KR101596166B1 - A Use of microRNA for Ddiagnosing and Treating Brest Cancer - Google Patents

Abstract

The present invention relates to a p130Cas control function of a specific micro RNA (miRNA) relating diagnosis and treatment of breast cancer. Specifically, the present invention relates to usage of three types of miRNAs (miR24, miR362, and miR329) which are specific micro RNAs controlling the function of p130Cas, and an anti-cancer usage according to a breast cancer diagnosing ability, a breast cancer treating ability upon decreasing of p130Cas, and a resistance controlling ability of a tamoxifen anti-cancer agent. An anti-cancer pharmaceutical composition against a cancer activating p130Cas comprises a micro RNA-24 (miR24) or a micro RNA-362 (miR362) as an active component, wherein the micro RNA-24 and the micro RNA-361 control expression of p130Cas.

Description

[0001] The present invention relates to a microRNA for diagnosis and treatment of breast cancer,

The present invention relates to the ability to regulate p130Cas of certain microRNAs (miRNAs) associated with breast cancer diagnosis and treatment. More specifically, the present invention relates to the use of three kinds of miRNAs (miR24, miR362, miR329) which are specific microRNAs (microRNA, miRNA) that regulate the function of p130Cas and include the ability to diagnose breast cancer, And tamoxifen anticancer drug resistance-inhibiting ability.

Breast cancer (BCa) is the most common cancer diagnosis in women and is the second leading cause of cancer-related death among women (Ries LAG, et al. (eds). SEER Cancer Statistics Review, 1975-2003, National Cancer Institute, Bethesda, MD]. A major advance in breast cancer treatment over the past two decades has significantly improved disease-free survival (DFS) rates. For example, therapies using antibodies reactive to tumor-associated antigens have been used to block certain cellular processes to slow disease progression or prevent disease recurrence. Despite recent advances in breast cancer treatment, a considerable number of patients are eventually killed by recurrent disease. Therefore, it is necessary to prevent, slow, or prevent progression of recurrent disease.

Preoperative chemotherapy for breast cancer patients is generally an expensive treatment that causes toxic side effects to the human body and is economically costly. In particular, since the significant improvement in survival rate is observed only in a very small number of patients, there is an ongoing effort to screen patients who respond to anticancer drugs from a personalized therapy perspective.

In recent years, preoperative chemotherapy has become more and more popular as a platform for basic research on drug development and resistance to cancer, as well as improving quality of life. The pathologic complete response (pCR) after preoperative chemotherapy in the NSABP B-18 study was reported to be the only predictor of the patient's prognosis. The use of pCR as a surrogate marker for the efficacy of the new therapy in a short period of time is under consideration.

In addition, it is possible to identify an acquired resistance-related marker or an intrinsic resistance-related marker inherent in a tumor immediately after the use of an anticancer agent that can confirm the efficacy of preoperative chemotherapy at an earlier stage , And these studies are mostly performed on preoperative chemotherapy platforms (plarforms)

In addition, recent attempts to predict tumor cell changes, pathologic complete remission, and the progression of tumors before surgery by using preoperative chemotherapy using functional molecular imaging have.

Toxic side effects of anticancer drugs vary according to individual and ethnic characteristics. Depending on the therapeutic dose used, the toxic side effects may be life-threatening. Therefore, the toxicity and side effects should be carefully observed and controlled when using anticancer drugs. For this reason, a number of studies have been conducted on the prognostic factors of the patients, and based on these studies, attempts were made to determine the selective administration of anticancer drugs,

Therefore, research and development of anticancer drugs is expected to accelerate the researches on 'molecular biology' and 'genome project' genetic information in order to overcome side effects, metastasis and tolerance, which are problems of existing anticancer drugs.

In particular, tolerance to anticancer drugs is frequently encountered in cancer treatment, and it is known that curing is difficult. Therefore, it is as important as chemotherapy to remove cancer primarily in cancer treatment, how to control the expression of resistance by the use of chemotherapeutic drugs, and the problem of when chemotherapeutic drugs can not be effectively used due to resistance to chemotherapy It should be solved.

Accordingly, there is a need in the art to develop resistant diagnostic markers and vaccines that will provide reliable protection against the recurrence of disease in breast cancer patients with clinical tolerance, which are resistant to anti-cancer agents.

As a result of intensive efforts to overcome the limitations of the prior art as described above, the present inventors have found a new use of a specific microRNA as a regulator of p130Cas and can use it to diagnose breast cancer, The present invention has been completed.

Korean Patent Application No. 2014-0067001

It is an object of the present invention to provide an anticancer composition for cancer in which p130Cas is activated, based on a novel use of a specific microRNA as a regulator of p130Cas.

It is an object of the present invention to provide a composition for diagnosing breast cancer comprising specific miRNAs and uses thereof.

