KR20230102595A - Nucleic acid fragment specifically binding to the 3' untranslated region of the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene and applications thereof - Google Patents

Abstract

The present invention relates to a nucleic acid fragment of SEQ ID NO: 1 that specifically binds to the 3' untranslated region (UTR) of a phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene and cervical cancer treatment comprising the nucleic acid fragment as an active ingredient, and It relates to the use of diagnostic compositions and the like.

Description

Nucleic acid fragment specifically binding to the 3' untranslated region of the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) genes and applications thereof}

The present invention relates to a nucleic acid fragment that specifically binds to the 3' untranslated region (UTR) of the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene and its applications.

Cervical cancer is the fourth most common cancer in women worldwide. In 2020, approximately 598,000 new cases and 338,800 deaths from cervical cancer were reported worldwide. The main cause of cervical cancer is known to be persistent high-risk human papillomavirus (HR-HPV) infection.

For cervical cancer screening, the HR-HPV test and the Papanicolaou test are performed on a single sample called the HPV/Pap cotest. This enables early diagnosis and treatment in the precancerous stage. Nevertheless, according to FIGO (International Federation of Gynecology and Obstetrics), about 50% of cervical cancer patients worldwide are at an advanced stage that cannot be treated with surgery.

Currently, the standard treatment for advanced cervical cancer is chemoradiation. However, patients with advanced disease have a high recurrence rate even with treatment. Treatments for patients with ongoing advanced stages only serve to relieve symptoms and prolong survival because the patients have recurrent or metastatic disease. Therefore, new therapies for advanced cervical cancer are needed.

Recently, oncogene-targeted therapy, which is a method of targeting and inhibiting oncogenes, has been intensively studied. As a result, there are several oncogene-targeted therapies that have received FDA approval. For example, trastuzumab, also known as Herceptin, treats breast and stomach cancer by selectively attacking the receptor tyrosine-protein kinase erbB-2 (HER-2) involved in tumor growth. In addition, vemurafenib is indicated for metastatic melanoma patients with serine/threonine-protein kinase B-Raf (BRAF) gene mutations.

The phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene is a major oncogene in cervical cancer. For example, cervical cancer patients with PIK3CA mutations had more mutations in cancer-related genes. In addition, according to The Cancer Genome Atlas (TCGA) database using the cBioPortal bioinformatics tool (http://www.cbioportal.org/), both gene mutation and copy number amplification frequencies of the PIK3CA gene are high in cervical cancer. Therefore, inhibition of PIK3CA expression seems to be useful for the treatment of cervical cancer. There are also preclinical studies showing that PI3K inhibitors reduce oncogenic activity in cervical cancer cells.

Recently, studies on microRNAs (miRNAs, miRs) have been actively conducted. MicroRNAs are small noncoding RNAs of 18-25 nucleotides that form miRNA-protein complexes called RNA-induced silencing complexes and bind to the 3'-untranslated region (UTR) of target mRNAs. In animals, the complementary sequence of the target mRNA is bound by 2-7 nucleotides at the 5' end of the miRNA, called the seed region. Perfect binding of the seed region to the target mRNA leads to degradation of the mRNA, whereas incomplete binding leads to translational inhibition of the mRNA. MicroRNAs are known to regulate the expression of oncogenes or tumor suppressor genes to promote or inhibit tumorigenesis. Tumor suppressor miRNAs are widely studied in therapeutic research.

Several studies have shown that miR-29a acts as an inhibitor of NAD-dependent deacetylase sirtuin-1 (SIRT1), cell division regulatory protein 42 homologue (CDC42), and cytokine signaling 1 (SOCS1). .

[Prior Patent Literature]

Republic of Korea Patent Publication No. 10-2016-0103949

The present invention has been made in response to the above needs, and an object of the present invention is to provide a nucleic acid fragment that specifically binds to the 3' untranslated region (UTR) of the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene.

Another object of the present invention is to provide a novel composition for diagnosing cervical cancer.

Another object of the present invention is to provide a novel composition for treating cervical cancer.

Another object of the present invention is to provide a novel method for providing information on cervical cancer.

In order to achieve the above object, the present invention provides a 3' untranslated region (UTR) of the phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (hereinafter referred to as 'PIK3CA') gene. Provides a nucleic acid fragment of SEQ ID NO: 1 (AGCACCA; SEQ ID NO: 1) that specifically binds to.

In addition, the present invention provides a composition for detecting phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene comprising the nucleic acid fragment (AGCACCA) of SEQ ID NO: 1 as an active ingredient.

In addition, the present invention provides a composition for diagnosing cancer comprising the nucleic acid fragment of SEQ ID NO: 1 as an active ingredient.

In one embodiment of the present invention, the cancer is preferably cervical cancer, but is not limited thereto.

In addition, the present invention provides a composition for treating cancer comprising the nucleic acid fragment of SEQ ID NO: 1 as an active ingredient.

In one embodiment of the present invention, the cancer is preferably cervical cancer, but is not limited thereto.

In another embodiment of the present invention, the composition preferably inhibits the ability of cancer cells to migrate and invade, but is not limited thereto.

