KR102190910B1 - Screening method for lung cancer treatment agent using miR-195 or miR-497 - Google Patents

Abstract

It relates to a composition for inhibiting SMURF2 activity of lung cancer cells using miR-195 or miR-497, and a method for screening a lung cancer therapeutic agent. Using a composition comprising miR-195 or miR-497 according to an aspect can effectively inhibit SMURF2 activity of lung cancer cells and enhance TGF-β signaling in vitro. In addition, if the activity of miR-195 or miR-497 is utilized, lung cancer treatments, especially early lung cancer treatments without metastasis, can be effectively found, which can be usefully used in related pharmaceutical and medical businesses.

Description

Screening method for lung cancer treatment agent using miR-195 or miR-497}

It relates to a composition for inhibiting SMURF2 activity of lung cancer cells using miR-195 or miR-497, and a method for screening a lung cancer therapeutic agent.

The role of TGF-β signaling appears differently depending on the stage of cancer progression. In early cancer cells, signaling activity acts as a cancer inhibitor that prevents cell division and induces death, but at the end stage, it functions as a cancer-inducing factor that promotes cell growth and metastasis. Therefore, accurately understanding the activity of TGF-β signaling is of great help in staged cancer treatment. TGF-β signaling begins with the activation of the receptor by binding of a ligand to the TGF-β receptor (TGF-β receptor I and TGF-β receptor II). The ligand-bound receptor phosphorylates the regulatory SMAD (regulatory SMAD; SMAD2 and SMAD3) protein, and when the phosphorylated regulatory SMAD protein binds to the signal transducer SMAD4 to form a complex, it enters the nucleus of the cell. The complex then binds to several transcriptional regulators and helps the expression of target genes for TGF-β signaling. SMURF2 ubiquitinase is known to attach to a complex consisting of a ligand-TGF-β receptor with SMAD7 inhibitory SMAD (SMAD) protein, causing ubiquitination of the receptor and degrading the receptor. In addition, it has been found that other factors involved in TGF-β signaling, such as SMAD1, SMAD2, and SMAD3, also cause proteolysis by ubiquitination.

MicroRNAs (miRNAs) are small non-coding RNA molecules that bind to the complementary sequence of target RNA and regulate gene expression. Through this, it is known to participate in various biological processes of cells such as cell growth, differentiation, and death.

Therefore, there is a need for research on miRNAs that exhibit anticancer activity by regulating cell proliferation through the regulation of TGF-signaling of cancer cells among miRNAs.

One aspect relates to a composition for inhibiting SMURF2 activity of lung cancer cells in vitro, including miR-195 or miR-497.

Another aspect relates to a method of inhibiting SMURF2 activity in lung cancer cells by incubating miR-195 or miR-497 in lung cancer cells in vitro.

Adding a test substance to lung cancer cells in which the expression of miR-195 or miR-497 is reduced compared to the negative control; Measuring the level of SMURF2 activity or TGF-β activity of the lung cancer cells; And selecting a test substance that inhibits SMURF2 activity or promotes TGF-β activity compared to a control group not treated with the test substance.

Another aspect is in vitro ( in vitro ), the step of contacting the lung cancer cells with miR-195 or miR-497 to inhibit SMURF2 activity or promote TGF-β activity of lung cancer cells; About.

One aspect provides a composition for inhibiting SMURF2 activity of lung cancer cells in vitro , including miR-195 or miR-497.

Hereinafter, the present invention will be described in more detail.

In the present specification, "miRNA (microRNA, miRNA)" refers to RNA that is a small non-coding RNA molecule that binds to a complementary sequence of a target RNA and controls the expression of a gene. The miRNA refers to an untranslated RNA of approximately 22 nt, which acts as a post-transcriptional repressor through base-linking with the 3'untranslated region (UTR) of the mRNA. The method for producing miRNA is not particularly limited, and a method known in the art may be used.

The present inventors found that miR-195 or miR-497 among miRNAs inhibited the expression of SMURF2 ubiquitinase, which is used in typical TGF-β signaling processes in lung cancer cells, and inhibited the ubiquitination of TGF-β receptor I by SMURF2. Therefore, it is possible to effectively inhibit SMURF2 activity of lung cancer cells in vitro by confirming through lung cancer cells that it promotes TGF-β signaling activity by preventing and stabilizing protein degradation of TGF-β receptor I. Was confirmed.

In addition, the composition inhibits the activity of the SMURF2 ubiquitinase, thereby preventing and stabilizing the TGF-β receptor I protein degradation to promote TGF-β signaling activity. Therefore, TGF-β in lung cancer cells in vitro It can promote activity.

The SMURF2 gene (Homo sapiens SMAD specific E3 ubiquitin protein ligase 2) can be identified as NCBI (National Center for Biotechnology Information) Gene Accession number NM_022739.

In addition to the active ingredients disclosed in the present specification, functional additives and components included in the composition may be additionally included, and further include conventional auxiliary agents and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, etc. can do. Other ingredients included include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation It may include accelerators, cooling agents, limiting agents, and purified water.

Another aspect provides a method of inhibiting SMURF2 activity in lung cancer cells by incubating miR-195 or miR-497 in lung cancer cells in vitro .

Since the miR-195 or miR-497 can inhibit the expression of SMURF2 ubiquitinase, which is used in typical TGF-β signaling processes in lung cancer cells, and inhibit the ubiquitination of TGF-β receptor I by SMURF2, lung cancer By incubating cells with miR-195 or miR-497, SMURF2 activity can be effectively inhibited in vitro .

In addition, as described above, the use of the method according to one aspect inhibits the activity of the SMURF2 ubiquitinase, thereby preventing and stabilizing the TGF-β receptor I protein degradation to promote TGF-β signaling activity. In vitro), it may be possible to promote TGF-β activity in lung cancer cells.

In addition, the promotion of TGF-β activity according to the above method may be due to a decrease in ubiquitination of the TGF-β receptor.

Adding a test substance to lung cancer cells in which the expression of miR-195 or miR-497 is reduced compared to the negative control; Measuring the level of SMURF2 activity or TGF-β activity of the lung cancer cells; And it provides a screening method for a lung cancer treatment comprising the step of selecting a test substance that inhibits the activity of SMURF2 or promotes TGF-β activity compared to the control group not treated with the test substance.

