Method for constructing liver cancer cell line through stable expression of DeltaNp63alpha and application of liver cancer cell line

Abstract

The invention belongs to the field of construction methods of liver cancer cell lines, and particularly relates to a method for constructing a liver cancer cell line through stable expression of DeltaNp63alpha and application of the liver cancer cell line. According to the invention, lentivirus transfection is adopted to obtain the liver cancer cell line capable of stably expressing DeltaNp63alpha, high expression of DeltaNp63alpha in liver cancer cells Huh7, PLC / PRF / 5, HCCLM3, SK-HEP-1, Hep3B, SMMC7721, SNU387, MHCC97H and BEL7402 is realized, and the obtained liver cancer cell line has high cell proliferation rate, supports drug screening, and can be used for researching the effect of novel drugs in liver cancer malignant development.

Description

Technical Field

[0001] The present invention belongs to the field of methods for constructing liver cancer cell lines, and specifically relates to a method for constructing liver cancer cell lines by stably expressing DeltaNp63alpha and an application thereof. Background Art

[0002] The transcription factor p63 is a member of the p53 protein family and is encoded by the Tp63 gene. TP63 derives a longer TA isomer (TAp63) and a shorter ΔN isomer (ΔNp63) according to the different transcription start points at its N-terminus (amino terminal). There are two protein subtypes of TA or ΔN isomers. The TA subtype contains a common transactivation isomer similar to p53, which can mimic the function of p53 and exert a strong tumor suppressor effect, while the ΔN subtype lacks this isomer and has a weak tumor suppressor function. Unlike TAp63, due to the lack of the amino terminal fragment, ΔNp63 loses its tumor suppressor function and becomes a transcription factor that promotes the growth and survival of cancer cells, thereby promoting the occurrence of cancer. Due to the different splicing methods of gene introns, the TA subtype and the ΔN subtype have different homologous isomers, and the selective promoters and splicing sites produce different protein subtypes, such as p63α, p63β, p63γ, p63δ and p63ε.

[0003] Existing studies have shown that overexpression of the p63 gene is common in head and neck squamous cell carcinoma, lung cancer, breast cancer, cervical cancer, prostate cancer and other tumors, while the role of p63 in liver cancer is controversial. Some studies have pointed out that p63 expression in liver cancer is associated with a worse prognosis, while other studies have pointed out that p63 expression has not been found in liver cancer, and previous studies have not further studied the role of specific subtypes. Therefore, the relationship between p63 and liver cancer is not clear. In addition, the existing genetic engineering technology to construct liver cancer cell lines is usually achieved by knocking out individual genes, such as a liver cancer cell line with stable knockout of the LAPTM5 gene and its construction method and application (publication number: CN119040323A). There has been no research on constructing liver cancer cell lines by overexpressing p63-related genes. Summary of the invention

[0004] Based on the above problems, the present invention studies the relationship between p63 and liver cancer, indicating that p63, as a pioneer transcription factor, opens the chromatin structure of oncogene regulatory elements, recruits chromatin structural proteins to form a promoter-enhancer loop, and ultimately upregulates the expression of multiple oncogenes, promoting liver cancer. High expression of p63 is significantly correlated with lower survival rates in some patients with hepatocellular carcinoma, and further characterizes that the DeltaNp63apha subtype plays a major role. Therefore, the present invention uses lentiviral transfection to obtain a liver cancer cell line that stably expresses DeltaNp63alpha. The obtained liver cancer cell line has a high cell proliferation rate, supports drug screening, and can also be used to study the role of new drugs in the malignant development of liver cancer.

[0005] On this basis, the present invention provides a method for constructing a liver cancer cell line by stably expressing DeltaNp63alpha, as follows: L1. Culture of liver cancer cells; L2. Construction of recombinant plasmid pCDH-EF1-MCS-DeltaNp63alpha-T2A-copGFP; L3. Preparation of lentivirus; L4. Construction of liver cancer cell line stably expressing DeltaNp63alpha.

