CN117904196A - Construction and application of a cell model of histone H1.4 lysine acetylation point mutation in non-small cell lung cancer - Google Patents

Abstract

The invention discloses construction and application of a non-small cell lung cancer connective histone H1.4 lysine acetylation mutant cell model. The invention discovers that the 75-position lysine of histone H1.4 in the non-small cell lung cancer A549 cells has acetylation modification for the first time. In order to study the function of the modification site, we mutated lysine into arginine (K75R) and glutamine (K75Q) which mimic deacetylation, respectively, thus constructing two mutants of H1.4 protein, and further analyzed the effect of the two mutants on proliferation and apoptosis of A549 cells. The invention discovers the relation between the post-translational modification of the 75 th lysine of the H1.4 protein and the growth and apoptosis of lung cancer cells, which has important significance in the aspects of deep exploration of the mechanism research, the treatment method, the drug screening and the like of lung cancer.

Description

Construction and application of a cell model of histone H1.4 lysine acetylation point mutation in non-small cell lung cancer

Technical Field

The invention relates to the technical field of molecular biology, and relates to the construction and application of a cell model of histone H1.4 lysine acetylation point mutation in non-small cell lung cancer.

Background technique

Lung cancer is the most common tumor disease. In my country, the number of deaths due to lung cancer accounts for 23.6% of the total number of cancer deaths, ranking first among cancers. Lung cancer (LC) is divided into small cell carcinoma (SCLC) and non-small cell carcinoma (NSCLC). NSCLC is more common than SCLC, accounting for about 85% of the total number of lung cancers. Lung adenocarcinoma and squamous cell carcinoma are the most common subtypes of NSCLC. The occurrence and development of lung cancer are accompanied by a large number of genetic and epigenetic changes. These changes lead to the activation of multiple tumor driving factors or the silencing of tumor suppressor factors, which are crucial for the malignant transformation of tumors.

Epigenetics refers to the heritable changes in gene expression patterns without changing the DNA sequence, which is an important mechanism for regulating gene expression. It includes DNA methylation, histone modification and chromatin remodeling. Histone modification refers to post-translational modification of histones, including acetylation, methylation, ubiquitination, etc., which can affect the structure and compactness of chromatin and thus affect gene expression.

In eukaryotic organisms, chromatin is coiled and stacked by its basic unit, the nucleosome, in which DNA is entangled with core histones (H2A, H2B, H3, H4) and linker histone H1 to form nucleosomes. The linker histone H1 family includes 11 variants, of which the H1.4 subtype accounts for 5% of the total H1 and is composed of 219 amino acids. Linker histone H1.4 (also known as HIST1H1E, H1F4 and H1e, etc.) belongs to the H1 histone family and is located in the histone gene cluster region on human chromosome 6 with the H1.1-H1.5 genes, and is involved in the regulation of processes such as the cell cycle. Currently, most studies are on the post-translational modification of core histones, while there are fewer studies on linker histone H1.

Summary of the invention

In view of the above problems, the present invention discloses the construction and application of a cell model of histone H1.4 lysine acetylation point mutation in non-small cell lung cancer.

The present invention includes the following technical solutions:

The invention discloses an amino acid acetylation modification site, which exists at the 75th amino acid of histone H1.4 in A549 cells.

Furthermore, the present invention discloses the use of the above-mentioned amino acid acetylation modification site in preparing a non-small cell lung cancer histone H1.4 acetylation point mutation cell model or in preparing a drug for treating non-small cell lung cancer.

The present invention also discloses a method for constructing a non-small cell lung cancer histone H1.4 acetylation point mutation cell model, which comprises mutating the above-mentioned acetylation modification site.

Furthermore, the mutation comprises the following steps:

S1. Design and construct mutation primers for the target gene site of the H1.4 gene that needs to be mutated;

S2, site-directed mutation of lysine at position 75 of H1.4 gene in lung cancer A549 cells;

S3. Transfect the mutant plasmid into A549 cells to obtain an A549 cell model with H1.4 gene mutation.

Furthermore, in the above construction method, the mutation can increase/maintain or decrease the acetylation level of the site.

Furthermore, in the above construction method, the mutation is H1.4 K75R that reduces the acetylation level or H1.4 K75Q that increases/maintains the acetylation level.

Furthermore, in the above construction method, the mutation uses one or more of the following primers:

H1.4 K75R-F: 5’-GGCTATGACGTGGAGAGGAACAACAGC-3’

H1.4 K75R-R: 5’-CTCTCCACGTCATAGCCAGCGGCTGC-3’

H1.4 K75Q-F: 5’-GGCTATGACGTGGAGCAGAACAACAG-3’

H1.4 K75Q-R: 5’-GCTCCACGTCATAGCCAGCGGCTGCC-3’.

