Non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model as well as construction method and application thereof

Abstract

The invention provides a construction method of a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model. The construction method comprises the following steps: designing mutation primers H1.3 R80A-F and H1.3 R80A-R; the method comprises the following steps: by taking a pCDH-HA-H1.3-Flag plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification by adopting a mutation primer, digesting a product by DMT enzyme, and converting a competent cell to obtain a mutant plasmid; and co-transfecting the mutant plasmid and a helper plasmid to a packaging cell, collecting a virus solution, filtering, infecting an A549 cell, adding Polybrene to assist infection, culturing, and screening a stably transfected cell strain by using puromycin to obtain the recombinant plasmid. The invention also provides application of the mutant cell model obtained by the construction method. According to the invention, the blank of histone H1.3 methylation research is filled, the 80th arginine is clear as a core modification site, and the established model provides a new tool for lung cancer mechanism research and drug research and development.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical and oncology, and particularly relates to a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model and a construction method and application thereof. BACKGROUND

[0002] Lung cancer is a malignant tumor with the highest morbidity and mortality in the world, and has become a major public health problem that seriously threatens human life and health. Lung cancer is mainly divided into two categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), of which non-small cell lung cancer accounts for 85% of the total number of lung cancer, and lung adenocarcinoma and lung squamous cell carcinoma are the most common subtypes. In the occurrence and malignant progression of lung cancer, a large number of genetic and epigenetic changes occur, which directly lead to the activation of tumor drivers or the silencing of tumor suppressors, and are the key molecular mechanisms of malignant transformation of tumors, and also provide an important direction for targeted therapy research of lung cancer.

[0003] Epigenetics is a core field of studying the regulation of gene expression levels caused by non-genetic sequence changes, and its regulation methods mainly include DNA methylation, histone modification, chromosome remodeling, and non-coding RNA regulation, etc. Through accurate regulation of gene transcription or translation process, it deeply affects the physiological functions and pathological characteristics of biological individuals. Histone modification, as an important part of epigenetics, refers to the post-translational chemical modifications of histones such as acetylation, methylation and ubiquitination. Such modifications can directly change the structure and compactness of chromatin, and then realize the dynamic regulation of gene expression, and play a key role in tumor occurrence and development.

[0004] In the chromatin structure of eukaryotes, the nucleosome is the basic functional unit, which is assembled by DNA and core histones (H2A, H2B, H3, H4) and linker histone H1. The linker histone H1 family contains 11 variants, of which the H1.3 subtype accounts for 10-15% of the total amount of H1. Although it is an H1 subtype with relatively low expression in somatic cells, its function is crucial. The H1.3 subtype is composed of 221 amino acids and has a molecular weight of about 22 kDa, and is located in the nucleus and chromosome. It participates in chromatin compression and the formation of high-order structure by binding to the connecting DNA between nucleosomes. Its C-terminal disordered region (IDR) can mediate the phase separation process and drive the condensation of heterochromatin, and plays an irreplaceable role in maintaining chromatin stability and gene silencing.

[0005] Although histone modification is of great concern in tumor research, the current research focus is mainly on the post-translational modification of core histones (H2A, H2B, H3, H4), and the modification research on the linker histone H1 family is still relatively scarce, especially the systematic research on the methylation modification of H1.3 subtype. In view of the core function of H1.3 in chromatin regulation and the high incidence and treatment demand of non-small cell lung cancer, it is of great significance to develop new lung cancer treatment targets and perfect the theoretical system of tumor epigenetic therapy by deeply mining the methylation modification characteristics of H1.3, and clarifying its modification sites and regulatory role in the occurrence and development of non-small cell lung cancer. However, it is still unknown whether H1.3 has methylation modification, its potential modification sites and the influence on the biological behavior of non-small cell lung cancer cells, and the lack of related cell models seriously limits the research progress in this field.

[0006] Therefore, it is urgent to construct a H1.3 methylation-related point mutation cell model and explore its function and application value in the field. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model and a construction method and application thereof.