Another object of the present invention is to provide a composition for treating breast cancer comprising specific miRNAs and uses thereof.

It is yet another object of the present invention to provide a method for inhibiting resistance to the anticancer agent Tamoxifen.

In order to solve the above problems,

There is provided a diagnostic breast cancer marker composition comprising a primer sequence specific to at least one microRNA selected from among microRNA-24 (miR24), microRNA-362 (miR362) and microRNA-329 (miR329).

The present invention also provides an anticancer composition for p130Cas-activated cancer, comprising at least one microRNA selected from miR24, miR362 and miR329 or an expression or action enhancer of said microRNA as an active ingredient.

one or more microRNAs selected from miR24, miR362 and miR329, and the expression or action enhancer of said microRNA are characterized by inhibiting the expression of p130Cas. At this time, the expression form or action promoter of at least one gene selected from among miR24, miR362 and miR329 is most representative of the vector form into which these genes are expressed.

In particular, the miR24, miR362 and miR329 genes may each be, for example, a sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3

The p130Cas-activated cancer may be selected from the group consisting of breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, non-small cell lung cancer, brain cancer, laryngeal cancer, gallbladder cancer, pancreatic cancer, rectal cancer, pituitary cancer, thyroid cancer, , Stomach cancer, bronchial cancer, and most preferably breast cancer.

The anticancer composition of the present invention is useful for: (i) lowering the activity of cancer cells; (ii) decreased colony forming ability; (iii) decreased cell mobility; (iv) inhibition of early development of the cancer (tumor formation ability deterioration).

In another aspect of the present invention, there is provided a method for expressing or activating one or more genes selected from among microRNA-24 (miR24), microRNA-362 (miR362) and microRNA- A composition for inhibiting resistance to tamoxifen, an anticancer agent, is provided.

Here, the miR24, miR362 and miR329 genes are described as described above.

In particular, the anticancer drug Tamoxifen is resistant to tamoxifen, and when p130Cas is activated, the expression of p130Cas is induced by promoting the expression of miRNA of the present invention. And tamoxifen (tamoxifen), respectively.

The present invention also provides a method for screening a substance for treating breast cancer comprising the steps of:

(a) treating an animal cell expressing at least one gene selected from among microRNA-24 (miR24), microRNA-362 (miR362) and microRNA-329 (miR329) with an anticancer candidate substance; And

(b) promoting expression of at least one gene selected from among microRNA-24 (miR24), microRNA-362 (miR362) and microRNA-329 (miR329) as compared with the control group in which the anti- Selecting a substance as a substance for treating breast cancer.

In this case, it is further preferable that the method further comprises the step of confirming the expression of p130Cas according to the presence or absence of the anticancer candidate substance and selecting the substance for inhibiting the expression of p130Cas as a substance for treating breast cancer.

The present invention relates to a method of inhibiting the expression of p130Cas by up-regulating the miR24, miR362 and / or miR329 genes, thereby inhibiting the development and growth of cancer cells. Diagnostic uses, anticancer uses and resistance to tamoxifen, and the genetic treatment of p130Cas-related cancer diseases It will be useful.

FIG. 1 is a schematic diagram and sequence diagram of a prepared outer genome for confirming a binding site of a microRNA of the present invention.
FIG. 2 shows photographs and expression of p130Cas over-expressing cell line and control group.
FIG. 3 is a Western blot analysis result showing a decrease in p130Cas expression after treatment with the microRNA of the present invention.
FIG. 4 shows the results of confirming the expression of p130Cas mRNA by injecting microRNA of the present invention into MCF7.
FIG. 5 is a Western blot analysis result of the degree of GFP expression, a reporter protein whose expression is inhibited when the microRNA of the present invention is bound.
FIG. 6 shows the results of confirming cell activity according to the decrease of p130Cas expression.
FIG. 7 shows the results of confirming the ability of the MCF cell line to colonize by overexpression of the microRNA of the present invention.
FIG. 8 shows the results of comparing the cell survival rate according to tamoxifen treatment for the test group and the control group overexpressing the microRNA of the present invention.
FIG. 9 shows the result of Scratch wound healing assay for observing changes in cell mobility due to a decrease in p130Cas expression.
10 shows the results of migration assay and invasion assay analysis to examine the effect of the microRNA of the present invention on cell mobility and invasiveness.
11 is a xenograft analysis result to confirm whether the microRNA of the present invention actually affects cancer development in the body.

The terms used in the present invention are defined as follows.

"Cancer "," tumor ", or "malignant" refers to or represents the physiological condition of a mammal that is generally characterized by unregulated cell growth. Examples of cancers in the present invention include cancers that are affected by p130Cas modulation, particularly those in which p130Cas is overexpressed and activated.

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included.

