KR20180092378A - COMPOSITION FOR ENHANCING RADIATION SENSITIVITY COMPRISING miR-130a AS AN ACTIVE INGREDIENT - Google Patents

Abstract

The present invention relates to a composition for enhancing radiation sensitivity comprising micro RNA, specifically, miR-130a as an active ingredient, wherein the miR-130a according to the present invention enhances the radiation sensitivity of cancer cells, suppresses the expression in cancer cells having radiation resistance, and when treated with cancer cells having the radiation resistance, sensitivity to the radiation is restored, thereby being useful for the treatment of cancer resistant to radiation therapy.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for enhancing radiation sensitivity comprising miR-130a as an effective ingredient. [0002] The present invention relates to a composition for promoting radiation sensitivity,

The present invention relates to a composition for promoting radiation sensitivity comprising microRNA, specifically, miR-130a as an active ingredient.

Throughout the world, cancer is a serious problem that threatens human health. Currently, many methods and techniques have been developed to treat or diagnose cancer, but research continues to find safer and more effective treatments.

Statistically, about 35% of patients in Korea and about 50% of patients in the US are receiving radiation therapy. The number of cancer patients receiving radiation therapy in Korea is increasing every year, and the importance of radiation therapy for cancer treatment is increasing. Cancers that are generally treated with radiation therapy include head and neck cancer, laryngeal cancer, cervical cancer, parietal cancer, lung cancer, brain cancer, breast cancer, rectal cancer and colon cancer. Although these carcinomas may be the main targets of radiation therapy, the prognosis for radiation therapy is often poor, so a strategy to improve treatment efficiency is needed. In addition, although the initial response to radiation therapy is good, the cells having resistance to recurrence or radiation may be a big problem, so it is important to take measures to improve the efficiency of the treatment. Because there is a difference in susceptibility to radiation therapy in cancer cells, customized therapy can be provided by developing techniques that can predict or increase susceptibility to individual radiation before radiation therapy. This can reduce the burden of social and economic costs as well as the suffering and economic burden of cancer patients.

Therefore, in order to examine the sensitivity of cancer cells to radiation before radiation therapy, A method of analyzing the degree of cancer cell death after irradiating isolated cancer cells is necessary. The most direct and ideal method known is to stain cells with a dye such as trypan blue and count the number of living cells using a microscope and a cell counter, but this requires time and too much effort , And can not be used when the number of specimens is large. Even in the case of cancer patients with histologic findings or stage, the radiation sensitivity is very different, and it is known that there are many factors influencing the radiation sensitivity.

The most important reason for the tumor to have radiation resistance is the intrinsic radiosensitivity of tumor cells. Cancer cells exposed to radiation are killed or restored by complicated molecular biologic changes. The genes and proteins involved in these mechanisms have been identified, and all the genes and proteins revealed can be targets for promoting the effects of radiation therapy 2004, 22: S7), which has been reported in the literature (Park HJ, J Korean Soc. Ther. Radiol. Oncol., 2004, 22: S6, Choi EK., J. Korean Soc. Ther. Radiol.

Studies on radiation resistance have been continuing, and these studies are the basis for the development of radiation sensitizers to overcome radiation resistance. Factors associated with the radiation resistance so far identified include cyclooxygenase-2 (Terakado N et al., Oral Oncol., 2004; 40: 383-9), epithelial growth factor receptor (EGFR) (Milas Let al., Int J Radiat Oncol Biol Phys., 2004; 58 (3); 966-71), cyclin D1 (Milas L et al., Int J Radiat Oncol Biol Phys., 2002; 52; 514-21) IGFBP-1 receptor, manganese superoxide dismutase, and survivin.

On the other hand, microRNAs (miRNAs) are a class of small noncoding RNAs that regulate recently expressed gene expression. Mature miRNAs have 18 to 25 nucleotides and are produced by hairpin structure transcripts. miRNA acts complementarily to target mRNA and functions as an inhibitor of the transcribed gene. It acts by inhibiting mRNA translation, inhibiting gene expression, or inducing destabilization by catalyzing mRNA cleavage. These miRNAs also act to regulate cell proliferation and metabolism, timing of development, apoptosis, hematopoiesis, neuronal development, human tumor development, DNA methylation and chromatin transformation.

