CN114107507A

CN114107507A - Application of ZSQIM 1 protein in regulation and control of lung adenocarcinoma cell proliferation and metastasis

Info

Publication number: CN114107507A
Application number: CN202111485057.1A
Authority: CN
Inventors: 何庆瑜; 高学娟; 张弓; 官柏烨; 连琼华; 刘朗夏
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2021-09-29
Filing date: 2021-12-07
Publication date: 2022-03-01
Anticipated expiration: 2041-12-07
Also published as: CN114107507B

Abstract

The invention belongs to the technical field of protein science, and particularly relates to application of ZSQIM 1 protein in regulation and control of lung adenocarcinoma cell proliferation and metastasis. The invention provides an application of ZSQIM 1 protein in regulation and control of cancer cell proliferation and metastasis, and the ZSQIM 1 protein can be used as a new target for regulation and control of cancer cell proliferation and metastasis; the development of more anticancer drugs is promoted by detecting the reduction of the expression level of the ZSQIM 1 protein or the dephosphorylation condition of ERK 1/2.

Description

Application of ZSQIM 1 protein in regulation and control of lung adenocarcinoma cell proliferation and metastasis

Technical Field

The invention belongs to the technical field of protein science, and particularly relates to application of ZSQIM 1 protein in regulation and control of lung adenocarcinoma cell proliferation and metastasis.

Background

The morbidity and mortality of malignant tumors such as lung cancer continue to increase, seriously threatening the health of patients. The excessive proliferation of lung cancer cells and their metastasis to distant sites such as brain, bone, lymph nodes are important causes of high mortality and poor prognosis. Despite recent advances in the diagnosis and treatment of tumors, such as non-small cell lung cancer (NSCLC), the 5-year survival rate of NSCLC patients remains low. The screening of new antitumor drugs from the phenotype of inhibiting the proliferation and metastasis of tumor cells provides a new scheme for tumor treatment.

Scientists completed the sequencing analysis of chromosome 20 in 2001, annotating 895 genes, many of which had little known function. In recent years these genes have been highlighted by tumor researchers and their regulation of tumor development has begun to be revealed, with the functions of two genes, FAM210B and GID8, published in journal "Cell Death Dis" and "Cell Res", respectively. These studies indicate that the genes with unknown functions probably play an important role in the occurrence and development of tumors, and will provide new clues for the targeted therapy of tumors.

The ZSQIM 1 gene is located on chromosome 20, and the function is almost unknown. The ZSQIM 1 has the transcription length of 2787bp, the length of the encoded protein is 485 amino acids, and only one SWIM functional domain is predicted to be contained. In 2002, researchers such as Makarova KS defined SWIM domains with the amino acid sequence of the SWIM motif: CxCxnCxH, n-5-39, contains cysteine and histidine, and researchers predict that they have a zinc finger-like structure and function by binding to DNA or protein. In 2014, researchers firstly proved the expression of the ZSQIM 1 in leukocytes, the expression level of the ZSQIM 1 is very sensitive to differentiation signals, and the expression quantity is remarkably down-regulated in the T cell differentiation process, which indicates that the ZSQIM 1 has the potential of participating in the cell life activities. However, the effect of ZSQIM 1 in tumors has not been reported at all.

Disclosure of Invention

The technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide an application of the ZSWIM1 protein in cancer cell proliferation and metastasis and as a cancer cell proliferation and metastasis regulation target.

The technical problem to be solved by the invention is realized by the following technical scheme:

the first purpose of the invention is to provide the application of the ZSQIM 1 protein in screening an agent for regulating the proliferation and the metastasis of cancer cells.

Preferably, the agent inhibits zswum 1 protein activity or down regulates zswum 1 gene expression.

Preferably, the cancer cell is a lung adenocarcinoma cell.

The invention also provides application of the ZSQIM 1 protein as a target for regulating lung cancer proliferation and metastasis.

Firstly, the invention provides application of ZSQIM 1 protein in regulating cancer cell proliferation and metastasis, wherein the ZSQIM 1 protein is increased in expression level in lung adenocarcinoma cells, participates in regulation of lung adenocarcinoma proliferation and migration invasion, increases expression level of ZSQIM 1, promotes proliferation and migration invasion of tumor cells, and inhibits proliferation and migration invasion of tumor cells by reducing expression of ZSQIM 1 protein.

