CN114377135A - Application of PPM1G in diagnosis and treatment of lung cancer - Google Patents

Abstract

The invention discloses application of PPM1G in diagnosis and treatment of lung cancer. In the specific embodiment of the invention, PPM1G is highly expressed in a lung cancer patient, PPM1G with high expression obviously shortens the survival time of the lung cancer patient, and PPM1G can be knocked down or knocked out to effectively prevent the growth, proliferation, migration and invasion of lung cancer cells. The invention proves that PPM1G can be used for diagnosing and treating lung cancer for the first time, and provides a new way for diagnosing, preventing or treating lung cancer.

Description

Application of PPM1G in diagnosis and treatment of lung cancer

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of PPM1G in diagnosis and treatment of lung cancer.

Background

Research has shown that cancer is a major public health problem worldwide, with lung cancer being one of the most common malignancies. According to statistics, 209 thousands of new lung cancer cases are added in 2018 all around the world and are positioned at the leaders of all cancer types. Lung cancer is largely divided into non-small cell lung cancer and small cell lung cancer, with non-small cell lung cancer accounting for 85% of the total lung cancer, and the most common non-small cell lung cancer is lung adenocarcinoma, accounting for about 40% of all lung cancers. The etiology of lung cancer is mainly divided into four, firstly, smoking is the leading cause of lung cancer, and 80% -90% of male lung cancer is related to smoking; second, occupational exposure may lead to lung cancer; thirdly, the occurrence of lung cancer, such as passive smoking, fuel burning and cooking processes, can also be caused by environmental pollution; fourth, chronic infection of the lung is another cause of lung cancer. Although populations at risk for lung cancer are readily identified, the current state of the art does not support large-scale screening for lung cancer. For non-small cell lung cancer, patients with early diagnosis can be cured by surgical resection and postoperative chemotherapy, and a few patients with locally advanced stages can be treated by preoperative chemotherapy and radiotherapy to reduce the stage of tumors, so that the possibility of operation is increased. However, most of lung cancer patients are in advanced stage at the time of initial diagnosis, but no effective treatment means for advanced lung cancer exists at present, so that it is of great significance to find a new method for early diagnosis and treatment of lung cancer.

Disclosure of Invention

The invention aims to provide a novel method for diagnosing, preventing or treating lung cancer, and adopts the following technical scheme for realizing the aim:

the invention provides an application of a composition for enhancing the phosphorylation level of p38 in preparing a medicament for preventing or treating lung cancer or inhibiting growth, proliferation, migration and invasion of lung cancer cells, wherein the composition comprises a substance for inhibiting the activity of PPM1G protein or a substance for inhibiting or silencing the expression of PPM1G gene.

In the present invention, PPM1G (gene ID: 5496) includes gene and its encoded protein and its homologues, mutations, and isoforms.

The level of PPM1G gene expression can be inhibited or reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% using an agent that inhibits PPM1G gene expression as described herein.

"prevention" means preventing the appearance of lung cancer or the recurrence of disappeared lung cancer in a subject at risk of the disease. By "treating" is meant controlling, reducing or ameliorating the pathological progression of lung cancer and prolonging the survival of the diseased subject. By "inhibiting the growth, proliferation, migration, or invasion of lung cancer cells" is meant preventing or slowing the growth, proliferation, migration, or invasion of lung cancer cells.

The present invention is not limited in any way to the species of the subject having or at risk of developing lung cancer, and preferably human and non-human mammals such as mice, rats, guinea pigs, cats, dogs, cows, horses, sheep, pigs, monkeys, etc.

Further, the lung cancer comprises non-small cell lung cancer and small cell lung cancer, preferably, the lung cancer is non-small cell lung cancer.

Further, the non-small cell lung cancer comprises squamous cell lung cancer, adenocarcinoma lung cancer, large cell carcinoma and carcinoid, and is preferably adenocarcinoma lung cancer.

Furthermore, the substance for inhibiting the activity of the PPM1G protein comprises a substance for inhibiting the synthesis of the PPM1G protein, a substance for promoting the degradation of the PPM1G protein or a substance for inhibiting the function of the PPM1G protein.

Further, the substance for inhibiting or silencing PPM1G gene expression comprises a substance for interfering PPM1G gene expression or a substance for knocking out PPM1G gene or a substance for mutating PPM1G gene.

Further, the substance includes synthetic small molecules, chemical agents, antisense oligonucleotides, siRNA, miRNA, ribozymes, polypeptides, proteins. In a specific embodiment of the invention, the agent is an siRNA.

The term "antisense oligonucleotide" refers to a short chain of nucleic acid (consisting of about 15 to 25 nucleotides) that has been chemically modified to have a base sequence complementary to a particular target sequence and which, upon entry into a cell, forms a duplex with the target sequence according to Watson-Crick base-complementary pairing rules.

In the present invention, "complementary" means that two nucleotides can be paired under hybridization conditions, for example, the relationship between adenine (A) and thymine (T) or uracil (U), and the relationship between cytosine (C) and guanine (G).

The term "ribozyme" refers to an RNA molecule that functions to catalyze a specific biochemical reaction.

The term "siRNA" refers to a ribonucleic acid (RNA) capable of inhibiting the expression of a target gene, including a region of a sense RNA fragment and a region of an antisense RNA fragment.

The term "miRNA" refers to a ribonucleic acid (RNA) molecule of about 21 to 23 nucleotides, widely found in eukaryotes, which regulates the expression of other genes.

The conventional design method of siRNA can be referred to the published data of company websites such as Reynoldsa, et al, Nature Biotechnology, 2004, Vol.22: 326-330) or Amhion, Qiagen, etc. The conventional design method of miRNA can be referred to in literature (Lo HL et al, Gene therapy, 2007, 14: 1503-1512), the method for selecting target sequence is similar to the design method of siRNA, for example, the designed sense strand containing target sequence and corresponding antisense strand can be replaced on pri-microRNA, so that the constructed miRNA can prevent the expression of mRNA containing target sequence. The conventional design of ribozymes can be found in the literature (Haseloff J et al, Nature, 1988, 334: 585-591), for example, by placing nucleotide sequences complementary to the sequences around the target sequence before and after the conserved core sequence of the ribozyme (e.g., hammerhead structure) so that the constructed ribozyme can cleave the nucleic acid containing the target sequence at the target sequence. Conventional design methods for antisense oligonucleotides are described in the literature (Matveeva OV et al, nucleic acids research, 2003, Vol.31: 4989-.

In the present invention, the polypeptide or protein includes hormones, cytokines, antibodies and fragments thereof.

As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination of the foregoing, through at least one antigen binding site. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, antibody fragments (such as Fab, Fab ', F (ab')2, and Fv fragments), single chain Fv (scfv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen binding site, so long as the antibody exhibits the desired biological binding activity. The antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2). The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies may be naked or conjugated to other molecules, including but not limited to toxins and radioisotopes.

The term "antibody fragment" refers to a portion of an intact antibody and refers to the epitope variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. As used herein, an "antibody fragment" comprises at least one antigen binding site or epitope binding site. The term "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. The variable region of a heavy or light chain is typically composed of four Framework Regions (FRs) connected by three Complementarity Determining Regions (CDRs), also referred to as "hypervariable regions". The CDRs in each chain are held together in close proximity by the framework regions and contribute to the formation of the antigen-binding site of the antibody.

Further, the substance for knocking out the PPM1G gene also comprises a gene editing tool for knocking out the PPM1G gene.

Further, the gene editing tools comprise Cre-lox recombination technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology and CRISPR/Cas9 technology. In a specific embodiment of the invention, the gene editing tool is CRISPR/Cas9 technology.

CRISPR/Cas9 is a technology for specific DNA modification of targeted genes by RNA-guided nuclease Cas9 protein. The principle of the technology is that crRNA (CRISPR-derived RNA) is combined with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, and the complex guides nuclease Cas9 protein to cut double-stranded DNA at a sequence target site paired with the crRNA, so that the genome DNA sequence is edited.

Further, the target sequence for specific cleavage of the CRISPR/Cas9 technology includes the sequence shown as SEQ ID No. 5.

Furthermore, the medicine also comprises a pharmaceutically acceptable carrier and/or an auxiliary material.