Recent evidence suggests that modulation of gene expression by miRNAs is associated with the development of cancer. It was found in lung cancer, breast cancer, glioblastoma, hepatocellular carcinoma, thyroid papillary carcinoma, rectal cancer and colorectal cancer. Furthermore, miRNA expression has been reported to be related to the clinical outcome of certain diseases, and it has been found that miRNAs are associated with the development of various human diseases.

The present inventors have found that miR-130a, a kind of microRNA, enhances the radiation sensitivity of cancer cells and induces expression in cancer cells having radiation resistance, while trying to find a substance capable of enhancing sensitivity to radiation therapy , And confirming that increasing the expression of miR-130a in the cells increases the radiation sensitivity again, thus completing the present invention.

 Park HJ., J Korean. Soc. Ther. Radiol. Oncol., 2004, 22: S6; Choi EK., J. Korean. Soc. Ther. Radiol. Oncol. 2004, 22: S7  Terakado N et al., Oral Oncol., 2004; 40: 383-9  Milas L et al., Int J Radiat Oncol Biol Phys., 2004; 58 (3); 966-71  Milas L et al., Int J Radiat Oncol Biol Phys., 2002; 52; 514-21

It is an object of the present invention to provide a composition for enhancing radiation sensitivity comprising miR-130a or an anti-cancer adjuvant.

Another object of the present invention is to provide a diagnostic, prognostic or monitoring method for radiation therapy by measuring the expression level of miR-130a.

It is another object of the present invention to provide a method for screening a substance that enhances radiation sensitivity by selecting a substance that changes the expression level of miR-130a.

In order to achieve the above object, the present invention provides a composition for promoting radiation sensitivity comprising miR-130a as an active ingredient.

The present invention also provides a radiation-sensitive anticancer adjuvant containing miR-130a as an active ingredient.

In addition, the present invention provides a kit for diagnosis, prognosis, or monitoring for radiation therapy comprising an agent that measures the level of expression of miR-130a.

Also, the present invention provides a method for detecting miR-130a comprising: 1) measuring the expression level of miR-130a in a biological sample separately from an experimental group and a normal control group; And 2) selecting a sample whose expression level of miR-130a in step 1) is lower than that of a normal control.

In addition, the present invention provides a method for producing a miR-130a comprising: 1) treating a test substance to a cell or tissue expressing miR-130a; 2) measuring the expression of miR-130a from the cell or tissue of step 1); And 3) selecting the test substance whose expression of miR-130a in the step 2) is increased compared to the untreated control group. The present invention also provides a substance screening method for enhancing sensitivity to radiation.

The miR-130a of the present invention enhances the radiation sensitivity of cancer cells and suppresses expression in cancer cells having radiation resistance. When cancer cells having radiation resistance are treated, they are resistant to radiation therapy by restoring sensitivity to radiation Can be useful for the treatment of cancer.

1 is a graph comparing the radiation sensitivity of the cell line by irradiating the rectal cancer cell line with radiation and confirming the cell survival rate.
FIG. 2 is a graph comparing irradiation of a rectal cancer cell line with radiation and confirming the extent of DNA damage by histone H2AX phosphorylation
Figure 3 is a graph of RT-qPCR results for identifying and screening miRNAs associated with radiation sensitivity of cancer cell lines.
FIG. 4 is a graph showing the process of screening cells having radiation resistance by irradiating SNU-70 cell line with radiation and the expression level of hsa-miR-130a-3p in SNU-70-P2 cells having radiation resistance (B) Fig.
FIG. 5 is a photograph (a) showing the cell death rate according to an increase in radiation sensitivity due to overexpression of hsa-miR-130a-3p in SNU-70-P2 cells and a change in the expression of a protein associated with apoptosis ).
FIG. 6 shows the result of alignment of miR-130a with the SOX4 gene selected by bioinformatics analysis, (a) inhibiting the expression of SOX4 by hsa-miR-130a-3p (b) (C) is recovered when the sponge is processed. The graph is obtained through the Luciferase assay

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for promoting radiation sensitivity comprising miR-130a as an active ingredient.