In addition, in the present invention, when the gene pool obtained from the sample in vitro has a gene that binds to the nucleic acid fragment of SEQ ID NO: 1 by processing the nucleic acid fragment of SEQ ID NO: 1, the sample is phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA ) Provides a method for providing information on the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene, which is determined to contain the gene.

In one embodiment of the present invention, the binding of the nucleic acid fragment to the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene is preferably performed by a dual luciferase assay, but is not limited thereto.

In addition, the present invention processes the nucleic acid fragment of SEQ ID NO: 1 in the gene pool obtained from the sample in vitro and determines that the sample contains a cervical cancer-related gene when there is a gene that binds to the nucleic acid fragment of SEQ ID NO: 1 Provides a method for providing information on cervical cancer.

The pharmaceutical composition for treating cancer comprising the nucleic acid fragment of the present invention as an active ingredient may further include a pharmaceutically acceptable carrier and may be formulated together with the carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate organisms and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

The composition for preventing or treating cancer comprising the nucleic acid fragment and a pharmaceutically acceptable carrier of the present invention can be applied in any formulation containing the nucleic acid fragment as an active ingredient, and can be prepared as an oral or parenteral formulation. The pharmaceutical formulations of the present invention may be taken oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). and intravenous) or forms suitable for administration by inhalation or insufflation.

Formulations for oral administration containing the composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do. For formulation into dosage forms such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, massene stearate Lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be included, and in the case of a capsule formulation, a liquid carrier such as fatty oil may be further included in addition to the above-mentioned materials.

The compositions described herein can be formulated as pharmaceutical compositions and administered to mammalian hosts, such as human patients, in a variety of forms. The form may be particularly suitable for the selected route of administration, eg oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.

Formulations for parenteral administration containing the composition of the present invention as an active ingredient include injection forms such as subcutaneous, intravenous, or intramuscular injections, suppository injections, or sprays such as aerosols that enable inhalation through the respiratory tract. can be formulated into In order to formulate an injectable formulation, the composition of the present invention may be mixed in water with a stabilizer or buffer to prepare a solution or suspension, which may be formulated for unit administration in an ampoule or vial. For injection as a suppository, it may be formulated into a composition for rectal administration such as a suppository or an enema containing a conventional suppository base such as cocoa butter or other glycerides. When formulated for spraying, such as aerosols, propellants and the like may be blended with additives so that the concentrated concentrate or wet powder dispersed in water is dispersed.

Useful dosages of the compositions described herein can be determined by comparing their in vitro and in vivo activities in animal models. Methods for extrapolating effective amounts to humans in mice and other animals are known in the art; See, eg, US Patent No. 4,938,949 (Borch et al.). The amount of the active ingredient of the present invention required for use in treatment depends not only on the particular compound or salt selected, but also on the route of administration, the nature of the disease being treated, and the age and condition of the patient, and ultimately the discretion of the participating physician or clinician.

In general, however, a suitable dosage ranges from about 10 to about 75 mg/kg/day, such as about 0.05 to about 100 mg/kg of body weight per day, for example about 0.1 to about 50 mg/kg of body weight of the recipient per day. would.

As can be seen through the present invention, the result of the present invention is that the 3' UTR of PIK3CA mRNA is one of the target mRNAs of the nucleic acid fragment of SEQ ID NO: 1 of the present invention, comprising the nucleic acid fragment of SEQ ID NO: 1 of the present invention It was demonstrated that overexpression of the gene down-regulated the expression level of the PIK3CA gene.

Moreover, the results of cell migration and invasion indicated that the overexpression of miR-29a comprising the nucleic acid fragment of SEQ ID NO: 1 of the present invention inhibits tumor formation in cervical cancer through the effect of regulated expression of PIK3CA.

Therefore, the results of the present invention suggest that the nucleic acid fragment of SEQ ID NO: 1 of the present invention can be used as a new therapeutic agent for cervical cancer by targeting the 3' UTR of PIK3CA.