[0008] The present application solves the above technical problems by adopting the following technical solutions: A construction method of a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model, comprising the following steps: (1) Designing mutation primers The mutation primers include forward primer H1.3 R80A-F and reverse primer H1.3 R80A-R, and the sequences are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively; (2) Constructing a mutant plasmid Using the pCDH-HA-H1.3-Flag plasmid as a template, the mutation primers of step (1) are used for PCR amplification, and the PCR product is digested by DMT enzyme and then transformed into competent cells, and positive clones are selected and verified by sequencing to obtain the H1.3 R80A mutant plasmid; (3) Constructing a mutant cell strain The H1.3 R80A mutant plasmid and the helper plasmids PLP1, PLP2 and VSVG are co-transfected into packaging cells, the virus liquid is collected and filtered, and then A549 cells are infected, Polybrene is added to assist the infection, and after culture, the stable transfection cell strain is screened with puromycin, and the point mutation cell model is obtained.

[0009] As one of the preferred modes of the present application, in the step (2), the pCDH-HA-H1.3-Flag plasmid is constructed based on the vector pCDH-CMV-MCS-EF1-Puro, and the construction method is as follows: the full-length sequence of the H1.3 gene CDS region is obtained, as shown in SEQ ID NO. 1, and a specific amplification primer is designed accordingly; the HA tag coding sequence is introduced into the forward primer to realize the fusion of the HA tag at the N terminus of the H1.3 protein, and the FLAG tag coding sequence is introduced into the reverse primer to realize the fusion of the FLAG tag at the C terminus of the H1.3 protein; the H1.3 CDS region cDNA is used as a template for PCR amplification to obtain the fusion HA and Flag tagged H1.3 target fragment; and the obtained fusion tagged H1.3 target fragment is connected with the linearized pCDH-CMV-MCS-EF1-Puro vector to obtain the pCDH-HA-H1.3-Flag plasmid.

[0010] As one of the preferred modes of the present application, the H1.3 protein amino acid sequence corresponding to the H1.3 gene CDS region full-length nucleotide sequence shown in SEQ ID NO. 1 is shown in SEQ ID NO. 2; the nucleotide sequence of the specific amplification forward primer into which the HA tag coding sequence is introduced is shown in SEQ ID NO. 3; and the nucleotide sequence of the specific amplification reverse primer into which the FLAG tag coding sequence is introduced is shown in SEQ ID NO. 4.

[0011] As one of the preferred modes of the present application, in the step (2), the PCR amplification system is as follows: Plasmid 10 ng, forward primer 1 μL, reverse primer 1 μL, 2×TransStart FastPfu Fly PCR SuperMix 25 μL, and ddH2O is added to 50 μL; the amplification program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 20 s, 62℃ annealing for 20 s, 72℃ extension for 2 min, 25 cycles; 72℃ post-extension for 10 min; 4℃ incubation.

[0012] As one of the preferred modes of the present application, in the step (2), the competent cells are DH5α competent cells, and after transformation, single clones are selected by ampicillin resistance, and positive clones are obtained by sequencing verification.

[0013] As one of the preferred modes of the present application, in the step (3), the packaging cells are 293FT cells, and PEI transfection reagent is used to co-transfect 6 μg of mutant plasmid and 2 μg of each of the helper plasmids PLP1, PLP2 and VSVG at a ratio of DNA:PEI=1:4, and the lentivirus liquid is collected after 48 h of 37℃, 5% CO2 culture.

[0014] As one of the preferred modes of the present application, the lentivirus liquid is filtered through a 0.45 μm filter membrane, and then A549 cells are assisted in infection with 8 μg / mL Polybrene, and after 48 h, a stable cell strain is obtained by screening with 2 μg / mL puromycin.

[0015] A non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model is obtained by the above construction method.

[0016] Application of the above-mentioned mutant cell model in verifying the function of methylation modification of the 80th arginine of histone H1.3.

[0017] Application of the above-mentioned mutant cell model in screening non-small cell lung cancer treatment drugs targeting the methylation modification of the 80th arginine of histone H1.3.

[0018] The present application has the following advantages compared with the prior art: The present application fills the blank in the field of histone H1.3 methylation research, and for the first time determines that histone H1.3 is subjected to methylation modification and that the 80th arginine is the methylation modification site through experiments such as immunoprecipitation, thereby solving the technical bottleneck of unclear H1.3 methylation sites and missing related research tools in the prior art; on this basis, the site is mutated to demethylated alanine to construct an H1.3 R80A site-directed mutant plasmid, and further, a stable mutant cell model is obtained by transfecting non-small cell lung cancer A549 cells mediated by a lentivirus, the precise mutation is realized by using specific mutant primers in the construction method, and the model mutation characteristics are stably inherited by combining the high-efficiency integration characteristics of the lentivirus vector and puromycin screening, thereby avoiding the problems of unstable and poor reproducibility of conventional models, and the double-tag design can improve the accuracy of subsequent verification.