The lung cancer refers to a malignant tumor that grows rapidly while infiltrating the surrounding tissues occurring in the lungs, spreads or metastases to each part of the body, and threatens life. Specifically, the lung cancer may be primary lung cancer. The primary lung cancer is cancer that originates in the lungs, excluding metastatic lung cancer, and specifically refers to early cancer in the present specification, before metastasis occurs, i.e., 1) an increase in cellular activity due to EMT 2) invasion into blood vessels 3) Migration to secondary tissues 4) Reversal processes in secondary tissues, that is, mesenchymal-to-epithelial transition (MET), 5) processes such as settlement and growth in secondary tissues This means cancer that has not yet appeared. The primary lung cancer is largely divided into peya-type and closed-gate type at the site on the chest X-ray, and histopathologically, squamous cell carcinoma, adenocarcinoma, large cell undifferentiated cancer, and small cell undifferentiated cancer. It is known to be difficult to detect early. It is a cancer that develops a lot in men after middle age, and coughs and bleeding are the main symptoms.

The step of measuring the activity or TGF-β activity level of the test substance may be measured from a biological sample isolated from a normal person or a lung cancer patient, and the biological sample is 1 selected from the group consisting of blood, plasma, serum, urine, and saliva. It may be more than a species, but is not limited thereto.

Specifically, the screening method is a method of measuring and comparing the level of SMURF2 activity or TGF-β activity in the presence and absence of a test substance, which is a candidate substance for treating lung cancer, and can be usefully used for screening a substance for treating lung cancer. Substances that indirectly or directly inhibit the activity of SMURF2 or promote TGF-β activity may be selected as the substances for treating lung cancer disclosed in the present specification.

That is, in the case of treatment with a candidate substance for treating lung cancer, if the substance inhibits the activity of SMURF2 and promotes TGF-β activity in lung cancer cells, it can be predicted as an initial substance for treating lung cancer cells.

Another aspect is in vitro (in vitro) from, contacting the miR-195 or miR-497 in cancer cell stage to promote SMURF2 inhibitory or TGF-β activity of the cancer cells; how cancer cytostatic containing to provide.

Using the method according to one aspect, it is possible to inhibit the expression of SMURF2 ubiquitinase used in the process of TGF-β signaling and inhibit the ubiquitination of TGF-β receptor I by SMURF2. Therefore, the TGF-β receptor I Since protein degradation can be prevented and stabilized to promote TGF-β signaling activity, in particular, it can effectively inhibit the proliferation of lung cancer cells.

Using a composition comprising miR-195 or miR-497 according to an aspect can effectively inhibit SMURF2 activity of lung cancer cells and enhance TGF-β signaling in vitro. In addition, if the activity of miR-195 or miR-497 is utilized, lung cancer treatments, especially early lung cancer treatments without metastasis, can be effectively found, which can be usefully used in related pharmaceutical and medical businesses.

1 is a result of confirming the relative expression values in normal persons and lung cancer patients (Fig. 1A), the results of confirming the relative expression values of miR-195/497 confirmed by GSE17681 data (Fig. 1B), and normal tissues and lung cancer tissues among GSE21933 data. The results of confirming the differential expression values of the SMURF2 gene for each group compared (Fig. 1C), the results of arranging the relative expression values of miR-195/497 for each sample (Fig. 1D), and the expression amount of the relative SMURF2 mRNA by sample. 1E), a result of confirming the correlation between the expression of the mir-195/497 or SMURF2 gene using qRT-PCR in A549, H157, H1299, H1703 cells, which are lung cancer cells, and L132 cells, which are normal lung epithelial cells ( 1F and 1G) ( *p <0.05, **p <0.01, **p <0.001).
2 is a result of comparing the expression of the SMURF2 gene through qRT-PCR by treating cancer cells with a control or inhibitor of miR-195/497, or a control or mimics (FIGS. 2A and 2B ), as a result of Western blot confirmation at the protein level (Fig. 2C), showing the binding sequence of miR-195/497 potential in the 3'UTR region of the SMURF2 gene and a sequence in which some of the potential binding sequences are modified into other sequences (Fig. 2D), A549 lung cancer cells containing 25, 50, 75, 100 nM miR-195/497 mimic agent and pmir-GLO dual luciferase enzyme protein expression vector or a sequence in which some of its binding sequences are modified It is a diagram showing the result of comparing the degree of reaction between the luciferase enzyme protein and the substrate after transfection with the luciferase enzyme protein expression vector ( *p <0.05, **p <0.01). , **p <0.001).
3 is a Western blot confirming whether the decrease in the amount of SMURF2 protein by miR-195/497 affects the expression of the TGF-β receptor I protein present on the cell surface (FIG. 3A ), miR-195/ in HEK293T cells. After transfection of 497 control or inhibitor together for 48 hours, the degree of ubiquitination of TGF-β receptor I was confirmed by Western blot (left of Fig. 3B). As a result, the amount of β-actin protein was used as a standard for the total TGF- The result of quantifying the level of β receptor I and SMURF2 protein, and the ubiquitinated TGF-β receptor I protein level (Fig. 3B right), the result of comparing the amount of expression of SMURF2 gene using qRT-PCR (Fig. 3C), HEK293T Cells were transfected with a control or mimic agent of miR-195/497 for 48 hours, and then the degree of ubiquitination of TGF-β receptor I was confirmed by Western blot (Fig. 3D), as a result of comparing the amount of expression of the SMURF2 gene. It is a figure showing (FIG. 3E).
Figure 4 is a result of confirming the reactivity between the control or miR-195/497 miR-195/497 and the luciferase enzyme protein and the substrate (Figure 4A), SMAD2 protein and phosphorylated SMAD2 protein (phosphor-SMAD2), SMAD3 protein and Phosphorylated SMAD3 protein and p21 protein levels were compared through Western blot (Figure 4B), phosphorylated SMAD2 protein and SMURF2 protein levels were compared through Western blot (Figure 4C), using qRT-PCR technique. It is a diagram showing the result of confirming the expression amount of the CDKN1A (p21) gene (Fig. 4D) ( *p <0.05, **p <0.01, **p <0.001).
5 is a result of measuring the proliferation ability of cancer cells when miR-195/497 mimic agent was added (FIG. 5A ), and the result of confirming the ability to inhibit colony formation of lung cancer cells when miR-195/497 mimic agent was treated (FIG. 5B And 5C), a diagram showing the results of confirming the effect of inhibiting metastasis of lung cancer cells (FIGS. 5D and 5E) ( *p <0.05, **p <0.01, **p <0.001).
6 is a result of measuring the proliferative ability of cancer cells when an inhibitor of miR-195/497 is added (FIG. 6A), and a result of confirming the ability to promote colony formation of lung cancer cells when treated with miR-195/497 mimic agent (FIG. 6B and 6C), is a diagram showing the results of confirming the metastasis effect of lung cancer cells (Figs. 6D and 6E) ( *p <0.05, **p <0.01, **p <0.001).
7 is a result of confirming the cancer growth inhibitory ability of lung cancer cells due to xenotransplantation (FIG. 7A), a result of measuring the growth of cancer tissue for 30 days every 3 days (FIG. 7B), a control or mimic agent of miR-195/497 The result of measuring the weight of the cancer tissue of the mouse after the injection (Fig. 7C), the result of measuring the cell death in the cancer tissue in which the control or miR-195/497 of miR-195/497 was injected using the TUNEL staining method (Fig. As a result of checking a fluorescence microscope after staining a control group of miR-195/497 or a part of cancer tissue injected with a mimic agent using SMURF2 protein antibody (Fig. 7E), a control group of miR-195/497 or a part of cancer tissue injected with a mimic agent It is a diagram showing the result of confirming the expression of the SMURF2 gene in (Fig. 7F) and the result of confirming the SMURF2 protein level by Western blot (Fig. 7G) ( *p <0.05, **p <0.01, **p <0.001).

