KR101737706B1 - a tumor suppressor containing miR-5582-5p - Google Patents

Abstract

The present invention relates to an anticancer agent comprising miR-5582-5p.
In the present invention, miR-5582-5p, a novel miRNA having a cancer-suppressing function, has been demonstrated. miR-5582-5p induces apoptosis and stops the cell cycle in cancer cells, but does not show this change in normal cells. GAB1, SHC1 and CDK2 were identified as target proteins of miR-5582-5p.

Description

A tumor suppressor containing miR-5582-5p comprising miR-5582-5p

The present invention relates to an anticancer agent using miR-5582-5p.

In the present invention, miR-5582-5p, a novel miRNA having a cancer-suppressing function, has been demonstrated. miR-5582-5p induces apoptosis and stops the cell cycle in cancer cells, but does not show this change in normal cells. GAB1, SHC1 and CDK2 were identified as target proteins of miR-5582-5p.

Similar to the function of miR-5582-5p, inhibition of GAB1 / SHC1 or CDK2, respectively, leads to apoptosis or cell cycle arrest. It was confirmed that the growth of cancer cells was inhibited by intra-tumor injection of miR-5582-5p or Tet-miR-5582-5p in a mouse xenograft model using HCT116 cells. These results show the applicability of miR-5582-5p, a novel miRNA with cancer-inhibiting function, as a cancer treatment agent.

MicroRNAs (miRNAs) have been attracting much attention as important regulators of gene expression with small RNAs of ~ 22 nucleotides (Endogenous small non-coding RNAs). miRNAs complementarily bind to the 3 'UTR sequence of mRNA, resulting in degradation or translational repression of target gene mRNA at post-transcriptional stages.

Because one miRNA can control many target genes at the same time, it can affect overall gene expression. In many studies, miRNA plays an important role throughout most of the biological processes, including cell growth, development, division and death, and is associated with many types of diseases, especially cancer. In cancer, many miRNAs exhibit abnormal expression. Several miRNAs function as cancer-inducing or cancer-suppressing genes by regulating genes important in a variety of cancers or by participating in the signaling pathway of tumorigenesis.

The most well-known cancer-suppressing miRNAs are the miR-34 family, the target of the tumor suppressor gene TP53. Because miR-34 targets a variety of cancer-causing genes such as MYC, BCL2, CDK4 and MET, it is considered to be a major regulator of cancer suppression and thus acts as an anti-cancer in a variety of cancers. In addition, several miRNAs such as miR-29 and miR-193a-3p are known to inhibit p85α and Mcl-1, respectively, thereby inducing apoptosis and inhibiting cancer. Some miRNAs, on the other hand, are highly expressed in a variety of cancers, and they have carcinogenic effects by targeting cancer suppressor genes. For example, miR-21, a miRNA involved in a typical carcinogenic process, targets phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4), while miR-17-92 cluster targets BIM and PTEN.

This characteristic of miRNAs involved in forming or inhibiting cancer can be a useful tool or method for cancer treatment. Therefore, studies are currently under way to determine if miRNA mimics or inhibitors are clinically available for cancer treatment as miRNA-based therapies.

In the present invention, a novel miRNA that regulates the growth of cancer cells for development of miRNA therapeutic drug was identified by confirming the function of miR-5582-5p, a new miRNA that induces apoptosis and cell cycle arrest in cancer cells. As far as we know, this is the first study of miR-5582-5p. Therefore, the present invention presents a high possibility as a novel anti-cancer miRNA therapeutic agent of miR-5582-5p which is a strong cancer suppressor gene.

As already mentioned in the foregoing, it is an object of the present invention to provide a novel anticancer miRNA therapeutic.

The present invention is characterized by being an anticancer agent comprising miR-5582-5p.

The miR-5582-5p according to the present invention has a cancer suppressing function and can be used as a powerful cancer treatment agent.