Furthermore, the present invention discloses the use of the above method in preparing a drug for treating non-small cell lung cancer.

Furthermore, the present invention discloses a method for changing the growth or apoptosis of A549 cells for non-diagnostic or therapeutic purposes, the method comprising changing the acetylation level of the 75th amino acid of histone H1.4 of A549 cells; the change is a single point mutation of the amino acid.

Furthermore, the present invention discloses a therapeutic drug for non-small cell lung cancer, wherein the drug takes the acetylation modification site disclosed in the present invention as a target and can reduce the acetylation level of the target.

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

The present invention first enriches H1.4 by immunoprecipitation, and then detects the presence of acetylation modification of lysine at position 75 by mass spectrometry. In order to explore whether this modification is related to the growth and apoptosis of non-small cell lung cancer cells, we mutated this site to deacetylated arginine and simulated acetylated glutamine, and constructed plasmids for site-directed mutation, namely H1.4 K75R and H1.4 K75Q. Next, we transfected the mutant plasmid into non-small cell lung cancer A549 cells, and detected the proliferation of A549 cells and the expression of apoptosis-related proteins by experiments such as MTT and Western Blot. The present invention will provide a theoretical basis for the treatment of NSCLC and provide a new target for the treatment of NSCLC.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of immunoprecipitation of histone H1.4 followed by Coomassie Brilliant Blue staining;

FIG2 is the result of mass spectrometry identification of acetylation of lysine 75 of histone H1.4;

FIG3 is the result of successful sequencing of the lysine 75 point mutation of histone H1.4;

FIG4 shows the results of cell death caused by mutation of the histone H1.4 lysine acetylation site;

FIG5 is the result of MTT cell proliferation assay of cells with histone H1.4 lysine acetylation site mutation;

FIG6 is the result of Western Blot detection of apoptosis-related protein expression in cells with histone H1.4 lysine acetylation site mutation.

Detailed ways

The present invention is further described below in conjunction with the accompanying drawings and embodiments, and the contents of the embodiments are not intended to limit the protection scope of the present invention.

Example 1

Acetylation of lysine at amino acid 75 of histone H1.4 was found

First, by inserting the H1.4 target fragment with Flag-tag into the purchased empty vector, this embodiment designed and constructed an overexpression vector for H1.4 protein, namely pCDH-H1.4-Flag, in which the empty vector is a commercial vector named pCDH-CMV-MCS-EF1-Puro. Subsequently, the pCDH-H1.4-Flag vector was stably transfected into A549 cells, and then the histones of the cells were extracted, and the histone lysate was incubated with Flag-Beads at 4°C for 2h for immunoprecipitation, thereby enriching the H1.4 protein. SDS-polyacrylamide gel electrophoresis and Coomassie brilliant blue staining were then performed, and the results are shown in Figure 1. Finally, the enriched H1.4 was subjected to mass spectrometry to detect all modification sites, and it was found that only the lysine at site 75 was acetylated, as shown in Figure 2.

Example 2

The construction steps of the A549 cell histone H1.4 lysine acetylation site mutation cell line are as follows:

1. Design of mutation primer sequences

In order to obtain the target sequence, the wild-type genome sequence information was first found. The following sequence is the amino acid sequence and corresponding base sequence of the CDS region of the H1.4 gene. The underlined sequence is the lysine at position 75 and its corresponding base.