The present invention will be described in more detail by the following examples. However, the following examples are only intended to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Experimental Example 1. Culture of cells used in the experiment

L132 cells (normal lung epithelial cells), A549 cells, H157 cells, H1299 cells, H1730 cells (lung cancer cells), and HEK293T cells were purchased from Korea Cell Line Bank. All lung cancer cells and normal lung epithelial cells are cultured with RPMI-1640 cell culture solution (Hyclone, USA) containing 10% FBS (Hyclone, USA) and 1% penicillin/streptomycin (Hyclone, USA). I did. HEK293T cells were cultured in DMEM cell culture solution (Hyclone, USA) containing 10% FBS and 1% penicillin/streptomycin, and all cells were cultured in an incubator supplying 5% carbon dioxide at 37 °C. Was done.

Experimental Example 2. MicroRNA transfection

Both inhibitors and mimetics of MicroRNA-195/497 were purchased from Genolution. Each inhibitor and mimic agent were used at a concentration of 50 nM, and transfected into cells according to the manufacturer's manual using the reagent Lipofectamine 2000 (Invitrogen, USA).

Experimental Example 3. Luciferase Reporter plasmid construction and luciferase reporter analysis

The binding sequence of miR-195/497 in the SMURF2 gene was designed using TargetScan, miRanda, and miRWalk programs. The binding sequence of miR-195/497 present in the 3'-UTR portion of the SMURF2 gene was synthesized (Cosmogenetech, Korea) and inserted into the pmirGLO luciferase enzyme protein expression vector (Promega Corp., USA). The binding sequence of miR-195/497 present in the SMURF2 gene is as follows: the prototype 3'-UTR of SMURF2 ; 5′-AAA CTA GCG GCC GCT AGT ATG AGG CCA CAT TCA GCT GCT ATT TAA T-3′, 5′-CTA GAT TAA ATA GCA GCT GAA TGT GGC CTC ATA CTA GCG GCC GCT AGT TT-3′. A sequence in which some of the target sequences are modified; 5′-AAA CTA GCG GCC GCT AGT ATG AGG ACC CCT TCA TCG GAT ATT TAA T-3′, 5′-CTA GAT TAA ATA TCC GAT GAA GGG GTC CTC ATA CTA GCG GCC GCT AGT TT-3′. The dual-luciferase assay was performed by modifying the pmirGLO dual luciferase enzyme protein expression vector containing the binding sequence of miR-195/497 present in the 3'-UTR region of the 200 ng SMURF2 gene, or a portion of the binding sequence. The luciferase enzyme protein expression vector containing the sequence and the control or mimetic of mir-195/497 of 25, 50, 75, and 100 nM were transfected into A549 cells together using the Lipofectamine 2000 reagent.

To confirm the TGF-β signaling activity in response to TGF-β1, 100 ng of SMAD-responsive reporter vector contained in the Cignal SMAD Reporter Assay Kit (Qiagen, USA) and mir-195/497 were used in A549 cells. The control or mimetic was transfected together at a concentration of 50 nM for 24 hours. After this, 5 ng/ml of TGF-β1 was treated for 24 hours. The activity of all Luciferase enzymes was measured using a dual-luciferase assay kit (Promega Corp., USA).

Experimental Example 4. Western Blot Assay

Each cell was obtained to prepare a protein using protein lysis buffer (50 mM Tris-Cl, 150 mM NaCl. 0.5% NP40, 1 mM EDTA, pH 7.5). 20-50 μg of protein was boiled for 5 minutes at 100 °C with 5X SDS sample buffer, and then separated by SDS-PAGE with 6-15% gel. The protein in the gel was transferred to a nitrocellulose membrane (Pall Corporation, USA), blocked with 5% fat-free milk, and incubated overnight at 4 °C with the primary antibody. Thereafter, the membrane was incubated with a secondary antibody for 1 hour at room temperature and reacted with an ECL solution (Santa Cruz Biotechnology, USA), and visualized using ChemiDoc (ATTO Technology, USA). The primary antibodies used in this experiment were as follows: Primary antibody-SMAD2 (catalogue no. 5339) purchased from Cell Signaling Technology (USA); phosho-SMAD2 (catalogue no. 3108); SMAD3 (catalogue no. 9523); p21 (catalogue no. 2947), (Santa Cruz Biotechnology, USA): SMURF2 (catalogue no. sc-25511); Na+/K+ ATPase α (catalogue no. sc-48345), antibody-β-actin (catalogue no. LFPA0207) purchased from AbFrontier (Korea), antibody-HA (catalogue no. H6908) purchased from Sigma-Aldrich (USA) ); Flag (catalogue no. F1804), antibody purchased from Abcam (UK)-TGF-β receptor 1 (catalogue no. ab31013); phospho-SMAD3 (catalogue no. ab52903). As secondary antibodies, Goat anti-Rabbit and Goat anti-Mouse (Santa Cruz Biotechnology, USA) were used.