[0019] The model of the present application not only can be used to explore the correlation between the methylation modification of the 80th arginine of H1.3 and the growth and apoptosis of non-small cell lung cancer cells through experiments such as MTT and Western Blot, thereby providing a core tool for analyzing the related molecular mechanism, but also can be used for screening treatment drugs targeting the modification site, thereby providing a new target and theoretical basis for the treatment of non-small cell lung cancer, and has important basic research value and clinical transformation potential; at the same time, the construction process is clear in steps and clear in key parameters, the cell lines and reagents used are conventional materials, no special and complex equipment is needed, and the model is easy to operate repeatedly in different laboratories, and has strong practicability and generalizability. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a Western Blot graph of the methylation modification of H1.3 in the H1.3 single / double-tag overexpression cell line of the present application; Figure 2is the sequencing verification alignment chart of the H1.3 R80A mutant plasmid of the application; Figure 3 is the verification result Western Blot chart of the A549 cell histone H1.3 80th arginine mutant cell strain of the application; Figure 4 is the result chart of the influence of the methylation of the 80th arginine of the histone H1.3 on the proliferation ability of A549 cells; Figure 5 is the result chart of the influence of the methylation of the 80th arginine of the histone H1.3 on the migration ability of A549 cells (in the chart, A is the microscopic observation chart of the scratch test, and B is the migration rate statistical column chart); Figure 6 is the result Western Blot chart of the influence of the methylation of the 80th arginine of the histone H1.3 on the expression of the apoptosis-related protein P53 of A549 cells. DETAILED DESCRIPTION

[0021] The following detailed description of the embodiments of the application is based on the premise of the technical solution of the application, and detailed implementation modes and specific operation processes are given, but the protection scope of the application is not limited to the following embodiments. Meanwhile, the experimental methods used in the following embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0022] Example 1, construction of pCDH-H1.3-Flag single-tag and pCDH-HA-H1.3-Flag double-tag overexpression cell lines and discovery of histone H1.3 methylation modification: 1. According to the H1.3 CDS sequence obtained on the NCBI website (the CDS full-length sequence is shown as SEQ ID NO. 1, and the corresponding H1.3 protein amino acid sequence is shown as SEQ ID NO. 2), the following two groups of primers are designed: Double-tag forward primer: containing HA tag coding sequence, sequence as SEQ ID NO. 3; Double-tag reverse primer: containing FLAG tag coding sequence, sequence as SEQ ID NO. 4; Single-tag forward primer: no HA tag, sequence as SEQ ID NO. 5; Single-tag reverse primer: containing FLAG tag coding sequence, same as double-tag reverse primer.

[0023] The above primers are synthesized by General Biotech (Anhui) Co., Ltd., and the single-tag and double-tag H1.3 target fragments are amplified, respectively.

[0024] PCR products were separated by 1% agarose gel electrophoresis, purified with a DNA purification kit, and then ligated with linearized pCDH-CMV-MCS-EF1-Puro vector (Biosettia, item CD510B-1) at 16°C overnight under the action of T4 DNA ligase. The ligated plasmid was double enzyme-digested for verification, and was sent to a biological company for sequencing to obtain pCDH-H1.3-Flag single-tag plasmid and pCDH-HA-H1.3-Flag double-tag plasmid.

[0025] 2. Take well-grown 293FT cells (Invitrogen R70007), inoculate into a 6cm culture dish, and culture with DMEM medium containing 10% FBS. When the cells grow to 70%-80%, prepare lentivirus. Prepare transfection complex in two groups: Single-tag group: pCDH-H1.3-Flag plasmid 6μg + commercially available auxiliary plasmid PLP1 2μg + PLP2 2μg + VSVG 2μg; Double-tag group: pCDH-HA-H1.3-Flag plasmid 6μg + 2μg of each of the above auxiliary plasmids; Both groups were prepared according to the full-size FT401-02 transfection reagent instruction, mixed the corresponding plasmid with the transfection reagent to prepare the transfection complex, and stood at room temperature for 15 minutes. Then discard the old culture solution in the cell culture dish, add 5mL of fresh DMEM culture solution containing 10% FBS, drop the transfection reagent complex into the culture solution, shake gently, and then place the culture dish in a 37°C constant temperature incubator for 48h. Then collect the culture solution into a 15mL centrifuge tube to obtain single-tag and double-tag lentivirus solution.

