[0002] Lung cancer is a common malignant tumor that seriously threatens human life and health. Lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with 80%–85% being NSCLC. Treatment of NSCLC has entered the era of precision medicine, but approximately one-third of patients with advanced NSCLC still lack therapeutic targets or do not respond to immune checkpoint inhibitor therapy, primarily due to tumor heterogeneity. To promote and meet the needs of research on human NSCLC, an increasing number of lung cancer cell lines from various human sources with different oncogenic mutations are required. Summary of the Invention

[0003] Therefore, the primary objective of this invention is to establish a human non-small cell lung cancer cell line with double mutations in ARID1A and FAT1. This cell line exhibits mutations in both ARID1A and FAT1 tumor suppressor genes, and its cell morphology has undergone epithelial-mesenchymal transition (EMT), which is closely related to tumor drug resistance. The establishment of this non-small cell lung cancer cell line can be used to study the cancer biological mechanisms of ARID1A and FAT1 double-mutant non-small cell lung cancer, develop and evaluate the effects of anticancer drugs on ARID1A and FAT1 double-mutant non-small cell lung cancer, and construct related in vivo and in vitro tumor models.

[0004] The aforementioned human non-small cell lung cancer cell line, named human non-small cell lung cancer cell line HC3167, is deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China. The deposit date is March 24, 2023, and the accession number is CCTCC NO: C202369.

[0005] The cell line described is a human non-small cell lung cancer squamous cell carcinoma cell line.

[0006] The cell line of this invention was obtained from a lung surgical specimen of moderately to poorly differentiated squamous cell carcinoma in a 61-year-old male of East Asian descent. The tumor was digested with type II collagenase and cultured in modified ACL4 serum-free medium for 2 months, then expanded and cultured in medium containing 10% FBSRPMI 1640. This cell line exhibited mutations in both the ARID1A and FAT1 tumor suppressor genes, and simultaneously underwent epithelial-mesenchymal transition. It also showed positive expression of keratin (Pan-Keratin), N-cadherin, and Vimentin proteins.

[0007] The present invention conducted the following experiments:

[0008] (1) Culture of human non-small cell lung cancer cell line HC3167 in serum-free ACL4 medium.

[0009] (2) The human non-small cell lung cancer cell line HC3167 was cultured, digested and passaged in RPMI 1640 medium containing 10% FBS.

[0010] (3) Expression of Pan-Keratin, Vimentin and N-cadherin proteins in human non-small cell lung cancer cell line HC3167.

[0011] (4) Detect gene mutations in human non-small cell lung cancer cell lines HC3167 ARID1A and FAT1.

[0012] (5) Tumorigenicity of human non-small cell lung cancer cell line HC3167 in immunodeficient mice.

[0013] (6) Identification of the human non-small cell lung cancer cell line HC3167 STR.

[0014] A second objective of this invention is to provide the application of the ARID1A and FAT1 double-mutant human non-small cell lung cancer cell line HC3167 in screening or preparation of antitumor drugs.

[0015] The screening application for this drug is used for exclusion purposes, including treatment and diagnosis.

[0016] A third objective of this invention is to provide the application of the ARID1A and FAT1 double-mutant human non-small cell lung cancer cell line HC3167 in analyzing the sensitivity of antitumor drugs or evaluating antitumor drugs.

[0017] This analysis and evaluation is used for exclusion purposes, including treatment and diagnosis.

[0018] The fourth objective of this invention is to provide the application of the ARID1A and FAT1 double mutant human non-small cell lung cancer cell line HC3167 in the study of tumor drug resistance mechanisms and drug resistance reversal mechanisms.

[0019] The fifth objective of this invention is to provide the application of the ARID1A and FAT1 double mutant human non-small cell lung cancer cell line HC3167 in the study of the morphological and biological characteristics of human non-small cell lung cancer cells.

[0020] The sixth objective of this invention is to provide the application of the ARID1A and FAT1 double mutant human non-small cell lung cancer cell line HC3167 in constructing in vivo and in vitro tumor models of human non-small cell lung cancer.

[0021] The ARID1A and FAT1 double-mutant human non-small cell lung cancer cell line HC3167 of this invention can be used to study the morphological and biological characteristics of human ARID1A and FAT1 double-mutant non-small cell lung cancer cells, screen and evaluate anti-tumor drugs and their mechanisms of action, construct tumor models, study tumor drug resistance mechanisms, develop drugs to reverse tumor drug resistance, and study more effective tumor treatment methods. It can also be used to study the tumorigenic mechanism and related signaling pathways of human ARID1A and FAT1 double-mutant non-small cell lung cancer. It has high scientific research and production application value and is expected to generate good scientific research, economic, and social benefits. Attached Figure Description

[0022] Figure 1 This is a photograph of the human non-small cell lung cancer cell line HC3167 of the present invention taken under an inverted phase-contrast microscope. The cell morphology is a mixture of epithelial and fibrous types. Figure 1 A is a 100x magnified image. Figure 1 B is a 200x magnified image.