Figure 1 is a picture showing the expected miR-29a binding site in the 3' UTR of PIK3CA mRNA,
Prediction of the mammalian miR-29a target site in the PIK3CA gene using TargetScan software (http://www.targetscan.org/). The TargetScan algorithm searches the UTR of mRNA for segments that are numbered at the 5' end and have perfect complementarity to bases 2-8 of the miRNA, referred to as the miRNA seed region.
Figure 2 is a diagram showing multiple alignment of PIK3CA primer sets for detecting mRNA of PIK3CA,
In primer design, PIK3CA mRNA RT-qPCR primer candidates were selected from Integrated DNA Technologies (IDT, Coralville, IA, USA) Primer Quest Tool, Oligo Analyzer Tool, Basic Local Alignment Search Tool (BLAST by NCBI, Bethesda, MD, USA) and PIK3CA Selected by a sequence of multiple alignments to detect only the mRNA of
Figure 3 is a picture showing the expression level of miR-29a in human cervical cancer cell lines, RT-qPCR analysis was used with cDNA of cell line RNA. Relative miR-29a expression levels were compared with RNU6B Ct values. Data were expressed as mean with standard deviation (SD). P values were calculated using Student's t-test and Kruskal-Wallis nonparametric test. **P <0.01; ***P < 0.001,
Figure 4 is a picture showing the effect of LipofectamineTM on the cell viability of HeLa cells,
A 96-well plate was seeded at a density of 1 x 10 ⁴ HeLa cells/well. After 24 hours incubation, cells were treated with miR-29a and scrambled miRNA. Cell viability of Hela cells was assessed using the MTT assay. Absorbance was measured at 540 nm with a spectrophotometer (Tecan).
Figure 5 is a picture showing the gel retardation analysis for optimization of the concentration of miR-29a using Lipofectamine ™,
Each volume (0, 1, 3, 5, 7, 9 μl) of Lipofectamine ^TM complexed with a constant amount of miR-29a, 100 nM. Each miR-29a and Lipofectamine™ complex was loaded into wells of a 2% agarose gel, electrophoresed at 100V for 15 minutes, and visualized with the Gel Doc Imaging System.
Figure 6 is a diagram showing the effect of miR-29a on the cell viability of HeLa cells,
(A) HeLa cells (1 x 10 ⁴ cells/well) were transfected with 25 nM, 50 nM, 100 nM, and 200 nM of miR-29a and 1, 2, 3, 6, or 12 μl of Lipofectamine ^TM .
(B) 200 nM and 400 nM concentrations of scrambled miRNA and miR-29a, respectively, using Lipofectamine™ for 24 hours. MTT assay measured viable cells in HeLa cells. Absorbance was measured at 540 nm. All experiments were performed independently in triplicate. Data were expressed as mean with standard deviation (SD). P values were calculated using Student's t-test. ***P < 0.001,
7 is a diagram showing that miR-29a directly binds to the 3' UTR of PIK3CA mRNA. (A) Wild-type and mutant vectors of PIK3CA 3' UTR were constructed as potential binding sequences using a gene cloning service.
(B) Luciferase activity of PIK3CA wild type and PIK3CA mutant plasmids was measured in HeLa cells cotransfected with miR-29a or scrambled miRNA. Relative luciferase activity was calculated as firefly luciferase activity versus Renilla luciferase activity. All experiments were performed independently in triplicate. Data were expressed as mean with standard deviation (SD). P values were calculated using Student's t-test. ***P < 0.001,
Figure 8 is a diagram showing PIK3CA expression using scrambled miRNA and miR-29a treatment in HeLa cells at the mRNA level,
HeLa cells were transfected with miR-29a and scramble miRNA for 24 hours, and total RNA of HeLa cells was synthesized into cDNA using an oligo dT primer. The relative expression level of PIK3CA mRNA was compared to the GAPDH expression level.
All experiments were independently performed in triplicate and data are presented as mean with standard deviation (SD). P values were calculated using Student's t-test. ***P < 0.001,
Figure 9 is a figure showing the expression level of PIK3CA mRNA in SiHa and C33A cells,
(A) SiHa and (B) C33A cells were transfected with miR-29a and scrambled miRNA for 24 hours. The relative expression level of PIK3CA mRNA was compared to the GAPDH expression level.
All experiments were independently performed in triplicate and data are presented as mean with standard deviation (SD). P values were calculated using Student's t-test. **P <0.05; ***P < 0.001,
Figure 10 is a picture showing PIK3CA expression with scrambled miRNA and miR-29a treatment in HeLa cells at the protein level.
(A) HeLa cells were transfected with miR-29a and scrambled miRNA for 24 hours, and 30 μg of cell lysate was used for Western blotting.
The protein expression level of PIK3CA in HeLa cells transfected with miR-29a was compared with scrambled miRNA through Western blotting. The PIK3CA protein expression level was calculated as the GAPDH protein expression level serving as an internal control.
(B) ImageJ measured the intensity of protein bands.
Figure 11 is a diagram showing the effect of miR-29a on the cell migration ability of HeLa cells.
HeLa cells were transfected with the miR-29a mimic and after miRNA scrambled for 24 hours, the cells were scratched to scratch a 500 μm wide wound gap. Wound gaps were (A) photographed under a microscope at 0, 24, 48 and 72 hours and (B) graphed using ImageJ software.
Data were expressed as mean with standard deviation (SD). All experiments were performed independently in triplicate.
Figure 12 is a picture showing the effect of miR-29a on cell invasion of HeLa cells,
After HeLa cells were transfected with miR-29a mimics and scrambled miRNAs, the cell invasion ability was analyzed using a transwell invasion assay. Cell invasion was counted after 24 hours and the percentage of invaded cells compared to the negative control was calculated.
The lower surface of the transwell insert was (A) photographed in a random field under a microscope and (B) infiltrated cells were counted using ImageJ software.
All experiments were performed independently in triplicate. Data were expressed as mean with standard deviation (SD). P values were calculated using Student's t-test. ***P < 0.001

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1. Cell culture

Human cervical cancer cell lines (SiHa, ATCC HTB-35; HeLa, ATCC CCL-2; C33A, ATCC HTB-31) were purchased from the American Type Culture Collection (ATCC, VA, USA).

SiHa, HeLa and C33A cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% streptomycin-penicillin (Gibco) . All cell lines were incubated at 37° C. in a humidified 5% CO2 incubator. Cells were subcultured at 80-90% confluence.

Table 1 shows the biological characteristics of cervical cancer cell lines used in the present invention.

