CN114592006A - New application of MEMO1 gene - Google Patents
New application of MEMO1 gene Download PDFInfo
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- CN114592006A CN114592006A CN202210462276.6A CN202210462276A CN114592006A CN 114592006 A CN114592006 A CN 114592006A CN 202210462276 A CN202210462276 A CN 202210462276A CN 114592006 A CN114592006 A CN 114592006A
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Abstract
The invention discloses a new application of MEMO1 gene, namely an application of MEMO1 gene high expression in screening drugs for treating non-small cell lung cancer, the invention utilizes CRISPR/cas9 technology to knock out MEMO1 in non-small cell lung cancer A549 cells, and experimental detection finds that the knock-out MEMO1 in A549 can obviously influence the expression of a PI3K/Akt signal channel and enhance the cell proliferation capacity, and simultaneously has promotion effect on migration, invasion and dryness of cells, can reduce the adhesion capacity of the cells, can improve the sensitivity of the cells to cis-platinum, 5-FU and etoposide, can increase the expression of fn11 in the cells after transcription, and the change of Slfn11 protein amount is negatively regulated and controlled by an inhibitor ZSTK474 of PI3K, and the invention is favorable for better treating lung cancer and improving the life quality of lung cancer patients clinically.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a new application of a drug for treating non-small cell lung cancer, which is screened by taking high expression of MEMO1 gene as a target.
Background
Lung cancer belongs to the most mortality and morbidity of the world, and is classified into Small Cell Lung Cancer (SCLC) and Non-Small cell lung cancer (NSCLC). Even though the treatment methods are various, the molecular targeted therapy has unique advantages in various treatment methods, plays an important role in the treatment scheme of the lung cancer and has great significance. However, the survival rate of 5 years after the lung cancer is diagnosed is 15.6%, which is lower than that of the prostate cancer, the breast cancer or the colon cancer, and the clinical treatment effect of the patient with the non-small cell lung cancer is not obviously improved. Therefore, it is crucial to find better clinical treatment for non-small cell lung cancer and understand the mechanism of action thereof.
The MEMO1 protein is a cell movement medium 1 driven by ErbB2(Human epidermal factor receptor 2), and is called hepatitis C virus NS5A transactivator 7 or C21orf 19-like protein, which is called MEMO1 for short. The MEMO1 gene contains 15 exons totally, and is located on homo sapiens chromosome 2 at the position 31865060bp-32011230bp of 2p 22.3. The MEMO1 protein is composed of 297 amino acids, and the three-dimensional structure of the MEMO1 protein has been determined so far. There are studies that indicate that increased levels of MEMO1 protein expression in breast and colon cancer patients are associated with poor prognosis in the patients and reduce patient survival. The over-expression of the MEMO1 protein promotes the migration, proliferation and invasion of cancer cells, participates in the metastasis of the cancer cells, inhibits the apoptosis and regulates the malignant behavior of the cancer cells. The mechanism research of the MEMO1 gene in breast cancer is relatively deep, but the detailed effect and the mechanism of action of the MEMO1 gene on non-small cell lung cancer are not correspondingly elucidated in relevant documents at present.
Disclosure of Invention
The invention provides a new application of MEMO1 Gene, namely an application of screening a medicament for treating non-small cell lung cancer with the aim of high expression of MEMO1 Gene, wherein the nucleotide sequence of the MEMO1 Gene is shown in Gene ID 51072 in NCBI.
In order to achieve the purpose, the invention provides the following technical scheme:
1. construction of MEMO1 gene knockout plasmid PX459-KO-MEMO1
CHOPCHOP is selected on ZHANG LAB (https:// zlab. bio/guide-design-resources) website, and the optimal pair of sgRNAs is selected for synthesis after comprehensive consideration in multiple aspects in all the sgRNAs;
phosphorylating and annealing the sgRNA by using a PCR technology, then connecting the sgRNA to a PX459 plasmid, and hydrolyzing linear DNA to obtain a PCR product, namely the PX459-KO-MEMO1 plasmid connected with the sgRNA; transforming, selecting single colony, extracting plasmid, detecting plasmid concentration and purity by agarose gel electrophoresis and ultraviolet spectrophotometer, cutting off the gel block corresponding to the target fragment, and sequencing by biological company; the sequence of the sequencing result of the biological company is compared with the sequence of the sgRNA, and the sgRNA on the PX459-KO-MEMO1 plasmid of the sequencing result is completely consistent with the sequence of the sgRNA, namely the PX459-KO-MEMO1 knock-out plasmid is successfully constructed.
2. Construction of A549-KO-MEMO1 knockout cell line
Stably transfecting PX459-KO-MEMO1 plasmid in A549 cells, and screening an A549 stable cell line with a MEMO1 gene knocked out by utilizing puromycin; extracting total protein in cells by 1 xSDS lysate (added with beta mercaptoethanol), and verifying whether a MEMO1 gene knockout cell line in A549 is successfully constructed by using a protein immunoblotting (Western blotting) experiment;
entering a CDS region of the gene of MEMO1 found by NCBI, selecting a proper base sequence as an amplification primer, performing blast on the primer, and then sending the primer to a biological company for synthesis; extracting a whole genome of a suspected knockout successful cell, performing agarose gel electrophoresis on an obtained PCR product by using a PCR technology, cutting a gel block corresponding to a target fragment, sending the gel block to a biological company for sequencing, and verifying whether a knockout MEMO1 gene cell line is successfully constructed in A549 at the mRNA level;
comparing the sequence of the knockout cell line in the sequencing result with the sequence of a wild cell line by DNAMAN analysis software, finding that a great amount of base mutation occurs in the sequence of the knockout cell line, and successfully knocking out the translated MEMO1 gene due to the change; namely, the A459-KO-MEMO1(AKM) knockout cell line is successfully constructed.
3. Detecting the effect of MEMO1 gene on KEGG signal channel enrichment
Extracting RNA from wild A549 cells and knockout AKM cells, and respectively detecting the concentration and the purity and the integrity of an RNA sample by using agarose gel electrophoresis and Agilent 2100; performing an experiment on the RNA sample qualified for detection to obtain mRNA, and then separating and randomly interrupting the mRNA; synthesizing a first strand and a second strand of the cDNA by using the first strand and the second strand as templates, and then carrying out double-strand purification on the cDNA; then, carrying out end repair on the purified double chains, adding an A base at the 3' end of the fragment and connecting a specific sequencing adaptor to obtain a cDNA library; then, performing quality evaluation on the transcriptome sequencing library, and performing on-machine sequencing on a sample meeting the conditions by using high-throughput sequencing Illumina HiSeq; filtering an original sequencing sequence, and aligning clean reads obtained by filtering to a reference genome sequence by using Hisat 2;
based on the selected reference genomic sequence, transcript reconstruction was performed on the Mapped Reads using StringTie software, and new transcripts and new genes of the species were discovered after analysis; based on the comparison result, the new gene and the current known gene are subjected to quantitative analysis, expression analysis of differential genes is performed on different gene expression levels in different sample groups, and then KEGG signal path enrichment analysis of the differential expressed genes is performed, so that the influence of the MEMO1 gene on the KEGG signal path enrichment is detected.