The term "miR "," miRNA ", or "microRNA ", or" microRNA ", as used herein, refers to a noncoding RNA that modulates gene expression after transcription by promoting degradation of target RNA, . The sequence of the miRNA used in the present invention can be obtained from the miRNA database (http://www.mirbase.org). As of January 2017, the miRNA database (version 19, miRBase) has registered 25,141 miRNAs from 193 species. Generally, microRNAs can be transcribed into precursors of about 70-80 nucleotides in length with a hairpin structure called pre-miRNA and then matured by truncation by the RNAse III enzyme Dicer. The microRNA may form a ribonucleoside complex called miRNP to cleave a target gene through complementary binding to a target site or inhibit translation. More than 30% of human miRNAs are present in clusters and can be transcribed into a single precursor and then cleaved to form the final mature miRNA.

The miRNA may be a mutant or a fragment of a nucleic acid molecule having a different sequence by deletion, insertion, substitution, or a combination thereof of the nucleotide sequence within a range that does not affect the function of the nucleic acid molecule constituting the miRNA. The miR-130a used may functionally equivalent to the miRNA nucleic acid molecule even if a functional equivalent of the nucleic acid molecule constituting it, for example, a partial nucleotide sequence of the miRNA nucleic acid molecule, is modified by deletion, substitution or insertion ≪ / RTI >

Specifically, the miRNA may have a homology of 80% or more, specifically 90% or more, more specifically 95% or more, to the nucleotide sequence of the corresponding sequence number. Such homology can readily be determined by comparing the sequence of the nucleotide with the corresponding portion of the target gene using computer algorithms well known in the art, such as Align or BLAST algorithms. In one embodiment of the present invention, miR-130a may be a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1.

Also, the nucleic acid molecule of miR-130a may exist in a single stranded or double stranded form. Although mature miRNA molecules are predominantly single stranded, precursor miRNA molecules may contain a partial self-complementary structure (e.g., a stem-loop structure) that can form double strands. In addition, the nucleic acid molecule of the present invention may be in the form of RNA, peptide nucleic acids (PNA), or locked nucleic acid (LNA). The nucleic acid molecule may be isolated or prepared using standard molecular biology techniques, for example, a chemical synthesis method or a recombinant method, or a commercially available nucleic acid molecule may be used.

Such nucleic acid molecules are known in the art for base exchange with polynucleotides that do not alter their activity globally. And may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, or farnesylation in some cases. Specifically, the miR-130a may include one or more phosphate groups at the 5 'terminus. Specifically, the phosphoric acid group may be 1 to 10, 2 to 5, or 2 to 4, and may be triphosphate in one embodiment of the present invention.

The term "radiation sensitivity" as used herein also means the sensitivity of the organism to radiation. The major factors involved are the size of the target (DNA amount), cell kinetics (cycle of cell division), the state of the cell (spore, etc.), and the repair of radiation damage. In the multicellular biosystem, cells that are generally active in cell division, such as all the organs of the embryo at the beginning of development, root tips or growth tips of plants, germ cells, hematopoietic tissues, intestinal epithelium, immune function cells or cancer cells, Cells that are sensitive and do not undergo cell division, such as adult nerves, most of the brain, muscle, liver, and mature blood cells, may also be resistant to radiation. However, even in a radiation sensitive tissue, radiation resistance can be obtained for various reasons.

In a specific embodiment of the present invention, we screened hsa-miR-130a-3p as an miRNA promoting sensitivity to radiation in a rectal cancer cell line (Figure 3)

Sensitizing SNU-70 cell lines were irradiated with radiation to make the cells resistant to radiation (SNU-70-P2). The expression of hsa-miR-130a-3p was reduced in SNU-70-P2 cells (see Fig. 4b) and overexpression of hsa-miR-130a-3p in the cells restored sensitivity to radiation 5A).