[0026] 3. Take logarithmically growing human non-small cell lung cancer A549 cells (purchased from the Chinese Academy of Sciences Cell Bank, number CBP60084), centrifuge after trypsin digestion, resuspend the cells in RPMI-1640 culture medium containing 10% FBS, and count the cell suspension with a hemocytometer. According to the experimental requirements, 2×10 5 cells were inoculated in a 6cm culture dish, and fresh culture medium and corresponding lentivirus solution (single-tag / double-tag) filtered with a 0.45μm filter were added at a ratio of 1:2. Control culture dishes were also set up with the same amount of cells without adding virus.

[0027] After 48 hours of culture, discard the old culture medium in the culture dish and add fresh RPMI-1640 culture medium containing 10% FBS. Add 2 mg / mL puromycin solution (i.e., final concentration of 2 μg / mL) at a volume ratio of 1:1000. Continue culturing and observe the cell status regularly until the cells in the control culture dish die completely while the cells in the experimental group remain viable, indicating that the transfection experiment was successful.

[0028] 4. Remove wild-type A549 cells and the two overexpression cell types from the incubator and place them stably on ice. Wash twice with pre-chilled PBS, then add 1 mL of RIPA lysis buffer containing PMSF (BioSharp, catalog number BL507A-1), protease inhibitor (ABBKINE, catalog number BMP1001), and phosphatase inhibitor (MCE, catalog number HY-K0022) (PMSF, protease inhibitor, and phosphatase inhibitor ratio 1:100). Collect the cell lysate with a clean cell scraper and transfer it all to a 2 mL EP tube. Centrifuge at 12000 rpm for 20 min at 4°C. Add the supernatant to pre-washed PBS-treated protein G agarose purification beads (YEASEN, catalog number 36405ES08) and rotate at 4°C. After 1 h, centrifuge the sample at 8000 rpm for 1 min.

[0029] The sample supernatant was added to TBS-pre-washed anti-flag affinity purification gel beads (YEASEN, catalog number 20584ES08) and rotated at 4°C. After 2 hours, the sample was centrifuged at 8000 rpm for 1 minute. The supernatant was discarded, and the gel beads were washed three times with TBS. Finally, 100 μL of 1× loading buffer was added, and the mixture was boiled in a 100°C water bath for 4 minutes to obtain the protein sample. SDS-polyacrylamide gel electrophoresis and Western blotting were then performed.

[0030] Finally, methylation modification of histone H1.3 was found. Experimental results are shown below. Figure 1 .

[0031] Example 2: Construction of A549 cell line with histone H1.3 arginine methylation site mutant: 1. Based on the H1.3 protein immunoprecipitated sample obtained in Example 1, the methylation modification region was preliminarily located by tandem mass spectrometry analysis, and it was found that the signal corresponded to the fragment of the arginine residue in the H1.3 protein amino acid sequence (SEQ ID NO. 2); further analysis of the H1.3 protein sequence showed that it only contained 4 arginine residues. In order to accurately determine the modification site, site-directed mutants covering all 4 arginine sites were constructed (all arginine was mutated to alanine which had no methylation modification potential), and were respectively transformed into A549 cells for overexpression, and the methylation signal was detected by Western Blot in the subsequent. The experimental results showed that the methylation signal of the remaining 3 arginine site mutants was still clear, and only the methylation signal of the 80th arginine mutant (R80A) completely disappeared, thus it was determined that the methylation modification site of histone H1.3 was the 80th arginine.

[0032] 2. Based on the above-mentioned determined 80th arginine methylation site, combined with the H1.3 CDS full-length sequence (SEQ ID NO. 1), site-directed mutation primers targeting the site were designed, namely H1.3 R80A mutant forward primer (SEQ ID NO. 6) and H1.3 R80A mutant reverse primer (SEQ ID NO. 7).

[0033] The above-mentioned primers were synthesized by General Biological (Anhui) Co., Ltd., and the core mutation site was designed to replace the codon CGT encoding the 80th arginine with the codon GCT encoding alanine, realizing the demethylation mutation of the site.