In addition, the invention also provides a method for screening a cancer treatment drug, which comprises the following steps: when the drug to be detected is added into a tumor cell culture medium, the expression degree of the ZSQIM 1 gene or protein in the cell is further detected, or the phosphorylation level of ERK1/2 is jointly detected, so that the aim of drug screening is fulfilled. When the drug to be detected inhibits the expression of the ZSQIM 1 gene or protein, or simultaneously dephosphorylates ERK1/2, the drug to be detected is indicated to be an anti-cancer drug.

The sequence information of the ZSQIM 1 protein gene can refer to the website:

https://www.ncbi.nlm.nih.gov/nuccore/NM_080603.5；

compared with the prior art, the invention has the following beneficial effects:

the invention provides an application of ZSQIM 1 protein in regulation and control of cancer cell proliferation and metastasis, and the ZSQIM 1 protein can be used as a new target for regulation and control of cancer cell proliferation and metastasis; the development of more anticancer drugs is promoted by detecting the reduction of the expression level of the ZSQIM 1 protein or the dephosphorylation condition of ERK 1/2.

Drawings

FIG. 1 is a graph showing the results of TCGA data analysis;

FIG. 2 is a Kaplan Meier survival plot;

FIG. 3 is a graph of expression of ZSQIM 1 in lung adenocarcinoma cells;

FIG. 4 is a graph of the results of over-expression or knockdown of ZSQIM 1 on adenocarcinoma cell proliferation;

FIG. 5 is a graph showing the results of over-expression or knockdown of ZSQIM 1 in the migration and invasion of lung adenocarcinoma cells;

FIG. 6 is a graph of PD98059 treatment for changes in ERK1/2 phosphorylation, ZSQIM 1 expression;

fig. 7 is a graph of the effect of PD98059 in combination with treatment with over-expressing zswum 1 on cancer cell proliferation.

Detailed Description

The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the subject matter of the present invention is limited to the examples. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The lung epithelial cells HBE were purchased from ATCC, CRL-2741; the lung adenocarcinoma cells a549 were purchased from shanghai institute of sciences cell bank under catalog No.: TCHU 150; the lung adenocarcinoma cells H1299 were purchased from shanghai institute of midges cell bank under catalog No.: TCHU 160; the H1299 cells were purchased from the chinese academy of cell resources center; the PD98059 is available from MedChemexpress Biotech corporation (MCE) USA, Cat. No.: HY-12028; the siRNA of the ZSQIM 1 is purchased from Shanghai Jima pharmaceutical technology GmbH; the PVDF membrane is polyvinylidene fluoride membrane, Millipore, USA, from Guangzhou Cheng ammonium Biotech limited, Cat #: ISEQ 00010; the Transwell cell was purchased from Corning, usa; the serum and DMEM media were purchased from Gibco, usa; the zskim 1 antibody was purchased from Novus, usa under the cat number: NBP 2-13606; the Vimentin antibody was purchased from Proteintech, usa, cat #: 10366-1-AP; the E-cadherin antibody was purchased from CST (cell Signaling technology) Inc., USA, cat #: 3195T; the BETA-ACTIN antibodies were purchased from Proteitech, Inc., USA under the trade designation: 66009-1-Ig; the murine secondary antibody was purchased from Jackson Immuno Research, usa, cat #: 115-035-003; the rabbit secondary antibody was purchased from Jackson Immuno Research, usa, cat #: 111-035-003; the pCMV-N-Flag vector is purchased from Shanghai Bintian biotechnology limited; the puromycin is purchased from kulybo technologies ltd, beijing, cat #: SL 4130; the pTSB02-GFP-PURO and pTSB 02-ZSQIM 1-Flag-GFP plasmids were purchased from Shanghai Yangyang Biotech, Inc.; the 293T cells were purchased from Shanghai national academy of sciences cell bank; the ZSQIM 1 lentivirus plasmid was purchased from Shanghai Yangyang Biotech Limited;

the EBC lysate formula is as follows: 1mM PI, 1mM PMSF, 1mM NaF, 1mM Na₃VO₄。

Example 1 expression of ZSFIM 1 in Lung adenocarcinoma cells and Lung epithelial cells

1.1 download analysis 57 pairs of lung adenocarcinoma and paracancerous tissue mRNA sequencing results and 10 additional lung adenocarcinoma tissue mRNA sequencing results (download site references: https:// portal. gdc. cancer. gov/subjects/TCGA-LUAD), the gene expression data of which were quantified by FPKM of "FPKM. txt. gz" file type, the expression results are shown in Table 1 below.