Thus, miR-130a can be usefully used to enhance radiation sensitivity.

The present invention also provides a radiation-sensitive anticancer adjuvant containing miR-130a as an active ingredient.

The miR-130a may have the above-described characteristics. For example, the miR-130a may comprise one or more phosphate groups at the 5 'terminus. Specifically, the phosphoric acid group may be 1 to 10, 2 to 5, or 2 to 4, and may be triphosphate in one embodiment of the present invention. In addition, miR-130a may be a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1.

The anticancer adjuvant may increase the expression of miR-130a in cells, thereby enhancing sensitivity to radiation and promoting the death of cancer cells. The cancer in which the anticancer adjuvant may be used may be blood cancer or solid cancer. Specifically, the blood cancer may be selected from the group consisting of lymphoma, acute leukemia, multiple myeloma and combinations thereof. The solid cancer may be selected from the group consisting of lung cancer, colon cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, Thymus cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary cancer, pancreatic cancer, and combinations thereof. According to an embodiment of the present invention, the cancer may be rectal cancer.

In a specific embodiment of the present invention, the present inventors have found that the increased expression of hsa-miR-130a-3p increases the radiation sensitivity of colorectal cancer cells to induce apoptosis (see FIG. 5A) Lt; RTI ID = 0.0 > (FIG. 5b). ≪ / RTI >

The composition for enhancing radiation sensitivity according to the present invention may contain 10 to 95% by weight of miR-130a as an active ingredient with respect to the total weight of the composition. In addition, the composition of the present invention may further comprise at least one active ingredient which exhibits the same or similar functions in addition to the above-mentioned effective ingredients.

Compositions of the present invention may include carriers, diluents, excipients or mixtures thereof conventionally used in biological preparations. Pharmaceutically acceptable carriers can be used as long as they are suitable for delivering the composition in vivo. Specifically, the carrier is selected from Merck Index, 13th ed., Merck & Sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. If necessary, conventional additives such as an antioxidant, a buffer, a bacteriostatic agent and the like may be added.

When the composition is formulated, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient may be added.

The compositions of the present invention may be formulated into oral or parenteral formulations. Oral preparations may include solid preparations and liquid preparations. The solid preparation may be a tablet, a pill, a powder, a granule, a capsule, or a troche. The solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or a mixture thereof. In addition, the solid preparation may comprise a lubricant, examples of which include magnesium stearate, talc, and the like. On the other hand, the liquid preparation may be a suspension, a solution, an emulsion or a syrup. At this time, the liquid preparation may contain an excipient such as a wetting agent, a sweetener, a fragrance, a preservative, and the like.

The parenteral preparation may contain injections, suppositories, respiratory inhalation powders, aerosol formulations for spraying, powders and creams. The injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspending agent, an emulsion, and the like. As the non-aqueous solvent or suspension, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate may be used.

The composition of the present invention can be administered orally or parenterally according to the desired method. Parenteral administration may include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathecal injection.

The composition may be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, severity, activity of the drug, sensitivity of the patient to the drug, administration time, route of administration, duration of treatment, However, for a desired effect, the amount of the active ingredient contained in the composition according to the present invention may be 0.0001 to 100 mg / kg, specifically 0.001 to 10 mg / kg. The administration may be several times per day.

The composition of the present invention may be administered alone or in combination with other therapeutic agents. When administered concomitantly, the administration can be sequential or simultaneous.

In addition, the present invention provides a kit for diagnosis, prognosis, or monitoring for radiation therapy comprising an agent that measures the level of expression of miR-130a.

The kit for diagnosis, prognosis or monitoring of radiation therapy according to the present invention can be useful for diagnosis of cancer, prediction of recurrence, and measurement of prognosis. In this respect, the present invention specifically binds to miR-130a, Can be measured. The agent may be any one selected from the group consisting of primers, probes, antisense nucleotides, or aptamers that specifically bind miR-130a.

The primer is complementary to the base sequence of miR-130a, and any primer designed to amplify it can be used.