1 is a diagram showing a screening experiment of miR-5582-5p effective for inhibiting growth through functional screening,
2 is a diagram showing the cell death induction and cell cycle termination experiments of miR-5582-5p,
FIG. 3 is a diagram showing an experiment for inhibiting the direct expression of GAB1, SHC1 and CDK2 of miR-5582-5p,
FIG. 4 is a graph showing the cell death and cell cycle arrest effect of miR-5582-5p through GAB1 / SHCl and CDK2 inhibition,
5 is an experimental view showing miR-5582-5p expression which is higher in normal cells than cancer cells and miR-5582-5p which does not cause apoptosis in normal cells,
Figure 6 is an experimental view of inhibition of tumor growth of miR-5582-5p in mice,
FIG. 7 is a simplified schematic diagram of the miR-5582-5p cancer suppressing function that inhibits the growth of cancer cells and the progression of the cell cycle by simultaneously targeting GAB1, SHC1, and CDK2.
A more specific description of each drawing should be provided.
<Drawing 1>
A. Primary screening in 267 miRNA pools synthesized. Each miRNA was overexpressed in HCT116 cells and the growth rate was measured 3 days later by MTS assay. * P < 0.005 (n = 3).
B. Secondary screening. Twenty-nine miRNAs, which regulate the growth of cancer cells significantly, were screened and re-verified.
C. Selection of miRNAs with growth inhibitory effect through primary and secondary screening. Each miRNA was overexpressed in HCT116 cells and the growth rate was measured 3 days later by MTS assay. The error range is ± SEM. * P < 0.005 (n = 3).
2,
A. Verification of growth inhibitory effect of miR-5582-5p. The control or miR-5582-5p cells were overexpressed in HCT11 and SW480 cells in 6-wells and the number of cells was measured every 24 hours. * P < 0.005 (n = 3).
B. Inhibition of colony forming ability of miR-5582-5p * P &lt; 0.05 (n = 3).
C. Cell cycle arrest of miR-5582-5p. In HCT116 cells, control or miR-5582-5p was overexpressed and PI staining was performed and confirmed by flow cytometry.
D. induction of apoptosis of miR-5582-5p. The control or miR-5582-5p was overexpressed in HCT11 and SW480 cells, and stained with Annexin V-FITC / PI three days later and confirmed by flow cytometry.
E. Measurement of intracellular reactive oxygen species by miR-5582-5p. The control or miR-5582-5p was overexpressed in HCT116 cells and stained with DCF-DA and confirmed by flow cytometry.
F. Induction of TP53-independent cell death by miR-5582-5p. TP53-wild-type ( TP53 ^{+ / +} ) and TP53-null ( TP53 ^{- / -} ) HCT116 cells overexpressing control or miR-5582-5p were stained with Annexin V-FITC / PI staining.
3,
A. Inhibition of GAB1, SHC1 and CDK2 Expression in miR-5582-5p. Each colon cancer cell line was overexpressed with miR-5582-5p, followed by western blotting 48 hours later.
B. Reduced expression of GAB1, SHC1 and CDK2 by miR-5582-5p. Expression of mRNA of GAB1, SHC1 and CDK2 was measured using qRT-PCR after HCT116 cell line was overexpressed in control or miR-5582-5p. β-Actin was used as a normalizer. * P < 0.05, ** P < 0.01 (n = 3).
C. Reporter production of 3'UTR sequences of GAB1, SHC1 and CDK2 complementary to miR-5582-5p. The portion of each GAB1, SHC1 and CDK2 binding site nucleotide was changed to gray. (3T UTR-1, miR-5582-5p, Mut 3'UTR-1, WT 3'UTR-2, Mut 3'UTR-2, WT 3'UTR, Mut 3 'UTR, WT 3' UTR, and Mut 3 'UTR are attached to Sequence Listing 1 to 9).
D. Direct confirmation of miR-5582-5p and 3'UTR of GAB1, SHC1 and CDK2. The HCT116 cell line was overexpressed with MIR-5582-5p and each reporter, and luciferase activity was measured 48 hours later. Firefly luciferase activity was normalized to Renilla luciferase activity. * P &lt; 0.05 (n = 3).
4,
A. Inhibition of AKT and ERK subpathway activation by miR-5582-5p in the cell growth signaling pathway. EGF (100 ng / mL) was added to the HCT-116 cell line for 24 hours in the absence of serum or a control or overexpression of miR-5582-5p.
B. Protein expression was confirmed 48 hours after overexpression of siRN of miR-5582-5p or GAB1, SHC1 in HCT116 cell line.
C. Induction of apoptosis by simultaneous inhibition of GAB1 and SHC1. GAB1, and SHC1, respectively, or siRNA were overexpressed and inhibited, and analyzed 3 days later using a flow cytometer.
D. Expression of cell cycle-related proteins in CDK2 inhibitory effect of miR-5582-5p. Expression of miR-5582-5p, CDK2 siRNA was overexpressed in HCT116 cell line for 48 hours.
E. Cell cycle arrest following siCDK2 and miR-5582-5p overexpression.
F. Expression of miR-5582-5p by doxycycline in Tet-5582 cell line. Total RNA was obtained from Tet-C and Tet-5582 cells with doxycycline (500 ng / mL) at each time point and the expression level was measured by qRT-PCR.
G. Changes in apoptosis-related protein expression by miR-5582-5p induction. Cells were harvested at each time point after treatment with doxycycline, and western blot was performed to observe changes in protein expression.
H. Cell death by miR-5582-5p induced. Cell death was performed after doxycycline treatment for 72 hours.
I. Changes in cell cycle-related protein expression by miR-5582-5p induction.
J. Cell cycle arrest effect of induced miR-5582-5p.
5,
A. Comparison of miR-5582-5p expression in normal and cancer cells. mature miR-5582-5p and primary miR-5582 (pri-5582) were subjected to qRT-PCR.
B. miR-5582-5p inhibiting growth of normal cell line CCD-18co and HPF. Cell counts were performed at defined times after overexpression of the control (α-cont) or miR-5582-5p inhibitor (α-5582-5p), respectively. * P &lt; 0.05 (n = 3).
C. Increased expression of target protein by over-expression of α-5582-5p in normal cell lines. 48 hours after overexpression of the control (α-cont) or miR-5582-5p inhibitor (α-5582-5p), cells were obtained and protein expression was confirmed.
D. miR-5582-5p which does not inhibit cell growth in normal cell line CCD-18co and HPF. Cell numbers were measured at defined time intervals after overexpression of control or miR-5582-5p * P &lt; 0.05 (n = 3).
E. Identification of target protein expression by miR-5582-5p in normal cell line. Cells were obtained 48 hours after overexpression of control or miR-5582-5p in CCD-18co and HPF to confirm the change.
F. miR-5582-5p, which does not cause apoptosis in normal cell line CCD-18co and HPF. Three days after overexpression of control or miR-5582-5 was confirmed using a flow cytometer.
6,
AF. Induction of miR-5582-5p by Doxycyclin inhibits tumor growth in a mouse xenograft model.
A. Observation of tumor growth over time. Tet-C or Tet-5582 cell lines were subcutaneously inoculated into the hind legs of the mice, and the size and weight of the tumors were measured and compared between the feeding and non-feeding groups containing doxycycline (2 mg / ml). * P &lt; 0.05 (n = 6).
B. Tumor photograph taken from mouse.
C. Comparison of tumor weight between groups. The weight of the tumor removed from the mouse was measured and plotted.
D. Representative picture of immunochemical staining analysis performed on tumor tissue.
E. Verification of miR-5582-5p expression of tumors isolated from mice inoculated with Tet-5582 cell line. Total RNA isolated from tumor tissues was analyzed by qRT-PCR. * P &lt; 0.005 (n = 3).
F. Decreased expression of target protein in tumor by inducing expression of miR-5582-5p with Doxycycline.
GL. Intratumoral injection of miR-5582-5p in a mouse xenograft model inhibits tumor growth.
G. Tumor growth over time. After miR-5582-5p or control miRNAs were administered intratumorally using in vivo-jet PEI, the tumor size was measured and compared over time. * P < 0.01 (n = 6).
H. Typical photograph of tumor treated with miR-5582-5p or its control.
I. Analysis of tumor weights. The tumor weighed from mice inoculated with miR-5582-5p (n = 6) or control (n = 6) was weighed and displayed as a graph. * P < 0.05.
J. Photograph of tumor tissue by immunohistochemical staining analysis.
Verification of the expression level of miR-5582-5p in K. miR-5582-5p or a control-treated tumor. * p &lt; 0.005 (n = 3).
L. Reduced expression of target protein in tumors administered miR-5582-5p.