Further, the pharmaceutically acceptable carrier and/or adjuvant comprises diluent, adhesive, surfactant, humectant, adsorption carrier, lubricant, filler, and disintegrant.

In a second aspect, the invention provides an application of a substance for inhibiting the activity of PPM1G protein or a substance for inhibiting or silencing the expression of PPM1G gene in inhibiting the phosphorylation level of cell p38 in vitro.

In a third aspect, the invention provides the use of an agent for detecting PPM1G expression level in the preparation of a product for diagnosing lung cancer or predicting the prognosis of lung cancer.

The term "diagnosis" as used herein refers to the identification or classification of a molecular or pathological state, disease or disorder. For example, "diagnosing" may refer to identifying a risk of developing lung cancer, either by the involved tissue/organ (e.g., lung), or by a molecular characteristic (e.g., expression characterized by a particular gene or one or a combination of the proteins encoded by that gene). The term "diagnosing" includes determining whether a subject has lung cancer, determining the risk of a subject to have lung cancer.

Furthermore, the lung cancer comprises non-small cell lung cancer and small cell lung cancer.

Further, the lung cancer is non-small cell lung cancer.

Further, the non-small cell lung cancer comprises squamous cell lung cancer, adenocarcinoma lung cancer, large cell carcinoma and carcinoid, and is preferably adenocarcinoma lung cancer.

Further, the reagents include probes, primers, and/or protein binding agents specific for PPM 1G.

The term "primer" refers to an oligonucleotide that hybridizes to a sequence in a target nucleic acid ("primer binding site") and is capable of serving as a point at which synthesis is initiated along a complementary strand of the nucleic acid under conditions suitable for such synthesis.

The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

A binding agent for a protein is, for example, a receptor for a protein, a lectin that binds a protein, an antibody against a protein, a peptide antibody (peptidebody) against a protein, a bispecific dual binding agent, or a bispecific antibody format.

Further, the product comprises a chip, a kit, test paper or a high-throughput sequencing platform.

In a fourth aspect, the present invention provides a method of screening a candidate compound for the treatment of lung cancer, said method comprising:

(1) administering a test compound to a subject to be tested in a test group, and detecting the expression level of PPM1G in a sample derived from the subject in the test group, V1; in a control group, a blank control is applied to a subject to be tested, and the expression level V2 of PPM1G in a sample derived from the subject in the control group is detected;

(2) comparing the expression level V1 and the expression level V2 detected in the previous step, thereby determining whether the test compound is a candidate compound for treating lung cancer.

Furthermore, the lung cancer comprises non-small cell lung cancer and small cell lung cancer.

Further, the lung cancer is non-small cell lung cancer.

Further, the non-small cell lung cancer comprises squamous cell lung cancer, adenocarcinoma lung cancer, large cell carcinoma and carcinoid, and is preferably adenocarcinoma lung cancer.

Drawings

FIG. 1 is a graph showing the results of PPM1G expression in lung adenocarcinoma, wherein, panel A is a statistical graph of PPM1G expression level in lung adenocarcinoma patients, panel B is a statistical graph of PPM1G expression level in different levels of patient tissues, panel C is a graph showing the results of Kaplan-Meier Plotter online database analysis of PPM1G correlation with patient survival time, panel D is a statistical graph of the mRNA expression level of PPM1G detected by Realtime-PCR in lung adenocarcinoma cell lines, and panel E is a graph showing the results of Western blot detection of protein expression level of PPM1G in lung adenocarcinoma cell lines;

FIG. 2 is a result diagram of construction and protein expression of pcDNA3.1-myc-His-hPPM1G plasmid, wherein, diagram A is a result diagram of PCR amplification PPM1G, diagram B is a result diagram of bacteria liquid PCR identification PPM1G, diagram C is a result diagram of PPM1G sequencing, and diagram D is a result diagram of Western blot identification PPM1G plasmid protein expression;

FIG. 3 is a result graph of CCK8 proliferation experiment and plate clone formation experiment for detecting NCI-H1299 cell proliferation and clone formation ability of instantaneously knocked down PPM1G, wherein, graph A is a result graph of detecting mRNA expression level of instantaneously knocked down PPM1G by Realtime-PCR, graph B is a result graph of detecting protein expression level of instantaneously knocked down PPM1G by Western blot, graph C is a result graph of detecting NCI-H1299 cell proliferation ability of instantaneously knocked down PPM1G by CCK8 proliferation experiment, and graph D is a result graph of detecting NCI-H1299 cell clone formation ability of instantaneously knocked down PPM1G by plate clone formation experiment and statistical analysis of plate clones;

FIG. 4 is a graph showing the result of verification of NCI-H1299 monoclonal cell strain stably knocking out PPM1G, wherein, a graph A is a graph showing the result of detecting the mRNA level stably knocking out PPM1G by Realtime-PCR, and a graph B is a graph showing the result of detecting the protein level stably knocking out PPM1G by Western blot;

FIG. 5 is a graph showing the results of a CCK8 proliferation assay and a plate clone formation assay for detecting the proliferation and clone formation ability of NCI-H1299 cells stably knocking out PPM1G, wherein, graph A is a graph showing the results of a CCK8 proliferation assay for detecting the proliferation ability of NCI-H1299 cells stably knocking out PPM1G, graph B is a graph showing the results of a plate clone formation assay for detecting the clone formation ability of NCI-H1299 cells stably knocking out PPM1G, and graph C is a graph showing the results of statistical analysis of plate clones;

FIG. 6 is a graph showing the results of testing the cell migration and cell invasion ability of NCI-H1299 cells stably knocking out PPM1G in scratch healing test, Transwell migration test and Matrigel invasion test, wherein, A is a graph showing the results of testing the lateral migration and statistical analysis of NCI-H1299 cells stably knocking out PPM1G in scratch healing test, B is a graph showing the results of testing the longitudinal migration and statistical analysis of NCI-H1299 cells stably knocking out PPM1G in Transwell migration test, and C is a graph showing the results of testing the cell invasion ability and statistical analysis of NCI-H1299 cells stably knocking out PPM1G in Matrigel invasion test;

FIG. 7 is a graph of the verification result of an A549 cell strain over-expressing PPM1G, wherein the graph A is a graph of the result of detecting the mRNA level over-expressing PPM1G by Realtime-PCR, and the graph B is a graph of the result of detecting the protein level over-expressing PPM1G by Western blot;

fig. 8 is a graph of the results of scratch healing experiments, Transwell migration experiments and Matrigel invasion experiments detecting a549 cell migration and cell invasion abilities of overexpressing PPM1G, wherein, the graph a is a graph of the results of lateral migration and statistical analysis of a549 cells of stably overexpressing PPM1G detected by scratch healing experiments, the graph B is a graph of the results of longitudinal migration and statistical analysis of a549 cells of overexpressing PPM1G detected by Transwell experiments, and the graph C is a graph of the results of invasion and statistical analysis of a549 cells of overexpressing PPM1G detected by Matrigel invasion experiments;

FIG. 9 is a graph showing the effect of PPM1G on the in vivo proliferation of lung adenocarcinoma cells, in which Panel A is a visual image of tumors (upper: NCI-H1299, lower: A549), Panel B is a weight monitor of nude mice (upper: NCI-H1299, lower: A549), Panel C is a statistical image of tumor weights (upper: NCI-H1299, lower: A549), and Panel D is a volume monitor of tumors (upper: NCIH1299, lower: A549);

FIG. 10 is a graph showing the result of detecting the phosphorylation levels of key proteins in lung adenocarcinoma cells in which PPM1G is knocked out/overexpressed by Western Blot, wherein, the graph A is a graph showing the result of detecting the phosphorylation levels of key proteins in A549 cells in which PPM1G is knocked out by Western Blot, and the graph B is a graph showing the result of detecting the phosphorylation levels of key proteins in NCI-H1299 cells in which PPM1G is overexpressed by Western Blot; FIG. C is a graph showing the results of detecting the phosphorylation levels of MEK3/MEK6 in A549 cells knocked out of PPM1G by Western Blot, and FIG. D is a graph showing the results of detecting the phosphorylation levels of MEK3/MEK6 in NCI-H1299 cells overexpressing PPM1G by Western Blot;

fig. 11 is a graph of in vitro kinase assay results showing that PPM1G can inhibit p38 activity by removing MEK6 phosphorylation, wherein, graph a is a graph of in vitro kinase assay results showing that PPM1G is affected by MEK6 on p38 phosphorylation, graph B is a graph of in vitro kinase assay results showing that PPM1G is affected by MEK3 on p38 phosphorylation, graph C is a graph of Co-IP assay results showing that whether PPM1G and MEK6 interact with each other, graph D is a graph of GST pull-down assay results showing that PPM1G and MEK6 interact with each other, and graph E is a graph of in vitro kinase assay results showing that PPM1G can remove MEK6 phosphorylation.