[0034] 3. The pCDH-HA-H1.3-Flag double-tag plasmid constructed successfully in Example 1 was used as the template, and the above-mentioned mutation primers were used for site-directed mutation PCR amplification. The amplification system was: 10 ng of template plasmid, 1 μL of mutant forward and reverse primers, 25 μL of 2×TransStart FastPfu Fly PCR SuperMix, and ddH2O was added to 50 μL; the amplification program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 20 s, 62℃ annealing for 20 s, 72℃ extension for 2 min, 25 cycles; 72℃ post-extension for 10 min; 4℃ incubation.

[0035] 4. 1 μL of DMT enzyme (full form gold, product number FM111-01) was added to the PCR amplification product, and incubated at 37℃ for 1 h. The digested product was directly transformed into DH5α competent cells (50 μL), and after ice bath for 30 min, 42℃ heat shock for 45 s, immediately ice bath for 2 min; 1 mL of LB medium was added, and cultured at 37℃, 200 rpm for 1 h.

[0036] Take 200 μL of bacterial solution and spread it on LB solid medium containing ampicillin, and incubate it at 37°C overnight. Pick a single colony from the plate and expand it. Then extract the plasmid using a plasmid extraction kit. Sequence the extracted plasmid to verify the mutation. The sequencing results show that only the 80th arginine codon is replaced from CGT to GCT, and the rest of the sequence is unchanged. The sequence and restriction site changes before and after the mutation are shown in Figure 2 Thus, the pCDH-HA-H1.3(R80A)-Flag methylation site mutant plasmid is obtained.

[0037] 5. Refer to the lentivirus preparation method of Example 1, and perform lentivirus packaging of the mutant plasmid. Take well-grown 293FT cells, seed them in a 6 cm dish, and incubate them to 70-80% confluence. Take 6 μg of the pCDH-HA-H1.3(R80A)-Flag mutant plasmid, and mix it with 2 μg of the helper plasmid PLP1, 2 μg of the helper plasmid PLP2, and 2 μg of the VSVG. Prepare the transfection complex according to the instructions of the full-size gold FT401-02 transfection reagent. Incubate the complex at room temperature for 15 min, and then transfect the 293FT cells. Incubate the cells at 37°C for 48 h, collect the culture medium, and filter it through a 0.45 μm filter to obtain the H1.3 R80A mutant lentivirus solution.

[0038] 6. Refer to the cell infection and screening method of Example 1, and perform A549 cell infection and stable strain screening. Take log-phase human non-small cell lung cancer A549 cells, trypsinize them, and seed them in a 6 cm dish at 2×10 5 cells per well. Add fresh RPMI-1640 culture medium containing 10% FBS and the mutant lentivirus solution at a ratio of 1:2. Set up a blank control group without infection. Replace the culture medium with fresh medium containing a final concentration of 2 μg / mL puromycin after 48 h, and continue to culture until the control cells die completely. The surviving cells in the experimental group are the A549-HA-H1.3(R80A)-Flag methylation site mutant stable cell strain.

[0039] 7. Take the successfully constructed mutant cell strain, prepare the total protein sample according to the protein extraction method of Example 1, and then perform SDS-polyacrylamide gel electrophoresis and membrane transfer (0.22 μm PVDF membrane). After membrane transfer, block the membrane with 5% skim milk (dissolved in TBST solution) at room temperature for 2 h. Prepare three kinds of primary antibody diluents: MMA antibody (CST, item number 8015S), dilution ratio 1:500; Actin antibody (proteintech, item number 66009-1-Ig), dilution ratio 1:2000; HA antibody (SANTA, item number sc-7392), dilution ratio 1:2000; After blocking, the membranes were incubated with the above three primary antibody dilutions, respectively, on a 4°C shaker overnight. The next day, the membranes were washed with TBST solution for 3 times, 15 min each time. Then, the HRP-labeled secondary antibody (rabbit anti-antibody for MMA, proteintech, item number RGAR001, dilution ratio 1:5000; mouse anti-antibody for Actin and HA antibody, proteintech, item number AS003, dilution ratio 1:5000) was incubated on a shaker at room temperature for 2 h, and the membranes were washed with TBST for 3 times. Finally, the ECL chemiluminescence substrate (proteintech, item number PK10003) was incubated at room temperature, and the developer was used for development.

[0040] The results showed that the HA-H1.3(R80A)-Flag fusion protein could be stably expressed in the mutant cell strain, proving that the A549 cell histone H1.3 arginine methylation site mutant cell strain was successfully constructed, and the experimental results are shown in Figure 3 .