Hereinafter, the present invention will be described in detail with reference to examples.

<Materials and Methods>

Cell culture conditions and reagents

The human colon cancer cell lines HCT116, SW480, DLD-1 and HCT-15 and the arsenic lung cancer cell lines A549 and H460, the large intestine fibroblast CCD-18Co and the lung fibroblast HPF were purchased from the American Type Culture Collection And cultured in RPMI medium containing 10% FBS and penicillin (100 U / mL) at 37 ° C in a 5% CO ₂ environment. The HCT116 (TP53 + / +) and HCT116 (TP53 - / -) cell lines were obtained from Dr. B Vogelstein (Johns Hopkins University, MD). RB1, phospho-RB1, AKT, phospho-RB1, and B-ACTIN were purchased from Santa Cruz Biotechnology, Inc. GAB1, SHC1, CDL2, SOS1, BAX, BAK1, BCL2, E2F1, cyclin E, AKT, ERK, phospho-ERK, XIAP, BCL2L1 and GRB2 were purchased from Cell signaling Technology.

RNA oligonucleotides and over-expression

The synthetic miRNA mimic pool was synthesized by RNA duplex in Genolution Pharmaceuticals (Seoul, Korea), which refers to the sequence registered in the miRBase database. version 16 and 267 miRNAs registered in version 17, a newly registered miRNA. The inhibitor for miR-5582-5p was also synthesized by Genolution Pharmaceuticals as a single ribonucleotide with a 2'-o-methyl group attached. sequence is 5'-GCTATAACTTTAAGTGTGCCTA-3 '(SEQ ID NO: 10). SiRNA oligonucleotides targeting GAB1, SHC1, and CDK2, and negative control siRNA oligonucleotides used as negative controls were purchased from Santa Cruz Biotechnoloty. To perform functional analysis, all miRNA mimic or miR-5582-5p inhibitors and siRNAs were overexpressed using G-fectin (Genolution Pharmaceuticals) at a concentration of 10 nM each of fianl.

Determination of cell growth

2.5 x 10 ³ cells were overlaid on 96-well plates and overexpressed miRNAs. After 96 hours, MTS assay was performed to measure the number of living cells by absorbance at 490 nm. Cell growth rate through cell counting was determined by trypan blue method. All these experimental results were determined through three independent experiments.