Amino acid sequence: SEQ ID No.1=

MSETAPAAPAAPAPAEKTPVKKKARKSAGAAKRKASGPPVSELITKAVAASKE

RSGVSLAALKKALAAAGYDVE K NNSRIKLGLKSLVSKGTLVQTKGTGASGSF

KLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPKKSAKKTP

KKAKKPAAAAGAKKAKSPKKAKAAKPKKAPKSPAKAKAVKPKAAKPKTAK

PKAAKPKKAAAKKK

Corresponding base sequence: SEQ ID No.2=

ATGTCCGAGACTGCGCCTGCCGCGCCCGCTGCTCCGGCCCCTGCCGAGAA

GACTCCCGTGAAGAAGAAGGCCCGCAAGTCTGCAGGTGCGGCCAAGCGC

AAAGCGTCTGGGCCCCCGGTGTCCGAGCTCATTACTAAAGCTGTTGCCGCC

TCCAAGGAGCGCAGCGGCGTATCTTTGGCCGCTCTCAAGAAAGCGCTGGC

AGCCGCTGGCTATGACGTGGAG AAG AACAACAGCCGCATCAAGCTGGGTC

TCAAGAGCCTGGTGAGCAAGGGCACCCTGGTGCAGACCAAGGGCACCGG

CGCGTCGGGTTCCTTCAAACTCAACAAGAAGGCGGCCTCTGGGGAAGCCA

AGCCTAAGGCTAAAAAGGCAGGCGCGGCCAAGGCCAAGGCCAAGAAGCCAGCAGG

AGCGGCGAAGAAGCCCAAGAAGGCGACGGGGGCGGCCACCCCCAAGAAG

AGCGCCAAGAAGACCCCAAAGAAGGCGAAGAAGCCGGCTGCAGCTGCTG

GAGCCAAAAAAGCGAAAAGCCCGAAAAAGGCGAAAGCAGCCAAGCCAA

AAAAGGCGCCCAAGAGCCCAGCGAAGGCCAAAGCAGTTAAACCCAAGGC

GGCTAAACCAAAGACCGCCAAGCCCAAGGCAGCCAAGCCAAAGAAGGCG

GCAGCCAAGAAAAAGTAG

In order to mutate K at position 75 to R, the AAG base needs to be changed to AGG, and in order to mutate K to Q, the AAG base needs to be changed to CAG. According to the primer design principles, the following primers are designed:

H1.4 K75R-F: 5'-GGCTATGACGTGGAGAGGAACAACAGC-3'=SEQ ID No.3

H1.4 K75R-R: 5'-CTCTCCACGTCATAGCCAGCGGCTGC-3' = SEQ ID No.4

H1.4 K75Q-F: 5'-GGCTATGACGTGGAGCAGAACAACAG-3'=SEQ ID No.5

H1.4 K75Q-R: 5'-GCTCCACGTCATAGCCAGCGGCTGCC-3' = SEQ ID No. 6

2. Construction of mutant plasmid

The designed primers were used for PCR according to the system in Table 1 and the program in Table 2. The plasmid template used was pCDH-H1.4-Flag, so as to amplify the required mutant plasmids, namely H1.4 K75R and H1.4K75Q. Then 1 μL of DMT enzyme was added to the PCR product and incubated at 37°C for 1 hour. 5 μL of DMT enzyme digestion product was added to 50 μL of DH5α competent cells, ice bathed for 30 minutes, and then heat-shocked in a 42°C water bath for 45 seconds. 1 mL of LB medium equilibrated to room temperature was added and placed in a shaking incubator at 200 rpm and 37°C for 1 hour. The bacterial solution was evenly spread on a plate with ampicillin resistance and placed in a 37°C incubator for overnight culture. The next day, a single clone was picked for culture, and the plasmid was extracted and sent for testing. After the sequencing was correct, the correct mutant plasmid was obtained, as shown in Figure 3.

Table 1 Amplification system

Table 2 PCR program

3. Construction of mutant cell lines

One day in advance, 293FT was transferred to the corresponding number of 6cm cell culture dishes and cultured with DMEM medium containing 10% FBS. The cell density after attachment the next day was 70%, which was the best. Using PEI transfection reagent, the transfection reagent and plasmids were added to 293FT cells at a ratio of DNA: PEI = 1:4, including 1μg each of auxiliary plasmids PLP1, PLP2, and VSVG, and 3μg of the target plasmid. After 8h of transfection, fresh DMEM medium was replaced, and the culture medium containing the virus was collected after 48h. Subsequently, the collected virus was filtered with a 0.45μm filter membrane, added to the counted A549 (1×10 ⁵ /6-well plate per well) cells, and Polybrene (8μg/mL) was added. After returning to the incubator for 48h, puromycin was used to screen the stable transfected cell lines. After successful transfection, we found that some dead cells appeared in the mutant cell H1.4 K75R, as shown in Figure 4, so we performed MTT proliferation and WB apoptosis-related protein expression detection experiments.

Example 3

Detection of proliferation ability of mutant cell lines

The wild-type A549 in the logarithmic growth phase and the two mutant cells constructed were digested with 0.25% trypsin, the cells were collected and counted, and the cell concentration was adjusted to 3×10 ⁴ /mL with 1640 culture medium containing 10% FBS. The three types of cells were inoculated in 5 96-well plates, and each cell was repeated in 6 wells, with a total volume of 100μL per well, so that the number of cells in each well was 1×10 ^3. After 4 hours of culture, 10μL MTT (5mg/mL) was added after the cells adhered to the wall. After continuing to culture for 4 hours, the culture medium was aspirated, 150μL DMSO was added to each well, and the light-proof shaking table was shaken for 10 minutes until the crystals were completely dissolved. Finally, the absorbance value at 490nm was detected with an enzyme marker. A 96-well plate was taken out at the same time for the next four days and the above operations were performed.