[0041] Example 3, function verification of mutant cell strain: To clarify the effect of histone H1.3 arginine methylation at position 80 on the biological behavior of A549 cells, the A549-HA-H1.3(R80A)-Flag mutant cell strain constructed in Example 2 was used as the experimental group, and the A549-HA-H1.3-Flag overexpression cell strain constructed in Example 1 was used as the control group, and the following function verification was performed: 1. Cell proliferation capacity The experimental group, control group cells and wild type A549 cells in the logarithmic growth phase were digested with 0.25% trypsin, collected and counted. Resuspend with RPMI-1640 culture medium containing 10% FBS, adjust the cell concentration to 3x10 4 6 / mL. Three kinds of cells were inoculated into 5 96-well plates, each cell repeated 6 holes, the total volume of each hole was 100 μL, and finally the number of cells in each hole was 500. After 4 h of culture, the cells adhered, 10 μL of MTT (5 mg / mL) (Solarbio life sciences, item number M8180) was added, and after 4 h of continuous culture, the culture medium was removed, 150 μL of DMSO was added to each hole, and the shaking table was shaken in the dark for 10 min until the crystals were completely dissolved. Finally, the absorbance at 490 nm was detected by a microplate reader. Four days later, a 96-well plate was taken out at the same time for the above operation.

[0042] The cell proliferation rate was calculated according to the formula: cell proliferation rate (%) = (experimental group OD mean - zero adjustment group OD mean) / (control group OD mean - zero adjustment group OD mean) x 100%.

[0043] The experimental results are shown in Table 1. Figure 4 The results show that the proliferation ability of H1.3 R80A mutant cells is decreased compared with wild type cells, which indicates that the methylation modification of H1.3 at its 80th site enhances the proliferation ability of A549 cells.

[0044] 2. Cell migration ability Select the experimental group, control group cells and wild type A549 cells with a growth density of 80% in a six-well plate, aspirate the culture waste liquid, wash once with PBS, use 200 μL of sterilized gun head to draw a straight "cross" in the cells, wash with PBS for three times after drawing, remove the floating cells, add culture solution, and take pictures at the fixed point as 0h. Take pictures at the corresponding position after 12h, 24h.

[0045] The experimental results are shown in Table 2. Figure 5 (A is the micrograph of the scratch test, and B is the statistical column chart of migration rate). The results show that the migration ability of H1.3 R80A mutant cells is decreased compared with wild type cells, which indicates that the methylation modification of H1.3 at its 80th site enhances the migration ability of A549 cells.

[0046] 3. Apoptosis-related protein expression detection Take the experimental group, control group cells and wild type A549 cells covered in a 6cm dish out of the incubator and place them on ice, and prepare total protein samples according to the protein extraction method of Example 1.

[0047] Denature the protein sample and perform SDS-polyacrylamide gel electrophoresis, then transfer the membrane at a constant current of 300mA for 2 hours (0.22μm PVDF membrane), after the protein electrophoresis is transferred to the membrane, use 5% skim milk (dissolved in TBST solution) to block at room temperature for 2h; then use the corresponding primary antibody diluent (Actin antibody, proteintech product number 66009-1-Ig, dilution ratio 1:2000; P53 antibody, Santa product number sc-126, dilution ratio 1:1000) at 4°C for 2h; the next day, wash the membrane with TBST solution for 3 times, each for 15min; incubate with the corresponding HRP secondary antibody diluent (mouse anti, proteintech product number AS003, dilution ratio 1:5000) at room temperature for 2 hours, wash the membrane with TBST for 3 times, incubate with ECL chemiluminescence substrate (proteintech product number PK10003) at room temperature, and develop using a developing instrument.

[0048] The experimental results are shown in Table 3. Figure 6The results show that the expression of apoptosis-related proteins in the H1.3 R80A mutant cells is increased compared with the wild type cells, which indicates that the methylation modification of the 80th site of H1.3 inhibits the apoptosis of A549 cells.

[0049] To sum up, the histone H1.3 single / double tag overexpression cell line is successfully constructed, and it is first confirmed that the 80th arginine of H1.3 is the core site of methylation modification. Further, the site demethylation mutant stable cell strain is obtained through the site-directed mutagenesis technology, and it is verified through the function that the methylation of the 80th arginine of H1.3 can significantly promote the proliferation of A549 cells, enhance the migration ability of cells and inhibit the apoptosis of cells. The technical scheme of the present application provides a key experimental model for analyzing the molecular mechanism of the methylation modification of histone H1.3 regulating the biological behavior of tumor cells, and provides a new theoretical basis and research direction for the development of non-small cell lung cancer targeted therapy target.