TABLE 1 expression of ZSQIM 1 in lung adenocarcinoma and paracarcinoma tissues

The experimental results are shown in fig. 1, p <0.0001, Student's t-test, and it can be seen that the zwim 1 protein has a significant high expression trend in paired lung adenocarcinoma tissues; as shown in FIG. 2, Kaplan Meier survival curve analysis showed that the survival of lung adenocarcinoma patients with high expression of ZSQIM 1 was significantly shortened, indicating that ZSQIM 1 is negatively correlated with the overall survival of the patients. Accordingly, it is speculated that the protein may be involved in the development of lung adenocarcinoma.

1.2 detecting the expression of the ZSQIM 1 protein at the cellular level, and the specific steps are as follows:

western Blot experiment

a. Normal lung epithelial cells HBE, ZSQIM 1 on lung adenocarcinoma cells A549 and H1299, washing residual culture medium with 1XPBS, directly adding EBC lysate on the plate, lysing the cells on ice for 30min, and shaking the six-well plate for several times every 10min to make EBC lysate fully contact with the cells.

b. The cell lysate was transferred to a low speed centrifuge and centrifuged at 12000rpm at 4 ℃ for 30 min.

c. The supernatant was taken and transferred to a new EP tube, and the bottom pellet was discarded.

Measuring the protein concentration by using a BCA method;

the adding amount of each component in the protein concentration measured by the BCA method is shown in the following table:

TABLE 2 BCA assay for protein concentration component addition Table

Protein standard substance/. mu.L	0	1	2	4	8	12	16	20
									EBC lysate/. mu.L	20	19	18	16	12	8	4	0
BCA working solution/. mu.L	200	200	200	200	200	200	200	200

Wherein the concentration of the protein standard substance is 0.5 mg/mL; A. the ratio of the B liquid is 50: 1.

e. Preparing a sample: corresponding volumes of samples were taken according to concentration and mixed with 5 x SDS loading buffer and heated in a boiling water bath for 10min to fully denature the protein.

f. Preparing PAGE (polyacrylamide gel electrophoresis) glue, loading a sample into glue holes of the PAGE glue, running the PAGE glue for 30min at a voltage of 80V, and regulating the voltage to 100V for running the PAGE glue for 1h after a Marker is separated.

g. Film transfer: cutting 5.5 × 8.0cm PVDF membrane with aperture of 0.22 μm, and activating with 100% anhydrous methanol for 3 min; cutting corners on the left side after taking out the PAGE gel, marking the PAGE gel in sequence, then attaching the PVDF films together on the PAGE gel according to the condition that the gel blocks are arranged below the PAGE gel, and scraping bubbles generated in the contact surface by using a scraper; the gel block and the membrane are placed in a clamping groove, the membrane is rotated for 90min under the voltage of 100V, and the membrane rotating time can be properly adjusted according to the molecular size of the needed protein.

h. And (3) sealing: after the membrane conversion is completed, the PVDF membrane is put into 5% milk and sealed for 2 hours.

i. Incubating a primary antibody; cutting the corresponding strip according to the size of the needed protein molecule, and incubating at 4 ℃ and 20rpm for one-time overnight; the primary antibody includes ZSFIM 1 antibody, Vimentin antibody, E-cadherin antibody and BETA-ACTIN antibody.

j. The primary antibody was recovered and the membrane was washed 3 times with 1 × TBST for 5min each time.

k. Hatching a secondary antibody; incubating the homologous source secondary antibody for 2h according to the corresponding species source of the primary antibody; the secondary antibody comprises a mouse secondary antibody and a rabbit secondary antibody.

l, washing the membrane: the secondary antibody is recovered, and the membrane is washed 3 times by 1 × TBST, wherein the time duration of each time is 10 min.

m. developing: ECL luminous liquid A, B liquid ratio is 1:1, and the light-proof configuration is adopted.

The experimental result is shown in fig. 3, compared with the normal lung epithelial cells HBE and ZSWIM1, the expression level of both lung adenocarcinoma cells a549 and H1299 is obviously high, and the two lung adenocarcinoma cells are known to have increasing metastatic capacity, which suggests that ZSWIM1 may be related to the malignant potential of lung adenocarcinoma metastasis and the like.