4. Detection of influence of MEMO1 gene on proliferation capacity of non-small cell lung cancer
Wild type A549 cells and knockout type AKM cells are inoculated into a 96-well plate, PI3K inhibitor (PI 3KI: ZSTK 474) is added into all the wells after the cells are attached to the wall for treatment, the difference value between the OD630 value and the OD450 value of the cells for four consecutive days is collected for experiment and the proliferation capacity of the cells is analyzed, and the influence of the MEMO1 gene on the proliferation capacity of the non-small cell lung cancer is detected by detecting the OD value of the cells.
Inoculating wild type A549 cells and knockout type AKM cells into a 12-hole plate, adding 500 cells per hole, uniformly mixing with 1mL of complete culture medium (high-glucose DMEM culture medium containing 10% fetal bovine serum and 1% streptomycin), and then putting into an incubator for culturing; observing the growth state of the cells every three days, and changing the liquid; when each clone contains about 50 cells, fixing and staining the cells; the influence of the MEMO1 gene on the proliferation capacity of the non-small cell lung cancer is detected by detecting the cell clone formation rate.
5. Detecting the influence of MEMO1 gene on migration capability of non-small cell lung cancer
Starvation treatment is carried out on wild type A549 cells and knockout type AKM cells for 6 hours; 100 μ L of a resuspension containing 10000 cells was added to the upper chamber of a Transwell chamber containing serum-free medium; add 550. mu.L of complete medium along the inner wall of the 24-well plate, bringing the Transwell chamber into complete contact with the complete medium level; after 36 hours, taking out the Transwell chamber, gently removing the serum-free culture medium in the chamber, and fixing and staining the cells; randomly taking 3-5 visual fields under a microscope, and photographing cells outside the chamber;
paving the scratch plug-in into a 6-hole plate one day in advance, and normally culturing wild A549 cells and knockout AKM cells; adding 200 mu L of a resuspension solution containing 30000 cells into each cell of the insert, scratching the insert in a 6-well plate, adding 1mL of complete culture medium outside the insert, and culturing the cells overnight; taking out the 6-hole plate, slightly pulling out the scratch plug-in by using a sterilization forceps, sucking out the complete culture medium, washing off a small amount of suspended cells by using phosphate buffer solution, adding the complete culture medium containing 20 mu g/mL mitomycin C, continuously culturing for 2 hours, and then changing the culture solution by using the complete culture medium; taking a picture of scratches under a microscope and recording the picture as 0 hour; photographs were taken in the same manner at 24-hour and 48-hour time points, respectively.
6. Detecting influence of MEMO1 gene on non-small cell lung cancer invasion capacity
Starvation treatment is carried out on wild type A549 cells and knockout type AKM cells for 6 hours; preparing a serum-free culture medium and matrigel in advance according to the volume ratio of 1:3, and placing the mixture in a refrigerator at 4 ℃; adding 50 μ L of diluted matrigel into the upper chamber of the chamber, placing in an incubator at 37 ℃ for 2 hours to wait for gelation, and then hydrating the basement membrane; adding 100 μ L of a suspension containing 10000 cells into a chamber, and using a serum-free culture medium in the chamber; add 550. mu.L of complete medium along the inner wall of the 24-well plate, bringing the Transwell chamber into complete contact with the complete medium level; after 36 hours, taking out the Transwell chamber, slightly removing the complete culture medium in the chamber, and fixing and staining the cells; the outside of the chamber was photographed by taking 3-5 fields at random under the microscope.
7. Detecting influence of MEMO1 gene on dryness of non-small cell lung cancer
Resuspending wild type A549 cells and knockout AKM cell precipitates by using special DMEM/F12 culture medium for balling, and adding 20ng/mL EGF, 10ng/mL bFGF and 2% B27 supplement into DMEM/F12 culture medium; adding 5000 cells into each hole of a 12-hole plate, setting 3 multiple holes for each cell, uniformly mixing, and putting into a cell culture box for normal culture; observing the cell balling condition, supplementing liquid, finishing the culture when the cell ball diameter is more than 50 μm, photographing and counting the number of the suspension cell balls with the ball diameter more than 50 μm.
8. Detection of influence of MEMO1 gene on adhesion capability of non-small cell lung cancer
Performing a cell adhesion experiment by using wild type A549 cells and knockout AKM cells; coating a 96-well plate with 10 mu g/mL bovine fibrinogen, 10 mu g/mL rat tail collagen I and polylysine respectively for 30 minutes at 37 ℃ according to experimental requirements, polymerizing the matrigel into gel, and then hydrating a basement membrane; adding 100 mu L of cell suspension containing 10000 cells into each hole of a 96-hole plate, and putting the cell suspension into an incubator for 2 hours; removing cells with weak adherence, adding 100 mu L of complete culture medium into each hole, and then adding 10 mu L of CCK-8 reagent; after incubation for 1 hour in the incubator, absorbance values were measured using a microplate reader.
9. Detection of influence of MEMO1 gene on drug sensitivity of non-small cell lung cancer
Carrying out chemotherapy drug sensitivity experiments on wild A549 cells and knockout AKM cells; uniformly inoculating the counted cells into a 96-well plate according to a system containing 5000 cells in 100 mu L of complete culture medium per well, wherein at least 3 parallel multiple wells exist in each experimental group, and putting the 96-well plate paved with the cells into a cell culture box for normal culture;
after the cells are completely attached to the wall after being paved for about 24 hours, taking out the 96-hole plate from the cell culture box to absorb the complete culture medium, and sequentially adding 100 mu L of complete culture medium containing the chemotherapeutic drugs with corresponding concentrations; the nonspecific medicine acting on the cell cycle is cisplatin inhibiting cell DNA synthesis, mainly acts on the S phase of the cell cycle, inhibits 5-fluorouracil synthesizing cell DNA, generates doxorubicin inhibiting topoisomerase II, and inhibits etoposide for DNA repair by inhibiting topoisomerase II; the drug treatment time of 5-fluorouracil and cisplatin is 36 hours; the drug treatment time for doxorubicin was 48 hours; the drug treatment time of etoposide is 24 hours; after the drug treatment time is up, adding 10 mu L of CCK-8 reagent into each hole and continuously culturing for 1 hour; OD450 and OD630 values were determined using a microplate reader.