[0023] Figure 2 This invention relates to the expression of Pan-Keratin, Vimentin, and N-cadherin proteins in the human non-small cell lung cancer cell line HC3167; wherein... Figure 2 A-2C represent the immunofluorescence staining results of Pan-Keratin, Vimentin, and N-cadherin expression, respectively. All three are green fluorescence, while the blue fluorescence represents the cell nucleus. Figure 2 D represents the expression of Pan-Keratin, Vimentin, and N-cadherin proteins in the human non-small cell lung cancer cell line HC3167 as detected by western blot. GAPDH is the internal reference protein, and small muscle cells, H3122, and A549 are control cells.

[0024] Figure 3 Sanger sequencing results for the ARID1A and FAT1 genes of HC3167;

[0025] in Figure 3 A: Sanger sequencing of the HC3167 ARID1A gene revealed deletion and insertion mutations, specifically NM_006015.6:c.6792_6833delinsTGG, NP_006006.3:p.[Ile2135_Leu2147del; Tyr2148Trp]; the blue arrows indicate the three inserted bases, F indicates forward primer sequencing, and R indicates reverse primer sequencing;

[0026] Figure 3B: Sanger sequencing of the HC3167 FAT1 gene revealed an insertion mutation in the FAT1 gene, specifically NM_005245.4:c.12239_12240insA, NP_005236.2:p.Phe4010TyrfsTer41; the blue arrows indicate the inserted bases; F indicates forward primer sequencing, and R indicates reverse primer sequencing.

[0027] Figure 4 The cell growth curve and population doubling time of the human non-small cell lung cancer cell line HC3167 are shown.

[0028] Figure 5 Tumors that grew in immunodeficient mice NU / NU Mice after the human non-small cell lung cancer cell line HC3167 was inoculated; indicated by the red arrow.

[0029] Figure 6 The results of the identification of the human non-small cell lung cancer cell line HC3167 STR. Detailed Implementation

[0030] To illustrate the technical content, structural features, objectives, and effects of this invention in detail, the following description, in conjunction with the embodiments, is provided without limiting the scope of protection of this invention.

[0031] The existing experimental methods, testing methods and solution preparation methods involved in this invention can be referred to "Cellular Experimentation Guide", ISBN: 7-03-007598-6 / Q.891, original author: [US] DL Specter RD Goldman & L.A. Reinvand.

[0032] The RPMI1640 culture medium, reagents, antibodies, and kits mentioned in the examples are all commercially available products.

[0033] Example 1: Preparation of human non-small cell lung cancer cell line HC3167

[0034] The steps are as follows:

[0035] (1) A lung tumor specimen of moderately to poorly differentiated squamous cell carcinoma of a 61-year-old male of East Asian descent (from the Second Xiangya Hospital of Central South University) was placed in cold RPMI 1640 culture medium. Necrotic tissue was aseptically cleaned in a biosafety cabinet, minced, digested with 400u / ml type II collagenase, and cultured in modified ACL4 serum-free medium for 2 months. Then, it was amplified and cultured in RPMI 1640 medium containing 10% FBS.

[0036] (2) Morphological observation of human non-small cell lung cancer cell line HC3167: Human non-small cell lung cancer cell line HC3167 was seeded in 25cm cells. 2In the culture flask, after the cells have grown to the logarithmic growth phase, observe the morphology of the live cells under an inverted phase-contrast microscope and take photographs, such as... Figure 1 As shown, the cell line HC3167 of this invention exhibits a mixed cell morphology of epithelial and fibroblast-like structures, adhering to the cell wall and growing.

[0037] (3) Digestion of the human non-small cell lung cancer cell line HC3167 of the present invention: Human non-small cell lung cancer cell line HC3167 was seeded in a 25cm... 2 In the culture flask, when the cells have reached 80% confluence, aspirate the culture medium, add 5 mL of calcium- and magnesium-free phosphate buffer (D-PBS buffer) for washing, add 0.25% trypsin and 1 mL of 0.25% Trypsin-EDTA for digestion at room temperature, observe under a microscope, and when the cells become round and resemble quicksand, add 5 mL of medium containing 10% FBS RPMI 1640 to neutralize, gently pipette, aspirate into a 15 mL centrifuge tube, centrifuge at 800 rpm for 5 minutes, discard the supernatant, add medium containing 10% FBS RPMI 1640, gently pipette to mix, and passage at a ratio of 1:6-8.