[0006] Preferably, the specific contents of step L1 are as follows: The hepatoma cell line is one of Huh7, PLC / PRF / 5, HCCLM3, SK-HEP-1, Hep3B, SMMC7721, SNU387, MHCC97H, and BEL7402; SNU387 is preferred for liver cancer cells; Hepatoma cells were cultured in RPMI-1640 medium at 37°C and 5% carbon dioxide.

[0007] Preferably, the specific contents of step L2 are as follows: L2.1 The total RNA of liver cancer cells obtained in step L1 was extracted by Trizol method, and cDNA was obtained by reverse transcription using a cDNA synthesis kit; L2.2 Use the cDNA obtained in step L2.1 as a template to perform PCR reaction. The upstream and downstream primers involved in the PCR reaction are as follows: DeltaNp63alpha forward primer: 5′-CGGAATTCCTTGCCCAGGAAGAGACAGG-3′; DeltaNp63alpha reverse primer: 5′-CGCCGCTTCGTACCATCACCGTT-3′; GAPDH forward primer: 5′-AAGAGAGGCAATCCTCACCCT-3′; GAPDH reverse primer: 5′-TACATGGCTGGGGTGTTGAA-3′; The PCR reaction system is as follows:

[0008] PCR reaction conditions: 94°C for 5 min; 94°C for 40 s, 50°C for 45 s, 72°C for 90 s, 33 cycles; 72°C for 10 min; The PCR product was recovered from L2.3 purification gel, digested with EcoR I and BamH I restriction endonucleases, and pCDH-EF1-MCS-T2A-copGFP and DeltaNp63alpha were ligated with T4 DNA ligase to obtain the recombinant plasmid pCDH-EF1-MCS-DeltaNp63alpha-T2A-copGFP.

[0009] Preferably, the specific content of step L3 is as follows: 293T cells were cultured in L3.1 until the cell confluence reached 70-80%, and then digested with trypsin and resuspended, and then inoculated in 12-well plates at a density of 1×10 5 -1×10 6 100 cells / well, culture for 18-20 hours, replace with serum-free DMEM culture medium to obtain 293T cells for use; L3.2 Mix the recombinant plasmid pCDH-EF1-MCS-DeltaNp63alpha-T2A-copGFP, Del8.9, VSVG, and serum-free DMEM culture medium obtained in step L2 according to a mass volume ratio of 1μg: 0.35-0.4μg: 0.7-0.8μg: 0.3-0.5mL to obtain mixed solution 1, add 0.2 times the volume of transfection reagent to mixed solution 1, mix well, let stand at room temperature for 5-10 minutes, and then add to the stand-by 293T cells obtained in step L3.1. After completion, culture in an incubator for 6 hours. After taking out, replace the culture medium with DMEM culture medium containing 10% FBS, and continue to culture for 24-36 hours. After completion, take out, collect the supernatant, and centrifuge at 3000-4000g for 8-10 minutes. The supernatant is the lentivirus.

[0010] Preferably, the specific content of step L4 is as follows: Hepatoma cells were obtained by culturing in the same manner as in step L1 until the cell confluence reached 60-70%, and the lentivirus obtained in step L3 was infected at a titer of MOI 200. After 72 hours, the culture medium was replaced with RPMI-1640 culture medium containing 10% FBS, and 1.5 μg / mL puromycin was added for screening for 1-2 weeks, and the culture medium was replaced every 2-3 days. During the screening process, the changes in GFP in the cells were continuously observed under a fluorescence microscope until all cells were green under a fluorescence microscope, and the fluorescence intensity did not weaken after continuous culture for several generations, and a hepatoma cell line stably expressing DeltaNp63alpha was obtained.

[0011] The present invention also provides an application of stably expressing DeltaNp63alpha to construct a liver cancer cell line, the specific contents of which are as follows: Liver cancer cell lines that stably express DeltaNp63alpha can be used for drug screening and to study the role of new drugs in the malignant development of liver cancer.