10. Detecting the influence of the MEMO1 gene on the PI3K/Akt signal channel
Extracting total protein of wild A549 cells and knockout AKM cells by using a strong RIPA lysate (containing 1% protease inhibitor), and carrying out quantitative analysis; western blotting experiments are carried out by using primary antibodies of pAkt, Akt and Gapdh, the expression conditions of pAkt and Akt on the level of the cell protein are verified, and the influence of the MEMO1 gene on a PI3K/Akt signal channel is detected.
11. Detecting the influence of the MEMO1 gene on Slfn11
Treating wild A549 cells and knockout AKM cells with ZSTK474, extracting total protein and carrying out quantitative analysis; carrying out Western blotting experiments by using primary antibodies of Slfn11 and Gapdh proteins, verifying the expression condition of the Slfn11 protein on the level of a cell protein, and detecting the influence of the MEMO1 gene on Slfn 11;
mRNA in a wild A549 cell and a knockout AKM cell is extracted, the mRNA is subjected to reverse transcription by PCR to form cDNA, the cDNA is used as a qRT-PCR template to perform qRT-PCR technology, a qRT-PCR specific primer of an Slfn11 gene is designed, the expression condition of the Slfn11 gene in the mRNA level of the cell is verified, Gapdh is used as an internal reference to calculate and analyze an experiment result, and the influence of the MEMO1 gene on Slfn11 is detected.
12. Detecting the influence of the MEMO1 gene on the IRS4 signal pathway
Extracting total protein of wild A549 cells and knockout AKM cells, carrying out Western blotting experiment by using primary antibodies of IRS4, Tublin, ISR and Gapdh, verifying the expression conditions of IRS4, Tublin, ISR and Gapdh on the level of cellular protein, and detecting the influence of MEMO1 gene on IRS4 signal channel.
The invention has the advantages and technical effects that:
the experimental result shows that the knockout of the MEMO1 gene in A549 can generate obvious influence on the expression of a PI3K/Akt signal pathway, can promote the proliferation capacity of single cells and cell totality, has promotion effect on migration, invasion and dryness of cells, can reduce the adhesion capacity of cells, can improve the sensitivity of cells to cis-platinum, 5-FU and etoposide, can increase the expression of Slfn11 in cells after transcription and translation, and the change of the Slfn11 protein amount can be negatively regulated and controlled by an inhibitor ZSTK474 of PI3K, can increase the expression of an insulin receptor (ISR) in cells, and can increase the expression of an insulin receptor substrate 4(IRS 4); namely, the MEMO1 protein can inhibit the expression of SLfn11 in A549 cells after transcription and translation, and then activate a PI3K/AKT pathway, so that the occurrence and development processes of lung cancer are influenced in various aspects of cell proliferation, migration, invasion, adhesion, dryness, drug sensitivity and the like, as shown in figure 1.
The experimental result shows that the reagent for promoting the expression of the MEMO1 gene can be used for preparing the medicine for treating the non-small cell lung cancer, and the invention provides a new way for treating the non-small cell lung cancer medicine.
Drawings
FIG. 1 is a schematic diagram of the signal pathway of the MEMO1 gene triggering PI3K/AKT in the present invention;
FIG. 2 shows the expression status of MEMO1 protein in wild type A549 cells and knockout AKM cells tested by Western blotting;
FIG. 3 is a schematic diagram showing the result of detecting the expression of the MEMO1 gene amplification product in the whole genomes of wild type A549 cells and knockout AKM cells by agarose gel;
FIG. 4 shows the sequencing results of the amplified products of MEMO1 gene in the whole genomes of wild type A549 cells and knockout AKM cells;
FIG. 5 is a schematic diagram showing the effect of detecting the MEMO1 gene on the enrichment of KEGG signal channel in the present invention;
FIG. 6 shows the results of CCK-8 assay for the proliferation of wild-type A549 cells and knockout AKM cells;
FIG. 7 shows the results of CCK-8 assay to examine the proliferation of wild-type A549 cells and knockout AKM cells treated with different ZSTK474 concentrations;
FIG. 8 is a graph (bottom) and a graph (top) showing the proliferation of wild-type A549 cells and knockout AKM cells tested in a colony formation assay;
FIG. 9 shows the results of examining the influence of MEMO1 gene on the migration ability of non-small cell lung cancer, wherein A is a graph showing the migration of wild-type A549 cells and knockout AKM cells tested by the Transwell migration test (left) and a quantitative graph (right); the B picture is a drawing (left) and a quantitative drawing (right) for testing the migration condition of wild type A549 cells and knockout AKM cells by using a scratch test;
FIG. 10 shows the results of examining the influence of MEMO1 gene on the invasive ability of NSCLC, wherein the left middle panel is the photographed result and the right panel is the statistical result;
FIG. 11 shows the results of examining the influence of the MEMO1 gene on the sternness of non-small cell lung cancer; wherein: the A picture is a graph (in the left middle picture) and a quantification picture (in the right picture) of the balling capacity of A549 and AKM which are tested by using a balling experiment; the B picture is a picture (left picture) and a quantitative picture (right picture) for testing the expression condition of the CD44 protein in wild A549 cells and knockout AKM cells by Western blotting;
FIG. 12 is a graph showing the effect of the MEMO1 gene on the ability of non-small cell lung cancer to adhere; wherein the left figure uses polylysine as matrix gum, the middle figure uses bovine fibrinogen as matrix gum, and the right figure uses rat tail collagen I as matrix gum;
FIG. 13 is a graph showing the results of drug susceptibility assays for the survival of wild-type A549 cells and knockout AKM cells treated with cisplatin at different concentrations (Panel A) and their respective IC' s50FIG. (B view);
FIG. 14 is a graph showing the survival of wild type A549 cells and knockout AKM cells treated with 5-fluorouracil at different concentrations (graph A) and their respective IC's tested by drug susceptibility assay50FIG. (B view);
FIG. 15 is a graph showing the results of drug susceptibility assays for the survival of cells treated with doxorubicin at different concentrations in wild-type A549 cells and knockout AKM cells (Panel A) and their respective IC' s50FIG. (B view);
FIG. 16 is a graph showing the survival of wild type A549 cells and knockout AKM cells treated with etoposide at different concentrations (graph A) and their respective IC' s50FIG. (B view);
FIG. 17 is a diagram showing the experimental results for detecting the effect of MEMO1 gene on PI3K/Akt signaling pathway;
FIG. 18 is a schematic diagram showing the results of an experiment for detecting the effect of the MEMO1 gene on Slfn 11; wherein A is the expression condition of Slfn11 protein in wild type A549 cells and knockout AKM cells detected by Western blotting; b, Western blotting is utilized to detect the expression conditions of Slfn11 proteins in wild type A549 cells and knockout type AKM cells after the wild type A549 cells and the knockout type AKM cells are respectively treated by PI3KI and MEKI; c is a quantization diagram of the A diagram; d is a quantization diagram of the B diagram;
FIG. 19 is a graph showing the mRNA level expression of the Slfn11 gene in wild-type A549 cells and knockout AKM cells tested by qRT-PCR assay;
FIG. 20 is a schematic diagram showing the experimental results for detecting the effect of MEMO1 gene on IRS4 signaling pathway; wherein A is the protein level expression condition of IRS4 in wild type A549 cells and knockout AKM cells detected by Western blotting; and B, Western blotting is used for testing the protein level expression conditions of ISR in wild type A549 cells and knockout type AKM cells.