[0038] (4) Cryopreservation of the human non-small cell lung cancer cell line HC3167 of the present invention: The obtained cells were placed in the cryopreservation solution of cryovials, and then the cryovials were placed in liquid nitrogen for preservation. The cryopreservation solution consisted of 95% RPMI 1640 medium containing 10% FBS and 5% dimethyl sulfoxide (DMSO). The cryopreservation process involved placing the cryovials in a nalgene programmed cooling box at -80°C overnight and then placing them in liquid nitrogen.

[0039] (5) Thawing of the human non-small cell lung cancer cell line HC3167 of this invention: Remove the cryovial containing the cells from liquid nitrogen and immediately place it upright in a 37-40℃ water bath, gently shaking to ensure the cryovial cap is on the water surface, allowing the frozen contents to thaw within 1-2 minutes. Wipe the outer wall of the cryovial with alcohol and then place it in a clean bench. Place the thawed cell suspension in a 15mL sterile centrifuge tube, add 5mL of D-PBS buffer, centrifuge at 800 rpm for 5 minutes, discard the supernatant, add 5mL of RPMI 1640 medium containing 10% FBS, gently pipette to mix, and aspirate the mixture into a 25cm aerator. 2 Place the cell culture flasks in a CO2 incubator with the caps loosened. Culture the cells at 37°C, 5% CO2, and saturated humidity. Change the culture medium every 2-3 days. When the cell density reaches 80-90%, digest and passage the cells.

[0040] Example 2: Expression of Pan-Keratin, Vimentin and N-cadherin proteins in the human non-small cell lung cancer cell line HC3167 of the present invention.

[0041] (1) Immunofluorescence detection of the expression of Pan-Keratin, Vimentin and N-cadherin proteins in human non-small cell lung cancer cell line HC3167: HC3167 cells cultured in 12-well plates were pooled to 40-50% concentration. The original culture medium was removed, and the cells were washed once with cold D-PBS buffer. Immunofluorescence staining was performed according to standard procedures. The results are shown in the figure. Figure 2 AC,HC3167 expresses Pan-Keratin, Vimentin, and N-cadherin proteins.

[0042] (2) Western blot detection of Pan-Keratin, Vimentin, and N-cadherin protein expression in human non-small cell lung cancer cell line HC3167: HC3167 cells cultured in 6-well plates to 80-90% confluence were collected. The original culture medium was removed, and the cells were washed once with cold D-PBS buffer. Cell lysis buffer containing protease inhibitors and phosphatase inhibitors was added. Cells were immediately scraped off on ice, and the extracted cell extract was transferred to an EP tube. The tube was sonicated for 10-15 seconds, cooled on ice, and centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant was collected, and the protein concentration was measured using a BCA kit. 25 μg of total protein was loaded with sample buffer, and the mixture was subjected to routine electrophoresis, transfer to a membrane, and incubation with antibody according to the Western blot method. The membrane was then exposed to the microplate. Results are shown in [Figure number missing]. Figure 2 D. Western blot results showed that HC3167 cells highly expressed Pan-Keratin, Vimentin, and N-cadherin proteins. Small muscle cells expressed Vimentin, H3122 cells expressed Pan-Keratin, and A549 cells expressed both Pan-Keratin and N-cadherin.

[0043] Example 3: Reverse transcription polymerase chain reaction and DNA Sanger sequencing

[0044] Total RNA was extracted from HC3167 cells in logarithmic growth phase using the RNeasy Mini Kit. 2 µg of total RNA was used to synthesize first-strand cDNA using the RevertAid First-Strand cDNA Synthesis Kit. PCR amplification was performed using the Platinum™ SuperFi II GreenPCR Master Mix kit. The primer pairs used were ARID1A-F (5′-GCAGTTGGACCTATCTCCATACC-3′), ARID1A-R (5′GGCAAGGCTGTCCTCTAGGAA-3′), FAT1-F (5′-AGCTATGCACACATCGAAGAGT-3′), and FAT1-R (5′-AGGAACACGGATTGACGCTTATC-3′). After amplification, the cells were sent to BGI Genomics in Beijing for Sanger sequencing. Sequencing results are shown below. Figure 3 The ARID1A gene sanger sequencing revealed deletion and insertion mutations, specifically NM_006015.6:c.6792_6833delinsTGG, NP_006006.3:p.[Ile2135_Leu2147del; Tyr2148Trp]. The HC3167 FAT1 gene sanger sequencing revealed insertion mutations, specifically NM_005245.4:c.12239_12240insA, NP_005236.2:p.Phe4010TyrfsTer41.

