YY1 S247A mutant lung cancer cell strain and application thereof

Abstract

The invention discloses a lung cancer cell strain with YY1S247A mutation and application of the lung cancer cell strain. According to the present invention, phosphorylation modification of serine at the 247th site of YY1 is detected through mass spectrometry, and in order to explore the influence of the phosphorylation modification at the site on the function of YY1 in non-small cell lung cancer cells, a lung cancer cell strain in which a YY1S247A mutant gene is knocked is constructed through a CRISPR / Cas9 gene editing technology; dephosphorylated YY1 (YY1S247A mutation) is detected in a constructed lung cancer cell line to block the promotion effect of third-generation EGFR-TKI on CD24 expression, and the cell strain can be widely applied to research on the function of YY1 in non-small cell lung cancer and research on the drug resistance mechanism of the third-generation EGFR-TKI, and can also be used for developing therapeutic drugs for targeted inhibition of phosphorylation of YY1S247 to sensitize EGFR-TKI.

Description

Technical Field

[0001] The present invention relates to a YY1 S247A mutated lung cancer cell line and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Lung cancer is the most common tumor disease. 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 all lung cancers. Therefore, the establishment and application of non-small cell lung cancer cell lines are of great significance for studying the pathophysiological mechanisms and drug resistance mechanisms of lung cancer, screening effective drugs and treatment methods, and evaluating the effectiveness of treatment plans.

[0003] YY1 (Yin Yang 1) is a multifunctional transcription factor widely present in eukaryotes. It regulates the expression of multiple genes and is closely related to cell proliferation, differentiation, apoptosis, and tumor resistance. The activity of YY1 is regulated by a variety of post-translational modifications, including phosphorylation and acetylation. Phosphorylation of the 247th serine residue (S247) of the YY1 protein has rarely been reported, and its function has not yet been fully revealed, but it may play an important role in YY1 dimer formation, enhancer-promoter loops, expression regulation of multiple key genes, and tumor resistance.

[0004] Therefore, in-depth research on S247 phosphorylation will not only help to more fully understand the post-translational modification regulatory network of YY1, but may also reveal new functions of YY1 in specific cellular processes and their relationship with the occurrence and development of diseases. In this context, the development of lung cancer cell lines that can stably express YY1 with the serine at position 247 mutated to alanine (S247A) can provide a powerful tool for studying the effects of S247 phosphorylation on YY1 function. By comparing the differences in the intracellular behaviors of wild-type YY1 and S247A mutant YY1, we can further explore the specific role of S247 phosphorylation in regulating YY1 activity, affecting cell fate, and participating in the progression of related diseases. At the same time, the YY1 S247A lung cancer cell line can also be used to detect the effects and specificity of various methods targeting YY1S247 phosphorylation.

[0005] However, there is currently no cell model suitable for studying the function and impact of YY1 S247 phosphorylation in lung cancer. Summary of the invention

[0006] The purpose of the present invention is to construct a lung cancer cell line with YY1 S247A mutation, so as to study the influence of post-translational modifications such as phosphorylation of serine 247 of YY1 on the function of YY1.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an sgRNA for knocking in a YY1 S247A mutation, the sequence of the sgRNA being as shown in SEQ ID NO: 1, and the sgRNA being capable of knocking in a YY1 S247A mutant gene together with a Cas9 protein and a homologous recombination sequence of a YY1 S247A mutant gene based on CRISPR / Cas9 gene editing technology, wherein the YY1 S247A mutant gene refers to a gene encoding a mutation in which the serine at position 247 of the YY1 protein is mutated to alanine.

[0009] Preferably, the double-stranded DNA template sequence of the sgRNA is shown in SEQ ID NO: 1-2.

[0010] In a second aspect, the present invention provides a composition comprising the sgRNA and Cas9 protein as described above, wherein the composition is used to knock in the YY1 S247A mutation in lung cancer cells, and regulate the phosphorylation or inhibition of the YY1 247 site in lung cancer cells.

[0011] Preferably, the Cas9 protein is recombinantly expressed via the GV392 plasmid.

[0012] Preferably, it further comprises detection primers, and the sequences of the detection primers are shown in SEQ ID NOs: 4-5.

[0013] In a third aspect, the present invention provides the use of the sgRNA or composition as described above, wherein the use is selected from the following group:

[0014] a) used to construct a cell model for the YY1 S247A mutation;

[0015] b) used to regulate the phosphorylation of YY1 247 site in lung cancer cells in vitro;

[0016] c) for preparing a reagent for regulating phosphorylation of YY1 247 site in lung cancer cells;

[0017] d) for preparing a product or reagent for increasing the sensitivity of the third generation EGFR-TKI.

[0018] In a fourth aspect, the present invention provides a lung cancer cell line with a YY1 S247A mutation, wherein the lung cancer cell line is selected from PC-9, HCC827 or H1975 cells, wherein the PC-9, HCC827 or H1975 cells express a gene and / or protein with a YY1 S247A mutation, and the YY1 S247A mutation refers to the mutation of serine at position 247 of the YY1 protein to alanine.