Detailed Description
The present invention is further described in detail with reference to the following drawings and examples, but the scope of the present invention is not limited to the above description, and reagents and methods used in the examples, unless otherwise specified, are conventional reagents and conventional methods;
example 1: construction of MEMO1 gene knockout plasmid PX459-KO-MEMO1
1. Design and Synthesis of sgRNA
Searching ZHANG LAB (https:// zlab. bio/GUIDE-DESIGN-resources) on the network to enter the website, selecting CHOPCHOP by TOOLS FOR GUIDE DESIGN, filling out a Target gene MEMO1 with the category of Homo sapiens (hg38/GRCH38), using CRISPR/Cas9 to perform knock-out, then clicking Find Target Sites to enter the next step, and selecting a pair of sgRNAs (MEMO 1-sgRNAs-F: 5' -caccgTGC) in all the sgRNAs according to the comprehensive consideration of the internal ranking, efficiency, the number of located exons, GC content, self-complementation, miss rate and the like
ATTCAGCTGCGGTCCTG-3’，MEMO1-sgRNA-R:5’-aaacCAGGACCGCAGC
TGAATGCAc-3'), sent to the organism company for synthesis.
2. sgRNA phosphorylation and annealing
Preparing a phosphorylation and annealing reaction system according to the table 1; transferring the PCR tube with the reaction system into a PCR instrument, and using the temperature of 37 ℃ for 30 minutes; phosphorylating and annealing sgrnas at 95 ℃ for 5 min; then cooling to 25 ℃ at the speed of 5 ℃/min;
table 1: sgRNA phosphorylation and annealing reaction system
3. sgRNA was ligated to plasmids
Diluting phosphorylated sgRNA in a volume ratio of the product of step 2: rnase-free water =1:199, shaking up after dilution; preparing a reaction system for connecting sgRNA and plasmid according to the table 2, transferring a PCR tube filled with the connection system into a PCR instrument, and performing reaction for 5 minutes at 37 ℃; carrying out six times of circulating reactions at 21 ℃ for 5 minutes;
TABLE 2 sgRNA and plasmid ligation System
Then hydrolyzing the linear DNA which is not successfully connected, wherein the hydrolysis system is shown in the table 3, and transferring the PCR tube filled with the reaction system into a PCR instrument for 30 minutes at 37 ℃; PCR was carried out at 70 ℃ for 30 minutes; the obtained PCR product is PX459-KO-MEMO1 plasmid which is successfully connected with sgRNA in PX459 plasmid;
TABLE 3 hydrolysis Linear DNA System
4. Px459-KO-MEMO1 plasmid transformation competenceE.Coli
Taking out from a low-temperature refrigerator at-80 deg.CE.ColiPlacing on ice for later use; taking 2 mu L and 50 mu L of hydrolysate in the step 3 for competenceE.ColiAs for the EP tube; flicking the bottom of the EP tube to mix the hydrolysate with competence, and standing on ice for 30 minutes; heat shock in 42 ℃ water bath for 90 seconds; standing on ice for 5 minutes; adding 800 μ L LB liquid medium, shake culturing for 50 minutes (250r/min, 37 ℃); taking the bacterial liquid EP tube from the shaking table, centrifuging (6000r/min, 3 min), sucking and discarding 700 mu L of supernatant on a sterile operating platform, and blowing and shaking the remaining 100 mu L of supernatant and thalli evenly; uniformly coating the bacterial liquid on the surface of an Amp-LB solid culture medium; after inoculation is completed and the surface of the plate is dried, inverting the Amp-LB solid culture medium and culturing for 13.5 hours in an incubator at 37 ℃; meanwhile, the bacterial liquid transformed into PX459 plasmid is used as a reference;
in a clean bench, adding ampicillin (Amp) with the concentration of 100mg/mL into LB liquid culture medium according to the volume ratio of 1:1000 to obtain Amp-LB liquid culture medium; selecting full, single and non-overlapping colonies, picking single colony to Amp-LB liquid culture medium by 10 μ L of gun head (picking 3 colonies containing PX459 plasmid and 3 colonies containing PX459-KO-MEMO1 plasmid respectively); culturing in shaking table at constant temperature of 37 deg.C at 250r/min for 5 hr; taking 600 mu L of bacterial liquid respectively and sending to biological sequencing; and discarding the bacterial liquid which is not successfully connected according to the sequencing result, and selecting the bacterial liquid which is successfully connected for carrying out the next experiment.
5. Extraction of PX459-KO-MEMO1 plasmid
Preparing 7.5 muL Amp (100mg/mL) +7.5 muL PX459-KO-MEMO1 plasmid bacterial liquid +7.5mL LB liquid culture medium in a super clean bench, uniformly mixing in a 50mL centrifuge tube, and preparing bacterial liquid containing PX459 plasmid by the same operation; slightly unscrewing the cover of the centrifugal tube, transferring the centrifugal tube into a constant-temperature shaking table at 37 ℃, and culturing for 14.5 hours at a speed of 250 r/min; extracting plasmids according to the specification of the small and medium endotoxin-free plasmid extraction kit in Biyun, detecting the extracted products by agarose gel electrophoresis, and sending the gel blocks corresponding to the target fragments to a biological company for sequencing; sequencing results show that sgRNA on PX459-KO-MEMO1 plasmid is completely consistent with sgRNA sequence, namely PX459-KO-MEMO1 plasmid construction is successful.