"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

The term "gene expression" generally refers to a cellular process in which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in the cell. In this sense, gene expression includes post-transcriptional and post-transcriptional processes that not only involve transcription and translation processes, but can also affect the biological activity of the gene or gene product. Such procedures include, but are not limited to, RNA synthesis, processing and transport as well as polyp peptide synthesis, transport and post-translational modification of the polypeptide. In the case of a gene that does not encode a protein product, such as an miRNA gene, the term "gene expression" refers to a process in which a precursor miRNA is produced from a gene. Generally, this process is referred to as transcription, although the transcription product of the miRNA gene is not translated to produce a protein, unlike the transcription induced by RNA polymerase II on the protein coding gene. Nevertheless, the generation of mature miRNAs from miRNA genes is encompassed by the term "gene expression" as that term is used herein

"Diagnosis" means identifying the presence or characteristic of a pathological condition. Among them, the present invention is particularly useful for diagnosis of breast cancer. The present invention includes prediction of recurrence progression and metastasis of breast cancer.

"Diagnostic markers or diagnostic markers are substances that can distinguish breast cancer cells from normal cells and include polypeptides or nucleic acids (eg, mRNA) that show increased patterns in cells with breast cancer as compared to normal cells, Lipids, glycolipids, glycoproteins, sugar (monosaccharides, disaccharides, polysaccharides, etc.), and the like.

"Protein" also includes fragments, analogs, and derivatives of proteins that retain essentially the same biological activity or function as the reference protein

"Label" or "label " means a compound or composition that facilitates the detection of a reagent, such as a reagent conjugated, conjugated, conjugated, or fused to a nucleic acid probe or antibody. The label may itself be detected (e. G., A radioactive isotope label or a fluorescent label), in the case of an enzyme label, to catalyze the chemical modification of the detectable substrate compound or composition.

The term "modulator" refers to a polypeptide, nucleic acid, macromolecule, complex, molecule, small molecule, compound, species (spontaneous or non-naturally occurring), or a bacteria, plant, fungus, Refers to an extract made from a biological material such as a cell or tissue. The modulator may be included in the assay and used to determine whether it is an inhibitor or activator (direct or indirect) of a functional property, biological activity or process, or a combination thereof (such as an active agent, a partial agonist, a partial agonist, - inhibitors of microbial agents, microbial infection or proliferation, etc.).

"Transfection or transfection" refers to the process by which extracellular DNA enters host cells in the absence and presence of entities. "Transfected cell" refers to a cell in which extracellular DNA is introduced into a cell and contains extracellular DNA. DNA can be introduced into the cell and the nucleic acid can be inserted into the chromosome or replicated as an extrachromosomal material. In the present specification, the terms transfection and transfection are used in combination.

The term "vector" refers to any nucleic acid that is inserted into a host cell, recombined with the host cell genome, inserted into it, or contains a competent nucleotide sequence that replicates spontaneously as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, and viral vectors.

"microRNA (microRNA, miRNA)" refers to a small RNA that plays a role in controlling the expression of genes in a living organism. It targets the mRNA translation by complementary base pairing with the target mRNA 3'UTR (untranslated region) Is a small RNA containing 20-25 nucleotides that can play an important regulatory role in the gene expression process. The microRNA plays an important role in cell function including proliferation, differentiation, and cell death, and is an evolutionarily conserved regulatory substance present in all animals. Some microRNAs have a proliferative, Histone modification, DNA methylation, etc.).

By "inhibitor" is meant a substance that inhibits, blocks or reduces the expression or activity of a particular gene. The p130Cas inhibitor used in the present invention is a substance that inhibits, blocks or reduces the expression or activity of p130Cas, respectively. The mechanism of activation of the inhibitor is not particularly limited. Examples include organic or inorganic compounds, proteins, carbohydrates, polymeric compounds such as lipids, and composites of various compounds.

"Activator" means a substance that promotes, enhances or increases the expression or activity of a particular gene. The miR24, miR362 or miR329 p130Cas expression or activity promoter used in the present invention is a substance that promotes, enhances or increases the expression or activity of each microRNA. The activation mechanism of the promoter is not particularly limited. Examples include organic or inorganic compounds, proteins, carbohydrates, polymeric compounds such as lipids, and composites of various compounds.

The expression "up-regulation" means that the expression of a specific gene into mRNA or the expression level of a protein is markedly increased by gene transcription or translation in activated cells compared to normal tissue cells do.

"Antibody" is used in its broadest sense and specifically includes intact monoclonal (monoclonal) antibodies, polyclonal antibodies, multispecific antibodies (e. G. Bispecific antibodies) formed from at least two intact antibodies, and And antibody fragments exhibiting the desired biological activity.

The term "anticancer effect" includes the above-mentioned cancer cell suppressing effect, and also includes the inhibition, blocking or reduction of the activity of cancer cells in tumorigenesis, malignancy and / or metastasis, And the like.