Example 2. Isolation of total RNA from cells

Total RNA was isolated from cervical cancer cell lines using the RNeasy Mini Kit (Qiagen, Hilden, Germany).

Briefly, supernatants of cell cultures were removed, and cells in 6-well culture plates were washed three times with phosphate buffered saline (PBS; Gibco). After removing the PBS, 600 μl of lysis buffer RLT supplemented with 6 μl of β-mercaptoethanol was added to the cells and mixed well by pipetting. The cell lysate was then transferred to a 1.5 ml microcentrifuge tube, 600 μl of 70% ethanol was added and mixed by pipetting.

Then, 600 μl of the lysate was loaded onto the RNeasy mini column in a 2 ml collection tube and centrifuged at 8,000 x g for 15 seconds (seconds).

The flow-through was discarded and an additional 600 μl of lysate was loaded onto the RNeasy mini column and centrifuged again at 8,000 x g for 15 seconds. Then, 700 μl of Buffer RW1 was added to the RNeasy column and centrifuged at 8,000 x g for 15 seconds. The flow through was discarded and 500 μl of Buffer RPE was added to the RNeasy mini column and centrifuged at 8,000 x g for 15 seconds. The flow-through was scrapped. 500 μl of buffer RPE was added back to the RNeasy mini column and centrifuged at 8,000 x g for 2 minutes (min). The RNeasy mini column was transferred to a new collection tube and centrifuged for 1 minute at maximum speed. The RNeasy mini column was then placed in a new 1.5 ml microcentrifuge tube.

50 μl of DEPC-treated water was added to the mini column and centrifuged at 10,000 x g for 1 minute to obtain total RNA. The purity and concentration of total RNA was determined by the absorbance ratio at 260 and 280 nm using an Infinite 200 spectrophotometer (Tecan, Austria). The processing procedure of total RNA was performed in a laminar flow hood under RNase-free conditions. Isolated total RNA was stored at -80 ℃ until use.

Example 3. DNase treatment and complementary DNA (cDNA) synthesis

The isolated RNA was treated with a Turbo DNase kit (Life Technologies, Carlsbad, CA, USA) to digest the dsDNA template and used for cDNA synthesis.

Briefly, 1.5 μl of DEPC, 2.5 μl of TURBO DNase buffer and 1 μl of TURBO DNase were added to 20 μl of total RNA sample. After incubation at 37 °C for 30 min, 3 μl of DNase inhibitor was added and tapped for 5 min in a flow hood. Thereafter, centrifugation was performed briefly at full speed and the supernatant was transferred to a 1.5 ml microcentrifuge tube.

For miRNA, complementary DNA (cDNA) reverse transcription was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA).

In brief, 1–10 ng of RNA was used for cDNA synthesis. The reverse transcriptase (RT) mixture consisted of 0.15 μl of 100 mM dNTP mixture (100 mM each of dATP, dGTP, dCTP and dTTP at neutral pH), 0.25 μl of 200 U/μl murine moloney leukemia virus reverse transcriptase (MMLV-RT; Invitrogen, Carlsbad). , CA, USA), 1.5 μl of 10X reverse transcriptase buffer, 0.19 μl of 20 U/μl RNase inhibitor and 3 μl of 5X miRNA-specific primer were mixed and the volume of the RT mixture was adjusted to 15 μl with nuclease-free water. The following TaqMan small RNA assay (Applied Biosystems) primers were applied: RNU6B and hsa-miR-29a.

The cDNA synthesis reaction was performed at 16°C for 30 minutes, 42°C for 30 minutes, and 85°C for 5 minutes.

For mRNA, cDNA was synthesized by PrimeScript RT Master Mix (PrimeScript RTase, RNase Inhibitor, random 6-mers, Oligo dT Primer, dNTP mixture and buffer, Takara, Shiga, Japan).

Briefly, 10 μl of Prime Script RT Master Mix, RNase free water and 2500 ng of total RNA were mixed. Incubate the PCR reaction in 50 μl for 60 min at 37 °C and 5 s at 85 °C. Cooling was done at 4°C.

Example 4. RT-qPCR for miRNA-29a expression analysis

MicroRNA expression was quantified using a TaqMan small RNA assay (Applied Biosystems) using miRNA-specific primers to determine the cycle threshold (Ct), which is the number of PCR cycles required for fluorescence to exceed a value significantly above background fluorescence. .

Briefly, 2 μl of cDNA was added to 10 μl of 2x Thunderbird probe qPCR mix (Toyobo, Osaka, Japan) along with 1 μl of 20x miRNA-specific primers and 7 μl of nuclease-free water in a final volume of 20 μl. . RT-qPCR reactions were performed on a CFX96 Real-Time PCR System Detector (Bio-Rad, Hercules, CA, USA). Samples were run in duplicate for each experiment.

Data were calculated with the comparative Delta Ct method (2-ΔCt) using RNU6B on cells as an endogenous control [Pfaffl, MW, et al. , Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Research, 2002. 30(9): p. e36; Livak, KJ, et al. , Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001. 25(4): p. 402-8].

A negative control was included for each primer pair to monitor for reagent contamination. PCR cycling conditions were repeated 40 times: 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 60 seconds.