Detailed Description

The following detailed description of embodiments of the present application will be made with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present application, are given by way of illustration and explanation only, and are not intended to limit the present application.

Experimental Material

First, experimental reagent

A549 cells, NCI-H1299 cells, 293T cells (ATCC); dmem (hyclone); puromycin (GIBCO); FBS (PAN); trypsin (melphalan organism); dimethyl i Sulfoxide (Solarbio); ethanol (Anhuite food Co., Ltd.); Opti-MEM (GIBCO); polybrene (Solarbio); BCA protein concentration assay kit (Thermo Fisher); tris Solambio, Tween-20 Solambio, Triton X-100, SDS (Solambio); n, N-methylenebisacrylamide (SIGMA); ammonium persulfate (SIGMA); glycine, HEPES (Solarbio); cell Counting Kit-8(CCK8) (bimake); NaCl (chemostat); skimmed milk powder (illite); paraformaldehyde (Tianjin Seiyuan chemical Co.); matrigel (corning); gel Extraction Kit, Endo-free Plasmid Mini Kit II (OMEGA); a total RNA rapid extraction kit (Fastagen); t4 DNA Ligase (TaKaRa); anti-c-myc magnetic Beads, Anti-flag magnetic Beads, protein marker (thermo Fisher); secondary antibody-HRP (goat anti-rabbit), secondary antibody-HRP (rabbit anti-mouse) (SIGMA); PPM1G Rabbit Ab (proteintech); Myc-Tag Rabbit Ab, DYKDDDDK Tag Rabbit Ab, P38 MAPK Rabbit Ab, P-P38 MAPK (T180/Y182) Rabbit Ab, P44/42MAPK (Erk1/2) Rabbit Ab, P-P44/42MAPK (T202/Y204) Mouse mAb, AKT Rabbit Ab, P-AKT (S308) (D25E6) Rabbit, P-AKT (S473) (D9E) Rabbit mAb, P-MEK3, MEK6 Rabbit mAb, MEK3 Rabbit mAb, MEK6 Rabbit mAb (Cell Signaling Technology).

Second, experimental equipment

A small vertical protein electrophoresis tank, a protein wet-to-membrane conversion tank, a horizontal electrophoresis tank (Bio-Rad); gel imaging analysis System (BIO-RAD Molecular Imager Gel Doc)^TMXR +; pH adjuster, analytical balance (Startorius); a stirrer, a horizontal shaker (guangzhou IKA); an ice maker (Scotsman); a ultramicro full-wavelength spectrophotometer, a constant temperature incubator, a biological safety cabinet and a micro centrifuge (Thermo Fisher); a chemiluminescent imaging system (CLINX); vortex shaker (Thermo Fisher); PCR instrument (sensuest); water purification instruments (Merck Millipore); cytometers (COUNTSTAR); a high-speed low-temperature centrifuge, a 7500 type real-time fluorescence quantitative PCR instrument, a conventional high-speed centrifuge and a liquid nitrogen tank (Thermo Fisher); a water bath (GD100, Grant); microscope (Nikon TS 100); cell cryopreservation cassettes (Thermo Fisher).

Third, plasmid construction primer (Forward, Reverse,5'to 3')

TABLE 1 plasmid construction primers

Experimental methods

First, cell recovery

Setting the temperature of a water bath kettle at 37 ℃ in advance, preheating a whole culture medium and a culture dish, putting cells to be recovered into the water bath kettle, transferring the cells to the preheated culture medium after melting, centrifuging at 500rpm for 5min, removing supernatant, re-suspending the cells with the whole culture medium, uniformly dropwise adding the cells to the culture dish, placing the cells at 37 ℃ after shaking uniformly, and adding 5% CO₂The constant temperature incubator of (1) determines whether to change the liquid or passage according to the cell condition after 12 hours.

II, cell culture

Human adenocarcinoma alveolar basal epithelial cells (A549, NCI-H1299) and human embryonic kidney epithelial cells (293T) were purchased from ATCC and cultured in DMEM high-glucose complete medium containing 10% FBS, 100U/mL penicillin and 0.1mg/mL streptomycin at 37 ℃ with 5% CO₂Culturing in a constant temperature incubator.

Thirdly, cell passage

The medium and other articles required for cell passage are taken out in advance and the temperature is restored to room temperature (the whole process of treating cells is in a biological safety cabinet, and the aseptic operation is strictly followed). Taking out cells to be treated, abandoning the culture medium, washing the surface with 1mL of PBS, digesting the cells with 1mL of pancreatin, adding 1mL of DMEM complete culture medium to terminate digestion, pinching the cover of the dish and the bottom of the dish with the left hand, beating the culture dish with the right hand, continuously beating in the rotating direction, blowing down the cells after basically dropping, centrifuging for 5min at 500rpm, abandoning the supernatant, suspending the cells with 1mL of DMEM high-sugar complete culture medium, uniformly dropping the cells into two dishes added with 3mL of DMEM high-sugar complete culture medium, and each dish is 0.5 mL. Marking relevant information on the dish cover, placing at 37 ℃ and 5% CO after shaking uniformly₂The constant temperature incubator.

Fourthly, freezing and storing cells

The medium and other articles required for cell treatment are taken out from the refrigerator in advance and returned to room temperature. Preparing cell freezing medium with the proportion of FBS: DMSO at 9:1, mixed and cooled at 4 ℃. Taking out cells to be cryopreserved, centrifuging at 500rpm for 5min after digestion, discarding supernatant, resuspending cells in 1mL of cell cryopreservation solution, transferring to a cryopreservation tube, marking related information on the tube wall, cryopreserving in a-80 ℃ ultra-low temperature refrigerator, and transferring the cells to a liquid nitrogen tank after 24 h.

Fifth, cell counting

The cells to be counted were removed, digested and centrifuged at 500rpm for 5min, the supernatant discarded, 1mL of complete medium was used to resuspend the cells, 20 μ L of which was taken in conjunction with trypan blue 1: fill the pond after 1 abundant mixing, make cell suspension full of whole counting cell, insert the counting board the count appearance and begin to detect, select the beginning of counting board, middle, 3 fields of end and detect, the average in 3 fields is cell concentration, washs the cell counting board after the count, dries reserve, closes at last count appearance and computer.

Sixthly, total RNA extraction

(1) (use Fastagen total RNA rapid extraction kit, care to avoid RNase contamination) open the metal bath set temperature to 60 ℃ in advance and put DEPC H2O for preheating. Taking out the cell of RNA to be extracted, washing the surface with 1mL of PBS, quickly adding 500 mu L of RA2, shaking uniformly, standing for 1-2min, and blowing and beating the cell; passing cell lysate through column at room temperature and rotation speedCentrifuging at 8000rpm for 1min, and discarding the filtrate; adding 500 mu LWash Buffer, centrifuging at room temperature and 8000rpm for 1min, discarding the filtrate, and repeating the process once; performing air separation at the room temperature at the rotating speed of 13400rpm for 2min, discarding the filtrate, uncovering and standing for 5min (covering a layer of PE gloves on the cover to prevent pollution), and drying the ethanol in the Wash Buffer in the air; discarding the cannula, replacing with a new tube without RNase, adding 35 μ L of 60 deg.C preheated DEPC H₂O, covering a tube cover and standing for 5 min; centrifuging at 13400rpm for 2min at room temperature, and measuring the concentration on a machine.