Experimental Example 5. Nickel-pulldown assay

For an experiment to confirm the degree of ubiquitination of the protein, TGF-β receptor I expression vector containing HA peptide (pCMV-HA-TGF-β receptor I) and circular SMURF2 expression vector containing Flag peptide (pCMV5B-) were used in HEK293T cells. Flag-SMURF2 WT), and a ubiquitin expression vector containing His peptide were transfected with a control or inhibitor/mimetic agent of miR-195/497, followed by treatment with 10 μM MG132 for 4 hours. pCMV-HA-TGF-β receptor I vector and pCMV5B-Flag-SMURF2 WT vector (Addgene plasmid, #11746) were used, and then cells were obtained and urea protein lysis buffer (8 M Urea, 0.3 M NaCl, 50 mM Na). ₂ HPO ₄ , 50 mM Tris-HCl, 1 mM PMSF, 10 mM Imidazole, pH 8) was added and the cells were lysed using a sonicator to obtain a protein. 100 μl of Ni-NTA agarose (QIAGEN, USA) was added to 3-5 mg protein and incubated at 4 °C for 4-6 hours. After washing, 5X sample buffer was added to the remaining agarose and boiled at 100° C. for 10 minutes. Each sample was visualized via Western blot.

Experimental Example 6. Cell surface protein extraction

A549 lung cancer cells were transfected with miR-195/497 control or inhibitor/mimetic agents together with several plasmids, and then cell surface proteins were extracted using Pierce Cell Surface Protein Isolation Kit (ThermoFisher, USA). The protein on the cell surface was bound to EZ-LINK Sulfo-NHS-SS-biotin for 4 to 30 minutes. Remaining biotin suppressed the reaction using a quenching buffer. After obtaining the cells, adding a protein lysis buffer to obtain a protein, reacted with the immobilized NeutrAvidin agarose slurry, and finally, a sample was extracted using a sample buffer containing DTT. Cell surface proteins were visualized via Western blot.

Experimental Example 7. Quantitative real-time PCR

To obtain cells, RNA was extracted using Trizol Reagent (Ambion. USA). In order to confirm the expression of miR-195/497, cDNA was synthesized using the Mir-X miRNA First-Strand Synthesis Kit (Takara, China). The sequence of primers used to amplify miR-195/497 is as follows: F-5′-UAG CAG CAC AGA AAU AUU GGC-3′, 5′-CAG CAG CAC ACU GUG GUU UGU-3′ and Mir- X miRNA qRT-PCR universal reverse primer (R) provided by SYBR Kit (TOYOBO, Japan). Primers for the amplification of U6 small nuclear RNA were also used in the Mir-X miRNA qRT-PCR SYBR Kit.

To confirm the expression of the SMURF2 gene and the CNKN1A gene, cDNA was synthesized using the ReverTra Ace q PCR RT Kit (Toyobo, Japan). The sequence of primers used for amplification of each gene is as follows: SMURF2 gene; F-5′- TTG GCT CTG CAG AAA GGA TT-3′ and R-5′- CCA CAG CTT TCC AGA ACC AT-3′. CDKN1A gene; F-5′- CAC CAC TGG AGG GTG ACT TC-3′ and R-5′- ATC TGT CAT GCT GGT CTG CC-3′. β-actin gene; F-5′-CTC TTC CAG CCT TCC TTC CT-3′ and R-5′-AGC ACT GTG TTG GCG TAC AG-3′. Quantitative real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Japan), and the expression of each microRNA and gene was normalized to the amount of expression of U6 small nuclear RNA and β-actin gene.

Experimental Example 8. Cell proliferation assay

After growing A549 lung cancer cells in a 96-well plate, a control or inhibitor/mimetic agent of miR-195/497 was transfected. Cell proliferation was measured over time using the WST-1 Assay Kit (Dogen, Korea). After removing the culture solution from the cells, 10 μl of the WST-1 solution and 100 μl of the cell culture solution were mixed, added to the cells, and reacted for 30 minutes. Thereafter, the plate with cells at 450 nm wavelength was read using a Microplate Absorbance Reader (Bio-Rad, USA) to measure the absorbance.

Experimental Example 9. Cell metastasis analysis (Invasion Assay)

For cell metastasis analysis, 6.5 mm Transwells (8.0 m pore size) (Corning Incorporation, USA) were used. After coating the surface of the transwell invasion chamber with a polycarbonate filter with Matrigel, 1×10 ³ cells were laid on it. The lower chamber was filled with a cell culture solution containing 10% FBS and 5 μg/ml of fibronectin, and after 48 hours, the remaining cells on the filter surface were removed, and only the cells that passed the filter were stained with Giemsa dye. .

Experimental Example 10. Colony formation reaction (Colony formation)

After transfection of the cells with a control or inhibitor/mimetic of miR-195/497, only 500 cells were transferred to a 35 mm cell dish and grown for 10 days. Thereafter, the cells were fixed with 4% formaldehyde and stained with a crystal violet stain.

Experimental Example 11. Xenograft

To create cancerous tissue resulting from xenotransplantation of A549 lung cancer cells, 1x10 ⁷ A549 cells were implanted into the subcutaneous fat of the thigh of a rat whose immune system was compromised. When the size of the resulting cancer tissue reached about 100 mm ³ , a control or mimic agent of miR-195/497 was injected into the cancer tissue in an amount of 4 mg/kg. Once every 3 days, a miR-195/497 control or mimic agent was injected into the cancer tissue for a total of 2 weeks, and the size of the cancer tissue was measured every 3 days.

Experimental Example 12. Analysis of immunostaining and TUNEL staining of cancer tissue

Immunostaining was performed using SMURF2 antibody (Santa Cruz, 1:100) and Alexa Fluor 549-conjugated goat anti-rabbit IgG (Thermo Fisher, 1:500) to measure the expression of SMURF2 protein in cancer tissues.

TUNEL staining was performed using a cell death measurement kit (Roche, USA) to measure cell death in cancer tissues. Visualization was performed using a ZEISS LSM 700 confocal microscope.