Example 2: construction of A549-KO-MEMO1 knockout cell line
1. Stable transfection of PX459-KO-MEMO1 plasmid in A549 cells
The complete medium for culturing A549 cells was changed to antibiotic-free medium (high-glucose DMEM medium containing 10% fetal bovine serum) six days in advance; a549 cells at a cell density of about 60% were plated in six-well plates; after the cells adhere to the wall for 24 hours, removing the culture medium, cleaning the cells by using phosphate buffer solution, then removing the phosphate buffer solution, and adding 1.5mL of optim culture medium into each hole to starve the A549 cells for 1 hour;
adding 200 mu L of optim culture medium and 2.0 mu L of lipo2000 transfection reagent into a No. 1 EP tube, uniformly mixing, and standing for 5 minutes; adding 200 μ L of optim culture medium and 1.0 μ g of PX459-KO-MEMO1 plasmid into No. 2 EP tube, mixing, and standing for 5 min; uniformly mixing the solutions in the two EP tubes to obtain a solution 1; placing the solution 1 for 20 minutes, and directly adding the solution into the A549 cells subjected to starvation treatment to obtain transfected cells; placing the transfected cells into an incubator for culturing for 5.5 hours, then changing the incubator into an antibiotic-free culture medium, and continuing culturing;
the next day after transfection, the transfected cells were digested and expanded to 10cm dishes for further culture.
2. Screening of A549 cell line with MEMO1 gene knockout function
On the third day after transfection, the antibiotic-free culture medium is replaced by a puromycin complete culture medium containing 0.8 mug/mL puromycin to carry out screening of positive clone cells; replacing a puromycin complete culture medium containing 0.8 mu g/mL puromycin once every five days of culture, and carrying out the liquid replacement 4 times to obtain a monoclonal cell mass by the transfected cells; picking the monoclonal cell masses into a 24-well plate by using a 10-mu-L pipette and culturing by using a complete culture medium; changing the liquid every 4 days, culturing the transfected cells for 20 days, and performing expansion culture on the transfected cells in a 12-hole plate for continuous culture; when the density of the transfected cells is estimated to be 80%, digesting the transfected cells, and taking A549 cells as control group cells;
3. protein level verification of whether a knockout MEMO1 gene cell line in A549 is successfully constructed
Extracting proteins of the transfected cells and A549 cells, and putting cell precipitates on ice at 4 ℃; adding 50 mu L of 1 xSDS lysate (added with beta mercaptoethanol) into each tube, cracking for 30 minutes on ice, and shaking once on a vortex shaking instrument every 10 minutes; boiling the sample in a metal bath at the temperature of 98 ℃ for 7 minutes, and storing the sample at the temperature of-20 ℃ for later use after the sample is cooled;
cleaning a glass plate for glue making, drying, performing leak detection by using sterilized water, and completely sucking filter paper for later use; preparing SDS polyacrylamide separation gel with the concentration of 10 percent and SDS polyacrylamide concentrated gel with the concentration of 5 percent; sampling the extracted protein, and then carrying out 80V for 35 minutes; electrophoresis at 120V for 80 min; pre-cooling the prepared membrane transferring liquid at 4 ℃, transferring the membrane by adopting a sandwich-wet transferring method, transferring the membrane for 1.5h at 290mA, and sealing the membrane for 1 h at normal temperature by using 5% of skimmed milk; carrying out shaking table incubation on the prepared primary antibody internally participating in the MEMO1 protein and the PVDF membrane at 4 ℃ overnight, rinsing the PVDF membrane for 7 minutes by using a 1 xTBST solution, and repeating for 3 times; adding a secondary antibody, incubating for 1 hour at room temperature, rinsing the PVDF membrane for 8 minutes by using a 1 xTBST solution, and repeating for 3 times; developing in a chemiluminescence apparatus;
the results are shown in FIG. 2, where M2 was suspected to be successful in knock-out.
4. mRNA level verification whether the MEMO1 gene knockout cell line in A549 is successfully constructed
Collecting A549 cells and precipitates of the transfected cells M2 in the step 3 to extract the whole genome of the cells, wherein the extraction step is according to a method in an instruction book of an animal genome DNA extraction kit of the Ongjingke company;
searching a CDS region of the MEMO1 gene in NCBI, designing an amplification primer (MEMO 1-Verify-F: 5'-ATGTCCAACCGAGTGGTCTGCCGAG-3', MEMO1-Verify-R: 5'-CGAAGGTCATACAGAGGTGTCCTAT-3') according to the position of the sgRNA sequence, carrying out blast and then sending the primer to a biological company for synthesis;
preparing an RCR reaction system according to the table 4, and carrying out target fragment amplification according to the PCR conditions of the table 5; the amplified PCR product was subjected to agarose gel electrophoresis, and the results of electrophoresis are shown in FIG. 3; and cutting off the gel block corresponding to the size of the target fragment, and sending the gel block to a biological company for sequencing. Comparing the sequence of the suspected knockout cell line with the sequence of the wild-type cell line in the sequencing result, and finding that a great amount of base mutations occur in the sequence of the knockout cell line, and the changes result in successful knockout of the translated MEMO1 gene, namely successful construction of an A459-KO-MEMO1 knockout cell line (AKM);
TABLE 4 RCR systems
TABLE 5 PCR amplification conditions
Example 3: detecting the effect of the MEMO1 gene on the enrichment of KEGG signal pathways
Extracting RNA of wild A549 cells and knockout AKM cells, and respectively carrying out concentration detection and purity and integrity detection on the RNA sample by using agarose gel electrophoresis and Agilent 2100; performing an experiment on the RNA sample qualified for detection to obtain mRNA, and then separating and randomly interrupting the mRNA; synthesizing a first strand and a second strand of the cDNA by using the first strand and the second strand as templates, and then carrying out double-strand purification on the cDNA; then, carrying out end repair on the purified double chains, adding an A base at the 3' end of the fragment and connecting a specific sequencing adaptor to obtain a cDNA library; performing quality evaluation on the transcriptome sequencing library, and performing on-machine sequencing on a sample meeting the conditions by using high-throughput sequencing Illumina HiSeq; filtering the original sequencing sequence, and comparing the filtered clean reads to a reference genome sequence (finished by Beijing Optimalaceae Biotechnology Co., Ltd.) by using Hisat 2;
based on the selected reference genome sequence, using StringTie software to reconstruct the transcript of the Mapped Reads, comparing the transcript with the original genome annotation information, searching the original unannotated transcription region, and discovering a new transcript and a new gene of the species so as to supplement and perfect the original genome annotation information; by filtering out sequences that encode peptide chains that are too short (less than 50 amino acid residues) or that contain only a single exon, 2482 new genes were discovered in total, of which 836 genes were differentially expressed, 284 genes were upregulated, and 552 genes were downregulated.