[0012] The beneficial effects of the present invention are as follows: The present invention clarifies the relationship between p63 and liver cancer, and the specific subtype is DeltaNp63apha. The high expression of DeltaNp63apha is significantly correlated with the lower survival rate of some hepatocellular carcinoma patients, and malignant liver cancer cells also often highly express DeltaNp63apha. On this basis, the present invention uses lentiviral transfection to obtain a liver cancer cell line that stably expresses DeltaNp63alpha, and achieves high expression of DeltaNp63apha in liver cancer cells Huh7, PLC / PRF / 5, HCCLM3, SK-HEP-1, Hep3B, SMMC7721, SNU387, MHCC97H, and BEL7402. The obtained liver cancer cell line has a high cell proliferation rate, supports drug screening, and can also be used to study the role of new drugs in the malignant development of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 The diagram shows the protein expression of DeltaNp63apha in different liver cancer cell lines; Figure 2 The figure shows the RNA expression results of DeltaNp63apha in different liver cancer cell lines; Figure 3 The protein expression results of liver cancer cell lines obtained in Examples 1-9 and Comparative Examples 1-9 are shown; Figure 4 The RNA expression results of the liver cancer cell lines obtained in Examples 1-9 and Comparative Examples 1-9 are shown; Figure 5 The results of the cell proliferation detection experiment of the liver cancer cell lines obtained in Example 1-9 and Comparative Example 1-9 are shown; Figure 6 The figure is a result of the clone formation experiment of the liver cancer cell lines obtained in Example 1-9 and Comparative Example 1-9; Figure 7 The results of the scratch test on the liver cancer cell lines obtained in Examples 1-9 and Comparative Examples 1-9 are shown; Figure 8 The phenotypic observation diagram of the liver cancer cell lines obtained in Example 1 and the comparative example. DETAILED DESCRIPTION

[0014] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0015] To further clarify the expression of DeltaNp63apha in liver cancer cells, the following Western Blot and RT-PCR experiments were performed: 1. Protein expression of DeltaNp63apha in different liver cancer cell lines (Western Blot): The cells involved are Huh7, PLC / PRF / 5, SNU182, HCCLM3, SK-HEP-1, Hep3B, SMMC7721, SNU387, MHCC97H, and BEL7402. The above cells are from ATCC, Chinese Academy of Sciences Cell Bank, etc., and are cultured in DMEM medium containing 10% FBS at 37°C and 5% carbon dioxide. (1) Extraction of cell proteins: Collect 1 × 10 6The cells were washed, resuspended, and centrifuged three times with PBS (to remove trypsin and cell culture medium), the supernatant was discarded, 200uL RIPA cell lysis buffer was added, lysed on ice for 30 minutes, centrifuged at 10000g for 30 minutes at 4°C, the supernatant was gently transferred to the EP tube, and the concentration of the extracted protein was determined by the BCA method. 2×SDS (equal volume to the supernatant) was added, and the EP tube was placed in boiling water for 30 minutes to denature the protein. Finally, it was placed on ice for loading; (2) Glue preparation: Dry the cleaned glass plates, electrophoresis apparatus and comb. Prepare 12% SDS polyacrylamide gel according to the linear separation range (KD). After the SDS polyacrylamide gel solidifies, remove the comb. After the gel plate is installed in the electrophoresis tank, add electrophoresis buffer and set aside. (3) Electrophoresis: Before loading, boil the protein extracted in (1) in water at 100°C for 3 minutes. Take 80 μg of protein for electrophoresis separation when loading. Set the voltage to 60V for the stacking layer and 80V for the separation gel. Refer to the marker band and end the electrophoresis after 1.5 hours. (4) Transfer: During electrophoresis, prepare 8.5cm×5.5cm filter paper and PVDF membrane; soak the PVDF membrane in an appropriate amount of methanol solution for 30s, then soak it in transfer buffer for 30min. Finally, stack the PVDF membrane and filter paper and soak them in transfer buffer (20mL 5×transfer buffer, 20mL methanol, add water to 100mL); Turn on the electrophoresis instrument, add a small amount of transfer buffer to the area to be transferred, and cover the soaked filter paper on the carbon plate. After the electrophoresis is completed, separate the two glass plates, cover the PVDF membrane on the gel from the middle, cut off the excess gel at the edge, and then pour a small amount of transfer buffer on the five pre-placed filter papers, and then place the gel on top and the membrane on the bottom on the five filter papers. Then pour a small amount of transfer buffer on the gel, cover the gel with another five filter papers, cover the transfer instrument lid, set 20V, 20min, and start the transfer; Ponceau staining; Closure: Rinse the membrane with 1× PBST. Block with blocking solution overnight at room temperature on a shaker; (7) Primary antibody incubation: Dilute the primary antibody with primary antibody diluent, rinse three times with PBST after blocking, and incubate with primary antibody for 2h; (8) Secondary antibody incubation: Dilute the secondary antibody in proportion, wash the membrane three times with PBST on a shaker for 10 min each time, and incubate with the secondary antibody for 1.5 h; (9) Development: Gel imaging.