Example 1. Expression of miR-195/497 and SMURF2 gene expression in lung cancer cells

An experiment was conducted to determine how the expression level of miR-195/497 changes among miRNAs expressed in lung cancer cells, and the expression level of the related SMURF2 gene also shows a difference between the gene expression levels in the samples of normal subjects and lung cancer cells. . Using the Gene Expression Omnibus (GEO) database provided by the National Center for Biotechnology Information (NCBI), tissue samples from 126 lung cancer patients and 5 normal individuals, as well as blood samples from 17 lung cancer patients and 19 normal individuals The expression of miR-195/497 was confirmed by analyzing, and the relative expression value of miR-195/497 confirmed by GSE1853 data is shown in Fig. 1A, and the relative expression value of miR-195/497 confirmed by GSE17681 data is shown in Fig. 1B. , From the mRNA microarray data comparing normal tissues and lung cancer tissues among GSE21933 data, the differential expression values of the SMURF2 gene per each group are shown in Fig. 1C, and the results of summarizing the relative expression values of miR-195/497 by sample are shown in Fig. Figure 1E shows the results of summarizing the relative SMURF2 mRNA expression levels for each sample. miR-195/497 or SMURF2 using qRT-PCR in lung cancer cells A549, H157, H1299, H1703 cells and normal lung epithelial cells L132 cells. The results of confirming the correlation between the expression of genes are shown in FIGS. 1F and 1G, respectively.

As shown in FIGS. 1A and 1B, it was confirmed that the expression of miR-195/497 was remarkably low in the tissues and blood of lung cancer patients. As confirmed in Figure 1C, through the analysis of the GEO database provided by NCBI, it was confirmed that the expression of the SMURF2 gene was high in lung cancer patients as opposed to the expression of miR-195/497. 1D and 1E, even when comparing the expression of miR-195/497 and the expression of SMURF2 gene using various lung cancer cell lines, the expression of miR-195/497 was remarkably in lung cancer cells compared to normal cells. It was confirmed that the expression of the SMURF2 gene was significantly low and the expression of the SMURF2 gene appeared high. 1F and 1G, it was confirmed that the expression of mir-195/497 and the SMURF2 gene were inversely proportional in lung cancer cells. Overall, it was confirmed that the expression of mir-195/497 and the expression of SMURF2 gene were associated with the occurrence of lung cancer.

Example 2. Confirmation of the ability of miR-195/497 to regulate SMURF2 gene expression

In order to confirm whether miR-195/497 can regulate the expression of SMURF2 gene, an experiment was performed to measure the amount of mRNA and protein of SMURF2 after transfection of the control or inhibitor/mimetic agent of miR-195/497 into cells. . We examined how miR-195/497 affects the expression of SMURF2 gene in A549 lung cancer cells. Cells were transfected with miR-195/497 control or inhibitors, or control or mimics for 48 hours, and then treated with 5 ng/ml of TGF-β1 for 4 hours to treat the SMURF2 gene. The expression of was compared through q RT-PCR, which is shown in FIGS. 2A and 2B. To analyze statistical significance, a two-way ANOVA method was used, and the error range of all data was expressed as ±SEM. A549 lung cancer cells were transfected with a control or mimetic of mir-195/497 for 48 hours, and then treated with 5 ng/ml of TGF-β1 for 24 hours to compare the SMURF2 protein level by Western blot. Indicated. In addition, the results showing the binding sequence of potential mir-195/497 in the 3'UTR region of the human SMURF2 gene and a sequence in which some of the potential binding sequences are modified into other sequences are shown in FIG. 2D.

Pmir-GLO dual lucifera containing the binding sequence of miR-195/497 present in the 3'-UTR region of the SMURF2 gene and 25, 50, 75, 100 nM miR-195/497 mimic agents in A549 lung cancer cells, respectively. Aase enzyme protein expression vector or a luciferase enzyme protein expression vector including a sequence in which a portion of its binding sequence is modified was transfected into cancer cells, and then the degree of reaction between the luciferase enzyme protein and the substrate was compared, and FIG. 2E And 2F. To analyze statistical significance, one-way ANOVA method was used, and the error range of all values was expressed as ±SEM.

2A and 2B, it was confirmed that the mRNA level of SMURF2 was increased by the miR-195/497 inhibitor and the mRNA level of SMURF2 was decreased by the mimic agent of mir-195/497. When cells are treated with TGF-β1, the expression of the SMURF2 gene is increased.At this time, the expression of the SMURF2 gene is further increased in the sample transfected with the miR-195/497 inhibitor, and conversely, the miR-195/497 mimic agent was transfected. One sample had a decreased expression of SMURF2 . In addition, as confirmed in Figure 2C, it was confirmed that the protein level of SMURF2 is also regulated by miR-195/497 in the same manner as the mRNA level.

In addition, in order to ensure that this can be miR-195/497 coupled to a portion of the 3'-UTR SMURF2 gene, also a combination of miR-195/497 present in the 3'-UTR part of SMURF2 gene sequence checking, as in the 2D The sequence and the sequence in which some of the binding sequences were modified were cloned, and each of the pmir-GLO dual luciferase enzyme proteins was inserted into the expression vector. In addition, as confirmed in 2E and 2F, after transfecting cells with the miR-195/497 mimic agent at various concentrations together with the vectors, the activity of luciferase enzyme was measured, and as a result, a circular binding sequence was inserted. In the case of the modified vector, the activity of luciferase enzyme was significantly lowered by the miR-195/497 mimic agent, whereas in the vector with a partially modified binding sequence inserted, the enzyme activity was inhibited by the miR-195/497 mimic agent. Did not appear. Therefore, it was confirmed that miR-195/497 could directly bind to the 3'-UTR portion of the SMURF2 gene and regulate the expression of the SMURF2 gene.

Example 3. Confirmation of the ability of miR-195/497 to modulate ubiquitination of TGF-β receptor I

SMURF2 Since it is known that TGF-β signaling can be inhibited through ubiquitination of TGF-β receptor I, it is confirmed that the regulation of SMURF2 expression by miR-195/497 can also affect TGF-β signaling. The following experiment was performed.