Determination of soft agar colony forming ability

Control cells and miR-5582-5p over-expressing cells were removed using TE, 1000 cells were mixed with the medium to be a final 0.35% low-melting agarose, and the final 0.5% low-melting agarose The colonies were observed after 2 weeks of overlaying on a 60 mm dish. Fresh medium was added to the agarose once a week. After 2 weeks, the cells were stained with 0.005% crystal violet. The number of stained colonies was counted to confirm colony forming ability.

Cell death measurement

Apoptosis was measured by flow cytometry using annexin V-FITC / Propidium iodide (PI) staining. Analysis of dead cells in xenograft tumor tissues was performed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method using the DeadEnd Fluorometric TUNEL system (Promega) kit.

Cell cycle measurement

Cells fixed in cold 70% ethanol for 1 hour were treated with RNase A (10 μg / mL) at 37 ° C for 30 minutes and washed with PBS. PI was then treated with a final concentration of 50 ug / mL and stained for 1 hour at room temperature. The distribution of the cell cycle was measured using a flow cytometer.

Measurement of active oxygen levels in cells

The level of active oxygen in the cells was determined by staining DCF-DA (2'-7'-dichlorofluorescein diacetate, Molecular Probes). After 48 hours of overexpression of the control or miR-5582-5p cells, DCF-DA was treated with DCF-DA at a final concentration of 5 μM and incubated at 37 ° C for 1 hour to stain intracellular reactive oxygen species. The intracellular levels of active oxygen were measured by measuring the fluorescence intensity of the cells using a flow cytometer.

Reporter assay (verification of target gene)

A reporter vector was cloned to verify the target gene. The 3'UTR portion of each target gene containing the binding site of miR-5582-5p was cloned into pGL3UC vector (Fig. 3C). The primer sequence for amplifying the 3'UTR of each target gene is shown in Table 1. Three nucleotides of the binding site of miR-5582-5p were transformed into other nucleotides using the QuickChange II site-directed mutagenesis kit (Agilent Technologies, CA) to generate a mutant reporter vector. These vectors were overexpressed in HCT116 cells using Lipofectamine 2000 (Invitrogen, CA) with pRL-pRL-CMV-Renilla (Promega) plasmid (2 ng) and each miRNA and reporter activity was measured after 48 hours. Firefly luciferase activity was measured using the Dual Luciferase Reporter Assay system (Promega) kit and normalized to renilla luciferase activity. This was experimented with triplicate.

(The sequence of Table 1 above is attached as SEQ ID NOS: 11-34).

RNA isolation and qRT-PCR analysis

RNA was isolated from the cells and tissues using Trizol as a culture medium and RNeasy Mini kit (Qiagen, Germany) as a frozen tumor. 2 μg of total RNA was quantitated using the KAPA SYBR FAST one-step qRT-PCR kit. The mRNA expression levels of various genes and the expression levels of primary miRNA transcripts were determined. The expression of mature miRNA was confirmed using Mir-X miRNA qRT-PCR SYBR kit. The primer sequence specific to each gene used was also attached to Table 1, and the expression level of all genes was normalized to β-actin and U6.

Western blotting (protein expression analysis)

Prepared overexpressed cells were boiled in SDS sample buffer and SDS-PAGE was performed to isolate the proteins. The separated proteins were transferred to nitrocellulose membranes (Whatman, PA). Then, the transferred membrane was treated with 5% skim milk solution (5% skim milk, 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 40 minutes, For 2 hours or overnight at 4 ° C. The next membrane was washed three times with TBST and the secondary antibody with HRP was treated at room temperature for 1 hour. After that, the amount of protein expression was measured using a chemiluminescence system (Amersham Pharmacia Biotech, NJ).

Production of cell line for miR-5582-5p induction expression

Cell lines were prepared using the Mir-X inducible miRNA System (Clontech) kit for the induction of miR-5582-5p conditions. Briefly, the precursor part (446 bp) of miR-5582-5p was amplified on genomic DNA using a specific primer and cloned into pmiR-mCherry vector to construct pTet-5582 vector. An HCT-116 cell line with a specific gene expression by Doxycyclin or Tetracycline was over-expressed in HCT-116 cells and selected by G418 (800 ㎍ / mL) concentration. The pTet-5582 vector or the negative control pMR-mCherry vector and hygromycin linear marker were overexpressed in the prepared Tet-on advanced HCT116 cells, and the hygromycin (200 ㎍ / mL) selection for 2 weeks was used for each double stable cell line Respectively. After selection, each clone was treated with Doxycycine to confirm the expression level of miR-5582-5p.

Immunohistochemical staining

Immunohistochemical staining was performed using Ready-to-use IHC / ICC kit (Biovision, CA). Briefly, tissue paraffin was removed using xylen, rehydrated with alcohol, and incubated for 30 min at room temperature with a specific 1 (100: 1) of GAB1 (1: 100), CDK2 The secondary antibody was treated. After washing with PBS, they were treated with One-Step HRP polymer for 20 minutes at room temperature. After washing with TBST, 3,3'-diaminobenzidine (DAB) was used for staining.