As used herein, the term "probe" refers to a nucleic acid fragment corresponding to several hundreds to several hundreds of bases that can be specifically bound to DNA or RNA, and may be used to confirm the presence or absence of a specific DNA or RNA . The probe may be prepared in the form of an oligonucleotide probe, a short-chain DNA probe, a double-stranded DNA probe, or an RNA probe. The probe may be labeled with biotin, FITC, rhodamine, DIG (digoxigenin) have.

The antisense nucleotides may be double helix or single helix DNA, double helix or single helix RNA, DNA / RNA hybrids, DNA and RNA analogs and bases, sugar or backbone variants.

The aptamer may be RNA, DNA, modified oligonucleotide or a mixture thereof as an oligonucleotide molecule having a binding activity to a predetermined target molecule, and may be in a linear or cyclic form.

The agent may be combined with a solid substrate to facilitate subsequent steps such as washing or separation of the complex. As the solid substrate, synthetic resin, nitrocellulose, glass substrate, metal substrate, glass fiber, microsphere or micro bead may be used. The synthetic resin may be polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, nylon, or the like.

In addition, the preparation may be a conjugate labeled with a detection element such as a chromogenic enzyme, a fluorescent substance, a radioisotope, or a colloid. The chromogenic enzyme may be a peroxidase, an alkaline phosphatase, or an acid phosphatase, and the fluorescent substance may be a fluorescein carboxylic acid (FCA), a fluorescein isothiocyanate (FITC), a fluorescein thiourea (FTH) 2 ', 7'-dichlorofluorescein-5-yl, 2', 7'-dichlorofluorescein-6 Yl, tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bor- Diaza-s-indacene-3-ethyl, 4,4-difluoro-5,7-diphenyl-4-bora-3a, , Cy3, Cy5, polyL-lysine-fluorescein isothiocyanate (FITC), rhodamine-B-isothiocyanate (RITC), PE (Phycoerythrin) or rhodamine.

The kit according to the present invention can be used for detecting the amount of a preparation, such as a fluorescence method in which a fluorescent substance is attached to a detector to detect fluorescence, or a fluorescence method in which a radioisotope is attached to a detection body to detect radiation, throughput screening (HTS) system, a surface plasmon resonance (SPR) method for measuring the surface plasmon resonance change in real time without a label of the detector, or a surface plasmon resonance imaging (SPRI) method for imaging and verifying the SPR system .

The kit may contain, in addition to the formulation as described above, distilled water or a buffer to keep their structure stable.

The kit may also be prepared by conventional methods known to those skilled in the art and may further include a positive control, a negative control, and instructions for use. Samples not containing miR-130a can be used as a negative control, and samples containing miR-130a as a positive control.

Also, the present invention provides a method for detecting miR-130a comprising: 1) measuring the expression level of miR-130a from a biological sample separated from an experimental group and a normal control group, respectively; And 2) selecting a sample whose expression level of miR-130a in step 1) is lower than that of a normal control.

The biological sample may include a biological sample capable of identifying a disease-specific polynucleotide that can be distinguished from a normal state such as organs, tissues, cells, urine, blood, serum or plasma derived from a biological organ, Lt; / RTI >

The sample can be pretreated using methods such as anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, sequential extraction, centrifugation or gel electrophoresis prior to measuring the expression level of miR-130a have.

In addition, the present invention provides a method for producing a miR-130a comprising: 1) treating a test substance to a cell or tissue expressing miR-130a; 2) measuring the expression of miR-130a from the cell or tissue of step 1); And 3) selecting the test substance whose expression of miR-130a in the step 2) is increased compared to the untreated control group. The present invention also provides a substance screening method for enhancing sensitivity to radiation.

The substance screening method can be used not only to identify human cells or tissues but also to identify compounds that regulate miR-130a expression in vivo or ex vivo in cancer models such as mice and rats. Examples of the test substance include peptides, Proteins, non-peptide compounds, active compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and plasma.

Expression of miR-130a in the above step 2) was determined by RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA ; RNase protection assay; Northern blotting; and DNA chip.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited thereto.