Based on the comparison result, carrying out quantitative analysis on the obtained new gene and the currently known gene, carrying out statistical analysis on the expression quantity of different genes in different sample groups and expression analysis on the different genes, then carrying out KEGG signal channel enrichment analysis on the previously screened different expressed genes, and further detecting the influence of the MEMO1 gene on the KEGG signal channel enrichment;
the results are shown in FIG. 5, where most of the signaling pathways are enriched by upregulated genes. Compared with the signal paths with more significant differences, the signal paths mainly concentrate the PI3K/Akt signal path, and the deletion of the MEMO1 gene has significant influence on the expression of the PI3K/Akt signal path; the pathway is related to the deletion of MEMO1 protein in non-small cell lung cancer, the high expression of Slfn11 and the increase of cell proliferation, migration, invasion, dryness and drug sensitivity in the occurrence and development process of the non-small cell lung cancer.
Example 4: effect of MEMO1 Gene on proliferative Capacity of non-Small cell Lung cancer
1. CCK-8 experiment: inoculating wild type A549 cells and knockout type AKM cells into a 96-well plate according to 2500 cells per well, wherein each group comprises 3 multiple wells, shaking, uniformly mixing, and lightly putting into an incubator; after 24 hours of plating, the cells were treated with different concentrations of ZSTK474 (concentration 0, 0.1, 1, 10 μ M), 10 μ L of CCK-8 reagent at a concentration of 10mg/mL was added to the wells on the first day, and the incubator was left for 1.5 hours; measuring OD450 value and OD630 value by using a microplate reader, wherein the measured values are the measurement results of the first day; thereafter, CCK-8 reagent was added to the other wells at the same time every day for measurement in the same manner as the first day for a total of 4 days;
the results are shown in fig. 6 and 7, and it can be seen from the figure that knocking out the MEMO1 gene in a549 can increase the proliferation capability of cells, but ZSTK474 can reverse the proliferation of a549 and AKM, i.e., deletion of MEMO1 in non-small cell lung cancer can affect the PI3K/Akt signaling pathway, thereby affecting the proliferation capability of cells in the development process of non-small cell lung cancer.
2. Cell clone formation experiment: inoculating wild type A549 cells and knockout type AKM cells into a 12-hole plate according to 500 cells per hole, adding 1mL of complete culture medium, uniformly mixing, and then putting into an incubator for culturing; observing the growth state of the cells every three days and changing the liquid; stopping cell culture when the number of the cells of the single cell clone block mass is more than 50, absorbing and discarding a complete culture medium, and washing the cells by using phosphate buffer saline solution; removing phosphate buffer solution by suction, and fixing cells for 30 minutes by using paraformaldehyde with the concentration of 4%; after the fixation is finished, removing paraformaldehyde by suction, washing cells by using phosphate buffer solution, and adding 0.5% crystal violet dye solution for dyeing for 15 minutes; after the crystal violet dye solution is absorbed and discarded, the 12-hole plate is washed for 5 times by phosphate buffer salt solution;
the result is shown in fig. 8, it can be seen from the figure that the proliferation capacity of the AKM cell is stronger than that of the a549 cell, and the knockout of the MEMO1 gene can promote the improvement of the proliferation capacity of the a549 cell, wherein the involved mechanism may also be the activation of the PI3K/Akt signaling pathway caused by the deletion of MEMO1 in the non-small cell lung cancer, thereby affecting the proliferation capacity of the cell in the development process of the non-small cell lung cancer.
Example 5: effect of MEMO1 Gene on migration Capacity of non-Small cell Lung cancer
1. Performing Transwell migration experiment on the wild A549 cell and the knockout AKM cell, and performing starvation treatment on the wild A549 cell and the knockout AKM cell 6 hours in advance; placing the Transwell chamber into a 24-well plate, adding 100. mu.L of a resuspension (serum-free medium) containing 10000 cells to the upper chamber of the Transwell chamber, and adding 550. mu.L of a normal medium along the inner wall of the 24-well plate to bring the Transwell chamber into complete contact with the surface of the medium; placing the treated 24-hole plate in an incubator to be cultured for 36 hours, taking out the Transwell chamber, removing a serum-free culture medium in the chamber, and washing cells by using phosphate buffer saline solution; removing phosphate buffer solution by suction, and fixing cells for 30 minutes by using paraformaldehyde with the concentration of 4%; after the fixation is finished, removing paraformaldehyde by suction, washing cells by using phosphate buffer solution, and adding 0.5% crystal violet dye solution for dyeing for 15 minutes; after absorbing and discarding the crystal violet dye solution, washing the small chamber with clear water until the liquid flowing through the small chamber is colorless, and drying the small chamber at room temperature; erasing cells in the upper chamber, randomly taking 3-5 visual fields, and taking pictures of cells outside the small chamber;
as shown in fig. 9A, it can be seen that the number of cells migrating to the lower chamber of AKM is much greater than that of a549 cells, and the migration ability of a549 cells is inhibited by the MEMO1 gene.
2. Performing a scratch experiment on the wild A549 cell and the knockout AKM cell, and putting a scratch plug-in into a 6-hole plate one day before the scratch experiment is performed; normally culturing a wild type A549 cell and a knockout type AKM cell; add 200 μ L of complete medium containing 30000 cells to each cell of the scratch insert; adding 1mL of complete culture medium outside the scratch plug-in of the 6-hole plate, and culturing the cells overnight; gently pulling out the scratch plug-in by using a sterilization forceps, sucking away the complete culture medium, and washing away a small amount of suspended cells by using phosphate buffer solution; adding a complete culture medium containing 20 mug/mL of mitomycin C for continuous culture for 2 hours, and then changing the culture solution by using the complete culture medium; the scratch was photographed under a microscope and recorded as 0 hour, after which the same method was used for photographing at 24 hour and 48 hour time points, respectively;
the results are shown in fig. 9B, and it can be seen from the figure that the knockout of the MEMO1 gene in a549 can increase the healing percentage and show stronger migration ability, and the results are also consistent with the results of the traswell migration experiment, that is, the knockout of the MEMO1 gene in a549 can promote the cell migration ability, and the MEMO1 gene can inhibit the migration ability of a549 cells.