Tumor xenograft model experiment

In Orientbio, a 4-week-old BABL / c nude mouse was purchased and grown in an SPF environment. At 5 weeks of age, 2x10 &lt; ⁶ &gt; colon cancer cell line HCT116 cells were subcutaneously transplanted (weight 18-20 g) into the right hind leg of the mice. When the size of the tumor reached 150 mm ³ , 10 μg of miR-5582-5p was administered twice daily at 3-day intervals using in vivo-jetPEI (Polyplus Transfection, Illkirch, France) 1.2 μL in vivo-jet PEI / 50 μL DW). In the case of miR-5582-5p-induced cell lines, the Tet-5582 cell line was also subcutaneously transplanted into the right hindlimb of the mouse and the next day, 10% of the cells were treated with Doxycycline (Sigma Aldrich, MO) After feeding on water containing crossover, mice were fed. Light was blocked to prevent the inhibition of the activity of the Doxycycline-containing material, which was changed once a day. The size of the tumor is twice weekly volume (V) mm ³ = (small diameter) ² x (largediameter) x (π / 6). According to the formula. All animal studies were conducted with the approval of the institutional animal care and use committee (IACUC) of the Korea Institute of Radiological and Medical Sciences (Seoul, Korea).

<Result>

Selection of miR-5582-5p effective for growth inhibition through functional screening

To find new miRNAs that regulate the growth of cancer cells, we synthesized 16 versions of the miRBase database and 267 miRNA pools registered in 17 versions. This was the most recently registered miRNA at the time of the start of the study. Each miRNA in the miRNA pool was overexpressed in the colon cancer cell line HCT116 and the cell growth rate was measured by performing the MTS assay. Ten primary miRNAs were selected and screened for secondary cell growth by primary screening. (FIGS. 1A and B). Primary and secondary screening were used to select five miRNAs that had the effect of inhibiting growth. Of the five selected miRNAs, miR-5582-5p, which most significantly inhibited the growth of HCT116 cells, was selected and other miRNAs selected to modulate cell growth proceeded in subsequent studies (Fig. 1C).

Cell death induction and cell cycle arrest by miR-5582-5p of cancer cells

The growth inhibitory effect of miR-5582-5p was once again verified by measuring the number of cells at regular intervals after overexpression. miR-5582-5p markedly inhibited the growth of two colon cancer cell lines HCT116 and SW480 (Fig. 2A). Colony forming ability was also decreased by miR-5582-5p (Fig. 2B). Cell cycle analysis was performed to confirm that miR-5582-5p caused a G1 arrest by increasing the population of the G1 group and decreasing the population of the S group in the HCT116 cell line (Fig. 2C). Next, the cells were stained with Annexin V / PI, and the apoptotic effect of miR-5582-5p was confirmed using a flow cytometer. Cell death was significantly increased in miR-5582-5p in HCT116 and SW480 cells (Fig. 2D). However, there was no difference in the amount of active oxygen in the cells at this time (Fig. 2E). This indicates that miR-5582-5p-induced apoptosis is not associated with intracellular reactive oxygen species. Since TP53 plays a major role in cell death by various stimuli, we have also examined whether miR-5582-5p-induced apoptosis is due to TP53 status. When miR-5582-5p was overexpressed in the HCT116 cell line with the genetic traits of TP53-wild type (TP53 + / +) or TP53-null (TP53 - / -) and their cell death rates were measured, (Fig. 2F). This suggests that miR-5582-5p-induced apoptosis is caused by the TP53 independent pathway.

Negative regulation of GAB1, SHC1 and CDK2 in miR-5582-5p

TargetScan (http://www.targetscan.org/), miRDB (http://mirdb.org/miRDB/) and DIANA-microT 3.0 (http : //diana.cslab.ece.ntua.gr/). (GAB1), Src homology 2 domain containing 1 (SHC1), and growth factor receptor-bound protein 2 (GRB2), which are involved in the regulation of the pro-mitogenic / survival pathway of receptor tyrosine kinases (RTKs) guanine nucleotide exchange factor SOS1, and cyclin-dependent kinase 2 (CDK2), which plays an important role in cell cycle progression. Therefore, we first determined the expression of target proteins by miR-5582-5p in several colorectal cancer cell lines.

As a result, GAB1, SHC1 and CDK2 were significantly changed by miR-5582-5p in all cell lines, but SOS1 did not change (Fig. 3A). On the other hand, the expression of GRB2 showed a tendency different for each cell line. GRB2 was decreased in HCT116 and HCT15 cell lines but increased in SW480 and DLD-1 cell lines. This suggests that regulation of GRB2 by miR-5582-5p is thought to be mediated through an indirect mechanism depending on the intracellular environment rather than by direct targeting, thereby eliminating GRB2 from direct target protein candidates. Similar to protein expression, mRNA expression of GAB1, SHC1 and CDK2 was also decreased when miR-5582-5p was overexpressed in HCT116 cells (Fig. 3B).