[0016] RNA expression of DeltaNp63apha in different liver cancer cell lines (RT-PCR): The cells involved are Huh7, PLC / PRF / 5, SNU182, HCCLM3, SK-HEP-1, Hep3B, SMMC7721, and SNU387, and their sources are consistent with those for protein detection; (1) Extraction of total RNA: After cell culture, grind in liquid nitrogen, transfer 50 mg into a glass homogenizer, add 1 mL TrizoI to beat the homogenate, then transfer into a 1.5 mL new centrifuge tube and aspirate with a 5 mL needle, incubate on ice for 5 min, centrifuge at 10000 g for 5 min at 4 ° C, take the supernatant and transfer into a 1.5 mL new centrifuge tube, add 0.2 mL chloroform, shake, incubate on ice for 5 min, centrifuge at 10000 g for 10 min at 4 ° C, transfer the upper liquid phase into a 1.5 mL new centrifuge tube, add 0.5 mL isopropanol, shake, incubate on ice for 5 min, centrifuge at 10000 g for 5 min at 4 ° C, add 1 mL of 75% ethanol to the precipitate, shake, centrifuge at 6500 g for 5 min at 4 ° C, discard the supernatant, dry the precipitate at room temperature for 10 min, then add 20 μL DEPC water to dissolve, and store at -80 ° C; (2) RNA content detection: Dilute 5 μL of the RNA obtained in (1) to 200 μL with ultrapure water, place in the cuvette of a UV spectrophotometer, measure the absorbance at 260 nm and 280 nm, and calculate the OD 260 / OD 280 (This value is between 1.8-2.0, indicating that the sample is not contaminated. If it is not within this range, repeat step (1)). Calculate the sample RNA concentration according to the following formula: Sample RNA concentration (mg / L) = OD 260 ×40×dilution factor; 6 μL of RNA sample was taken and electrophoresed on 2% agarose gel. The 18s and 28s bands were observed by RNA denaturing electrophoresis to identify the extracted total RNA. Primer design: The primers used are as follows: DeltaNp63alpha forward primer: 5′-CGGAATTCCTTGCCCAGGAAGAGACAGG-3′; DeltaNp63alpha reverse primer: 5′-CGCCGCTTCGTACCATCACCGTT-3′; GAPDH forward primer: 5′-AAGAGAGGCAATCCTCACCCT-3′; GAPDH reverse primer: 5′-TACATGGCTGGGGTGTTGAA-3′; The above primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. and used at a concentration of 100 μmol / L with sterile deionized water; Reverse transcription: Reverse transcription was performed using the EasyScript One-step gDNA Removal and cDNA Synthesis SuperMix kits to reverse transcribe 5 μg of the total RNA obtained in step (1) for 1 hour to obtain cDNA. The specific operation was referred to the instructions of the above kits. PCR reaction: PCR reaction conditions: 94°C for 5 min; 94°C for 40 s, 50°C for 45 s, 72°C for 90 s, 33 cycles; 72°C for 10 min, using the 50 μL system listed in the table;

[0017] Agarose gel electrophoresis: Add 50 mL 1×TAE solution to 1 g agarose, dissolve the agarose in a microwave oven at medium heat for 2 min, take out and shake well, then put in medium heat for 2 min to remove bubbles, keep warm in a 60°C water bath, add 2 μL EB, put in a comb, pour gel, and after the gel solidifies for 30 min, pull out the comb, take out the gel, put it in the electrophoresis instrument, add the RT-PCR reaction product to the gel well, add the marker to the gel well next to it for control, and electrophoresis at 120 V for 30 min; (7) Product analysis: The ratio of the optical density of the target PCR product to the optical density of the internal reference PCR product was taken as the relative expression level of the target gene, which was the semi-quantitative RT-PCR result.