Seventhly, PPM1G sgRNA CRISPR/Cas9 knockout plasmid construction

According to the gene sequence of PPM1G, a CRISPR/Cas9 sgRNA is designed, a PCR instrument is used for chelating a sense strand and an antisense strand to form a double strand, and the double strand is connected to a Lenti CRISPR/Cas 9V 2 vector by using T4 enzyme. After transformation, selecting a single colony, carrying out PCR verification and nucleic acid gel detection, sequencing the positive bacteria liquid, activating the bacteria liquid with successful sequencing, shaking the bacteria to extract plasmids, and placing at-20 ℃ for later use.

Eight, pcDNA3.1-myc-His-HPPM1G overexpression plasmid construction

Designing PPM1G upstream and downstream primers, carrying out PCR amplification according to a polymerase system, and recovering an amplified product; carrying out double enzyme digestion on the recovered amplification product and the pcDNA3.1-myc-His vector, and recovering after enzyme digestion; ligation of the fragment to the vector with T4 enzyme was performed overnight at 16 ℃; after transformation, selecting a single colony, carrying out PCR verification and nucleic acid gel detection, sequencing the positive bacteria liquid, activating the bacteria liquid with successful sequencing, shaking the bacteria to extract plasmids, and placing at-20 ℃ for later use.

Transfection of plasmid

One day in advance, cells were plated, the middle dish 293T cells were centrifuged and resuspended, 1/3 cells were plated, the cells were blown down evenly and placed at 37 ℃ in 5% CO₂The constant-temperature incubator of (1), wherein the transfection is carried out when the density is 75% on the next day; in transfection, take two 1.5mL EP tubes, labeled A and B, respectively, and add 100. mu.L of Opti-MEM medium, then add 4. mu.g of plasmid to be transfected into A tube, add 10. mu.L of PEI (plasmid mass: PEI volume: 1: 2.5) into B tube, mix the liquids gently in the two 1.5mL EP tubes, blow the same number of times, leave immediately and stand at room temperature for 5min, then mix A tube and B tube together (if multiple plasmids are transfected, blow the same number of times in different plasmid tubes),standing at room temperature for 18min after instantaneous separation; changing the old culture medium to 1.5mL new culture medium, standing, dropping 200 μ L mixed solution into the well, shaking, and placing at 37 deg.C and 5% CO₂The constant temperature incubator is changed to a new culture medium after 6 hours, timing is started, then the culture medium is changed every 12 hours, and protein is collected in 48 hours.

Ten, viral concentration and cell infection

And (3) virus concentration: co-transferring the lentivirus packaging plasmid and PPM1G overexpression/PPM 1G knockout plasmid to 293T cells; after transfection for 48h, collecting supernatant, centrifuging, filtering with a 0.45 μm filter, adding 7.5mL of 5 × PEG8000 to each 30mL, mixing for about 30min for 3-5 times, and shaking overnight at 4 deg.C; separating with 4 deg.C centrifuge 4000g for 20min the next day, removing supernatant, standing for about 2min, dissolving precipitate with 150 μ L empty DMEM medium, packaging into 3 tubes, quick freezing in liquid nitrogen, and storing at-80 deg.C.

Cell infection: digesting A549 cells and NCI-H1299 cells, centrifuging, resuspending, taking a small amount of cells, spreading the cells on a small dish, placing the dish at 37 ℃ and 5% CO₂The density of the culture box reaches 20 to 30 percent on the next day, and infection begins; changing the dish into a new culture medium, taking out 1 part (50 mu L) of concentrated virus packaged with PPM1G overexpression plasmid/1 part (50 mu L) of concentrated virus packaged with PPM1G knock-out plasmid from-80 ℃, thawing, adding 1 mu L polybrene into each concentrated virus, uniformly mixing, uniformly adding the concentrated virus packaged with PPM1G overexpression plasmid into A549 cells, adding the concentrated virus packaged with PPM1G knock-out plasmid into NCI-H1299 cells, uniformly shaking, placing at 37 ℃, and placing at 5% CO₂The constant temperature incubator of (1); and after infection for 12h, changing the culture medium into a new culture medium, and performing resistance screening when the fusion degree is 90%.

Eleven, screening the optimal concentration of puromycin

A549 cells and NCI-H1299 cells were digested and counted, and the cells plated in 96-well plates at 5000 cells/well at 37 ℃ with 5% CO₂The constant temperature incubator of (1); first, complete culture media with different concentrations of Puro are prepared: 0ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 1. mu.g/mL, 2. mu.g/mL, 3. mu.g/mL and 5. mu.g/mL, and 100. mu.L of Puro-added medium per well (3 replicate wells per Puro concentration) was changed after 24 hours of cell culture in the 96-well plate; show every 3h or soObserving the cell morphology and the cell survival rate under a microscope, changing the liquid twice every day, and changing dead cells in the culture medium; finally, the lowest Puro concentration for complete death of A549 cells was determined to be 1. mu.g/mLNCI-H1299 cells and 5 ng/mL.

Twelve, screening of monoclonal cells

Taking out infected cells, digesting and centrifuging 1mL of complete culture medium to resuspend the cells, and counting; diluting the cell mixture to a proper concentration, adding 100 cells into a 15mL centrifuge tube containing 10mL complete culture medium (containing Puro), fully and uniformly mixing, paving a 96-well plate, uniformly mixing cell suspension at 37 ℃ and 5% CO at 100 mu L per well, and continuously mixing the cell suspension₂The incubator of (1); when the cells are just attached to the wall, the holes with a plurality of cells are cut off by a microscope, only single cell holes are reserved, the temperature is continuously kept at 37 ℃ and 5% CO₂The culture box is used for timely supplementing liquid; transferring the cells to a small dish and a medium dish in sequence after the cells grow to be full, and taking half of the cells to extract protein for verification.

Thirteen, Western Blot

Protein extraction: firstly, preparing RIPA lysate for later use on ice, removing a culture medium of cells for extracting protein, washing 1mL PBS, adding a proper amount of RIPA lysate on ice after removing the PBS, scraping the cells, adding the cells into a 1.5mL EP tube, putting the EP tube into a 4 ℃ suspension instrument for lysis for 30min, adjusting a low-temperature high-speed centrifuge to 4 ℃ for cooling, centrifuging at a rotating speed of 12000rpm for 20min after the protein lysis is finished, transferring supernatant to the 1.5mL EP tube, and starting BCA protein quantification;

BCA protein quantification: using a BCA protein quantitative kit of Thermo, preparing a working solution (calculating loss) according to the proportion of A solution: solution B is 50:1, each well needs 200 mul of working solution, 22.5 mul of double distilled water is added into a 96-well plate, 2.5 mul of protein supernatant is added into the double distilled water, 200 mul of working solution is added, the reaction is carried out in an incubator at 37 ℃ for 30min, an enzyme-labeling instrument detects OD562nm, and the concentration is calculated according to protein standard curve; after the quantification is finished, adding 1/4 volumes of 5 Xloading buffer into the protein supernatant to dilute the 5 Xloading buffer into 1 Xloading buffer, boiling in boiling water for 10min, separating at 12000rpm for 5min, and placing at-20 ℃ for later use.