Therefore, we performed a reporter assay to determine whether the direct target gene of miR-5582-5p is GAB1, SHC1 and CDK2. We found the 3'UTR of each mRNA that would be complementary to the miR-5582-5p seed region. To determine if the sites were directly targeted by miR-5582-5p, we used the sequence of the complementary sequence in the miR-5582-5p seed region downstream of the firefly luciferase open reading frame of the modified pGL3 reporter vector A 3 'UTR portion of the target protein was cloned into a reporter vector (FIG. 3C).

To investigate the target-specificity of miR-5582-5p for each target protein, a mutant reporter was constructed in which three nucleotides were mutated at the binding site of the seed sequence. The luciferase activity of miR-5582-5p and miR-5582-5p were overexpressed in the HCT116 cell line. As predicted, all wild-type (3'UTR) reporters were reduced in luciferase activity by miR-5582-5p. (Fig. 3D). In contrast, all mutant-type 3'UTR reporters did not show any change in luciferase activity by miR-5582-5p. This means that the expression of GAB1, SHC1, and CDK2 is directly inhibited by miR-5582-5p. FIG. 3C schematically shows a specific sequence and region in which miR-5582-5p binds to control the expression of each target protein.

Cell death and cell cycle arrest effect of miR-5582-5p through GAB1 / SHC1 and CDK2 inhibition

Since GAB1 and SHC1 play a major role in Ras-MAP kinase activity and the downstream of activated RTKs is the PI3K-Akt / PKB pathway, we observed the effect of miR-5582-5p on ERK and AKT activity after growth factor EGF treatment .

When HCT116 cells were treated with EGF immediately after serum starvation, phosphorylation of ERK and AKT was decreased in the over-expression condition of miR-5582-5p (FIG. 4A). This shows that the survival pathway is inhibited by miR-5582-5p. To determine whether the role of miR-5582-5p, which inhibits the growth of cancer cells, is through the inhibition of the target gene, we artificially suppressed the target gene and examined whether it is similar to the effect of miR-5582-5p. First, inhibition of GAB1 and SHC1 using siRNA strongly induced apoptosis as shown by miR-5582-5p. On the other hand, when GAB1 and SHC1 were inhibited, apoptosis was weak (Fig. 4B). Overexpression of miR-5582-5p reduced the phosphorylation of ERK and AKT and their anti-apoptotic proteins, XIAP, BCL2, and BCL2L1. (Fig. 4C). However, the pro-apoptotic proteins BAX and BAK1, which are inhibited by ERK or AKT, have increased. Similar to the effect of miR-5582-5p, the inhibition of GAB1 and SHC1 resulted in decreased phosphorylation of ERK and AKT, thereby decreasing anti-apoptotic protein expression and pro-apoptotic protein expression. These results suggest that inhibition of ERK and AKT pathway by simultaneous inhibition of GAB1 and SHC1 is sufficient to induce apoptosis and that most of the apoptosis by miR-5582-5p occurs via inhibition of GAB1 and SHC1 . Inhibition of CDK2 using siRNA, as in miR-5582-5p overexpression conditions, resulted in a change in the cell cycle distribution in which the G1 population increased (Fig. 4D). Overexpression of miR-5582-5p or siCDK2 reduced phosphorylated retinoblastoma 1 (p-RB1) and PCNA, and E2F1 and cyclin E expression did not change (Fig. 4E). It can be seen that CDK2 regulates cell cycle arrest as a direct target of miR-5582-5p.

To further study the function of miR-5582-5p, Tet-5582 cell line was constructed in which miR-5582 expression was induced by Tetracycline. The expression level of miR-5582-5p after 72 hours of Doxycycline treatment in Tet-5582 cell line was 8 times higher than that of the control group. (Fig. 4F). The ability of miR-5582-5p to induce apoptosis was again verified by confirming that Doxycycline induces apoptosis in the Tet-5582 cell line (Fig. 4G). The apoptosis induced by Doxycycline-induced miR-5582-5p induces the inhibition of GAB1, SHC1 and GRB2 expression as well as the decrease of XIAP, BCL2L1 and BAX expression as previously confirmed at the cellular level (Fig. 4H). The expression of miR-5582-5p induced by Doxycycline treatment on Tet-5582 cells was also confirmed to increase the G1 phase by inducing phosphorylation of CDK2, RB1 and reduction of PCNA expression (Fig. 4 I, J), miR-5582- 5p-induced cell cycle arrest was caused by targeting of CDK2.

miR-5582-5p is more expressed in normal cells than cancer cells and does not induce apoptosis in normal cells

To investigate the possibility of miR-5582-5p as a cancer-suppressing gene, which causes apoptosis and inhibits the growth of cancer cells through cell cycle arrest. First, we compared miR-5582-5p expression in cancer cells and normal cells of the same origin. The expression of miR-5582-5p was significantly higher in the colon-derived normal cell CCD-18-co than in the colon cancer cell lines HCT116 and SW480. Human pulmonary fibroblasts (HPFs) also showed higher expression than lung cancer cell lines A549 and H460 (Fig. 5A, top). pri-miR-5582, an early transcription factor for miR-5582-5p, also showed a higher expression pattern in normal cells. (Fig. 5A, bottom). This suggests that the expression of miR-5582-5p in cancer cells is suppressed from the transcriptional stage.