[0018] The protein expression of DeltaNp63apha in liver cancer cells is shown in Figure 1 ,Depend on Figure 1 It can be seen that DeltaNp63apha is only expressed in SNU182 liver cancer cells, and the same result also appears in Figure 2 middle, Figure 2 The RNA expression of DeltaNp63apha in liver cancer cells indicates that the expression level of DeltaNp63apha itself is low in most liver cancer cells.

[0019] In previous studies, the public database TCGA found that high expression of p63 was significantly associated with lower survival rates in some patients with hepatocellular carcinoma. Further analysis found that these patients with hepatocellular carcinoma were more likely to be in the middle and late stages of liver cancer development than other patients, indicating that p63 may play a key role in the late progression of liver cancer. Further analysis of p63 subtypes showed that the most correlated was DeltaNp63apha. Based on this, DeltaNp63apha was overexpressed in the above liver cancer cells Huh7, PLC / PRF / 5, HCCLM3, SK-HEP-1, Hep3B, SMMC7721, SNU387, MHCC97H, and BEL7402. It was further found that liver cancer cells were indeed more malignant when DeltaNp63apha was highly expressed. Transcriptome sequencing data further identified genes with significant changes in expression levels, such as sulfatase 2 (SULF2), and ChIP-seq results showed that there were multiple p63 binding sites in the upstream / downstream enhancer and promoter regions of SULF2. Various experimental results show that DeltaNp63apha promotes the occurrence of liver cancer, and its high expression is closely related to the advanced progression of liver cancer and low survival rate.

[0020] Therefore, the preparation of liver cancer cell lines with stable expression of DeltaNp63alpha will be helpful for drug screening and can also be used to study the role of new drugs in the malignant development of liver cancer.

[0021] Example 1: This example provides a method for constructing a liver cancer cell line by stably expressing DeltaNp63alpha, the specific contents of which are as follows: L1. Culture of liver cancer cells: SNU387 hepatoma cells were cultured in RPMI-1640 medium at 37°C and 5% carbon dioxide; L2. Construction of recombinant plasmid pCDH-EF1-MCS-DeltaNp63alpha-T2A-copGFP: L2.1 The total RNA of SNU387 liver cancer cells obtained in step L1 was extracted by Trizol method, and cDNA was obtained by reverse transcription using cDNA synthesis kit; L2.2 Use the cDNA obtained in step L2.1 as a template to perform PCR reaction. The upstream and downstream primers involved in the PCR reaction are as follows: DeltaNp63alpha forward primer: 5′-CGGAATTCCTTGCCCAGGAAGAGACAGG-3′; DeltaNp63alpha reverse primer: 5′-CGCCGCTTCGTACCATCACCGTT-3′; GAPDH forward primer: 5′-AAGAGAGGCAATCCTCACCCT-3′; GAPDH reverse primer: 5′-TACATGGCTGGGGTGTTGAA-3′; The PCR reaction system is as follows:

[0022] PCR reaction conditions: 94°C for 5 min; 94°C for 40 s, 50°C for 45 s, 72°C for 90 s, 33 cycles; 72°C for 10 min; The PCR product was recovered from the L2.3 purification gel, digested with EcoR I and BamH I restriction endonucleases, and pCDH-EF1-MCS-T2A-copGFP and DeltaNp63alpha were ligated with T4 DNA ligase to obtain the recombinant plasmid pCDH-EF1-MCS-DeltaNp63alpha-T2A-copGFP; L3. Preparation of lentivirus: 293T cells were cultured in L3.1 until the cell confluence reached 70%, and then digested with trypsin and resuspended, and then inoculated in a 12-well plate at a density of 1×10 6 100 cells / well, culture for 18 h, replace with serum-free DMEM culture medium to obtain 293T cells for use; L3.2 Mix the recombinant plasmid pCDH-EF1-MCS-DeltaNp63alpha-T2A-copGFP, Del8.9, VSVG, and serum-free DMEM culture medium obtained in step L2 according to a mass volume ratio of 1 μg: 0.4 μg: 0.8 μg: 0.5 mL to obtain mixed solution 1, add 0.2 times the volume of transfection reagent to the mixed solution 1, mix well, let stand at room temperature for 10 min, and then add to the stand-by 293T cells obtained in step L3.1. After completion, culture in an incubator for 6 h. After taking out, replace the culture medium with DMEM culture medium containing 10% FBS, and continue to culture for 36 h. After completion, take out, collect the supernatant, and centrifuge at 4000g for 10 min. The supernatant is the lentivirus; L4. Construction of liver cancer cell line stably expressing DeltaNp63alpha: SNU387 liver cancer cells were obtained by culturing according to step L1 until the cell confluence reached 60%, and the lentivirus obtained in step L3 was infected at a titer of MOI 200. After 72 hours, the culture medium was replaced with RPMI-1640 culture medium containing 10% FBS, and 1.5 μg / mL puromycin was added for screening for 2 weeks, and the culture medium was replaced every 3 days. During the screening process, the changes of GFP in the cells were continuously observed under a fluorescence microscope until all cells were green under a fluorescence microscope, and the fluorescence intensity did not weaken after continuous culture for several generations, and the liver cancer cell line stably expressing DeltaNp63alpha was obtained.

[0023] Example 2: This example provides a method for constructing a liver cancer cell line by stably expressing DeltaNp63alpha, the specific contents of which are as follows: The difference between this embodiment and embodiment 1 is that the liver cancer cells used in the whole process are Huh7.

[0024] Example 3: This example provides a method for constructing a liver cancer cell line by stably expressing DeltaNp63alpha, the specific contents of which are as follows: The difference between this embodiment and embodiment 1 is that the liver cancer cells used in the whole process are PLC / PRF / 5.

[0025] Example 4: This example provides a method for constructing a liver cancer cell line by stably expressing DeltaNp63alpha, the specific contents of which are as follows: The difference between this embodiment and embodiment 1 is that the liver cancer cells used in the whole process are HCCLM3.

[0026] Example 5: This example provides a method for stably expressing DeltaNp63alpha to construct a liver cancer cell line, the specific contents are as follows: The difference between this embodiment and embodiment 1 is that the liver cancer cells used in the whole process are SK-HEP-1.

[0027] Example 6: This example provides a method for constructing a liver cancer cell line by stably expressing DeltaNp63alpha, the specific contents of which are as follows: The difference between this embodiment and embodiment 1 is that the liver cancer cells used in the whole process are Hep3B.

[0028] Example 7: This example provides a method for constructing a liver cancer cell line by stably expressing DeltaNp63alpha, the specific contents of which are as follows: The difference between this embodiment and embodiment 1 is that the liver cancer cells used in the whole process are SMMC7721.

[0029] Example 8: This example provides a method for stably expressing DeltaNp63alpha to construct a liver cancer cell line, the specific contents are as follows: The difference between this embodiment and embodiment 1 is that the liver cancer cell MHCC97H is used in the whole process.

[0030] Example 9: This example provides a method for constructing a liver cancer cell line by stably expressing DeltaNp63alpha, the specific contents of which are as follows: The difference between this embodiment and embodiment 1 is that the liver cancer cell BEL7402 is used in the whole process.

[0031] Comparative Example 1: This comparative example provides a liver cancer cell line without a target sequence, and the specific contents are as follows: The difference between this comparative example and Example 1 is that in this comparative example, the pCDH-EF1-MCS-T2A-copGFP plasmid not containing DeltaNp63alpha is directly transfected into SNU387 liver cancer cells, thereby obtaining the liver cancer cell line of this comparative example, and the other steps are performed with reference to Example 1.

[0032] Comparative Example 2-9 also provides a liver cancer cell line that does not contain the target sequence, which corresponds to Example 2-9, and can be obtained by transfecting the pCDH-EF1-MCS-T2A-copGFP plasmid that does not contain DeltaNp63alpha into the corresponding liver cancer cells.