Example 2 results of over-expression or knock-down of ZSQIM 1 protein to promote proliferation of lung adenocarcinoma cells

The regulation capacity of the ZWIM 1 protein to the proliferation level of lung adenocarcinoma H1299 cells is tested, and the specific test steps are as follows:

2.1 clone formation experiment:

a. inserting cDNA of ZSQ 1 into pCMV-N-Flag vector to construct FALG-ZSQ 1 plasmid, H1299 cell transiently transfected with FALG-ZSQ 1 plasmid and its control cell (FLAG-V), or H1299 cell stably knocked down ZSQ 1 (KD-ZSQ 1) and its control cell (KD-V), pancreatin digested cell, centrifuging at 2000g for 3min, discarding supernatant and re-suspending the cells with 1mL of culture medium, diluting 100 μ L of cell suspension by 50 times, and counting 10 μ L of diluted suspension in a blood count plate.

b. Laying 1500 cells in a six-hole plate, and adding 2mL of culture medium into each hole of the six-hole plate; PBS was added between wells to maintain a moist environment, 5% CO at 37 ℃₂The culture was carried out for 12 days.

c. The growth of the cells was observed daily during the culture period, and the cells were harvested when they grew to macroscopic clonal spots.

d. And (5) collecting the cloning plate. The medium was discarded from the plate, the cells were rinsed with 1XPBS and then fixed for 18min with a 4% paraformaldehyde solution. And recovering 4% paraformaldehyde, adding 0.1% crystal violet for dyeing for 10min, washing off excessive crystal violet with ultrapure water, drying at room temperature, and imaging with a 2D imager.

The H1299 cells and control cells (KD-V) with stably knockdown ZSQIM 1 (KD-ZSQIM 1) were constructed as follows: transfecting 293T cells with pLKO.1-shZWIM 1-EGFP and pLKO.1-EGFP plasmids for 72h, and collecting a supernatant culture medium to a 50mL centrifuge tube; centrifuging the collected supernatant culture medium at 3500rpm at room temperature for 10min, pouring the supernatant into a new centrifuge tube, and filtering the supernatant with 0.45 μm filter membrane for use in infecting tumor cells.

The lentivirus infected and screened cells:

1) h1299 cells were plated in 6-well plates overnight at a cell density of 75%;

2) and removing the supernatant. 2mL of fresh virus solution (supernatant from the 0.45 μm filter) was added to each well;

3) replacing with fresh culture medium after 24 h;

4) after 72h, cells were cultured in fresh medium containing 2. mu.g/mL puromycin;

5) after the cells were confluent, passaging was performed to 24-well plates and cells were maintained in fresh medium containing 2. mu.g/mL puromycin;

6) after the cells were confluent, the cells were digested, counted, plated in 96-well plates to control only 1 cell per well, and maintained in culture with fresh medium containing 2. mu.g/mL puromycin;

7) after the cells are full, the cells are subcultured into a 24-well plate, and a fresh culture medium containing 2 mug/mL puromycin is used for continuous culture until the cells are transferred into a 6-well plate for culture;

8) the next day, the supernatant was removed and replaced with fresh medium containing 5. mu.g/mL puromycin;

9) the expression of EGFP protein in the cells is observed by an inverted fluorescence microscope and the cells are collected for WB detection of the protein expression level.

2.2 MTT assay

a. Inserting cDNA of ZSQ 1 into pCMV-N-Flag vector to construct FALG-ZSQ 1 plasmid, transiently transfecting H1299 cell and its control cell (FLAG-V) of FALG-ZSQ 1 plasmid, or stably knocking down H1299 cell and its control cell (KD-ZSQ 1) of ZSQ 1 (KD-ZSQ 1), using pancreatin digestion cell, counting by blood counting plate.

b. 3000 cells were plated in 96-well plates, 3 replicates per set, and PBS was added to the empty wells around the samples to maintain a moist environment.

c.24h later, the 96-well plate is taken out, the culture medium is discarded, and the 10 percent MTT solution is replaced with 5 percent CO at 37 DEG C₂Incubating for 4h, and changing to 150 μ after 4hL (formazan) solution in DMSO (formazan dissolved in cells) is treated for 10min in a dark place at 20rpm, and then the absorbance value of OD490/OD570 is read.

d. Measurements were taken every 1 day interval for at least 3 time points.