Western Blot: selecting a WB thick plate, a thin plate and a comb which are matched in specification, washing the plate, airing, and then matching glue with the clamping plate; preparing lower layer separation gel with proper concentration according to experiment needs, filling the separation gel into a gap between a thick plate and a thin plate, adding the separation gel to a proper height, flattening the surface with isopropanol, removing the isopropanol on the upper layer after 30min, preparing upper layer concentrated gel, standing for about 30min, taking down the whole albumin gel, and soaking in single distilled water for storage at room temperature for later use; taking out a protein sample from-20 ℃, centrifuging for 5min at a rotating speed of 12000rpm after melting, loading according to a designed loading sequence and loading quantity, running the concentrated gel at 80V after loading is finished, adjusting the voltage after a 72-length strip of a protein Marker runs out, separating the gel at 120V, and finishing electrophoresis according to experiment requirements; preparing 1 Xmembrane transferring solution in advance, putting the membrane transferring solution into a tray for standby at 4 ℃, pouring the membrane transferring solution into the tray during membrane transferring, putting black sponge, white sponge, filter paper and NC membrane, putting the black surface of a membrane transferring clamp on the lowest layer of the tray after fully soaking, putting the black sponge, the white sponge and the three layers of filter paper from bottom to top, putting protein glue on the filter paper, putting the NC membrane, putting the three layers of filter paper, the white sponge and the black sponge in sequence, slightly clamping the membrane transferring clamp, inserting the membrane transferring clamp into a membrane transferring groove (black to black and white to red), reversing the membrane solution, cooling the ice block, and transferring the membrane solution for 2 hours at constant current of 280 mA; taking out the NC membrane after the membrane is transferred, rinsing the NC membrane in TBST, washing off methanol on the surface, putting 5% skimmed milk prepared by TBST, and sealing the NC membrane on a shaking table at a low rotating speed for 2 hours at room temperature; washing the sealed NC membrane with TBST, slightly absorbing residual TBST by absorbent paper, flatly placing on an antibody-sealed incubation plate, uniformly dropwise adding primary antibody (diluting the primary antibody with 5% skimmed milk prepared by TBST), covering with a cover, and standing overnight at 4 ℃; recovering primary antibody on the next day (about 14h of the primary antibody), washing the NC membrane with TBST for three times for 10 min/time, preparing secondary antibody in the period, preparing the secondary antibody with TBST, after the NC membrane is washed, absorbing residual TBST with absorbent paper, flatly placing the NC membrane on an incubation plate sealed with the antibody, uniformly dropwise adding the secondary antibody, covering the incubation plate with a cover, washing the NC membrane in TBST for three times for 10 min/time, and exposing after washing; opening a chemiluminescence apparatus in advance for precooling, preparing luminescence liquid, preparing ECL luminescence liquid A and B according to the ratio of 1:1, placing an NC film on a plate of the chemiluminescence apparatus with the front side facing upwards, uniformly dripping the luminescence liquid, carrying out exposure according to the operation instruction of the apparatus, marking the exposed strip clearly with the protein name, and storing the strip in a computer after the exposure is finished for a period of time.

Fourteen, CCK8 proliferation experiments

Taking out cells to be subjected to proliferation experiments, centrifuging at 500rpm for 5min after digestion, discarding supernatant, and counting after 1mL of complete culture medium is resuspended; diluting the cell mixture to appropriate concentration, spreading in 96-well plates (5) with 100. mu.L/well, spreading one well of culture medium for the cells as control, spreading 4 multiple wells for each cell, spreading 1000 cells for A549 cells and 750 cells for NCI-H1299 cells, adding PBS around the wells, slowing the evaporation rate of the culture medium, placing at 37 deg.C and 5% CO₂The culture box is taken out after 4 hours, CCK8(10 mu L/hole) is added, and the culture box is placed at 37 ℃ in the dark and 5% CO₂The incubator is used for reaction for 2 hours, then a multifunctional microplate reader is used for detecting OD450nm, then CCK8 is added according to the time of the first day every day, OD450nm is detected, and the result is analyzed after the last detection is finished.

Fifteen plate cloning experiment

Taking out cells to be subjected to plate cloning experiments, centrifuging at 500rpm for 5min after digestion, removing supernatant, and counting after 1mL of complete culture medium is resuspended; diluting the cell suspension to appropriate concentration, spreading in 6-well plate with 3 multiple wells per cell, spreading 500A 549 cells per well and 1000 NCI-H1299 cells per well, adding the calculated cell number (3 wells) into 15mL centrifuge tube, supplementing the culture medium to 6mL, mixing well, adding 2mL per well, shaking well, standing at 37 deg.C, and adding 5% CO₂The culture box of (1) is changed every 2 days; stopping culturing when the cells grow to be single colonies visible to naked eyes, collecting the A549 cells for 9 days generally, collecting the NCI-H1299 cells for 7 days generally, discarding the culture medium, washing the surface with PBS (phosphate buffer solution), adding 1mL of 4% paraformaldehyde into each hole for fixation, discarding after 20min, washing with PBS for three times, adding 1mL of 0.2% crystal violet dye into each hole for staining for 30 min; after dyeing is finished, discarding the crystal violet dye solution, indirectly cleaning the dye solution of the cell holes by using the gap of lightly punching holes by using tap water until the crystal violet dye solution is thoroughly cleaned, and taking a picture after drying; and (4) analyzing the result of Image J after the gel imaging analysis system takes a picture.

Sixteen test for healing scratch

Taking out cells to be subjected to the scratch healing experiment from the incubator, centrifuging at 500rpm for 5min after digestion, discarding supernatant, and completely culturing with 1mL of DMEM high-sugarSuspending the cells in nutrient medium, and counting the cells; 6-well plates were plated, and A549 cells were plated at 7X 10 cells per well⁵One, NCI-H1299 cells at 5X 10 cells per well⁵Adding the counted number of cells into 6-well plate, supplementing to 2mL with culture medium for culturing the cells, shaking well, placing at 37 deg.C, and adding 5% CO₂The incubator of (1), scratching is started after the cells grow full; sterilizing a ruler for scratching by irradiating ultraviolet in advance, taking out cells to be scratched from an incubator, opening a cover, placing the ruler on a 6-hole plate, vertically fixing the ruler, marking a vertical mark along the ruler by using a 200-microliter yellow gunpoint, slightly washing the marked cells by using a serum-free DMEM high-sugar medium, changing the medium into a DMEM high-sugar medium with the concentration of 3% FBS, and then taking a picture; selecting the visual field with the same scratch width as the visual field of the control group and the experimental group as the photographing result of 0h, marking the visual field of the photographing by using a marker pen, placing the 6-hole plate at 37 ℃ and 5% CO₂The healing conditions of the scratches of 12h, 24h, 36h and 48h in the same visual field are tapped until the scratches of a group of cells are completely healed, and the experiment is finished; analyzing the healing rate of the scratch by using Image J statistics;

(in 12h example) scratch healing rate (0h area-12 h area)/0 h area × 100%

Seventeen, Transwell experiment

Taking out cells to be subjected to a Transwell experiment, centrifuging at 500rpm for 5min after digestion, discarding supernatant, resuspending 1mL of serum-free culture medium, and counting; taking a 24-pore plate, adding 600 mu L of a serum-free empty culture medium, putting the 24-pore plate on a Transwell chamber, adding 200 mu L of the serum-free empty culture medium in an upper chamber, and fully wetting the chamber; changing the lower empty culture medium to 600 μ L of complete culture medium for culturing the cells, and discarding the upper empty culture medium; take 2.5X 10⁴Supplementing each cell (the number of A549 cells is the same as that of NCI-H1299 cells) to 200 μ L with bloodless clear culture medium, mixing, slowly spreading in upper chamber to avoid generating bubbles, standing, and standing at 37 deg.C and 5% CO₂The incubator of (1); collecting the A549 cells after 43H, collecting the NCI-H1299 cells after 28H, discarding the old culture medium, washing the upper and lower chambers with PBS 3 times, fixing with 4% paraformaldehyde, washing the upper and lower chambers with PBS 3 times after 20min, dyeing with 0.2% crystal violet dye solution, washing with tap water after 30min, and washing the chambers without washing the chambers all the timeThe outer surface of the lower chamber can be scrubbed; the chamber was photographed under a microscope with photographing software, 2 low power glasses and 5 high power glasses, and Image J was subjected to statistical analysis.

Eighteen, Matrigel experiments

Taking out the Matrigel from-20 deg.C 24h in advance, and melting at 4 deg.C. Matrigel and bloodless clear medium were diluted 1:6 (50 μ L per well) on ice for use; wetting Transwell chamber with serum-free DMEM medium in 24-well plate, changing the empty culture medium in the lower chamber to 600 μ L complete culture medium, discarding the empty culture medium in the upper chamber, spreading 50 μ L matrigel in the upper chamber, standing at 37 deg.C and 5% CO₂The culture box is used for solidifying the matrigel for more than 1 h; taking out cells to be subjected to a Matrigel experiment, centrifuging at 500rpm for 5min after digestion, discarding supernatant, and counting after 1mL of serum-free culture medium is resuspended; take 5X 10⁴Supplementing each cell (the number of A549 cells is the same as that of NCI-H1299 cells) to 200 μ L with serum-free DMEM high-sugar medium, mixing, slowly dripping cell suspension into the chamber, standing, and standing at 37 deg.C and 5% CO₂The incubator of (1); collecting the A549 cells after 86 hours, collecting the NCI-H1299 cells after 56 hours, discarding the old culture medium, washing the upper chamber and the lower chamber with PBS (phosphate buffer solution) for 3 times, fixing with 4% paraformaldehyde, washing the upper chamber with 200 muL and the lower chamber with 600 muL, washing the upper chamber with PBS for 3 times after 20min, dyeing with 0.2% crystal violet dye solution, washing with tap water after 30min, wherein the outer surface of the lower chamber cannot be scrubbed in the washing process; the chamber was photographed under a microscope with photographing software, 2 low power glasses and 5 high power glasses, and Image J was subjected to statistical analysis.