[0019] Preferably, the lung cancer cell line is a PC-9, HCC827 or H1975 cell in which the YY1 S247A mutation gene is knocked in by CRISPR / Cas9 gene editing.

[0020] Preferably, the lung cancer cell line has reduced phosphorylation modification level of YY1 compared to the lung cancer cell line without knock-in of YY1 S247A mutation, and reduced expression of CD24 after treatment with the third-generation EGFR-TKI.

[0021] In a fifth aspect, the present invention provides the use of the lung cancer cell line as described above, wherein the use is selected from the following group:

[0022] A) was used to study the effect of phosphorylation modification of serine 247 of YY1 on the function of YY1;

[0023] B) Used to study the resistance mechanism of anti-tumor drugs (such as the third-generation EGFR-TKI);

[0024] C) Used to screen or develop drugs that enhance the sensitivity of third-generation EGFR-TKI.

[0025] Preferably, the third-generation EGFR-TKI is selected from at least one of osimertinib, ametinib, vometinib, befortinib, rizitinib and rieutinib.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention first detects the presence of phosphorylation modification of serine at position 247 of YY1 by mass spectrometry. In order to explore the effect of phosphorylation modification of this site on the function of YY1 in non-small cell lung cancer cells, the present invention mutates the site to alanine and knocks it into the non-small cell lung cancer cell line by CRISPR / Cas9 gene editing technology. Since the inventor's recent research results show that upregulation of CD24 caused by third-generation EGFR-TKI treatment induces tumor immune escape, the present invention conducts experiments in the constructed lung cancer cell line to detect whether dephosphorylated YY1 (YY1 S247A mutation) blocks the promotion effect of third-generation EGFR-TKI on CD24 expression. The cell line of the present invention can be widely used to study the function of YY1 in non-small cell lung cancer and the drug resistance mechanism of third-generation EGFR-TKI, and can also be used to develop therapeutic drugs that target the inhibition of phosphorylation of YY1 S247 to sensitize EGFR-TKI. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1Mass spectra of YY1 S247 phosphorylation in PC-9 parental and resistant cells (A), Western blot results of YY1 S247 phosphorylation levels after third-generation EGFR-TKI treatment of lung cancer cell lines PC-9, HCC827, and H1975 cells (B), and changes in YY1 S247 phosphorylation levels in parental and osimertinib-resistant cells (C);

[0029] Figure 2 is the vector plasmid spectrum;

[0030] Figure 3 is the vector plasmid spectrum;

[0031] Figure 4 Sanger sequencing results of YY1 in PC-9, HCC827, and H1975 cells with wild-type and S247A mutant forms.

[0032] Figure 5 qRT-PCR (A) and Western blot (B) results showing that the YY1 S247A mutation blocked the third-generation EGFR-TKI's promoting effect on CD24 expression. DETAILED DESCRIPTION

[0033] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0034] The experimental methods in the following examples without specifying specific conditions were carried out according to conventional methods and conditions, or selected according to the product instructions; the materials, reagents, etc. used were conventional commercially available products unless otherwise specified.

[0035] Example 1

[0036] In this example, YY1 CoIP combined with mass spectrometry experiments were performed in PC-9 parental and drug-resistant cells. The analysis results showed that the S247 phosphorylation of YY1 was significantly increased in drug-resistant cells ( Figure 1 A). We further used a customized antibody targeting YY1 S247 phosphorylation and an antibody specific for the unique phosphorylated Ser-Pro-Pro motif around the S247 residue to detect the phosphorylation level of YY1 S247 in lung cancer cell lines PC-9, HCC827, and H1975 cells treated with third-generation EGFR-TKIs (Osimertinib (Osi), Aumolertinib (Alm), Fumertinib (Fur), and Befotertinib (Bef)) by Western blot. The results showed that the third-generation EGFR-TKI induced a significant increase in the phosphorylation of the YY1 S247 site ( Figure 1 BC).

[0037] Example 2

[0038] In this example, the CRISPR / Cas9 gene editing method was used to construct a YY1 S247A mutant lung cancer cell line, and the specific steps were as follows:

[0039] 1) Designing complementary sgRNA (sequence: 5'-GTGGTTGAAGAACAGATCAT-3', SEQ ID NO: 1) based on the gene sequence of the YY1 phosphorylation site mutation (YY1 S247A mutation);

[0040] 2) The vector GV392 was linearized by restriction endonuclease, and the target double-stranded sgRNA was connected. The double-stranded sgRNA included a coding strand (SEQ ID NO: 1) and a complementary strand (sequence 5'-ATGATCTGTTCTTCAACCAC-3', SEQ ID NO: 2). The sgRNA coding strand and the complementary strand were annealed to form a sgRNA double-stranded DNA; a CRISPR / Cas9 vector with the target sgRNA sequence and capable of expressing the Cas9 protein was constructed and coated with a lentivirus. The vector plasmid map is shown in Figure 2 As shown;