[0033] Experimental test: Protein and RNA expression test: Western Blot and RT-PCR experiments were performed on the liver cancer cell lines obtained in Examples 1-9 and Comparative Examples 1-9 to examine the protein and RNA expression of DeltaNp63alpha: Protein expression Figure 3 As shown by Figure 3 It can be seen that the liver cancer cell lines prepared in Examples 1-9 all highly expressed DeltaNp63alpha, while the liver cancer cell lines prepared in Comparative Examples 1-9 basically did not express DeltaNp63alpha, proving that the method for constructing liver cancer cell lines by stably expressing DeltaNp63alpha provided by the present invention is highly efficient. Figure 4 As for the RNA expression, it is also proved that the liver cancer cell line prepared in Example 1-9 stably expresses DeltaNp63alpha.

[0034] Cell proliferation assay: The cell proliferation detection experiment was performed on the liver cancer cell lines obtained in Examples 1-9 and Comparative Examples 1-9. Taking Example 1 as an example, a liver cancer cell line with an adhesion rate of 80% was selected, and trypsin and EDTA digestion solution were used for digestion. After dilution, 5000 cells / 200 μL / well were added, and 16 μL of FBS was added at the same time. The experiment was repeated three times, and cultured at 37° C. and 5% carbon dioxide for 48 h. Then, CCK-8 reagent was added, and incubated for 0 h, 24 h, 48 h, and 72 h. The absorbance value at 450 nm was detected by a microplate reader; The results of cell proliferation assay were as follows Figure 5 As shown by Figure 5 It can be seen that the liver cancer cell lines stably expressing DeltaNp63alpha obtained in Examples 1-9 all have better cell proliferation rates. In comparison, the liver cancer cell lines obtained in Comparative Examples 1-9 have lower absorbance values ​​at the same time.

[0035] Colony formation assay: The hepatoma cell lines obtained in Examples 1-9 and Comparative Examples 1-9 were subjected to a cloning experiment. Taking Example 1 as an example, 800 cells were seeded in a 6-well plate, the culture medium was replaced every 3 days, and the cells were cultured for 14 days, fixed with % paraformaldehyde for 30 min, stained with crystal violet for 7 min, washed with PBS 3 times, dried and photographed, and the colonies formed by cell clones were counted using Image J software; The results of the clone formation experiment are as follows Figure 6 As shown by Figure 6 It can be seen that compared with the liver cancer cell line obtained in comparative example 1-9, the liver cancer cell line stably expressing DeltaNp63alpha obtained in example 1-9 has a stronger proliferation ability.

[0036] 4. Scratch test: The hepatoma cell lines obtained in Examples 1-9 and Comparative Examples 1-9 were subjected to a scratch test. Taking Example 1 as an example, the cells were inoculated at 3×10 5 The cells were inoculated into 6-well plates with 10 μL of sterilized pipette tip and scratched vertically. The culture medium was discarded and washed three times with PBS. The floating cells in the 6-well plates were washed clean and 2 mL of basal culture medium was added into the well. The cells were photographed under a microscope at 0 h, 24 h, and 48 h, and statistical analysis was performed with Image J software. The results of the scratch test are as follows Figure 7 As shown by Figure 7 It can be seen that the liver cancer cell line stably expressing DeltaNp63alpha obtained in Example 1-9 has a faster scratch healing speed and a significantly increased cell migration ability.

[0037] 5. Cell phenotype observation: The liver cancer cell lines obtained in Example 1-9 and Comparative Example 1-9 were observed under a microscope. Figure 8 The cell surface obtained in Example 1 and Comparative Example 1 is shown. Figure 8 It can be seen that the cells in Example 1 are typical epithelial-like, and there is no obvious change in cell morphology compared with Comparative Example 1.

[0038] The above are only several embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention is disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for constructing a liver cancer cell line stably expressing DeltaNp63alpha, characterized in that: The content is as follows: L1. Culture of liver cancer cells; L2. Construction of recombinant plasmid pCDH-EF1-MCS-DeltaNp63alpha-T2A-copGFP; L3. Preparation of lentivirus; L4. Construction of liver cancer cell line stably expressing DeltaNp63alpha.