Nineteen, co-immunoprecipitation experiments

Taking 1000 mu g of protein supernatant in a 1.5mL EP tube, supplementing the protein supernatant to 1000 mu L with an IP lysate, adding a label magnetic bead according to the experimental requirement, and placing the label magnetic bead in a 4 ℃ suspension instrument for shaking for 4 hours; inserting an EP tube on a magnetic frame after 4h, discarding the lysate when the magnetic beads are completely adsorbed, adding 1mL of NETN high-salt Buffer to wash the magnetic beads, shaking the suspension instrument at 4 ℃ for 5min, and repeating the step twice; inserting an EP tube on a magnetic frame, abandoning the NETN high-salt Buffer when the magnetic beads are completely adsorbed, adding 1mL of NETN medium-salt Buffer to wash the magnetic beads, shaking the suspension instrument at 4 ℃ for 5min, and repeating the step once; inserting an EP tube on a magnetic frame, discarding a salt Buffer in the NETN when the magnetic beads are completely adsorbed, adding 1mL of low-salt Buffer in the NETN to wash the magnetic beads, shaking the mixture on a suspension instrument at 4 ℃ for 5min, and repeating the step twice; inserting an EP tube on a magnetic frame, discarding the NETN low-salt Buffer when the magnetic beads are completely adsorbed, taking down the EP tube, blowing down the magnetic beads by using 20 mu L of 2 multiplied Loading Buffer, boiling for 10min, cooling, centrifuging at the rotating speed of 12000rpm for 5min at room temperature, and placing at-20 ℃ for later use.

Twenty and GST pull-down experiment

Induced expression of GST fusion protein: the GST-MEK6 fusion protein expression vector is shaken to extract plasmids, transformed to BL21 competence, picked single colony is inoculated to 400 mu L culture medium and shaken at 37 ℃ overnight; inoculating 100 μ L of bacterial liquid into a glass tube containing 10mL of culture medium at a ratio of 1:100, and performing shake culture at 37 deg.C; detecting the bacterial liquid OD600 at 1.5h, when the value of the bacterial liquid OD600nm is 0.113-0.117, taking 100 mu L of bacterial liquid, centrifuging for 1min at the rotating speed of 12000g, discarding the culture medium, adding 10 mu L of 5 Xloading buffer after resuspending and precipitating with 40 mu L of PBS, boiling for 10min as a negative control, adding 5 mu L of 100mM PTG into the residual bacterial liquid of the glass tube to ensure that the final concentration of IPTG is 0.05mmol/L, and placing the mixture in a constant temperature shaking table at 16 ℃ and 220rpm for induction for about 24 h; taking 100 mu L of induced bacterial liquid, centrifuging for 1min at the rotating speed of 12000g, adding 10 mu L of 5 Xloading buffer after 40 mu L of PBS heavy suspension precipitation, and boiling in boiling water for 10min as a positive control;

purification of GST fusion protein: centrifugally collecting bacterial liquid, separating at the rotating speed of 4000g for 10min, and collecting thalli of GST fusion protein and GST protein; discarding the supernatant, adding 900 μ L of bacterial lysate to the sample, and blowing to fully crack the thallus; performing ultrasonic lysis on 320W bacteria, clarifying the solution (for detection, 50 mu L of mixed solution is taken in a 1.5mL EP tube, centrifuging and transferring supernatant to another EP tube, 50 mu L of balance buffer solution is used for resuspending the precipitate, 12.5 mu L of 5 Xloading buffer is respectively added in the supernatant tube and the precipitation tube, boiling in boiling water for 10min for SDS-PAGE, and detecting the expression of fusion protein in the supernatant and the precipitate), and centrifuging at the rotating speed of 12000rpm for 20min at the temperature of 4 ℃; transferring 900 μ L of the supernatant to an EP tube, and adding 45 μ L of 10% NP-40 to make the final concentration of NP-40 0.05%; add 10. mu.L of diluted GST beads, shake at 4 ℃ for 4h (10. mu.L of diluted beads per 900. mu.L of protein sample); after 4h, washing the magnetic beads for 3 times by using a bacterial lysate containing 0.05 percent NP-40, and then washing for 3 times by using a bacterial lysate containing no 0.05 percent NP-40, wherein the same product can be combined into a tube; the beads were resuspended in 40. mu.L PBS, 5. mu.L of each bead was taken from each tube, 15. mu.L PBS and 5. mu.L 5 Xloading buffer were added, boiled and used for SDS-PAGE to detect expression of GST fusion protein, while approximately quantifying with 2mg/mL BSA, and the remaining samples were stored at 4 ℃ until use (2 weeks).

Exogenous expression of eukaryotic proteins: spreading HEK293T cells, transfecting at a density of 80%, and collecting protein (IP lysate) after 48 h; protein quantification is carried out by using a BCA method, a small amount of protein supernatant is taken, 5 XLoading buffer is added for boiling for 10min, and the expression condition of the target protein is verified by Western Blot; the quantified protein is divided into 1mg of each EP tube, and the EP tubes are placed in a refrigerator at the temperature of 80 ℃ below zero for standby.

GST fusion protein-magnetic beads bind to the protein of interest: taking out the subpackaged EP tubes containing 1mg of protein from minus 80 ℃, putting the tubes on ice for melting, supplementing 1mL of the tubes with BufferA added with protease inhibitor and PMSF after complete melting, adding 5 mu g of GST fusion protein-magnetic beads, and putting the tubes on a 4 ℃ suspension instrument for shaking overnight to ensure that the GST fusion protein is fully combined with the target protein; inserting an EP tube on a magnetic frame on the next day, discarding liquid when the magnetic beads are completely adsorbed, adding 1mL of Buffer A to wash the magnetic beads, and repeating the step for 4 times; after Buffer A washing, the supernatant is discarded, 20 mu L of 2 × Loading Buffer is used for blowing magnetic beads to the bottom of an EP tube, boiling is carried out for 10min, and after cooling, the tube is centrifuged at 12000rpm for 5min and is used for detecting the binding condition of protein by Western Blot.

Twenty-one, in vitro phosphorylation experiment

0.2mg of the overexpression exogenous Myc-PPM1G and Flag-MEK6 proteins in HEK293T cells are taken, 5 mu L of Myc-magnetic beads and 5 mu L of Flag-magnetic beads are added for co-immunoprecipitation, and after 4h, the nonspecific binding (high salt for 3 times, medium salt for 2 times and low salt for 3 times, each time for 5min) is washed away by an IP washing buffer solution; wash 2 times with 500 μ L kinase buffer for 5 minutes each; add 40. mu.L kinase buffer, 1. mu.L ATP and 0.5. mu.g GST-p38 purified protein to each tube, react for 90 minutes in 30 ℃ metal bath; add 10. mu.L of 5 × Loading buffer to each tube, boil in boiling water for 10 minutes, centrifuge at 12000rpm for 5min after cooling, and detect the experimental results by Western Blot.

Twenty-two, statistical analysis

Using the t-test, P <0.05 indicates that the difference is statistically significant

Example 1 high expression of PPM1G to shorten survival time of patients with lung adenocarcinoma and difference in expression of PPM1G in lung adenocarcinoma cell lines

PPM1G was highly expressed in lung adenocarcinoma patients by TCGA database analysis (fig. 1A), and PPM1G expression was significantly higher in high grade patient tissues than in the low grade group (fig. 1B); the survival of patients with high expression of PPM1G was significantly shortened by Kaplan-Meier Plotter online database analysis (figure 1C); the invention detects the expression of PPM1G at the mRNA level and the protein level in a lung adenocarcinoma cell line, and the results of Realtime-PCR and Western blot show that PPM1G is low in the A549 cell of lung adenocarcinoma and high in the NCI-H1299 cell (figure 1D and figure 1E), so that PPM1G overexpression cell line construction is carried out in the A549 cell and PPM1G knockout cell line construction is carried out in the NCI-H1299 cell.