Example 6: effect of MEMO1 Gene on non-Small cell Lung cancer invasiveness
Carrying out a Transwell invasion experiment by using a wild type A549 cell and a knockout type AKM cell, wherein the wild type A549 cell and the knockout type AKM cell are subjected to starvation treatment 6 hours in advance; preparing a serum-free culture medium and matrigel according to the proportion of 1:3, adding 50 mu L of diluted matrigel into the upper chamber of the small chamber, and placing the small chamber in an incubator at 37 ℃ for 2 hours; then 20. mu.L of phosphate buffered saline was added to the upper chamber of the chamber and left in an incubator at 37 ℃ for 30 minutes; placing the Transwell chamber treated above into a 24-well plate, adding 100. mu.L of serum-free medium containing 10000 cells into the upper chamber of the Transwell chamber, and adding 550. mu.L of complete medium into the 24-well plate; after the cells are cultured for 36 hours, removing the serum-free culture medium in the Transwell chamber, and washing the cells by using phosphate buffer saline solution; removing phosphate buffer solution by suction, and fixing cells for 30 minutes by using paraformaldehyde with the concentration of 4%; after the fixation is finished, removing paraformaldehyde by suction, washing cells by using phosphate buffer solution, and adding 0.5% crystal violet dye solution for dyeing for 15 minutes; after absorbing and discarding the crystal violet dye solution, washing the small chamber with clear water until the liquid flowing through the small chamber is colorless, and drying the small chamber at room temperature; erasing cells in the upper chamber, randomly taking 3-5 visual fields, and taking pictures of cells outside the small chamber;
as shown in fig. 10, it can be seen that the number of cells of AKM is much greater than that of a549, that is, the knockout of the MEMO1 gene in a549 promotes the invasive ability of the cells, and the MEMO1 gene inhibits the invasive ability of the a549 cells.
Example 7: detecting influence of MEMO1 gene on dryness of non-small cell lung cancer
Carrying out cell balling experiments by using wild type A549 cells and knockout AKM cells, wherein a culture medium used in the balling experiments is a special balling DMEM/F12 culture medium, and 20ng/mL of EGF, 10ng/mL of bFGF and 2% of B27 supplement are added into a DMEM/F12 culture medium; inoculating wild type A549 cells and knockout type AKM cells into a 12-hole plate, wherein 5000 cells are arranged in each hole, and each cell is provided with 3 multiple holes, and putting the cells into a cell culture box for normal culture after uniformly mixing; gently shaking the 12-well plate every day, observing cells every 3 days, and supplementing 50 mu L of the culture medium; photographing after 14 days of cell culture and counting the number of the suspension cell balls with the ball diameter larger than 50 μm, wherein the result is shown in FIG. 11A;
western blotting experiment: total protein extraction was performed on a549 cells and AKM cells cultured in complete medium, and expression of CD44 protein was detected by Western blotting, with the results shown in fig. 11B;
as can be seen in fig. 11, the number of spheres or the diameter of the spheres of the AKM cells was significantly larger than that of the control group; the CD44 protein is considered as a cell surface marker of stem cells, the expression level of the CD44 protein in AKM cells is far higher than that of A549, in conclusion, the knockout of the MEMO1 gene in A549 increases the lung cancer cell population with typical cancer stem cell-like phenotype, and the MEMO1 gene inhibits the drying capacity of A549 cells.
Example 7: effect of MEMO1 Gene on adhesion Capacity of non-Small cell Lung cancer
Performing a cell adhesion experiment by using wild type A549 cells and knockout AKM cells, and respectively coating a 96-well plate with 10 mu g/mL bovine fibrinogen, 10 mu g/mL rat tail collagen I and polylysine for 30 minutes at 37 ℃; then 50. mu.L of phosphate buffered saline solution was added to each well, and left in an incubator at 37 ℃ for 30 minutes, and the excess phosphate buffered saline solution was aspirated away; adding 100 μ L of cell suspension containing 10000 cells into each well of a 96-well plate, and culturing at 37 ℃ for 2 hours; the complete culture medium is sucked away, the surface of the cells is gently cleaned by phosphate buffer saline solution, and the cells with weak adherence are removed; adding 100 mu L of complete culture medium into each well, and then adding 10 mu L of CCK-8 reagent; after incubation for 1 hour in an incubator, determining an OD value by using an enzyme-labeling instrument;
as shown in fig. 12, it can be seen that the number and effect of adherent cells of AKM are significantly weaker than those of a549 cells, the adhesion ability of the cells is reduced after knockout of the MEMO1 gene in a549, and the MEMO1 gene promotes the adhesion ability of a549 cells.
Example 8: detection of influence of MEMO1 gene on drug sensitivity of non-small cell lung cancer
Wild type A549 cells and knockout type AKM cells are adopted to carry out a chemotherapy drug sensitivity experiment, the wild type A549 cells and the knockout type AKM cells are inoculated into a 96-well plate according to 5000 cells per well and 100 mu L of complete culture medium, and each group comprises 3 parallel multiple wells; after the cells are completely attached to the wall in about 24 hours, the complete culture medium is removed by suction, and different chemotherapeutic drugs with different treatment times and different concentrations are sequentially added; the cisplatin drug concentration is 0, 1, 10, 50 and 100 mu M, and the treatment time is 36 hours; the drug concentration of 5-fluorouracil is 0, 1, 10, 100, 1000 mug/mL, the treatment time is 36 hours; the drug concentration of doxorubicin is 0, 100, 500, 1000, 2000nM, and the treatment time is 48 hours; the drug concentration of the etoposide is 0, 1, 10, 50 and 100 mu M, and the treatment time is 24 hours; after the drug treatment time is up, adding 10 mu L of CCK-8 reagent into each hole and continuously culturing for 1 hour; measuring an OD450 value and an OD630 value by using a microplate reader;
the results are shown in fig. 13, 14, 15 and 16, and it can be seen from the graphs that compared with a549, AKM has increased drug sensitivity, increased cell death rate and decreased survival rate under the treatment of 5-fluorouracil, cisplatin and etoposide, and has significant difference, especially etoposide, but doxorubicin does not show obvious difference between the two cells after the treatment of the two cells. Just IC50In the above, the knockout of the MEMO1 gene led to the IC of cisplatin and 5-fluorouracil50Values were reduced to 30. mu.M and 1. mu.g/mL, respectively, whereas for etoposide, IC was50The difference between the two drugs is particularly remarkable, namely the sensitivity of the A549 cells to the cisplatin, the etoposide and the 5-fluorouracil is in negative correlation with the expression quantity of the MEMO1 protein, and the three drugs are closely related to DNA, in conclusion, the knockout of the MEMO1 gene in the A549 can increase the sensitivity of the cells to the cisplatin, the etoposide and the 5-fluorouracil, and the MEMO1 gene inhibits the sensitivity of the A549 cells to the cisplatin, the etoposide and the 5-fluorouracil.