2. The method for constructing a liver cancer cell line stably expressing DeltaNp63alpha according to claim 1, characterized in that: In step L1, the liver cancer cell is one of Huh7, PLC / PRF / 5, HCCLM3, SK-HEP-1, Hep3B, SMMC7721, SNU387, MHCC97H, and BEL7402.

3. The method for constructing a liver cancer cell line stably expressing DeltaNp63alpha according to claim 2, characterized in that: The specific content of step L2 is as follows: L2.1 The total RNA of liver cancer cells obtained in step L1 was extracted by Trizol method, and cDNA was obtained by reverse transcription using a cDNA synthesis kit; L2.2 Use the cDNA obtained in step L2.1 as a template to perform PCR reaction. The upstream and downstream primers involved in the PCR reaction are as follows: DeltaNp63alpha forward primer: 5′-CGGAATTCCTTGCCCAGGAAGAGACAGG-3′; DeltaNp63alpha reverse primer: 5′-CGCCGCTTCGTACCATCACCGTT-3′; GAPDH forward primer: 5′-AAGAGAGGCAATCCTCACCCT-3′; GAPDH reverse primer: 5′-TACATGGCTGGGGTGTTGAA-3′; The PCR product was recovered from L2.3 purification gel, digested with EcoR I and BamH I restriction endonucleases, and pCDH-EF1-MCS-T2A-copGFP and DeltaNp63alpha were ligated with T4 DNA ligase to obtain the recombinant plasmid pCDH-EF1-MCS-DeltaNp63alpha-T2A-copGFP.

4. The method for constructing a liver cancer cell line stably expressing DeltaNp63alpha according to claim 3, characterized in that: The specific content of step L3 is as follows: 293T cells were cultured in L3.1 until the cell confluence reached 70-80%, and then digested with trypsin and resuspended, and then inoculated in 12-well plates at a density of 1×10 5 -1×10 6 100 cells / well, culture for 18-20 hours, replace with serum-free DMEM culture medium to obtain 293T cells for use; L3.2 Mix the recombinant plasmid pCDH-EF1-MCS-DeltaNp63alpha-T2A-copGFP, Del8.9, VSVG, and serum-free DMEM culture medium obtained in step L2 according to a mass volume ratio of 1μg: 0.35-0.4μg: 0.7-0.8μg: 0.3-0.5mL to obtain mixed solution 1, add 0.2 times the volume of transfection reagent to mixed solution 1, mix well, let stand at room temperature for 5-10 minutes, and then add to the stand-by 293T cells obtained in step L3.

1. After completion, culture in an incubator for 6 hours. After taking out, replace the culture medium with DMEM culture medium containing 10% FBS, and continue to culture for 24-36 hours. After completion, take out, collect the supernatant, and centrifuge at 3000-4000g for 8-10 minutes. The supernatant is the lentivirus.

5. The method for constructing a liver cancer cell line stably expressing DeltaNp63alpha according to claim 4, characterized in that: The specific content of step L4 is as follows: Hepatoma cells were obtained by culturing in the same manner as in step L1 until the cell confluence reached 60-70%, and the lentivirus obtained in step L3 was infected at a titer of MOI 200. After 72 hours, the culture medium was replaced with RPMI-1640 culture medium containing 10% FBS, and 1.5 μg / mL puromycin was added for screening for 1-2 weeks, and the culture medium was replaced every 2-3 days. During the screening process, the changes in GFP in the cells were continuously observed under a fluorescence microscope until all cells were green under a fluorescence microscope, and the fluorescence intensity did not weaken after continuous culture for several generations, and a hepatoma cell line stably expressing DeltaNp63alpha was obtained.

6. A use of constructing a liver cancer cell line stably expressing DeltaNp63alpha according to any one of claims 1 to 5, characterized in that: The specific contents are as follows: Liver cancer cell lines that stably express DeltaNp63alpha can be used for drug screening and to study the role of new drugs in the malignant development of liver cancer.

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