Example 2 PPM1G promotion of Lung adenocarcinoma cell proliferation and clonogenic

1. pcDNA3.1-myc-His-hDPPM 1G plasmid construction and protein expression success

The invention successfully constructs the human PPM1G plasmid. Firstly, inserting a fragment amplified by PCR (figure 2A) into a pcDNA3.1-myc-His (-) B vector, selecting bacteria after transformation to carry out bacteria liquid PCR, detecting by using a nucleic acid gel (figure 2B), selecting positive bacteria liquid to send a large gene for sequencing to obtain a correct sequence (figure 2C), transfecting the correct sequence to 293T cells to verify plasmid expression (figure 2D), and displaying the result that the construction of the human source PPM1G plasmid is successful.

2. Transient knockdown of PPM1G inhibits NCI-H1299 cell proliferation and clonogenic

The invention uses the NCI-H1299 cells with siRNA instantaneously knocking down PPM1G to carry out CCK-8 proliferation experiments and plate cloning experiments. Transient knockdown effect was first detected and the realtome-PCR results showed significant reduction in mRNA levels of PPM1G after siRNA treatment (fig. 3A); the Western blot result shows that the protein expression level of PPM1G is remarkably reduced after siRNA treatment (figure 3B); the invention then uses CCK8 proliferation experiment to detect the effect of transient knockdown of PPM1G on the proliferation of NCI-H1299 cells (FIG. 3C), and the result shows that transient knockdown of PPM1G significantly reduces the proliferation capacity of NCI-H1299 cells (P < 0.001); the effect of transient knockdown of PPM1G on clonogenic capacity of cells was examined by plate cloning experiments (fig. 3D), and the results showed that NCI-H1299 clones transiently knocked down of PPM1G had reduced volume, reduced number, and significantly reduced clonogenic capacity (P < 0.05).

3. NCI-H1299 cell line verification for stably knocking out PPM1G

The invention constructs the NCI-H1299 cell strain for stably knocking out PPM1G, and repeats the experiment by using the stably knocked-out cell strain. PPM1G CRISPR/Cas9 plasmid and helper plasmid were first transfected into HEK293T cells by lentiviral packaging to obtain viruses, polybrone (10mg/mL) was expressed as 1: 1000, adding a mixed solution of the virus and the complete culture medium, uniformly blowing and beating, then infecting NCI-H1299 cells, and after infecting for three times, culturing the cells by using a high-sugar complete culture medium with the puro concentration of 5 ng/mL. And (3) carrying out monoclonal screening on the mixed clone, extracting the control group cells and proteins of PPM1G stably knocked-out group cells when the density of the monoclonal cells is about 90%, marking the control group cells as ctrl, and marking the PPM1G stably knocked-out NCI-H1299 cells as sgRNA 1-14. The knockout effect is detected by using Realtime PCR and Western Blot technologies, the Realtime-PCR result shows that the mRNA level is obviously reduced after PPM1G is stably knocked out (figure 4A), and the Western Blot result shows that the protein level is obviously reduced after PPM1G is stably knocked out (figure 4B), which indicates that the NCI-H1299 cell strain for stably knocking out PPM1G is successfully constructed.

4. Stable knock-out PPM1G inhibits NCI-H1299 cell proliferation and clonogenic

The screened NCI-H1299 cell strain with PPM1G stably knocked out is used for repeated experiments, the CCK8 proliferation experiment detects the influence of PPM1G stably knocked out on cell proliferation (figure 5A), and the result shows that the NCI-H1299 cell proliferation capacity is remarkably reduced by stably knocking out PPM1G (P is less than 0.001); the influence of stable knockout of PPM1G on the clonogenic capacity of cells is detected by a plate cloning experiment (FIG. 5B and FIG. 5C), and the results show that the clone volume, the number and the clonogenic capacity of NCI-H1299 cells stably knocking out PPM1G are reduced (P < 0.05).

Example 3 PPM1G promotes cell migration and cell invasion

1. Stable knock-out PPM1G inhibits NCI-H1299 cell migration and cell invasion

To more fully study the effect of knocking out PPM1G on NCI-H1299 cell function, we tested the change of stable knocking out PPM1G on the migration ability of NCI-H1299 cells by a cell scratch healing experiment and a Transwell migration experiment, and tested the change of PPM1G on the invasion ability of NCI-H1299 cells by a Matrigel invasion experiment. The scratch healing experiment result shows (fig. 6A), scratches of NCI-H1299 cells in ctrl group are basically healed after 36H, and the NCI-H1299 cells in the experiment group with PPM1G stably knocked out are obviously weakened in transverse migration capacity, and the result is obviously different (P < 0.001); the results of Transwell migration experiments show (fig. 6B) that the number of cells in ctrl group migrated into lower chamber was large, the number of cells migrated after stably knocking out PPM1G was small, and stably knocking out PPM1G weakened the longitudinal migration ability of NCI-H1299 cells, with significant difference (P < 0.01); matrigel invasion assay results (fig. 6C) showed that the number of cells in ctrl group migrated into the lower chamber was greater, the number of cells migrated after stably knocking out PPM1G was smaller, and stably knocking out PPM1G reduced NCI-H1299 cell invasion ability, with significant difference (P < 0.001).

2. Verification of A549 cell strain over-expressing PPM1G

The invention constructs an A549 cell strain over expressing PPM1G, firstly transfects the constructed pCDH-PPM1G plasmid and helper particle to HEK293T cell by a slow virus packaging technology to obtain virus, and then transfects polybrone (10mg/mL) according to the ratio of 1: adding a mixed solution of the virus and the complete culture medium according to the proportion of 1000, and blowing to uniformly infect the A549 cells. After infection for three times, culturing the cells by using a high-sugar complete culture medium with puro concentration of 1 mu g/mL, extracting cells of a control group and PPM1G overexpression cell proteins when the cell density is about 90%, marking the cells of the control group as ctrl, and marking A549 cells overexpressing PPM1G as PPM 1G. The overexpression effect was detected by Realtime-PCR and Western Blot techniques. The realtome-PCR results showed significant increases in mRNA levels after overexpression of PPM1G (fig. 7A); the Western Blot result shows that the protein level is remarkably increased after PPM1G is over-expressed (FIG. 7B), which indicates that the A549 cell strain over-expressing PPM1G is successfully constructed.

3. Overexpression of PPM1G promotes migration and invasion of A549 cells

The invention detects the change of the over-expression PPM1G to the migration capacity of A549 cells through a cell scratch healing experiment and a Transwell migration experiment, and detects the change of the PPM1G to the invasion capacity of the A549 cells through a Matrigel invasion experiment. The cell scratch healing experiment result shows (fig. 8A), scratches of the PPM1G group A549 cells are basically healed after 36h, the lateral migration capability of the ctrl group A549 cells is obviously weakened, the lateral migration capability of the A549 cells is enhanced by over-expressing PPM1G, and the result is remarkably different (P < 0.001); the results of Transwell experiments show (fig. 8B) that the number of cells in the ctrl group that migrated to the lower chamber was small, the number of cells that migrated to the lower chamber after overexpression of PPM1G was large, and the longitudinal migration ability of a549 cells was enhanced by overexpression of PPM1G, with significant differences (P < 0.001); the Matrigel invasion experiment result shows (fig. 8C) that the number of cells in the ctrl group migrated to the lower chamber is small, the number of cells migrated after PPM1G was overexpressed is large, and the invasion capacity of a549 cells was enhanced by overexpression of PPM1G, and the result is significantly different (P < 0.001).