Example 5. Cell transfection of miRNA-29a and scrambled miRNA

HeLa, SiHa and C33A cells were seeded at a density of 4×10 ⁵ cells/well in 6-well culture plates and miRNA-29a mimics (miR-29a; Bioneer, Daejeon, Korea) and negative control mimics (scrambled miRNA, Bioneer, Korea) ) was transfected with. The sequence of the miRNA-29a mimic was 5'-ACUGAUUUCUUU UGGUGUUCAG-3' (SEQ ID NO: 2).

After culturing to 70-80% confluence, cells were transfected with miRNA-29a mimics and miRNA scrambled using Lipofectamine™ RNAiMAX reagent (Lipofectamine ^™ ; Invitrogen) and Opti-MEM (Gibco) with serum-free medium . After 24 hours (hrs) at 37° C. incubation, transfected cells were harvested and analyzed for RNA and protein extraction.

Example 6. Cell viability assay

A 96-well plate was seeded at a density of 1 x 10 ⁴ HeLa cells/well. After 24 hours, cells were treated with miRNA-29a and scrambled miRNA. After culturing for 4 hours at 37° C. in serum-free medium, the cells were further cultured with 10% FBS for 24 hours. Cell viability of Hela cells was assessed by MTT assay.

Briefly, after transfection cells were cultured overnight in 96-well culture plates. A total of 2 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; Sigma-Aldrich, Burlington, MA, USA) solution in PBS was gently loaded into each well. Incubate at 37°C in a humidified incubator with a volume of 200 μl and 5% CO2. After 2 hours incubation, the MTT solution in each well was removed, 200 μl of DMSO was added and mixed thoroughly. After incubation for 2 hours in a 37° C. incubator, absorbance was measured at 540 nm with an Infinite 200 spectrophotometer (Tecan).

Example 7. Gel Retardation Assay

An agarose gel retardation assay was performed to observe the complexation ratio of miRNA-29a and Lipofectamine ^TM transfection reagent.

Briefly, 200 nM miRNA-29a mimics were mixed with various concentrations of LipofectamineTM and the volume was adjusted to 20 μl with Opti-MEM. After 5 min incubation, the RNA:lipid complex solution was mixed with 2 μl of 5X loading buffer and loaded onto a 2% agarose gel containing ethidium bromide. Electrophoresis was performed in 1X TBE running buffer at 100V for 15 minutes. Images were taken using the Gel Doc Imaging System (Bio-Rad).

Example 8. Cell Migration Assay

Cell migration assay was performed with SPLScar™ (SPL, Pocheon, Korea). HeLa was inoculated into a 6-well culture plate at a density of 4 x 10 ⁵ cells/well. After 24 hours, cells at 70-80% confluence were transfected with miRNA-29a mimics and scrambled miRNAs. After culturing at 37° C. for 24 hours, the cells were washed three times with PBS, and the monolayer cells were scratched with SPLScar™ in a straight line through a 500 μm wide wound gap. The scratched cells were cultured in DMEM containing 10% FBS, and 0, 24, 48, and 72 hours after scratching, the wound gap was photographed using an optical microscope (Olympus, Tokyo, Japan) at 400x magnification. The percentage of migrated cells was graphed using ImageJ software (version 1,53k; Media Cybernetics, Rockville, MD, USA).

Example 9. Cell invasion assay

Cell invasion assay was performed with SPLInsertTM Hanging (SPL). After HeLa cells were transfected with miRNA-29a mimics and the miRNAs were scrambled for 24 hours, the transfected cell pellets were resuspended in serum-free culture medium. To simulate the basement membrane environment, the upper chamber of the transwell insert was pre-coated with Matrigel Matrix (50 μL/cm2 growth area, BD Biosciences, Sparks, MD, USA).

The resuspended cells were seeded into the upper chamber of the transwell insert. A culture solution containing 10% FBS as a chemoattractant was added to the lower chamber, and the transwell was cultured for 24 hours.

After incubation, cells on the top surface of the transwell insert were removed using a cotton swab. Infiltrated cells on the lower surface of the transwell membrane were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.05% crystal violet for 1 hour. After washing the transwell with PBS, the number of invaded cells in three random fields was observed using an optical microscope and counted using ImageJ software. Three fields were randomly selected from each membrane, and the number of infiltrated cells was counted.

Example 10. Dual Luciferase Assay

To quantify the binding affinity between miRNA-29a and PIK3CA, the Dual-Luciferase Reporter Assay System (Promega, San Luis Obispo, CA, USA) was used. Using the predicted 3' UTR binding site of PIK3CA mRNA (Fig. 2), the wild or mutant binding site to the firefly luciferase gene expression control region of the reporter plasmid pmiRGLO Dual-Luciferase miRNA Target Expression Vector (pmiRGLO; Promega) was inserted by the Bioneer Cloning Service System (Bioneer);

PIK3CA wild type: 5′-GTTTAAACAATATGTGGTGTTAATAGATTGUGGUGCUTTTACTATTTAAAGACAACTTTCATCTAGA-3′; SEQ ID NO: 3,

PIK3CA mutation type: 5'-GTTTAAACAATATGTGGTGTTAATAGATTGACCACGATTTACTATTTAAAGACAACTTTCATTCTAGA-3'; SEQ ID NO: 4.