"Treatment" means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, "treatment" may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. &Quot; Palliating " a disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression, It means to lose.

"Gene therapy" refers to a method of treating inherited or acquired gene abnormalities that are difficult to treat by conventional methods, by genetic engineering methods. Specifically, for the treatment and prevention of chronic diseases such as congenital or inherited genetic defects, viral diseases, cancer or cardiovascular diseases, genetic materials such as DNA and RNA are administered into the body to express therapeutic proteins As a therapeutic method for inhibiting the expression of a specific protein, it is expected to be an alternative means of existing medical treatment methods as well as overcoming incurable diseases because it can fundamentally treat diseases by analyzing the cause of diseases at a gene level.

An "effective amount" is an appropriate amount that affects a beneficial or desired clinical or biochemical outcome. An effective amount may be administered one or more times. For purposes of the present invention, an effective amount of an inhibitor compound is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, slow down, or delay the progression of a disease state. If the recipient animal is capable of enduring the administration of the composition, or the administration of the composition to the animal is suitable, the composition will be "pharmaceutically or physiologically acceptable ". If the dose administered is physiologically significant, it can be said that the formulation is administered in a "therapeutically effective amount ". The formulation is physiologically relevant if the presence of the formulation results in a physiologically detectable change in the recipient.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The disease to which the present invention is directed may include all cancers (tumors) associated with the regulation of p130Cas function, but most preferably it is a breast cancer.

The present inventors have found that the expression of miR24, miR362 and miR329 specific microRNAs in breast cancer tissues is closely related to the expression of p130Cas. That is, the function of the specific genes in breast cancer as p130Cas regulator was first identified.

In addition, based on the fact that p130Cas is relatively strongly expressed in a breast cancer cell line having an anticancer drug resistance, it was also first described for the use of anticancer drug tamoxifen in the prevention of resistance.

<miRNA>

Therefore, the present invention relates to miR24, miR362 and miR329 which are specific miRNAs as modulators of p130Cas.

miRNA (microRNA) is a single-stranded RNA molecule of 21-25 nucleotides (nt), a regulatory element that controls gene expression in eukaryotes. miRNAs are made up of two stages of processing. The first miRNA transcript is made in the nucleus by the RNase III type enzyme called Drosha, which is a 70-90 nt stem-loop structure, that is, a pre-miRNA which is then transferred to the cytoplasm and cleaved by an enzyme called Dicer It is made of 21-25 nt of mature miRNA. Mature miRNAs are processed from longer transcripts and have a length of ~ 22 nucleotides (nt).

The primary-miRNA (pri-miRNA) may be transcribed by RNA Pol II as an independent transcription unit or may originate from an intron connected to a host gene. Currently there are over 1,000 human miRNAs found. Only a small fraction of their unique biological significance has been identified. Studies of specific miRNAs may contribute to understanding of life events such as cell growth, death, differentiation, and development in various types of cells. Abnormal miRNA expression is closely related to the onset of various human diseases. In particular, For the first time, specific miRNAs that regulate p130Cas activity, which promotes growth and development.

MiR24, miR362 and miR329 in the present invention include nucleic acid molecules of about 17 to 24 nucleotides which are generated from their pri-miRNA, pre-miRNA, mature-miRNA or functional equivalent. Examples of miRNA sequences of SEQ ID NOS: 1 to 3 include:

3 'gacaaggacgaCUUGACUCGGu 5' (SEQ ID NO: 1)

3 'acuuaggaacuuAUCCACACAa 5' (SEQ ID NO: 2)

3 'uuucUCCAAUUGGUCCACACAa 5' (SEQ ID NO: 3)

MicroRNAs (miRNAs) can target specific messenger RNAs (mRNAs) and negatively regulate gene expression by inducing their degradation or translation inhibition. The microRNAs of the present invention are regulators of p130Cas and are characterized by having a negative regulatory function that targets p130Cas mRNA and suppresses its expression.

The present invention relates to miR24, miR362 and miR329, which are specific miRNAs capable of regulating cancer diagnostic and anticancer activities based on the regulation mechanism of p130Cas.

Overexpression of the miR24, miR362 or miR329 gene exerts the following functions:

(i) a decrease in p130Cas expression

It is known that the p130Cas protein binds to Smad3, a protein that mediates TGF-beta signal, thereby eliminating the cell proliferation inhibitory effect of TGF-beta. As a result, it is known that when p130Cas protein expression is inhibited, It is possible to suppress the proliferation of the cells.

The microRNA of the present invention is not involved in the direct destruction of p130Cas mRNA but directly binds to p130Cas mRNA and inhibits the expression of the target protein. In one embodiment of the present invention, it was confirmed that the expression of p130Cas protein was inhibited in all three experimental groups of MCF7 in which microRNA overexpression occurred.