We next examined the effect of inhibitors of miR-5582-5p on the growth of miR-5582-5p-expressing normal cell lines. When the miR-5582-5p inhibitor was overexpressed, the cell growth was significantly increased in both CCD-18Co and HPF cells (Fig. 5B), indicating that miR-5582-5p expression in normal cells was balanced The inhibitory effect of miR-5582-5p inhibitor on the expression of GAB1 and SHC1 in both cell lines was increased by the overexpression of miR-5582-5p inhibitor , Whereas there was no significant increase in CDK2 (Figure 5C).

Next, it was confirmed whether miR-5582-5p was effective for the growth of normal cells. Overexpression of miR-5582-5p did not inhibit the growth of CCD-18Co and HPF cells (Fig. 5D). Expression of the target protein, which was already low, was further reduced by miR-5582-5p (Fig. 5E). In addition, cell death by overexpression of miR-5582-5p did not occur in normal cell CCD-18Co and HPF (Fig. 5F). These results suggest that miR-5582-5p, a cancer suppressor gene, is an important cancer cell specific inhibitor of cell death and cell growth inhibition.

Inhibition of tumor growth of miR-5582-5p in mouse

To confirm the inhibitory effect of miR-5582-5p in vivo in cancer, two different methods increased the expression of miR-5582-5p in a mouse xenograft model of cancer cells. In the first method, a cell line in which miR-5582-5p expression was induced by Doxycycline was injected into a xenotransplantation model. Tet-5582 was designated as a control and Tet-C was designated as a control. Tet-5582 cells were injected and Dox-fed mice significantly decreased cancer cell growth compared to mice that did not feed Dox (Fig. 6A-C). On the other hand, Tet-C cell injected group did not differ in tumor size regardless of whether Dox was fed or not. Immunohistochemical analysis showed that PCNA expression was decreased and TUNEL staining rate was higher in the Dox-fed group after injection of Tet-5582 cells (FIG. 6D) than in the control group (FIG. 6D) And cell death was induced.

MiR-5582-5p and target protein were quantitated in tumor tissues isolated from mouse, and the expression of miR-5582-5p was examined in Dox-fed group after xenotransplantation of Tet-5582 cells. Expression of target protein (Fig. 6E and F). The miRNA delivery system was used as an alternative. The HCT116 cell line was injected into the tumor, and miR-5582-5p and control miRNAs were injected into the complex using in vivo over-expressing agents. After three injections at 3-day intervals, tumor growth was significantly reduced in the group overexpressing miR-5582-5p compared to the control group (Fig. 6G-I). Correspondingly, the expression of PCNA was decreased in the overexpressing group of miR-5582-5p, and the proportion of TUNEL-stained cells was increased (Fig. 6J). miR-5582-5p and target protein were measured in tumor tissues isolated from the mice 20 days after the last injection of miRNA. The expression of miR-5582-5p was significantly increased by 70-fold compared with that of the control, and the expression of the target protein was significantly decreased (Fig. 6K and L), indicating that miR- It has been clearly demonstrated that it has an effective function as a repressor gene, which shows a high possibility as an anticancer drug.

<Review>

Recent studies have shown that a number of miRNAs are abnormally expressed in a variety of cancers, and they play a very important role in the development of cancer as a cancer-inducing or cancer-suppressing gene. In the present invention, miR-5582-5p, a novel cancer-suppressing miRNA having the function of inducing apoptosis and cell cycle inhibition of cancer cells, was unearthed. To investigate miRNAs that can regulate the growth of cancer cells, miRNA pools of recently registered 267 miRNAs were synthesized and used at the beginning of the study to discover miRNAs that affect the growth of cancer cells without any previous studies. Functional screening was performed after overexpression of each miRNA mimic in cancer cells.