When miR-29a binds to the PIK3CA wild-type insertion site, the expression of firefly luciferase is suppressed, but Renilla luciferase is expressed independently of binding depending on the number of plasmids. Wild-type and mutant plasmids were co-transfected with miRNA-29a or scrambled miRNA, respectively, and introduced into HeLa cells.

Reporter activity was then performed 24 h after transfection using a microplate luminometer (Centro XS3 LB960; Berthold, Oak Ridge, TN, USA). Relative luciferase activity was calculated as firefly luciferase activity compared to Renilla luciferase activity.

Example 11. RT-qPCR for PIK3CA expression analysis

Reverse transcription quantitative PCR (RT-qPCR) for PIK3CA mRNA was performed using CFX96 (Bio-Rad). PIK3CA and GAPDH mRNA primers and probes were designed and synthesized (Genotech, Daejeon, Korea) (Table 2) (Fig. 2).

A total volume of 20 μl was used, containing 10 μl of 2X Thunderbird probe qPCR mix (Toyobo), 3 μl of primers, 5 μl of distilled water and 2 μl of template cDNA. Positive and negative controls were included for each procedure. The reaction conditions of real-time PCR were repeated 40 times: 3 minutes at 95 ° C, 3 seconds at 95 ° C, and 30 seconds at 63 ° C.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control and confirmation of mRNA degradation. Each sample was tested three times.

mRNA expression was quantified by determining the cycle threshold (Ct), which is the number of PCR cycles required for fluorescence to exceed a value significantly above background fluorescence.

The amount of PIK3CA was determined using the comparative Delta Ct method (2 ^-ΔCt ) using GAPDH as an endogenous control.

Table 2 shows primers and probes used for mRNA expression analysis in the present invention.

Example 12. Protein Isolation and Western Blotting Analysis

HeLa was inoculated into a 6-well culture plate at a density of 4 x 10 ⁵ cells/well. After HeLa cells were transfected with miRNA-29a mimics and the miRNAs were scrambled for 24 hours, the cells were washed twice with PBS and then lysed in 50 μl of cold RIPA lysis buffer (RIPA; Boster Biological Technology, Pleasanton, CA, USA) and It was dissolved in protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA).

Cell lysates were collected using a Cell Scraper (SPL), pooled into 1.5 ml microcentrifuge tubes, and centrifuged at 14,000 rpm and 4°C for 30 minutes.

The supernatant was used as protein. Protein levels were quantified with a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). A total of 30 μg of protein sample was used for Western blotting. Total protein was subjected to SDS-PAGE on a 10% running gel at 100 V for 90 min and then transferred to a nitrocellulose membrane (Bio-Rad) at 350 mA for 1 h. Thereafter, non-specific binding sites were blocked with 5% skim milk for 1 hour incubation.

The membrane was soaked in 5% skim milk in TBST. Membranes were then incubated with primary antibodies against PIK3CA (1:1000, Rabbit mAb; Cell Signaling, Beverly, MA, USA) and GAPDH (1:1000, Mouse mAb; Cell Signaling) overnight at 4 °C.

The following day, membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:1000; Cell Signaling) for 2-4 hours at room temperature. For visualization, protein bands were visualized using SuperSignal® WestPico Plus (Thermo Fisher Scientific). Band intensities were measured with a chemiluminescence imaging system (FUSION Solo; Vilber, Paris, France) and relative protein expression was calculated with ImageJ software.

Statistical analysis of the present invention was performed using GraphPad Prism software version 9.0 (GraphPad Software, La Jolla, CA, USA). All experiments were repeated at least three times, and one representative result is presented. A two-tailed Student's t deviation (Student's t-test) was used for comparison between the two groups. The Kruskal-Wallis test was used to determine statistical significance as a non-parametric statistic. A P value of less than 0.05 was considered statistically significant.

The results of the above examples are as follows.

Selection of cervical cancer cell lines for miR-29a overexpression

To select useful cervical cancer cell lines for overexpression of miR-29a, we tested the expression level of miR-29a in various cervical cancer cell lines (C33A, SiHa and HeLa). To this end, we determined the expression level of miR-29a using RT-qPCR analysis. The relative expression level of miR-29a was calculated by the delta Ct (2 ^-ΔCt ) method. To normalize miR-29a Ct values, the small nucleolar RNA RNU6B was used as a reference gene control.

Data from this experiment suggested that the expression level of miR-29a in HeLa (HPV type 18 positive) was significantly lower (P < 0.001) than in other cervical cancer cell lines (C33A and SiHa) (Figure 3). Therefore, HeLa cells were selected for subsequent experiments.

Transfection conditions of miR-29a and Lipofectamine™ complex

To select the concentration and duration of miR-29a by Lipofectamine™ treatment of the lipid vesicle transporter, HeLa cells, we tested the cytotoxicity of Lipofectamine™ at different doses and duration on HeLa cells using a cell viability assay ( Fig. 4). When cells were treated with 1-15 μl LipofectamineTM for 24 hours, cell viability remained almost 100% until 11 μl LipofectamineTM was used. On the other hand, when the cells were treated for 48 hours, the cell viability began to decrease even with 3 μl of LipofectamineTM treatment. Therefore, in the next experiment, treatment was performed for 24 hours with 1-9 μl of LipofectamineTM.