(ii) Degradation of cell activity

Overexpression of the microRNAs of the present invention decreases the activity of cancer cells. In particular, in the case of an experimental group in which miR-362-3p and 329 are overexpressed, the effect of reducing cell activity is significant.

In the example of the present invention, it was confirmed that the cell activity was decreased by about 20% as compared with the control group.

(iii) degradation of colony forming ability in breast cancer cell lines

The breast cancer cell line due to overexpression of the microRNA of the present invention, for example, a decrease in the ability of the MCF cell line to colonize. This is an effect of lowering the expression of p130Cas in microRNA.

In one embodiment of the present invention, it was confirmed that the colony forming ability was reduced by about 50 to 30% as compared with the control group. In particular, MCF7 overexpressing p130Cas, which is not affected by microRNAs, was found to have no effect on colony forming ability even when microRNAs were overexpressed. Thus, degradation of p130Cas in microRNAs affected the degradation of colony forming ability I could see.

(iv) Degradation of cell mobility

Overexpression of the microRNA of the present invention lowers cell mobility. In addition, overexpression of microRNAs in the cells directly inhibited the expression of p130Cas, resulting in changes in cell mobility.

In one embodiment of the present invention, in the case of the migration assay, the cell mobility decreased by 30% to 50% in the three experimental groups compared with the control group, and the cell mobility declined by 30% to 50% in the invasion assay . In addition, as compared with the case where p130Cas not affected by microRNA was overexpressed, it was confirmed that the overexpression of microRNAs directly inhibited the expression of p130Cas, because the changes of the mobility and invasiveness of the cells were observed.

(v) Inhibition of early development of cancer

Through the xenograft assay, it was confirmed that the decrease of p130Cas expression through overexpression of the microRNA of the present invention can induce a decrease in tumorigenicity in the body

(vi) Effect on drug reactivity

It is well known that p130Cas is expressed relatively strongly in MCF7 cell line having anticancer drug resistance.

The microRNA of the present invention induces a decrease in the expression of p130Cas, and thus can change the anticancer drug resistance, that is, the drug reactivity. Preferably, the anticancer agent is Tamoxifen. Therefore, the present invention can be applied to a method of overcoming tolerance of an anticancer agent or increasing use efficiency in chemotherapy used for breast cancer treatment.

&Lt; Diagnostic composition &

In one aspect, the present invention relates to the use of at least one gene selected from among miR24, miR362, and miR329 as diagnostic markers, wherein the expression of one or more genes selected from miR24, miR362, and miR329 is decreased and / or p130Cas overexpression The diagnosis can be made by predicting breast cancer.

As a specific example, the present invention provides a diagnostic marker comprising the diagnostic marker or diagnostic composition comprising the diagnostic marker; And provides a method for early diagnosis of breast cancer or information for early diagnosis of breast cancer using the same.

The diagnosis of breast cancer includes generation, recurrence, and progression.

The selection and application of significant diagnostic markers determines the reliability of diagnostic results. Significant diagnostic markers are those markers that are highly reliable with high validity and consistency in repeated measurements. The breast cancer diagnostic markers of the present invention show the same results in repeated experiments with genes whose expression always increases as a direct or indirect factor together with the onset of breast cancer and the difference in expression level is very large when compared with the control group, They are highly reliable markers with little probability. Therefore, the diagnosis result based on the result of measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable.

The specific genes of the present invention are characterized in that they are expressed upregulated in patients with breast cancer. Accordingly, it is possible to confirm the onset of breast cancer through reduction of expression of one or more genes selected from miR24, miR362 and miR329 and / or confirmation of overexpression of p130Cas. In particular, when using the markers, it is also possible to confirm the expression of individual genes, but it is preferable to measure the combination of two or more genes, more preferably, three miRNA combinations.

The level of gene expression may be determined by measuring the level of gene expression in a biological sample including, but not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine, Or confirming the amount of the protein.

The expression level of mRNA is measured by measuring the presence or the mRNA level of the marker genes in a biological sample in order to diagnose breast cancer. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) Northern blotting, DNA chips, and the like, but are not limited thereto.

Accordingly, in one specific embodiment of the present invention, a diagnostic marker composition for breast cancer including a primer sequence specific to at least one gene selected from miR24, miR362 and miR329 can be provided. At this time, it is preferable to use pri-miRNA 362 for miR362.

In addition, the protein expression level measurement is a process for confirming the presence and expression level of a protein expressed from a breast cancer marker gene in a biological sample in order to diagnose breast cancer. Preferably, an antibody that specifically binds to the protein of the gene Can be used to confirm the amount of protein. Methods for this analysis include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket, But are not limited to, immunoelectrophoresis, immunohistochemistry, immunoprecipitation assays, complement fixation assays, fluorescence activated cell sorters (FACS), protein chips, and the like. .