First, an experiment was conducted to confirm whether the decrease in the amount of SMURF2 protein by miR-195/497 affects the amount of TGF-β receptor I protein present on the cell surface. In A549 lung cancer cells, the expression vector of TGF-β receptor I containing the HA peptide and the original SMURF2 expression vector containing the Flag peptide, or the expression vector of the active variant of SMURF2 containing the Flag peptide, and the control or mimic of mir-195/497 After transfection with the agent for 48 hours, 5 ng/ml of TGF-β1 was treated for 4 hours. Thereafter, biotin protein was attached to the cell surface protein, and biotin protein was pulled using avidin-agarose beads to extract only the cell surface protein. Finally, the expression of the TGF-β receptor I protein on the cell surface was confirmed by Western blot analysis, and this was shown in FIG. 3A.

As shown in Fig. 3A, after TGF-β1 treatment, cell surface protein and total protein were extracted from the cells, and the amounts were compared by Western blot. As a result, TGF on the cell surface by miR-195/497 mimic agent It was confirmed that both the -β receptor I protein and the total TGF-β receptor I protein amount were increased. The increase in the amount of TGF-β receptor I protein by miR-195/497 was reduced again by overexpression of the prototype SMURF2, but it was confirmed that there was no change by the expression of the inactivated SMURF2 mutant. That is, when miR-195/497 targets the SMURF2 gene and decreases its expression, it was confirmed that the amount of the TGF-β receptor I protein increased.

To confirm that the increase in the amount of TGF-β receptor I protein by miR-195/497 was the result of controlling the ubiquitination of TGF-β receptor I by SMURF2, we examined the ubiquitination of TGF-β receptor I. The experiment was carried out. HEK293T cells were transfected with a TGF-β receptor I expression vector containing HA peptide, a ubiquitin expression vector containing His peptide, and a control or inhibitor of miR-195/497 for 48 hours, and then MG132, a proteasome inhibitor. Was treated at a concentration of 10 μM for 4 hours. Ni-NTA bead was used to pull down His peptide by performing nickel-pull-down to confirm the degree of ubiquitination of TGF-β receptor I by Western blot. This is shown on the left side of FIG. 3B, and the amount of β-actin protein is standardized in each sample. The total TGF-β receptor I and SMURF2 protein levels, and the ubiquitinated TGF-β receptor I protein levels were quantified and shown in a graph on the right of FIG. 3B. In addition, the results of comparing the amount of expression of the SMURF2 gene using qRT-PCR after transfecting HEK293T cells with a control or inhibitor of miR-195/497 for 48 hours are shown in FIG. 3C. 3B and 3C, it was confirmed that the ubiquitination of TGF-β receptor I was significantly increased in cells transfected with an inhibitor of miR-195/497, and at this time, the expression of the SMURF2 gene was also increased. . HEK293T cells with a TGF-β receptor I expression vector containing HA peptide, a SMURF2 expression vector containing a Flag peptide, a ubiquitin protein expression vector containing a His peptide, and a control or mimic agent of miR-195/497 for 48 hours After transfection during the period, the proteasome inhibitor MG132 was treated at a concentration of 10 μM for 4 hours. Ni-NTA beads were used to pull down the His peptide, and the degree of ubiquitination of TGF-β receptor I was confirmed by Western blot, which is shown in FIG. 3D as a control of miR-195/497 in HEK293T cells or After transfection of the mimic agent for 48 hours, the result of comparing the amount of expression of the SMURF2 gene using qRT-PCR is shown in FIG. 3E.

On the contrary, as confirmed in FIGS. 3D and 3E, it was confirmed that the expression of the SMUFR2 gene was reduced by the miR-195/497 mimic agent, and at the same time, the ubiquitination of the TGF-β receptor I was also reduced. In order to check whether the regulation of the ubiquitination of TGF-β receptor I by miR-195/497 is mediated through SMURF2, the ubiquitination of TGF-β receptor I by mir-195/497 after overexpressing the prototype SMURF2. When checking the change again in FIG. 3D, it was confirmed that the ubiquitination of TGF-β receptor I reduced by miR-195/497 was increased again by overexpression of SMURF2. Taken together, it was confirmed that miR-195/497 inhibited the expression of SMURF2 and, through this, affected the ubiquitination of TGF-β receptor I by SMURF2, thereby regulating the amount of protein in TGF-β receptor I.

Example 4. Activity regulation of TGF-β signaling by miR-195/497

To determine whether miR-195/497 can also be involved in overall TGF-β signaling activity, the expression vector of the TGF-β-responsive signal SMAD luciferase enzyme containing a sequence (SBE) capable of binding SMAD protein (Qiagen ) Was used to perform the experiment.

A549 lung cancer cells were transfected with a control or mimic agent of miR-195/497 and a luciferase protein expression vector containing the SMAD gene sequence that responds to TGF-β for 24 hours, and then 5 ng/ml of TGF. -β1 was treated for 24 hours, and the reactivity between the luciferase enzyme protein and the substrate was shown in FIG. 4A. To analyze statistical significance, a one-way ANOVA method was used, and the error range of all values was expressed as ±SEM ( *p <0.05, **p <0.01, **p <0.001). A549 lung cancer cells were transfected with miR-195/497 control or mimic agent for 24 hours and then treated with LY364947, an inhibitor of TGF-β receptor I, at a concentration of 100 ng/ml for 24 hours. Thereafter, after treatment with TGF-β1 for 4 hours, the levels of SMAD2 protein and phosphorylated SMAD2 protein (phosphor-SMAD2), SMAD3 protein and phosphorylated SMAD3 protein, and p21 protein were compared through Western blot, and the results are shown in FIG. 4B. Indicated. A549 lung cancer cells were transfected with miR-195/497 control or mimic agent and then treated with 5 ng/ml of TGF-β1 for 0, 1, 2, 4 hours, respectively, to compare phosphorylated SMAD2 protein levels through Western blot And shown in Figure 4C. A549 lung cancer cells were transfected with miR-195/497 control or mimetic, and then 5 ng/ml of TGF-β1 was treated for 4 hours. The amount of expression of the CDKN1A (p21) gene was compared using the qRT-PCR technique. To analyze the statistical significance, a two-way ANOVA method was used, and the error range of all data was expressed as ±SEM values ( *p <0.05, **p <0.01, **p <0.001), and the results of this experiment were It is shown in Figure 4D.