Example 9: influence of MEMO1 gene on PI3K/Akt signal pathway
Extracting total proteins of wild A549 cells and knockout AKM cells, spreading the wild A549 cells and the knockout AKM cells into a six-well plate one day in advance, culturing until the cell density is 80%, removing the complete culture medium, and adding 120 mu L of strong RIPA lysate (containing 1% protease inhibitor) into each well; performing cell lysis on ice for 30min, and placing the six-hole plate on an oscillator for shaking for 1min every 10 min; collecting the cell lysate sample in an EP tube, and centrifuging for 15 minutes at the temperature of 4 ℃ and at the speed of 15000 r/min; placing the centrifuged sample on ice, and taking the supernatant into a new EP tube which is pre-cooled and marked in advance;
protein quantification by pipetting standard solutions (0.5. mu.g/. mu.L) into 96-well plates and subsequently diluting the solutions in a concentration gradient (0, 0.5, 1, 2, 4, 6, 8, 10. mu.g/. mu.L) such that the final volume of solution per well is 20. mu.L and the volume deficiency is filled with phosphate buffer; adding 2 mu L of cell lysate sample and 18 mu L of phosphate buffer solution into each well of a 96-well plate in a one-to-one correspondence manner; adding 200 μ L BCA working solution (BCA working solution is prepared from solution A and solution B according to the ratio of 50: 1) into all the wells, and culturing 96-well plate in 37 deg.C incubator for 30 min; measuring an OD562 value by using a microplate reader, and calculating the mass of the protein of the sample; then 5 × loadin accounting for the total volume 1/5 is added; mixing the samples, instantly separating, boiling in a metal bath at 98 ℃ for 7 min, and storing at-20 ℃ after the samples are naturally cooled for later use;
carrying out Western blotting experiments by using primary antibodies of pAkt, Akt and Gapdh, verifying the expression conditions of pAkt and Akt on the level of the cell protein, and detecting the influence of the MEMO1 gene on a PI3K/Akt signal channel;
the result is shown in fig. 17, and it can be seen from the figure that knocking out the MEMO1 gene in a549 can reduce the expression of Akt in cells, and increase the phosphorylation level of Akt, that is, deletion of MEMO1 gene can cause activation of PI3K/Akt signaling pathway, thereby affecting the development process of non-small cell lung cancer.
Example 10: effect of MEMO1 Gene on Slfn11 protein
Wild-type A549 cells and knockout-type AKM cells are treated by ZSTK474 to extract total protein, protein quantitative analysis is carried out (the protein extraction and quantitative method is the same as that in example 9), Western blotting experiments are carried out by using primary antibodies of Slfn11 and Gapdh proteins to verify the expression state of the Slfn11 protein on the cell protein level, and the influence of the MEMO1 gene on the Slfn11 protein is detected, wherein the result is shown in figure 18;
mRNA in a wild A549 cell and a knockout AKM cell is extracted, cDNA is obtained by reverse transcription through a PCR technology, the cDNA is taken as a qRT-PCR template to carry out qRT-PCR technology to verify the expression state of the Slfn11 gene in the mRNA level of the cell, a qRT-PCR primer of Slfn11 is Slfn11-qPCR-F: 5'-GAAACGCTAAGGGGGCTTCG-3',
5'-ACGGGTAGAAACGCAACTCC-3' of Slfn11-qPCR-R, and Gapdh is taken as an internal reference to calculate and analyze the experimental result, and the influence of the MEMO1 gene on Slfn11 is detected, and the experimental result is shown in FIG. 19;
it can be seen from the figure that the Slfn11 protein is low expressed in A549, the knockout of MEMO1 gene in A549 can reverse the low expression of Slfn11 protein in A549 and has nothing to do with the transcription level of Slfn11 gene, namely MEMO1 gene influences the expression translated after Slfn11 is transcribed, and the change condition of the protein can be negatively regulated by TK ZS474 which is an inhibitor of PI3K, namely the deletion of MEMO gene only influences the expression of Slfn11 protein at the protein level, and PI3K/Akt signal channel is involved in the process to jointly influence the cancer process.
Example 11: effect of the MEMO1 Gene on the IRS4 Signal pathway
Extracting total proteins of wild A549 cells and knockout AKM cells, carrying out protein quantitative analysis (the protein extraction and quantitative method is the same as the embodiment 9), carrying out Western blotting experiment by using primary antibodies of IRS4, Tublin, ISR and Gapdh, verifying the expression conditions of IRS4, Tublin, ISR and Gapdh on the level of cell protein, and detecting the influence of MEMO1 genes on an IRS4 signal path;
the result is shown in fig. 20, and it can be seen from the figure that knocking out the MEMO1 gene in a549 can increase the expression of insulin receptor in cells and simultaneously increase the expression of insulin receptor substrate 4, that is, MEMO1 gene also affects IRS4 signaling pathway, and the involvement in non-small cell lung cancer is not a simple pathway, but a plurality of pathways jointly regulate the whole process.
According to the invention, MEMO1 gene is knocked out in a non-small cell lung cancer A549 cell by using a CRISPR/cas9 technology, bioinformatics analysis and experimental detection show that the MEMO1 gene knocked out in A549 can obviously influence the expression of a PI3K/Akt signal channel, can promote the proliferation capacity of a single cell and the cell as a whole, has promotion effects on migration, invasion and dryness of the cell, can reduce the adhesion capacity of the cell, can improve the sensitivity of the cell to cis-platinum, 5-fluorouracil and etoposide, can increase the expression of Slfn11 in the cell after transcription, and the change of Slfn11 protein can be negatively regulated and controlled by an inhibitor ZSTK474 of PI3K, can increase the expression of an insulin receptor in the cell, and can increase the expression of an insulin receptor substrate 4. Namely, the MEMO1 protein can inhibit the expression of SLfn11 in A549 cells after transcription and translation, and then activate a PI3K/AKT channel, so that the occurrence and development processes of lung cancer are influenced in various aspects of cell proliferation, migration, invasion, adhesion, dryness, drug sensitivity and the like.
The research contents show that the reagent for promoting the expression of the MEMO1 gene can be used for preparing the medicine for treating the non-small cell lung cancer, and also show that the reagent for inhibiting the expression of the Slfn11 gene can be used for preparing the medicine for treating the non-small cell lung cancer.
Sequence listing
<110> university of Kunming science
New application of <120> MEMO1 gene
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Claims (1)
1. The application of the medicine for treating the non-small cell lung cancer is screened by taking the high expression of the MEMO1 gene as the aim.
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