The experimental results are shown in FIG. 4, and as shown in FIGS. 4A-C, the clonogenic capacity of the cells was significantly increased in the H1299 cells transiently overexpressing ZSQIM 1; as shown in fig. 4D, MTT assay showed that ZSWIM1 promoted the proliferative capacity of lung adenocarcinoma cells; in contrast, as shown in fig. 4E-G, in H1299 cells transiently knocked-down or stably knocked-down zskim 1, the cells were significantly reduced in clonogenic capacity; MTT assay as shown in figure 4H showed that knockdown zswum 1 inhibited proliferation of lung adenocarcinoma cells. Therefore, over-expression of the ZSQIM 1 protein can promote the proliferation of lung adenocarcinoma cells, and the knock-down of the ZSQIM 1 protein can inhibit the proliferation of lung adenocarcinoma cells.

Example 3 results of over-expression or knockdown of ZSQIM 1 to promote migration and invasion of lung adenocarcinoma cells

The effect of SWIM1 on the metastatic capacity of lung adenocarcinoma cells was tested by the following specific experimental steps:

3.1 migration invasion assay

a. H1299 cells stably expressing ZSQ 1 (OE-FALG-ZSQ 1) and their control cells (OE-V) or H1299 cells and control cells (KD-V) stably knocked down ZSQ 1 (KD-ZSQ 1), digested with pancreatin (containing 2.5% EDTA), centrifuged, serum-free medium resuspended cells and counted with a blood count plate.

The construction of the H1299 cells stably expressing ZSQ 1 (OE-FALG-ZSQ 1) and their control cells (OE-V) was as follows: pTSB02-GFP-PURO and pTSB 02-ZSQIM 1-Flag-GFP plasmid were transfected into 293T cells, and after transfection for 72h, the supernatant medium was collected into a 50mL centrifuge tube; centrifuging the collected supernatant culture medium at 3500rpm at room temperature for 10min, pouring the supernatant into a new centrifuge tube, and filtering the supernatant with 0.45 μm filter membrane for use in infecting tumor cells.

b. Placing the chambers into a 24-well plate, paving 60000 cells in the chambers of the migration group, paving 120000 cells in the chambers of the invasion group after adding 100 mu L of matrigel, and arranging two multiple holes in the migration and invasion groups; then gently along the outer wall of the chamberAdd 650. mu.L of 10% FBS medium to the bottom of the chamber, notice that no air bubbles were formed, and place the 24-well plate at 37 ℃ with 5% CO₂Culturing for 12 h.

c. A collecting chamber. The cell is taken out, washed by 1xPBS gently once, soaked in 4% polyformaldehyde for fixing for 20min after washing, 4% paraformaldehyde (toxic) is recovered after 20min, the cell is transferred to 0.1% crystal violet for dyeing for 15min, and finally the cell is washed by 1 XPPBS, dried and photographed for storage.

The experimental results are shown in FIG. 5, and as shown in FIGS. 5A-B, in H1299 cells stably expressing ZSQ 1 (OE-FLAG-ZSQ 1), the results of transwell experiments show that over-expression of ZSQ 1 significantly promotes cell migration and invasion compared with the control group; in contrast, as shown in FIGS. 5C-D, in H1299 cells stably knockdown ZSQIM 1 (KD-ZSQIM 1), the results of transwell experiments showed a significant decrease in both migration and invasion capacity of the cells. Therefore, over-expression of ZSFIM 1 promotes migration and invasion of lung adenocarcinoma cells, and knock-down of ZSFIM 1 inhibits migration and invasion of lung adenocarcinoma cells. The expression amount of ZSQIM 1 in lung adenocarcinoma can be used as an index for evaluating the influence of drugs on the migration and invasion of lung adenocarcinoma cells.

Example 4 Effect of PD98059 treatment on ERK1/2 phosphorylation and on ZWIM 1 expression

Experiments were carried out on the effect of ZWIM 1 on the phosphorylation of lung adenocarcinoma cells ERK1/2, and the specific experimental steps were as follows:

4.1Western Blot experiment

a. In H1299 cells stably expressing ZSQ 1 (OE-FALG-ZSQ 1) and control cells (OE-V), experiments were directly performed, or PD98059 was added for 8 hours, the residual medium was washed out with 1XPBS, EBC lysate was added directly to the plates to lyse the cells on ice for 30min, and the six-well plates were shaken several times every 10min or so to allow the EBC lysate to sufficiently contact the cells.