In a similar aspect, the present invention relates to a kit for early diagnosis of human breast cancer. Based on the functions described above, it can provide information about the early stages of breast cancer development, i.e. early diagnosis

The kit is used to determine the presence or absence of breast cancer by analyzing the expression of one or more genes selected from miR24, miR362 and miR329, which can be performed in two ways: genetic analysis and immunoassay ).

To this end, the kit may comprise a primer or a probe specifically binding to at least one gene sequence selected from miR24, miR362 and miR329; Or an antibody that specifically binds to at least one protein selected from the proteins expressed by one or more genes selected from among miR24, miR362, and miR329.

When the human breast cancer diagnostic kit of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally include a reagent necessary for PCR amplification, such as a buffer, a DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus Thermostable DNA polymerases obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs. And, when the human breast cancer diagnostic kit of the present invention is applied to immunoassay, the kit of the present invention may optionally include a secondary antibody and a label substrate.

The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.

The methods for diagnosing breast cancer are directed at examining the expression of a particular marker and the methods disclosed herein are convenient, efficient, and cost effective methods of obtaining data and information useful in assessing appropriate or effective therapies for the treatment of breast cancer patients. Lt; / RTI &gt;

In another embodiment, the present invention relates to a method for screening a substance for treating breast cancer capable of promoting expression of at least one gene selected from miR24, miR362 and miR329 and / or inhibiting the expression of p130Cas .

The method comprises, in one embodiment,

(a) expressing one or more genes selected from miR24, miR362 and miR329 in the presence of a candidate substance; And

(b) selecting a candidate substance for breast cancer treatment as compared to the case where one or more genes selected from among miR24, miR362 and miR329 are expressed without a candidate substance, when the expression of the genes is promoted have.

At this time, it is preferable to confirm whether or not the expression of p130Cas is suppressed, and to select a candidate substance for suppressing the expression of p130Cas.

As such, the present invention may include various uses related to the diagnosis of breast cancer based on the over-expression characteristics of miR24, miR362 or miR329 gene, and in particular, it is possible to estimate the prognosis of the disease in the course of treatment after the onset of breast cancer, It may also be used as a prognostic factor.

In other words, the therapeutic effect can be predicted by analyzing the degree of expression of these miRNAs and the expression level of p130Cas in tissues / cells of the lesion / cell or lesion after treatment such as surgery or medication due to breast cancer. MiR24 , miR362 or miR329 overexpression in the expression of breast cancer or suppression of expression of p130Cas is predicted to be favorable due to appropriate treatment of breast cancer, whereas miR24, miR362 or miR329 The expression of p130Cas is similar to that of breast cancer at the onset of breast cancer, and the expression of p130Cas is similar to that of breast cancer.

<Anticancer composition>

On the other hand, the present invention provides anticancer applications based on the ability of the miR24, miR362 or miR329 gene to express p130Cas in a different aspect.

This is due to the suppression of p130Cas expression by overexpression of the miR24, miR362 or miR329 gene. That is, the present invention is based on the discovery that the effect of the expression of the microRNA of the present invention on the cancer cells is due to the regulation of p130Cas.

Therefore, the miR24, miR362 or miR329 gene of the present invention is characterized not only by the diagnostic function of p130Cas activity-related cancer, for example, breast cancer, but also by its own ability to treat breast cancer.

On the basis of the functions described above, such as lowering of cell activity, lowering of colony forming ability, lowering of cell mobility, inhibition of early development of cancer, lowering of tumor forming ability, and suppression of tamoxifen anticancer drug resistance as a result of lowering of p130Cas expression, As an example, there is provided an anticancer composition for a p130Cas-related cancer, that is, a cancer for which p130Cas is activated, comprising the expression or activity promoter of miR24, miR362 or miR329 gene as an active ingredient.

The cancer to which p130Cas is activated may be selected from the group consisting of breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, non-small cell lung cancer, brain cancer, laryngeal cancer, gallbladder cancer, pancreatic cancer, rectal cancer, parathyroid cancer, Cancer of the head and neck, cancer of the head and neck, colon cancer, gastric cancer, bronchial cancer, kidney cancer, basal cell carcinoma, ulcerative and papillary type squamous cell carcinoma, metastatic skin carcinoma, osteosarcoma, Ewing sarcoma, , Small cell lung tumor, gallstone, islet cell tumor, primary brain tumor, acute and chronic lymphocyte and granulocyte tumor, hair cell tumor, adenoma, hyperplasia, watery carcinoma, chromium-friendly neoplasm, mucosal neoplasm, Neoplasms, retinoblastomas, soft tissue sarcomas, malignant carcinoids, ovarian tumors, ovarian tumors, smooth muscle tumors, cervical dysplasia and intraepithelial carcinomas, neuroblastoma, retinoblastoma, soft tissue sarcoma, A malignant melanoma, and epidermal hemangioblastoma. The present invention also relates to a method for the treatment and / or prophylaxis of a malignant melanoma, a malignant melanoma, an epidermoid carcinoma, And may be at least one kind of cancer selected from the group consisting of carcinomas. Most preferably it is breast cancer.