Example 4 Effect of PPM1G on in vivo proliferation of Lung adenocarcinoma cells

The in vitro function experiment of the cell shows that PPM1G can promote the proliferation of lung adenocarcinoma cells, and in order to further detect whether PPM1G can promote the growth of tumors in vivo, the invention constructs a nude mouse subcutaneous tumor-bearing model by using stable cell strains (each nude mouse is inoculated with 6 multiplied by 10⁶Individual cells). Tumor volume (length x width) measured three days apart, starting from palpable tumor²And/2) and nude mouse body weight, continuously monitored for 22 days. After the experiment is finished, the nude mouse is anesthetized, and the posterior cervical vertebra is dislocated, taken out, weighed and statistically analyzed. The results show that the stable knockout of PPM1G significantly inhibits the growth rate of NCI-H1299 cells in vivo, obviously reduces tumor weight (on figure 9A, on figure 9C and on figure 9D), the overexpression of PPM1G significantly increases the growth rate of A549 cells in vivo, obviously increases tumor weight (under figure 9A, under figure 9C and under figure 9D), and the results are all statistically different (P9A, under figure 9C and under figure 9D)<0.001). However, neither cell had statistical significance for the body weight effects of nude mice (fig. 9B).

Example 5 PPM1G inhibition of phosphorylation of p38 by inhibition of MEK6 Activity

1. PPM1G influences the phosphorylation level of p38

The invention proves that knocking out/over-expressing PPM1G can affect lung adenocarcinoma cell proliferation and cell metastasis through a cell in-vitro function experiment and a nude mouse tumor-bearing experiment. PPM1G is protein phosphatase, and the main function of the protein phosphatase is dephosphorylation, so that the change of the phosphorylation levels of key proteins in PI3K and MAPK signal channels related to cell proliferation and cell transfer is detected by using Western Blot technology after PPM1G and A549 cell overexpression knocked out by NCI-H1299 cells. The results show that knocking out PPM1G obviously enhances the phosphorylation level of p38, the change of the phosphorylation level is consistent with the function of enzyme, the change of the phosphorylation level of AKT is small, and the phosphorylation level of ERK is not obviously changed (figure 10A); phosphorylation of p38 was significantly reduced after overexpression of PPM1G, with less change in phosphorylation of AKT and no significant change in phosphorylation of ERK (fig. 10B). Since p38 is a protein kinase and plays an important role in signal transmission of cells, the invention focuses on how PPM1G regulates the phosphorylation level of p38 and further influences the occurrence and development of lung adenocarcinoma as the next research.

2. PPM1G affects the phosphorylation level of MEK3/MEK6

The invention uses Western Blot technology to detect the phosphorylation of MEK3/MEK6 after NCI-H1299 cell knockdown of PPM1G and A549 cell overexpression of PPM1G, and the result shows that the phosphorylation level of MEK3/MEK6 is obviously enhanced by knocking down PPM1G (figure 10C), and the phosphorylation level of MEK3/MEK6 is obviously reduced by overexpression of PPM1G (figure 10D), which is consistent with the experiment result of NCI-H1299 cell.

3. PPM1G removal of phosphorylation of p38 by affecting MEK6 activity

In vitro phosphorylation experiment detection shows that when PPM1G is overexpressed, the capacity of exogenous MEK6 for activating p38 is weakened, and phosphorylation of p38 is reduced, which indicates that PPM1G can inhibit the kinase activity of MEK6 on p38, but PPM1G does not influence the kinase activity of MEK3 on p38 (FIGS. 11A and 11B). The invention subsequently examined the interaction between PPM1G and MEK6 using Co-IP experiments and GST pulldown experiments, and the results showed a direct interaction between the two (fig. 11C, 11D). Finally, in vitro kinase assays of the present invention revealed that PPM1G can remove the phosphorylation of MEK6 (fig. 11E).

The preferred embodiments of the present application have been described in detail with reference to the accompanying drawings, however, the present application is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the technical idea of the present application, and these simple modifications are all within the protection scope of the present application.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in the present application.

In addition, any combination of the various embodiments of the present application is also possible, and the same should be considered as disclosed in the present application as long as it does not depart from the idea of the present application.

Sequence listing

<110> university of Henan

Application of <120> PPM1G in diagnosis and treatment of lung cancer

<141> 2022-03-15

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atttgcggcc gcatgggtgc ctacctctcc cag 33

<210> 2

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgcggatcct ggtctcgctt ggccttcttc t 31

<210> 3

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctagctagca tgggtgccta cctctcccag 30

<210> 4

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atttgcggcc gctcacttat cgtcgtcatc cttgtaatcg tctcgcttgg ccttcttct 59

<210> 5

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

caccgctacc tctcccagcc caacacgg 28

<210> 6

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aaacccgtgt tgggctggga gaggtagc 28

<210> 7

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgcggatcca tggagtcgcc cgccgcgagc c 31

<210> 8

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ccgctcgagt gaatcctctc ccaggatctc ctt 33

<210> 9

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cgcggatcca tgtctcagtc gaaaggcaag a 31

<210> 10

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccgctcgagg tccccaagta tcagttttac aa 32

Claims (10)

1. Use of a composition for enhancing phosphorylation level of p38 in the preparation of a medicament for preventing or treating lung cancer, or inhibiting growth, proliferation, migration, invasion of lung cancer cells, wherein the composition comprises a substance that inhibits the activity of PPM1G protein, or a substance that inhibits or silences the expression of PPM1G gene, preferably, the lung cancer comprises non-small cell lung cancer and small cell lung cancer, preferably, the lung cancer is non-small cell lung cancer, preferably, the non-small cell lung cancer comprises lung adenocarcinoma, large cell carcinoma and carcinoid, preferably, the non-small cell lung cancer is lung adenocarcinoma.

2. The use of claim 1, wherein the substance that inhibits the activity of PPM1G protein comprises a substance that inhibits the synthesis of PPM1G protein or a substance that promotes the degradation of PPM1G protein or a substance that inhibits the function of PPM1G protein.

3. The use of claim 1, wherein the substance that inhibits or silences PPM1G gene expression comprises a substance that interferes with PPM1G gene expression or knocks out PPM1G gene or a substance that mutates PPM1G gene.

4. The use according to any one of claims 1 to 3, wherein the substance comprises a synthetic small molecule, a chemical agent, an antisense oligonucleotide, siRNA, miRNA, ribozyme, polypeptide, protein, preferably wherein the substance is siRNA.

5. The use of claim 3, wherein the substance for knocking out PPM1G gene further comprises a gene editing tool for knocking out PPM1G gene, preferably the gene editing tool comprises Cre-lox recombination technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, and CRISPR/Cas9 technology, preferably the gene editing tool is CRISPR/Cas9 technology.

6. The use according to claim 5, wherein the target sequence for specific cleavage of CRISPR/Cas9 technology comprises the sequence shown as SEQ ID No. 5.

7. The use according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier and/or adjuvant, preferably the pharmaceutically acceptable carrier and/or adjuvant comprises a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, a disintegrant.

8. The application of a substance for inhibiting the activity of PPM1G protein or a substance for inhibiting or silencing the expression of PPM1G gene in inhibiting the phosphorylation level of cell p38 in vitro.

9. Use of a reagent for detecting PPM1G expression level in the preparation of a product for diagnosing lung cancer or predicting the prognosis of lung cancer, preferably, the lung cancer comprises non-small cell lung cancer and small cell lung cancer, preferably, the lung cancer is non-small cell lung cancer, preferably, the non-small cell lung cancer comprises squamous cell lung carcinoma, adenocarcinoma lung carcinoma, large cell carcinoma and carcinoid, preferably, the non-small cell lung cancer is adenocarcinoma lung carcinoma;

preferably, the reagents include probes, primers and/or protein binding agents specific for PPM 1G;

preferably, the product comprises a chip, a kit, a test strip or a high-throughput sequencing platform.

10. A method of screening a candidate compound for treatment of lung cancer, said method comprising:

(1) administering a test compound to a subject to be tested in a test group, and detecting the expression level of PPM1G in a sample derived from the subject in the test group, V1; in a control group, a blank control is applied to a subject to be tested, and the expression level V2 of PPM1G in a sample derived from the subject in the control group is detected;

(2) comparing the expression level V1 and the expression level V2 detected in the previous step, thereby determining whether the test compound is a candidate compound for treating lung cancer;

preferably, the lung cancer comprises non-small cell lung cancer and small cell lung cancer, preferably, the lung cancer is non-small cell lung cancer, preferably, the non-small cell lung cancer comprises squamous cell lung cancer, adenocarcinoma lung cancer, large cell lung cancer and carcinoid, preferably, the non-small cell lung cancer is adenocarcinoma lung cancer.

Images