[0045] Example 4: Cell growth curve and doubling time determination of human non-small cell lung cancer cell line HC3167

[0046] The human non-small cell lung cancer cell line HC3167 of this invention was digested with 0.25% trypsin and 0.25% trypsin-EDTA to prepare a single-cell suspension, which was then seeded into 24-well plates at 1×10⁶ cells per well. 4 Cell counts were performed at 24, 48, 72, 96, and 120 hours, with three wells counted each time. The average value was used to plot a cell curve, and the doubling time (Td) was calculated. The population doubling time Td (h) during the logarithmic growth phase was calculated using the Patterson formula: Tlg2 / lg(N / NO), where T represents the time of logarithmic cell proliferation, N is the number of cells at the end of the logarithmic growth phase, and NO is the number of cells at the beginning of the logarithmic growth phase. The average population doubling time for HC3167 was calculated to be 30.08 hours. (See attached table). Figure 4 .

[0047] Example 5: Subcutaneous tumor formation in immunodeficient mice NU / NU Mice after inoculation with the human non-small cell lung cancer cell line HC3167 of the present invention.

[0048] The human non-small cell lung cancer cell line HC3167 of this invention, which is in the logarithmic growth phase, was digested with 0.25% trypsin and 0.25% trypsin-EDTA, respectively. The cells were washed twice with pre-cooled PBS to remove residual serum. The cell pellet was then pipetted to a suitable concentration in pre-cooled serum-free medium. HC3167 cells were then mixed with 1:1 4°C matrix gel at a ratio of 2-3 × 10⁻³. 6 One cell / mice was subcutaneously injected into a NU / NU Mice (Beijing Vital River Laboratory Animal Co., Ltd.) at 3-4 weeks of age, with an injection volume of 0.1 mL. Tumor formation was observed twice a week until tumor formation occurred. Figure 5 The tumor formed by the human non-small cell lung cancer cell line HC3167 under the skin of immunodeficient mice NU / NU Mice.

[0049] Example 6: STR identification of the human non-small cell lung cancer cell line HC3167 of the present invention.

[0050] The human non-small cell lung cancer cell line HC3167 of this invention was confluent to 80-90% concentration in a 25cm column. 2 One flask of cells was cultured, the culture medium was aspirated, and the cells were digested with 0.25% trypsin and 0.25% trypsin-EDTA. After centrifugation, the culture medium was discarded, 5 mL of phosphate buffer was added, the cells were rinsed once, centrifuged again, and the phosphate buffer was discarded. The cell pellet was sent to the company for human cell STR identification. Results are shown below. Figure 6 Comparison with cell bank databases such as ATCC and DSMZ revealed no identical STR test results, thus proving its uniqueness and that no cross-contamination with other cells occurred during primary culture.

[0051] The above description is merely an embodiment of the present invention and does not limit the scope of patent protection of the present invention. Any equivalent structural transformations made using the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. An ARID1A and FAT1 double mutant human non-small cell lung cancer cell line HC3167, with accession number CCTCC NO: C202369.

2. The ARID1A and FAT1 double-mutant human non-small cell lung cancer cell line HC3167 according to claim 1, characterized in that, The cell line described is a human non-small cell lung cancer squamous cell carcinoma cell line.

3. The ARID1A and FAT1 double-mutant human non-small cell lung cancer cell line HC3167 according to claim 1, characterized in that, The cell line in question exhibited mutations in both the ARID1A and FAT1 tumor suppressor genes, and the cell morphology underwent epithelial-mesenchymal transition.

4. The ARID1A and FAT1 double-mutant human non-small cell lung cancer cell line HC3167 according to claim 1, characterized in that, The cell line expressed Pan-Keratin, Vimentin, and N-cadherin proteins.

5. The application of the ARID1A and FAT1 double-mutant human non-small cell lung cancer cell line HC3167 according to any one of claims 1-4, characterized in that, Used for screening or preparation of anti-tumor drugs.

6. The application of the ARID1A and FAT1 double-mutant human non-small cell lung cancer cell line HC3167 according to any one of claims 1-4, characterized in that, Used to analyze the sensitivity of antitumor drugs or to evaluate antitumor drugs.

7. The application of the ARID1A and FAT1 double-mutant human non-small cell lung cancer cell line HC3167 according to any one of claims 1-4, characterized in that, Used to study tumor drug resistance mechanisms and drug resistance reversal mechanisms.

8. The application of the ARID1A and FAT1 double-mutant human non-small cell lung cancer cell line HC3167 according to any one of claims 1-4, characterized in that, Used to study the morphological and biological characteristics of human non-small cell lung cancer cells.

9. The application of the ARID1A and FAT1 double-mutant human non-small cell lung cancer cell line HC3167 according to any one of claims 1-4, characterized in that, Used to construct in vivo and in vitro tumor models of human non-small cell lung cancer.

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