"Accelerator" means a substance that promotes, enhances or increases the expression or activity of a gene of interest. The activation mechanism of the promoter is not particularly limited. Examples include organic or inorganic compounds, proteins, carbohydrates, polymeric compounds such as lipids, and composites of various compounds. For example, an &quot; enhancer &quot; may include a substance that promotes, enhances or increases the expression or activity of these miRNAs. As will be apparent to those skilled in the art, it may be chemically or biochemically modified, or it may be formulated into a composition and administered to the target cells by any of a number of means or through an expression construct.

In some embodiments, the miRNA molecule or miRNA precursor molecule is expressed from a transcription unit inserted into a nucleic acid vector (commonly referred to as a recombinant or expression vector). A vector can be used to target a specific gene by delivering a nucleic acid molecule encoding the miRNA into the cell. Examples of the recombinant vector include a DNA plasmid or a viral vector. A variety of expression vectors are known in the art. Selection of a suitable expression vector may be made based on a number of factors, including, but not limited to, the cell type for which expression is intended.

The nucleic acid encoding at least one of the miR24, miR362, and miR329 genes may be used without limitation as long as it has a nucleotide sequence known in the art. The nucleic acid may also have a nucleotide sequence encoding its functional equivalent. For example, nucleic acids represented by SEQ ID NOS: 1 to 3 can be used.

The "functional equivalents" are those having at least 70%, preferably 80%, more preferably 90% or more sequence homology with the microRNA sequences as a result of addition, substitution or deletion of amino acids, Refers to a polypeptide exhibiting homologous physiological activities. Here, 'substantially homogenous physiological activity' means an activity of inhibiting the expression of p130Cas and inhibiting the function of tumor cells. The nucleic acid can also be prepared by a recombinant method known in the art (Sambrook, Fritsch and Maniatis, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Short Protocols in Molecular Biology, John Wiley and Sons, 1992).

The expression vector comprising the nucleic acid according to the present invention may be prepared by methods known in the art, including, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation Liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electropora tion, ), A gene gun, and other known methods for introducing a nucleic acid into a cell.

Generally, the cells expressing the target gene are transfected. For the transfection, various methods such as, for example, electroporation, use of a cationic lipid or a cationic polymer as a helper for transfection are used . The cells are then cultured under suitable conditions to allow expression of the target gene. The expression of the target gene is then determined using suitable techniques such as, for example, RT-PCR or determination of the amount of reporter gene. Basically, although any type of cell can be used for transfection, in preferred embodiments the cell is a eukaryotic cell, preferably an animal cell, more preferably a mammalian cell, most preferably a human cell.

The anticancer composition according to the present invention comprises one or more miRNA selected from therapeutically effective amounts of miR24, miR362 and miR329 so that p130Cas is down-regulated; Or mimics of them (mimics). Therapeutically effective amounts may be provided by known routes of administration. Embodiments in accordance with the present invention include those miRNAs; Or their mimics into a cancer cell that is involved in p130Cas.

The composition of the present invention is not limited thereto, but may include or utilize any of the above-mentioned means for transfecting a genetic material into a target cell. Preferably the composition may comprise cationic lipids and may further comprise a cationic amphipathic substance such that the miRNA and analog thereof can be released into cancer cells.

Within the transfected cells, the formation of double-stranded RNA through the binding of miRNAs results in the degradation of the p130Cas mRNA transcript through a process similar to RNA interference (RNAi), blocking the protein translation system or causing degradation of the p130Cas mRNA Inhibiting protein translation, ultimately inhibiting p130Cas expression.

Therefore, as a specific example of the present invention, there is provided an anticancer pharmaceutical composition comprising the expression vector as an active ingredient, and a method of treating cancer. Specifically, the method may comprise the step of administering the expression vector of the present invention to a cancer patient. In another embodiment of the present invention, p130Cas-activated cancer cells, for example, breast cancer, etc., are treated by transfecting cancer cells into which p130Cas is activated with one or more genes of miR24, miR362 and miR329 genes to inhibit and decrease p130Cas expression &Lt; / RTI &gt;

Meanwhile, the nucleic acid molecule and the promoter of the present invention may be formulated into a pharmaceutical composition for anticancer produced according to a conventional pharmaceutical synthesis technique. The anticancer pharmaceutical composition has no cytotoxicity in cells that are not cancer cells.