< Example 1> Screening of radiation-sensitive rectal cancer cell lines

<1-1> Confirmation of cell death rate by radiation treatment

First, in order to select a cancer cell line having high sensitivity to radiation, 5 Gy or 10 Gy of radiation was irradiated to the SNU-70, SW-1463, SW-837 and SNU-1411 cell lines and MTT assay Were performed to compare the survival rates of the cells.

As a result, as shown in Fig. 1, the survival rate of the SNU-70 cell line was greatly decreased, confirming that the cell line had the highest sensitivity to radiation (Fig. 1).

<1-2> Histone by radiation treatment Of H2AX  Phosphorylation confirmation

First, the degree of DNA damage was examined by confirming phosphorylation of histone H2AX in a rectal cancer cell line irradiated with radiation under the same conditions and methods as those in Example <1-1>.

Rats were irradiated with 6 Gy of radiation. After 24 hours, the cells were stained with a histone H2AX phosphorylated antibody. DAPI was stained together to confirm the nuclei. The cells were stained in confocal microscope at the same position Respectively.

As a result, as shown in FIG. 2, the phosphorylation of histone H2AX was significantly increased in the SNU-70 cell line, confirming that DNA was most damaged in the cell (FIG. 2).

Therefore, SNU-70 cell line was confirmed to be the most sensitive colorectal cancer cell line to radiation, and it was used in the subsequent experiments by measuring cell death rate of rectal cancer cell line and phosphorylation of histone H2AX.

< Example  2> Promotes radiation sensitivity miRNA  Selection

To select and validate miRNAs associated with radiation sensitivity, we performed the following.

Sequencing of small RNAs of 100 bp or less isolated from the rectum cancer cell lines SNU-70, SW-1463, SW-837 and SNU-1411 cell lines was performed, and miRNAs were selected by comparison with the reference genome And classified as increased or decreased depending on the radiation sensitivity. Results were verified using the miRNA-specific qRT-PCR kit (Takara) with the RNU48 gene as a calibration group and miRNA-130a-3p was found to be highly expressed in radiation-sensitive cells and decreased in resistant cells Respectively.

As a result, as shown in FIG. 3, hsa-miR-130a-3p (5'-CAGUGCAAUGUUAAAAGGGCAU-3 '; SEQ ID NO: 1) was selected as an miRNA which promotes sensitivity to radiation in a rectal cancer cell line.

< Experimental Example  1> In a cell line having radiation resistance miR Decreased expression of -130a-3p

Expression of hsa-miR-130a-3p in radiation-resistant cells The following method was used.

Specifically, the SNU-70 cell line selected as the cell with the highest radiation sensitivity in Example 1 was irradiated with 6 Gy radiation on the 1st and 11th days of culture. After irradiation, surviving cells were selected and cultured and used as an experimental group as SNU-70-P2 cells having radiation resistance (Fig. 4A). On the other hand, SNU-70-P0 without irradiation was used as a control group.

RNA from the cells was extracted from the control and experimental cells using a TRIzol solution (Invitrogen), and the amount of the RNA was extracted with a has-miR-130a-selective qRT-PCR kit (Takara) and then corrected for the amount of RNU48 RNA.

As a result, the expression of hsa-miR-130a-3p was decreased in SNU-70-P2, a rectal cancer cell resistant to radiation therapy, as shown in Fig. 4B (Fig. 4B).

< Experimental Example  2> miR -130a-3p Expression of Radiation-Sensitive Cell Lines Confirm

<2-1> Visual confirmation of cell death

In SNU-70-P2, a cell showing radiation resistance in Experimental Example 1, changes in apoptosis caused by an increase in the expression level of hsa-miR-130a-3p With the naked eye Respectively.

Specifically, hsa-miR-130a-3p and scramble miRNA (control) were transfected into SNU-70-P2 cells using lipofectamine. After 6 hours, the medium was changed, and after 48 hours, 6 Gy of radiation was directly irradiated, and the degree of apoptosis was visually confirmed.