As shown in Figure 4A, it was confirmed that the activity of the luciferase enzyme was increased by the miR-195/497 mimic agent. In addition, when TGF-β1 is treated, the activity of luciferase enzyme is increased. At this time, it was confirmed that the activity of luciferase enzyme was increased in the sample transfected with miR-195/497 mimic agent. 4B, it was confirmed that phosphorylation of SMAD2 and SMAD3, which shows TGF-β signaling activity, was increased by the mimic agent of mir-195/497 in TGF-β1-treated cells. It was confirmed that the above effect disappeared when LY364947, a phosphorylase inhibitor of TGF-β signaling, was treated, and it was confirmed that the increase in phosphorylation of the two SMAD proteins by miR-195/497 is due to the increase in the activity of TGF-β signaling. In addition, since the increase in the amount of p21 protein by miR-195/497 was reduced again by the treatment of LY364947, when the activity of TGF-β signaling was increased by miR-195/497, TGF-β signaling was increased. It was confirmed that the expression of the target gene of CDKN1A was increased.

Next, the degree of phosphorylation of SMAD2 protein changes according to the treatment time of TGF-β1, and an experiment was conducted to confirm how miR-195/497 can function in this situation. As can be seen from Figure 4C, in the cells transfected with the miR-195/497 control, it was confirmed that the phosphorylation of SMAD2 increased the most when TGF-β1 was treated for 1 hour, and decreased thereafter. In cells transfected with miR-195/497 mimic agent, it was confirmed that the phosphorylation of SMAD2 by TGF-β1 treatment was maintained longer than that of the control group. Conversely, in the case of the amount of SMURF2 protein, it was confirmed that there was little change by the control group, whereas when transfected with the miR-195/497 mimic agent, the amount of TGF-β1 gradually decreased as the treatment time increased. Through this, it was confirmed that miR-195/497 can maintain the activity of TGF-β signaling through a decrease in SMURF2 gene expression, thereby increasing the phosphorylation of SMAD2 and SMAD3.

In order to confirm the regulation of the activity of TGF-β signaling by miR-195/497, the expression of the CDKN1A (p21) gene regulated by the activity of TGF-β signaling was confirmed. First, the CDKN1A gene was miR-195/497 Analysis of databases such as Targetscan, miRanda, and miRWalk 2.0 used previously to show that it is not a direct target gene of CDKN1A revealed that there is no sequence to which mir-195/497 can bind in the 3'-UTR portion of the CDKN1A gene. Confirmed. As shown in FIG. 4D, it was confirmed that the expression of the CDKN1A gene was increased when the cells were treated with TGF-β1. At this time, it was confirmed that the expression of CDKN1A was much more increased when the miR-195/497 mimic agent was transfected. Likewise, it was confirmed that the amount of p21 protein was also increased by treatment with TGF-β1, and was further increased by miR-195/497 mimic agent.

Therefore, it was confirmed that miR-195/497 inhibits the expression of SMURF2 gene, prevents ubiquitination of TGF-β receptor I, and thereby is an important regulator of TGF-β signaling activity.

Example 5. Confirmation of miR-195/497's ability to regulate lung cancer cell proliferation, colony formation, and metastasis

5.1 Confirmation of inhibition of growth and metastasis of A549 lung cancer cells by miR-195/497 mimic agents

Since TGF-β signaling is known to play an important role in cancer development, growth, and metastasis, an experiment to determine whether miR-195/497, which can regulate TGF-β signaling, can also affect cancer development. Was performed.

A549 lung cancer cells were used to detect changes in the phenotype caused by miR-195/497 mimic agents. After transfection of miR-195/497 control or mimic agents into A549 lung cancer cells, cells were used for 1 -3 days using WST assay. It was confirmed by measuring the proliferation ability of, and it is shown in FIG. 5A. As confirmed in FIG. 5A, it was confirmed that cells transfected with miR-195/497 mimics did not undergo cell proliferation when compared to control cells, and this was confirmed that the same appeared even when TGF-β1 was treated. I did. Next, a colony formation experiment was performed to find out how miR-195/497 affects cell growth. After transfection of the control or mimic agent of miR-195/497 into A594 lung cancer cells, the cells were stained with crystal violet. Thus, colony formation was observed and counted, which are shown in Figs. 5B and 5C.

5B and 5C, it was confirmed that the A549 lung cancer cells transfected with the miR-195/497 mimic agent had a lower colony-forming ability compared to the control group, which is the same even when treated with TGF-β1. It was confirmed.

A549 lung cancer cells were transfected with a control or mimic agent of miR-195/497, and then the cells that passed through Matrigel were stained with Kimja solution using Matrigel invasion assay, and the number thereof was counted. Shown in. 5D and 5E, as a result of examining the metastatic ability of cancer cells, it was confirmed that cells transfected with the miR-195/497 mimic agent did not undergo cell metastasis compared to the control group. In particular, it was confirmed that colony formation and reduction in cell metastasis by miR-195/497 mimics were all the same even in the situation where TGF-β signaling was activated by treatment with TGF-β1. Overall, it was confirmed that miR-195/497 can inhibit lung cancer by interfering with the growth and metastasis of cancer cells.

5.2 Confirmation of the effect of miR - 195 / 497 inhibitor on promoting the growth and metastasis of A549 lung cancer cells

An experiment was conducted to confirm the effect of miR-195/497 inhibitor on the proliferation, colony formation, and metastasis of A549 lung cancer cells. A549 lung cancer cells were transfected with a control or inhibitor of miR-195/497, and then cell proliferation was measured for 1 to 4 days using a WST assay, which is shown in FIG. 6A.

6A, it was confirmed that cells transfected with an inhibitor of miR-195/497 progressed very rapidly compared to the control cells, confirming that miR-195/497 inhibited the proliferation of cancer cells. Could

Next, a colony formation experiment was performed to find out how miR-195/497 affects cell growth. After transfecting A594 lung cancer cells with a control or inhibitor of miR-195/497, the cells were stained with crystal violet to observe colony formation and counting results are shown in FIGS. 6B and 6C. It was confirmed that colony formation occurred very well in A549 lung cancer cells transfected with an inhibitor of miR-195/497 compared to the control group.