Next, to determine the optimal complexing ratio between Lipofectamine™ and miR-29a, various volumes of Lipofectamine™ were complexed with 100 nM of miR-29a and gel retardation assays were performed.

As shown in FIG. 5, 3-9 μl of Lipofectamine™ seems to contain all of 100 nM of miR-29a compared to free miR-29a not mixed with Lipofectamine™. From these results, it was concluded that 3 μl or more of Lipofectamine ^TM could contain 100 nM of miR-29a.

Optimization for miR-29a overexpression in HeLa cells

To find optimal conditions for overexpression of miR29a in HeLa cells, HeLa cells were transfected with miR-29a complexed with Lipofectamine ^™ and cell viability was evaluated. To this end, miR-29a was transfected into HeLa cells at 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM concentrations with Lipofectamine ^TM 1, 2, 3, 6, and 12 μl, respectively.

As shown in Figure 6a, compared to cell viability when treated with Lipofectamine™ alone, miR-29a was transfected at 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM concentrations, respectively, by 102.5%, 101.1%, 96.0%, 75.6%, It showed a cell viability of 28.8%.

miR-29a significantly inhibited cell survival at concentrations of 200 nM and 400 nM (P < 0.001).

The above results of the present invention showed that miR-29a transfection at 200 nM or more affected the cell viability of HeLa cells, and thus could lead to a miR-29a overexpression state in HeLa cells.

Next, to confirm whether the decrease in cell viability by miR-29a is specific to miR-29a, scrambled miRNAs were incubated with 6 and 12 μl Lipofectamine ^TM and miR-29a at 200 nM and 400 nM concentrations for 24 hours in HeLa cells. during transfection.

Compared to the viability of cells transfected with Lipofectamine ^TM alone, the viability of cells transfected with scrambled miRNA was 89.6% and 31.7%, and 74.6% and 30.9% at 200 nM and 400 nM concentrations of miR-29a, respectively (Fig. 6b).

Cell viability by miR-29a transfection was significantly reduced at a concentration of 200 nM compared to scrambled miRNA (P < 0.001).

On the other hand, there was no significant difference in cell viability by 400 nM miR-29a and scrambled miRNA, indicating that 400 nM miR-29a and 12 μl of Lipofectamine ^TM caused miRNA-independent cytotoxicity.

Therefore, from these results, it is concluded that the decrease in cell viability of HeLa cells by 200 nM of miR-29a and Lipofectamine ^TM is specific to miR-29a overexpression.

Therefore, in subsequent experiments this condition was used for overexpression of miR-29a in HeLa cells.

Dual luciferase reporter assay for direct binding of miR-29a to the 3' UTR of PIK3CA mRNA

To investigate whether miR-29a binds to the 3' UTR of PIK3CA mRNA, a dual luciferase reporter assay was performed.

For the PIK3CA wild-type plasmid, the wild-type sequence of the 3' UTR (5'-UGGUGCU-3') in the PIK3CA gene was cloned. In addition, in the case of the mutant plasmid, the 3' UTR binding sequence of the PIK3CA gene was mutated to 5'-ACCACGA-3' (FIG. 7a).

Next, PIK3CA wild-type or PIK3CA mutant plasmids were co-transfected into HeLa cells with miR-29a or scrambled miRNA, respectively. The reporter activity of the two luciferases was measured 24 hours after transfection using a luminometer. Relative luciferase activity was then compared to Renilla luciferase activity and calculated as firefly luciferase activity (Figure 7b).

As a result, the PIK3CA wild-type and miR-29a co-transfected groups showed a significant decrease in relative luciferase activity compared to the PIK3CA wild-type and scrambled miRNA co-transfected groups (P < 0.001).

On the other hand, in the group co-transfected with PIK3CA mutant cells, there was no difference in relative luciferase activity when HeLa cells were treated with scrambled miRNA and miR-29a. These results seem to suggest that miR-29a binds directly to the 3' UTR of PIK3CA mRNA.

Effect of miR-29a on PIK3CA expression level

To investigate the effect of overexpressed miR-29a on PIK3CA expression, mRNA and protein expression levels of the PIK3CA gene were analyzed by RT-qPCR and Western blot, respectively.

After designing PIK3CA primers and probes to detect only PIK3CA mRNA, HeLa cells were transfected with miR-29a and scramble miRNA for 24 hours, and relative expression levels of PIK3CA mRNA were compared.

As a result, it was confirmed that the PIK3CA mRNA expression level of HeLa cells was significantly decreased in the miR-29a-treated group compared to the scrambled miRNA-treated group (FIG. 8) (P < 0.001).

In addition to the results of the PIK3CA expression level in HeLa cells, the PIK3CA mRNA expression level upon overexpression of miR-29a was also investigated in SiHa and C33A cells using the PIK3CA primers and probes designed in the present invention.

The results showed that the PIK3CA mRNA expression level was reduced by miR-29a in SiHa (P < 0.05) and C33A cells (P < 0.001) (FIG. 9).