The results showed that many miRNAs significantly promoted or inhibited cell growth, and at the same time, this functional screening was an effective method. This study was carried out by selecting miR-5582-5p, which most effectively inhibits the growth of HCT116, a colon cancer cell line. (Other miRNAs selected as candidates for regulating the growth of cancer cells are also being studied in other studies .) Several target genes have been identified that play an important role in inducing apoptosis and inhibiting growth of miR-5582-5p. Among the identified genes, GAB1 and SHC1 are constitutive proteins of the complex formed on the cytoplasmic side to mediate the downstream signaling pathways of RTKs such as EGFR. These proteins play an important role in promoting cell division by activating Ras-MAP kinase and PI3K-Akt / PKB pathway in combination with activated RTK. Although SHC1 is often referred to as promoting apoptosis as an oxidation promoting protein among its isoforms, many studies have suggested that GAB1 and SHC1 play an important role in carcinogenesis in various cancers. Recently, miRNAs that directly target GAB1 or SHC1 have been reported. miR-150 inhibits B-cell receptor signaling by inhibiting the target gene GAB1 in acute lymphoblastic leukemia. miR-409-3p was also reported to partially inhibit the metastasis of colorectal cancer by inhibiting GAB1 expression. miR-365 has both SHC1 and BAX as target genes that induce resistance to gemcitabine in pancreatic cancer. Simultaneously inhibiting GAB1 and SHC1 through direct targeting appears to play an important role in miR-5582-5p-induced apoptosis. Inhibition of GAB1 and SHC1 simultaneously inhibits the apoptotic proteins XIAP and BCL2L1 as well as molecular events such as increasing the expression of the pro-apoptotic protein BAK1, as well as cell death by miR-5582-5p (Fig. 4B). Compared to inhibiting GAB1 or SHC1 alone, simultaneous inhibition of two cancer proteins by miR-5582-5p could more effectively inhibit cancer-induced signaling and further increase cell death. This is a great advantage as a cancer-inhibiting miRNA. GRB2 complexed with GAB1 and SHC1 was also included in the target protein list of miR-5582-5p, but expression of GRB2 by miR-5582-5p was cell-line dependent (FIG. 3A). These results suggest that although GRB2 is unlikely to be a direct target protein of miR-5582-5p, changes in the expression of GRB2 will contribute to the function of miR-5582-5p by increasing or decreasing the activity of inhibiting RTK signaling in an environmentally dependent manner .

MiR-5582-5p also terminated the cell cycle by directly targeting CDK2. CDK2, a checkpoint regulator of G1, plays a crucial role in regulating cell cycle progression and has been studied to be inhibited by several miRNAs. For example, miR-885-5p inhibits CDK2 and inhibits cell proliferation and survival of neuroblastoma through activation of TP53. MiR-29b, a well-known cancer suppressor miRNA, also inhibits CDK2 and regulates the growth of epithelial cells. The cell cycle arrest induced by CDK2 inhibition contributes to the effective activity of miR-5582-5p, which inhibits the growth of cancer cells, in addition to apoptosis through inhibition of GAB1 and SHC1 expression. The advantage of miR-5582-5p as a cancer suppressor gene is the cancer cell specific effect. The expression of miR-5582-5p is higher than that of normal cells, CCD-18co and HPF, and its target protein shows lower expression in normal cells than cancer cells. Unlike cancer cells, miR-5582-5p overexpression in normal cells did not affect normal cell growth or apoptosis, but inhibition of miR-5582-5p facilitated normal cell growth (Fig. 5). The persistent activity of RTK signaling, which is related to overexpression of GAB1 and SHC1 for cancer cell growth, may contribute to the effect of miR-5582-5p and the presence of miR-5582-5p in normal cells lacking RTK signaling Can contribute to survival. However, it is not clear at this time whether this assumption is true or whether there are other factors contributing to the cancer cell-specific miR-5582-5p role. The mechanisms of cancer cell-specific miR-5582-5p need to be systematically analyzed in the future, and understanding these mechanisms is very important for establishing a more sophisticated strategy for treating cancer cells with minimal damage to normal cells It will be effective.

Because of the various advantages of miRNA as a therapeutic, many studies focus on the development of miRNA therapeutics for cancer treatment. MiRNAs that inhibit cancer The use of miRNA inhibitors with overexpression or cancer-inducing function of synthetic mimic is a key approach to developing anticancer miRNA therapeutics. A very good example of this is the clinical or preclinical miR-34 mimic and miR-21 inhibitors for the development of liver cancer therapies. Through this study, miR-5582-5p is also expected to be a good candidate for the development of miRNA therapeutics. We demonstrated the anticancer effect of miR-5582-5p using either of these two methods, overexpression of miR-5582-5p through miR-5582-5p over-expressing cell line or direct injection into tumor xenograft model. As a result, it has been demonstrated that miR-5582-5p effectively inhibits cancer growth and target protein expression in vivo as well as in cells. These results demonstrate the potential of miR-5582-5p as a tumor suppressor in patients (Figure 6). In contrast to miR-34, which requires TP53 activity to inhibit cancer, miR-5582-5p did not require TP53 activity in inducing apoptosis (Fig. 2F). Considering that TP53 is often inactivated or not expressed in cancer patients, the independence of TP53 is a good advantage for future clinical applications of miR-5582-5p.

This study uncovered miR-5582-5p as a novel cancer-inhibiting miRNA. The mechanism of miR-5582-5p is depicted in FIG. In brief, miR-5582-5p caused cancer cell death and cell cycle arrest through inhibition of GAB1 and SHC1 and inhibition of CDK2. The anti - cancer effect was verified by in vivo experiment using cell level and mouse. As far as we know, this is the first study to demonstrate the function of miR-5582-5p. miR-5582-5p has an advantage as an anticancer miRNA therapeutic agent having strong growth inhibitory activity because it inhibits various related proteins simultaneously, independent of the activity of TP53 and specific to cancer cells only. Through this study, miR-5582-5p is a powerful cancer-inhibiting miRNA that can be used to develop new anti-cancer miRNA therapeutics.

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Claims (1)

An anticancer agent comprising miR-5582-5p.

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