The cell proliferation rate was calculated according to the following formula:

Cell proliferation rate (%) = (mean OD of experimental group - mean OD of zero-adjustment group) / (mean OD of control group - mean OD of zero-adjustment group) × 100%. The experimental results are shown in Figure 5.

The results showed that compared with wild-type cells, the proliferation ability of H1.4 K75R mutant cells was reduced, while the H1.4K75Q mutation had no significant effect, indicating that acetylation modification of H1.4 at position 75 inhibited the proliferation ability of A549 cells.

Example 4

Detection of apoptotic proteins in mutant cell lines

Take the wild-type A549 and two mutant cells covering a 6cm dish out of the incubator and place them on ice. Wash them twice with pre-cooled PBS, add 150ul of the pre-prepared RIPA lysis buffer (with protease inhibitors added in advance), collect the cell lysate with a clean cell scraper and transfer it all to a 1.5mL EP tube. After lysis for 20 minutes, centrifuge at 12000rpm for 20 minutes at 4℃, take the supernatant and transfer it to a new EP tube, add 5× loading buffer with a volume of 0.25 times the protein, mix well, and boil in boiling water for 5 minutes to denature the protein.

The denatured protein samples were subjected to SDS-polyacrylamide gel electrophoresis, and then transferred to the membrane (0.22μm PVDF membrane) at a constant current of 300mA for 2 hours. After the protein was transferred to the membrane by electrophoresis, it was blocked with 5% skim milk (dissolved in TBST solution) at room temperature for 1 hour on a shaker, and then incubated with the corresponding primary antibody dilution at 4°C overnight on a shaker. The next day, the membrane was washed 3 times with TBST solution, each time for 10 minutes, and incubated with the corresponding HRP secondary antibody dilution at room temperature for 2 hours on a shaker, washed 3 times with TBST, incubated with ECL chemiluminescent substrate at room temperature, and developed using a developer. The results are shown in Figure 6.

The results showed that compared with wild-type cells, the expression of apoptosis-related proteins in H1.4 K75R mutant cells increased, while the expression of apoptosis-related proteins in H1.4 K75Q mutant cells had no significant difference from that in wild-type cells, indicating that acetylation modification at position 75 of H1.4 promoted the apoptosis of A549 cells.

In summary, acetylation modification of lysine 75 of histone H1.4 can inhibit the proliferation of A549 cells and promote their apoptosis. This indicates that the post-translational modification of H1.4 protein has an important influence on the growth and apoptosis of lung cancer cells, which provides a theoretical basis for the in-depth study of non-small cell lung cancer.

The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims (10)

1. An amino acid acetylation modification site, characterized by being present in a549 cells at amino acid 75 of histone H1.4.

2. The use of the acetylation modified site of claim 1 in the preparation of a non-small cell lung cancer histone H1.4 acetylation point mutation cell model or in the preparation of a medicament for treating non-small cell lung cancer.

3. A method for constructing a non-small cell lung cancer histone H1.4 acetylation point mutation cell model, comprising mutating the acetylation modification site according to claim 1.

4. A method according to claim 3, comprising the steps of:

s1, designing and constructing a mutation primer aiming at a target gene locus of H1.4 gene to be mutated;

S2, carrying out site-directed mutagenesis on lysine at 75 sites of H1.4 genes in lung cancer A549 cells;

S3, transfecting the mutant plasmid into an A549 cell to obtain an A549 cell model with the H1.4 gene mutation.

5. The method of claim 4, wherein the mutation increases/maintains or decreases the level of acetylation at the site.

6. The method of claim 4, wherein the mutation is a decrease in the level of acetylation of H1.4K 75R or an increase/maintenance of the level of acetylation of H1.4K 75Q.

7. The method of claim 6, wherein the mutation uses one or more of the following primers:

H1.4 K75R-F：5’-GGCTATGACGTGGAGAGGAACAACAGC-3’

H1.4 K75R-R：5’-CTCTCCACGTCATAGCCAGCGGCTGC-3’

H1.4 K75Q-F：5’-GGCTATGACGTGGAGCAGAACAACAG-3’

H1.4 K75Q-R：5’-GCTCCACGTCATAGCCAGCGGCTGCC-3’。

8. Use of the method of any one of claims 3-7 in the manufacture of a medicament for the treatment of non-small cell lung cancer.

9. A method of altering growth or apoptosis of a549 cells for non-diagnostic or therapeutic purposes, comprising altering the level of acetylation of amino acid 75 of histone H1.4 of a549 cells; the change is a single point mutation of an amino acid.

10. A therapeutic agent for non-small cell lung cancer, wherein the agent targets the site of acetylation modification of claim 1, and is capable of reducing or increasing the level of acetylation at the target.