The protein expression level of PIK3CA in HeLa cells was investigated by Western blot. After transfecting HeLa cells with miR-29a and scrambled miRNA for 24 hours, western blotting was performed (Fig. 10a), and the intensity of protein bands was measured using ImageJ (Fig. 10b).

The expression level of PIK3CA protein was significantly downregulated in HeLa cells treated with miR-29a compared to the scramble miRNA treated group. RT-qPCR and Western blot analysis showed that the mRNA and protein expression levels of PIK3CA were downregulated by the effect of overexpressed miR-29a.

Effects of miR-29a on HeLa cell migration

To investigate the effect of changes in PIK3CA expression level on the oncogenic ability of cervical cancer, cell migration and invasion activities were investigated in HeLa cells, two of the six oncogenic properties of cancer cells.

The effect of overexpressed miR-29a on HeLa cell migration was evaluated using a cell migration assay. Wound gaps were created after 24 h of transfection with miR-29a mimics and scrambled miRNAs and were photographed at 0, 24, 48 and 72 h after scratching (Fig. 11a).

The percentage of migrated cells was graphed using ImageJ software (FIG. 11B). Closure of the monolayer scratched with scrambled miRNA was 21.6%, 41.6% and 71.1% at 24, 48 and 72 hours, respectively. For miR-29a, the closure rates were 11.9%, 24.4%, and 38.9% at 24, 48, and 72 hours, respectively.

These results indicate that overexpression of miR-29a inhibits the migration ability of HeLa cells.

Effects of MiR-29a on HeLa cell invasion

Transwell invasion assay was performed to evaluate the effect of overexpressed miR-29a on HeLa cell invasion.

To simulate the basement membrane environment, the upper chamber of the transwell insert was coated with Matrigel Matrix (50 µL/cm2 growth area). After HeLa cells were transfected with the miR-29a mimic and the miRNA scrambled for 24 hours, the transfected cells were resuspended in serum-free culture medium and seeded in the upper chamber of the transwell insert.

A culture solution containing 10% FBS as a chemoattractant was added to the lower chamber, and the transwell was cultured for 24 hours.

The number of infiltrating cells in three random fields was observed under a microscope (FIG. 12A) and counted using ImageJ software (FIG. 12B).

In the group with scramble miRNA, about 1,500 HeLa cells invaded compared to the negative staining result of transwell membrane fixed and stained without cell inoculation. The number of cells that invaded and passed through the transwell membrane in the miR-29a treatment group was about 350 HeLa cells. The number of cells invaded by miR-29a through the basement membrane Matrigel was significantly lower than those treated with scramble miRNA (P < 0.001).

These results suggest that overexpression of miR-29a inhibits the cell invasion ability of HeLa cells.

<110> UNIVERSITY INDUSTRY FOUNDATION, YONSEI UNIVERSITY WONJU CAMPUS <120> Nucleic acid fragment specifically binding to the 3' untranslated region of the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene and applications thereof <130> DP21-0049HS <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> RNA <213> artificial sequence <220> <223> Nucleic acid fragment <400> 1 agcacca 7 <210> 2 <211> 22 <212> RNA <213> artificial sequence <220> <223> miRNA-29a mimics <400> 2 acugauuucu uuugguguuc ag 22 <210> 3 <211> 67 <212> DNA <213> artificial sequence <220> <223> PIK3CA <400> 3 gtttaaacaa tatgtggtgt taatagattg uggugcuttt actatttaaa gacaactttc 60 atctaga 67 <210> 4 <211> 68 <212> DNA <213> artificial sequence <220> <223> PIK3CA Mutant <400> 4 gtttaaacaa tatgtggtgt taatagattg accacgattt actatttaaa gacaactttc 60 attctaga 68

Claims (10)

A nucleic acid fragment of SEQ ID NO: 1 that specifically binds to the 3' untranslated region (UTR) of the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene. A composition for detecting phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene comprising the nucleic acid fragment of SEQ ID NO: 1 as an active ingredient. A composition for diagnosing cancer comprising the nucleic acid fragment of SEQ ID NO: 1 as an active ingredient. The composition for diagnosing cancer according to claim 3, wherein the cancer is cervical cancer. A composition for treating cancer comprising the nucleic acid fragment of SEQ ID NO: 1 as an active ingredient. The composition for treating cancer according to claim 5, wherein the cancer is cervical cancer. The composition for treating cancer according to claim 5, wherein the composition inhibits migration and invasion of cancer cells. When there is a gene that binds to the nucleic acid fragment of SEQ ID NO: 1 by processing the nucleic acid fragment of SEQ ID NO: 1 in the gene pool obtained from the sample in vitro, the sample contains the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene. A method for providing information on the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene, which is determined to be. The phosphatidylinositol 3-kinase catalytic subunit according to claim 8, wherein the binding of the nucleic acid fragment to the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene is performed by a dual luciferase assay. Method for providing information on alpha (PIK3CA) gene. When the gene pool obtained from the sample in vitro has a gene that binds to the nucleic acid fragment of SEQ ID NO: 1 by processing the nucleic acid fragment of SEQ ID NO: 1, the sample is considered to contain a gene related to cervical cancer. How to provide information.

Images