As a result, as shown in Fig. 5A, the number of cells killed by irradiation with SNU-70-P2 cells overexpressing miR-130a-3p was increased (Fig. 5A).

<2-2> Western Blotting

In addition, the expression of hsa-miR-130a-3p in the SNU-70-P2 cells was examined to determine the expression of the protein associated with apoptosis Western blotting.

Specifically, SNU-70-P2 cells were transfected with siRNA and irradiated with the same conditions and in the same manner as in Example <2-1>. The cells were eluted with RIPA solution and centrifuged to collect only the supernatant and quantify proteins using Bradford quantitation. The quantified protein was mixed with a sample buffer containing SDS, separated by size using a polyacrylamide gel, and then transferred to a PVDF membrane. The membrane was reacted with a primary antibody against Bid (Cell Signaling, 1: 1,000), Cell Signaling (1: 1,000) and β-actin protein (Sigma, 1: 5,000) Subsequently, the secondary antibody was further reacted and the protein band was detected by the color development of the ECL reagent. The detected protein bands were obtained and analyzed by X-ray film and digital image.

As a result, as shown in FIG. 5B, the expression of caspase-3 protein cleaved in SNU-70-P2 cells in which hsa-miR-130a-3p was overexpressed was increased as compared with the control (FIG. From this, it was found that increasing expression of hsa-miR-130a-3p in cancer cells increases radiation sensitivity of cancer cells and induces apoptosis.

< Experimental Example  3> miR -130a-3p target  Identify genes

It was confirmed by the following method as to which gene was targeted by the radiosensitivity-enhancing activity of hsa-miR-130a-3p identified above.

First, we compared the target genes of has-miR-130a-3p in TargetScan, PicTar and MirBase constructed as databases for bioinformatics analysis, and compared them with the target genes that are expected to be related to radiation sensitivity And the SOX4 gene was finally selected as a candidate gene (Fig. 6A). A plasmid containing the luciferase gene linked to the 3'UTR sequence of the SOX4 gene was constructed to confirm whether the selected target gene was matched. The plasmid was transfected with hsa-miR-130a-3p or scramble miRNA into SNU-70-P2 cells using lipofectamine. After 6 hours of infection, the cells were replaced with the medium, and after 48 hours, the cells were lysed and only the supernatant was extracted. Next, the mixture was mixed with a luciferase substrate, and the plate was placed in a 96-well plate, and the activity of luciferase was measured using a luminometer (Promega). At this time, b-galactosidase plasmid was added together to measure the activity, and the result was corrected.

As a result, the activity of luciferase was reduced by hsa-miR-130a-3p as shown in Fig. 6B (Fig. 6B). On the other hand, the reduced luciferase activity was restored by microRNA sponge, an inhibitor of microRNA (Fig. 6C).

Thus, it was found from the above that the target gene of hsa-miR-130a-3p is the SOX4 gene.

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Claims (9)

A composition for promoting radiation sensitivity comprising miR-130a as an active ingredient.
2. The composition of claim 1, wherein the miR-130a comprises at least one phosphate group at the 5 &apos; end.
2. The composition of claim 1, wherein the miR-130a comprises a triphosphate at the 5 &apos; end.
The composition for promoting radiation sensitivity according to claim 1, wherein the miR-130a is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.
A radiation-sensitive anticancer adjuvant containing miR-130a as an active ingredient.
A kit for diagnosis, prognosis, or monitoring for radiation therapy comprising an agent that measures the level of expression of miR-130a.
1) measuring the expression level of miR-130a in a biological sample separated from the experimental group and the normal control, respectively; And
2) A method for diagnosis, prognosis or monitoring for radiation therapy, comprising the step of selecting the sample whose expression level of miR-130a in step 1) is reduced compared to the normal control.
8. The method according to claim 7, wherein said biological sample is organs, tissues, cells, urine, blood, serum or plasma.
1) treating the test substance with cells or tissues expressing miR-130a;
2) measuring the expression of miR-130a from the cell or tissue of step 1); And
3) The method for screening substances promoting sensitivity to radiation, wherein the expression of miR-130a in step 2) is increased compared to the untreated control.

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