Finally, in order to examine the metastasis ability of cancer cells, A549 lung cancer cells were transfected with miR-195/497 control or mimetic, and then the cells that passed Matrigel were stained with Kimja solution using Matrigel invasion assay. After the cells were counted, they are shown in FIGS. 6D and 6E. It was confirmed that metastasis of cancer cells was significantly increased in the cells transfected with the inhibitor of miR-195/497 compared to the negative control that was not treated with the miR-195/497 inhibitor. These results support the fact that miR-195/497 can inhibit lung cancer by interfering with the growth and metastasis of cancer cells. Overall, it was confirmed that miR-195/497 can inhibit lung cancer cell proliferation and colony formation, and also significantly inhibit metastasis.

Example 6. Confirmation of inhibition of cancer growth caused by xenograft overexpression of miR-195/497

In order to confirm that miR-195/497 actually has remarkable anticancer activity, an experiment was conducted to confirm the ability to inhibit the growth of cancer tissues in mice that artificially caused cancer by injecting A549 lung cancer cells. A 549 lung cancer cells xenotransplanted mice were injected with a miR-195/497 control or mimic agent, and the mice were dissected on the 30th day of xenotransplantation, and the cancer tissue was cut out and its size was measured, and the results are shown in FIG. 7A. Done. In addition, the growth of cancer tissues of each week was measured every 3 days for 30 days after injecting a control or miR-195/497 control agent or a mimic agent to a mouse with cancer caused by the injection of A549 lung cancer cells, and is shown in FIG. 7B as a graph. After injecting a control or miR-195/497 miR-195/497 to mice with cancer due to the injection of A549 lung cancer cells, the weight of cancer tissues of each mouse was measured, and this is shown in FIG. 7C.

As confirmed in FIG. 7A, it was confirmed that the size of the cancer tissue cut out from the mice injected with the miR-195/497 mimic agent was remarkably small, about 50% compared to the control group. In addition, as confirmed in Figures 7B and 7C, it was confirmed that the volume and weight of the cancer tissue was also significantly lower than the control group.

Using the TUNEL staining method, apoptosis in the cancer tissues injected with the control or mimic agent of miR-195/497 was measured, and this is shown in FIG. 7D, and the control of miR-195/497 or a portion of the cancer tissue injected with the mimic agent were The image obtained by observing the fluorescence microscope after staining with the SMURF2 protein antibody is shown in FIG. 7E.

As confirmed in FIG. 7D, it was confirmed that cell death was significantly increased in cancer tissues of mice injected with miR-195/497 mimic agents through the TUNEL staining assay capable of measuring cell death compared to the control group. In addition, as confirmed in Figure 7E, it was confirmed that the expression of the SMURF2 gene was significantly reduced in the cancer tissues of mice injected with the miR-195/497 mimic agent.

In addition, after obtaining RNA and protein from cancer tissues injected with miR-195/497 control or mimic agent, the expression amount of SMURF2 gene and SMURF2 protein level were compared using qRT-PCR and Western blot, respectively, and the result of graphing Fig. 7F shows the results of Western blot in Fig. 7G.

As confirmed in FIGS. 7F and 7G, it was confirmed that the amount of expression of the SMURF2 gene was reduced by miR-195/497 in cancer tissues. In addition, it was confirmed that the amount of SMURF2 protein in cancer tissues was decreased by miR-195/497 through Western blot, and the amount of SMAD2 protein phosphorylated with TβRI was significantly increased. Therefore, it was comprehensively confirmed that miR-195/497 can effectively inhibit cancer growth even in vivo.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Screening method for lung cancer treatment agent using miR-195 or miR-497 <130> PN126562 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 46 <212> DNA <213> Unknown <220> <223> binding sequence of miR-195 on SMURF 2 gene <400> 1 aaactagcgg ccgctagtat gaggccacat tcagctgcta tttaat 46 <210> 2 <211> 50 <212> DNA <213> Unknown <220> <223> binding sequence of miR-497 on SMURF 2 gene <400> 2 ctagattaaa tagcagctga atgtggcctc atactagcgg ccgctagttt 50 <210> 3 <211> 46 <212> DNA <213> Unknown <220> <223> modified binding sequence of miR-195 on SMURF 2 gene <400> 3 aaactagcgg ccgctagtat gaggacccct tcatcggata tttaat 46 <210> 4 <211> 50 <212> DNA <213> Unknown <220> <223> modified binding sequence of miR-497 on SMURF 2 gene <400> 4 ctagattaaa tatccgatga aggggtcctc atactagcgg ccgctagttt 50 <210> 5 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Forward primer sequence of miR-195 <400> 5 uagcagcaca gaaauauugg c 21 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Forward primer sequence of miR-497 <400> 6 cagcagcaca cugugguuug u 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer sequence of SMURF2 gene <400> 7 ttggctctgc agaaaggatt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer sequence of SMURF2 gene <400> 8 ccacagcttt ccagaaccat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer sequence of CDKN1A gene <400> 9 caccactgga gggtgacttc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer sequence of CDKN1A gene <400> 10 atctgtcatg ctggtctgcc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer sequence of beta-actin gene <400> 11 ctcttccagc cttccttcct 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer sequence of beta-actin gene <400> 12 agcactgtgt tggcgtacag 20

Claims (8)

A composition for inhibiting SMURF2 activity of lung adenocarcinoma cells in vitro containing miR-195 or miR-497. The composition of claim 1, wherein the composition promotes TGF-β activity of lung adenocarcinoma cells. A method of inhibiting SMURF2 activity in lung adenocarcinoma cells by incubating miR-195 or miR-497 in lung adenocarcinoma cells in vitro . The method of claim 3, wherein the method promotes the activity of TGF-β in lung adenocarcinoma cells. The method of claim 4, wherein the promotion of TGF-β activity is due to reduction of ubiquitination of TGF-β receptors. Adding a test substance to lung adenocarcinoma cells in which the expression of miR-195 or miR-497 is reduced compared to the negative control;
Measuring the level of SMURF2 activity or TGF-β activity of the lung adenocarcinoma cells; And
A method for screening a lung adenocarcinoma treatment comprising the step of selecting a test substance that inhibits SMURF2 activity or promotes TGF-β activity compared to a control group not treated with the test substance. delete In vitro ( in vitro ), by contacting the lung adenocarcinoma cells miR-195 or miR-497 to promote the SMURF2 activity inhibition or TGF-β activity of the lung adenocarcinoma cells; Lung adenocarcinoma cell proliferation inhibition method comprising a.

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