[0041] 3) A vector plasmid expressing the homologous recombination sequence (SEQ ID NO: 3) of the YY1 S247A mutant gene was constructed by the In-Fusion cloning method. The obtained homologous recombination vector plasmid map is as follows: Figure 3 As shown;

[0042] SEQ ID NO: 3 is as follows:

[0043]

[0044] 4) The lentivirus of step 1) and the plasmid expressing the homologous recombination sequence in step 2) were simultaneously transfected into lung cancer cell lines (PC-9, HCC827 or H1975 cells), followed by puromycin selection. Afterwards, single clones were isolated and Sanger sequencing was performed to verify whether YY1 S247A was successfully knocked in ( Figure 4 The primers used for sequencing were: upstream primer 5'-GCTGAATGAGCCGTGATTGTGAT-3' (SEQ ID NO: 4), downstream primer 5'-TGGAGGAATACCTGGCATTGACC-3' (SEQ ID NO: 5).

[0045] Example 3

[0046] Based on the results of Example 1 and the recent research results of the inventors' team: CD24 upregulation caused by third-generation EGFR-TKI treatment induces tumor immune escape. In this example, in order to examine whether YY1 S247 phosphorylation is a key factor in the increase of CD24 expression induced by the third-generation EGFR-TKI, we conducted a control experiment in the YY1 S247A mutant lung cancer cell line constructed in Example 2. The results showed that the S247A mutation significantly reduced the expression of CD24 and blocked the increase of CD24 expression caused by the third-generation EGFR-TKI ( Figure 5 AB). These results indicate that the third-generation EGFR-TKI promotes CD24 expression by inducing phosphorylation of YY1 S247. Therefore, the YY1 S247A mutated lung cancer cell line of the present invention can be used as a control to develop drugs that target and inhibit phosphorylation of YY1 S247 to enhance the therapeutic effect of EGFR-TKI.

[0047] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A sgRNA for knocking in a YY1 S247A mutation, characterized in that: The sequence of the sgRNA is as shown in SEQ ID NO:

1. The sgRNA can be used together with the Cas9 protein and the homologous recombination sequence of the YY1 S247A mutant gene to knock into the YY1 S247A mutant gene based on the CRISPR / Cas9 gene editing technology. The YY1 S247A mutant gene refers to the coding gene in which the serine at position 247 of the YY1 protein is mutated to alanine.

2. The sgRNA according to claim 1, characterized in that The double-stranded DNA template sequence of the sgRNA is shown in SEQ ID NO: 1-2.

3. A composition, characterized in that The composition comprises the sgRNA and Cas9 protein according to claim 1 or 2, and is used to knock in the YY1 S247A mutation in lung cancer cells and regulate the phosphorylation or inhibition of the YY1 247 site in lung cancer cells.

4. The composition according to claim 3, characterized in that The Cas9 protein is recombinantly expressed via the GV392 plasmid.

5. The composition according to claim 3, characterized in that The invention also comprises detection primers, the sequences of which are shown in SEQ ID NOs: 4-5.

6. The sgRNA of claim 1 or 2, or the use of the composition of claim 3, wherein the use is selected from the group consisting of: a) used to construct a cell model for the YY1 S247A mutation; b) used to regulate the phosphorylation of YY1 247 site in lung cancer cells in vitro; c) for preparing a reagent for regulating phosphorylation of YY1 247 site in lung cancer cells; d) for preparing a product or reagent for increasing the sensitivity of the third generation EGFR-TKI.

7. A YY1 S247A mutant lung cancer cell line, characterized in that: The lung cancer cell line is selected from PC-9, HCC827 or H1975 cells, and the PC-9, HCC827 or H1975 cells express the YY1S247A mutant gene and / or protein, and the YY1 S247A mutation refers to the mutation of serine at position 247 of the YY1 protein to alanine.

8. The lung cancer cell line according to claim 7, characterized in that The lung cancer cell line is a cell line in which the YY1S247A mutation gene is knocked into PC-9, HCC827 or H1975 cells by using the CRISPR / Cas9 gene editing method.

9. The lung cancer cell line according to claim 7, characterized in that Compared with lung cancer cell lines without knock-in of the YY1S247A mutation, the lung cancer cell line has reduced phosphorylation modification levels of YY1, and reduced CD24 expression after treatment with the third-generation EGFR-TKI.

10. The use of the lung cancer cell line according to any one of claims 7 to 9, wherein the use is selected from the group consisting of: A) was used to study the effect of phosphorylation modification of serine 247 of YY1 on the function of YY1; B) Used to study the resistance mechanism of anti-tumor drugs (such as the third-generation EGFR-TKI); C) Used to screen or develop drugs that enhance the sensitivity of third-generation EGFR-TKI.

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