Protein concentration was measured by bca method.

e. And (5) preparing a sample. Corresponding volumes of samples were taken according to concentration and mixed with 5 x SDS loading buffer and heated in a boiling water bath for 10min to fully denature the protein.

g. And (5) transferring the film. A5.5X 8.0cm PVDF membrane (polyvinylidene fluoride membrane, Millipore, USA, available from Guangzhou Cheng ammonium Biotech Co., Ltd.: ISEQ00010) was cut into a pore size of 0.22 μm and activated with 100% anhydrous methanol for 3 min. The PAGE gel was removed and the left side was corner-cut to mark the order, and then the PVDF films were attached together with the gel pieces down and the contact surface was scraped with a spatula to remove the bubbles that were generated. The gel block and the membrane are placed in a clamping groove, the membrane is rotated for 90min under the voltage of 100V, and the membrane rotating time can be properly adjusted according to the molecular size of the needed protein.

h. And (5) sealing. After the membrane conversion is completed, the PVDF membrane is put into 5% milk and sealed for 2 hours.

i. Hatched primary antibody (FLAG primary antibody: MBL, Inc., Cat: M185-3S; ZWIM 1 antibody: Novus, USA, Cat: NBP 2-13606; Phospho-p44/42MAPK (Erk1/2) antibody: Cell Signaling Technology, Cat: 9101, and GAPDH antibody: Proteitech, Cat: 10494-1-AP, USA). The corresponding band was cut according to the size of the desired protein molecule, and incubated overnight at 4 ℃ at 20rpm for the primary antibody.

k. A secondary hatching antibody (a secondary murine antibody, Jackson Immuno Research, USA, Cat: 115-. And (4) incubating the secondary antibody of the same source for 2h according to the corresponding species source of the primary antibody.

l, washing the membrane. The secondary antibody is recovered, and the membrane is washed 3 times by 1 × TBST, wherein the time duration of each time is 10 min.

And m, developing. ECL luminous liquid A, B liquid ratio is 1:1, and the light-proof configuration is adopted.

The experimental results are shown in FIG. 6, and the over-expression of ZSQIM 1 in H1299 cells stably expressing ZSQIM 1 (OE-FLAG-ZSQIM 1) significantly promoted the phosphorylation of ERK1/2 compared to the control group as shown in FIG. 6A; in contrast, phosphorylation of ERK1/2 was significantly reduced in H1299 cells stably knockdown with ZSQIM 1 (KD-ZSQIM 1), as shown in FIG. 6B; as shown in FIG. 6C, when the inhibitor PD98059 of ERK1/2 was added to the cells, both groups of cells (OE-V and OE-FLAG-ZWIM 1) showed a significant decrease in phosphorylation of ERK1/2, and expression of ZWIM 1 increased first and then decreased. Thus, PD98059 treatment was able to produce an inhibitory effect on ERK1/2 phosphorylation and on ZSQIM 1 expression.

Example 5 Effect of PD98059 in combination with over-expression of ZSQIM 1 treatment on cancer cell proliferation

The effect of PD98059 in combination with treatment with over-expressed zsvim 1 on cancer cell proliferation was tested as follows:

5.1 MTT assay, the same procedure as for MTT assay in example 2

a. In H1299 cells stably expressing ZSQIM 1(OE-Z) and control cells (OE-V), PD98059 (20. mu.g/mL) was added for 10 hours, cells were trypsinized, and plates were counted.

c.24h later, the 96-well plate is taken out, the culture medium is discarded, and the 10 percent MTT solution is replaced with 5 percent CO at 37 DEG C₂After incubation for 4h, the cells were changed to 150. mu.L of DMSO solution (to dissolve formazan in the cells) and then treated with light at 20rpm for 10min before the absorbance value of OD490/OD570 was read.

d. Measurements were made every 1 day interval, 3 time points were examined.

As shown in FIG. 7, in H1299 cells stably expressing ZSQ 1 (OE-ZSQ 1) and control cells (OE-V), PD98059 (20. mu.g/mL) was added to each cell for treatment, and MTT was used to detect changes in cell activity; as shown in FIG. 7, the activity of the cells was significantly down-regulated after the control group was added with PD98059, whereas the activity of the cells was not down-regulated in the presence of the over-expressed ZSQIM 1 protein. Therefore, under the condition of higher expression level of ZSQIM 1, the effect of PD98059 on inhibiting tumor growth is inhibited.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

Use of the ZSWIM1 protein in screening for agents that modulate cancer cell proliferation and metastasis.
2. The use of claim 1, wherein the agent inhibits zsvim 1 protein activity or down regulates zsvim 1 gene expression.
3. The use of claim 1, wherein the cancer cell is a lung adenocarcinoma cell.
The application of the ZSQIM 1 protein as a target for regulating the proliferation and metastasis of lung cancer.