[0050] The above merely describes preferred embodiments of the present application but should not be used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for constructing a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model, characterized in that, Includes the following steps: (1) Design of mutation primers Designed mutation primers, including forward primer H1.3 R80A-F and reverse primer H1.3 R80A-R, with sequences shown in SEQ ID NO.6 and SEQ ID NO.7, respectively; (2) Constructing mutant plasmids Using pCDH-HA-H1.3-Flag plasmid as a template, PCR amplification was performed using the mutant primers described in step (1). The PCR product was digested with DMT enzyme and then transformed into competent cells. Positive clones were screened and sequenced for verification to obtain the H1.3 R80A mutant plasmid. (3) Constructing mutant cell lines The H1.3 R80A mutant plasmid was co-transfected with helper plasmids PLP1, PLP2, and VSVG into packaging cells. The viral fluid was collected, filtered, and then used to infect A549 cells. Polybrene was added for helper infection. After culturing, stable cell lines were selected with puromycin to obtain the point mutant cell model.

2. The construction method according to claim 1, characterized in that, In step (2), the pCDH-HA-H1.3-Flag plasmid is constructed based on the vector pCDH-CMV-MCS-EF1-Puro. The construction method is as follows: obtain the full-length sequence of the CDS region of the H1.3 gene, as shown in SEQ ID NO.1, and design specific amplification primers accordingly; introduce the HA tag coding sequence into the forward primer to achieve the fusion of the HA tag at the N-terminus of the H1.3 protein, and introduce the FLAG tag coding sequence into the reverse primer to achieve the fusion of the FLAG tag at the C-terminus of the H1.3 protein; perform PCR amplification using the cDNA of the H1.3 CDS region as a template to obtain the H1.3 target fragment fused with HA and Flag tags; ligate the obtained H1.3 target fragment with fused tags to the linearized vector pCDH-CMV-MCS-EF1-Puro to obtain the pCDH-HA-H1.3-Flag plasmid.

3. The construction method according to claim 2, characterized in that, The amino acid sequence of the H1.3 protein corresponding to the full-length nucleotide sequence of the CDS region of the H1.3 gene shown in SEQ ID NO.1 is shown in SEQ ID NO.2; the nucleotide sequence of the specific amplification forward primer with the HA tag coding sequence is shown in SEQ ID NO.3; and the nucleotide sequence of the specific amplification reverse primer with the FLAG tag coding sequence is shown in SEQ ID NO.

4.

4. The construction method according to claim 1, characterized in that, In step (2), the PCR amplification system is as follows: Plasmid 10ng, forward primer 1μL, reverse primer 1μL, 2×TransStart FastPfu Fly PCR SuperMix 25μL, and ddH2O added to make up to 50μL; the amplification program is as follows: 94℃ pre-denaturation for 5min; 94℃ denaturation for 20s, 62℃ annealing for 20s, 72℃ extension for 2min, 25 cycles; 72℃ extension for 10min; 4℃ incubation.

5. The construction method according to claim 1, characterized in that, In step (2), the competent cells are DH5α competent cells. After transformation, single clones are selected by ampicillin resistance screening, and positive clones are obtained after sequencing verification.

6. The construction method according to claim 1, characterized in that, In step (3), the packaging cells are 293FT cells. The mutant plasmid 6 μg and the helper plasmids PLP1, PLP2 and VSVG 2 μg are co-transfected with PEI transfection reagent at a ratio of DNA:PEI=1:

4. The cells are cultured at 37℃ and 5% CO2 for 48 h and the lentivirus solution is collected.

7. The construction method according to claim 6, characterized in that, The lentivirus solution was filtered through a 0.45 μm filter membrane and then used to infect A549 cells with 8 μg / mL Polybrene. After 48 hours, stable cell lines were selected using 2 μg / mL puromycin.

8. A non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model, characterized in that, Obtained by the construction method described in any one of claims 1 to 7.

9. The application of the mutant cell model as described in claim 8 in verifying the function of histone H1.3 arginine methylation modification.

10. The application of the mutant cell model as described in claim 8 in screening therapeutic drugs for non-small cell lung cancer targeting histone H1.3 arginine methylation modification.

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