The composition may comprise a pharmaceutically acceptable salt of the active or active agent. The composition may be administered simultaneously or sequentially. These compositions may contain, in addition to one active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials well known in the art. These materials must be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a variety of forms depending on the form of preparation desired for administration, for example, localized, intravenous, oral, brain, neural, or parenteral. Can generally be prepared by mixing with a diluent such as a filler, a diluent, a binder, a wetting agent, a disintegrating agent, a surfactant, or an excipient.

Further, it is contemplated that additional therapies may be used in the methods of the present invention. The one or more other therapies may be administered in combination with other therapies known in the art and specifically defined above as radiation therapy, surgery, immunotherapy, cytokine (s), growth inhibitor (s), chemotherapeutic (s) ), Tyrosine kinase inhibitors, rasparnesyl transferase inhibitors, angiogenesis inhibitors, and cyclin dependent kinase inhibitors. Among them, chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors, and antitumor agents. All of these drugs affect cell division or function in DNA synthesis and some pathways. Some formulations do not directly interfere with DNA, such as tyrosine kinase inhibitors (Gleevec).

The pharmaceutical composition of the present invention is preferably parenteral, and can be administered, for example, by intravenous administration, intraperitoneal administration, intratumoral administration, intramuscular administration, subcutaneous administration, or local administration, but is not limited thereto . In the case of breast cancer, it may be administered by injection directly into the tumor mass, but is not limited thereto.

The dosage ranges vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease. The daily dose is about 0.1 to 100 mg / kg, preferably 0.5 to 10 mg / kg for a compound, more preferably, but not necessarily, administered once a day or several times a day.

The pharmaceutical composition of the present invention can be used alone or in combination with adjuvant therapy such as surgical surgery. A chemotherapeutic agent that may be used with the compositions of the present invention is cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ), Ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, doxorubicin, but are not limited to, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5- 5-fluorouracil, vincristin, vinblastin and methotrexate, and the like.

In particular, by effectively overcoming (suppressing) tolerance to the tamoxifen anticancer agent, it can be usefully used as an active ingredient of an anticancer drug by inducing the inhibition of growth and death of cancer cells in breast cancer tissues

&Lt; Resistant inhibition &

Therefore, the present invention relates, in another aspect, to the ability of the miRNAs to inhibit resistance to anticancer agents, particularly to the ability to inhibit the resistance of one or more genes selected from miR24, miR362 and miR329 to tamoxifen, .

Anticancer drug "tolerance" means that cancer cells respond to anticancer drugs and cause genetic alteration, thereby weakening the effects of chemotherapy by attacking anticancer drugs. The term "resistant" may be used interchangeably with the term "resistant ".

By overexpressing at least one gene selected from miR24, miR362 and miR329 of the present invention, anticancer drug resistance can be suppressed by inducing a decrease in the expression of p130Cas, an anticancer drug resistance-related gene. More specifically, the miR24, miR362, and miR329 genes directly bind to p130Cas mRNA and inhibit expression of the target protein p130Cas, thereby suppressing resistance to tamoxifen.

Therefore, one or more genes selected from among miR24, miR362 and miR329 according to the present invention can be used as an anticancer adjuvant and can be used as an adjuvant for tamoxifen.

In addition, the present invention includes a method of using tamoxifen to inhibit the expression of p130Cas to reduce the side effects that may occur when using a high dose of tamoxifen and to enhance the anticancer effect.

<Screening>

In another aspect, the present invention provides a method for treating cancer, preferably for treating breast cancer, which promotes expression of at least one gene of miR24, miR362 and miR329 genes and / or suppresses expression of p130Cas, Screening method.

The method comprises, in one embodiment,

(a) treating an animal cell expressing at least one gene among miR24, miR362 and miR329 genes with an anticancer candidate substance; And

(b) selecting, as a substance for treating breast cancer, a substance in which expression of at least one gene among iR24, miR362 and miR329 genes is promoted as compared with a control group in which the anti-cancer candidate substance is not treated in the animal cell.

At this time, it is most preferable to confirm the p130Cas expression together. Materials with inhibition of p130Cas expression are selected compared to the control group not treated with anticancer candidate substance.

Examples of animal cells to be used include, for example, breast cancer cells or artificially produced breast cancer cells, and preferably human breast cancer cells. The cultivation is carried out under culture conditions and culture conditions that can be selected according to the conventional knowledge of those skilled in the art.

In this method, measuring the expression of miRNA and / or p130Cas does not limit the form of the biomarker target, such as RNA, DNA, protein, etc. associated therewith.

The various uses of the present invention, on the other hand, extend to a genetic approach involving the regulation of expression of p130Cas by upregulating or downregulating one or more of miR24, miR362 and miR329 genes.

That is, by inhibiting or destroying the translation of p130Cas RNA and by participating in post-transciptional gene silencing, it can inhibit the growth of cancer cells by inhibiting the expression of p130Cas mRNA.

< Example >

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Materials and methods

1. Organization preparation

The tissues used in this experiment were purchased from the Catholic Research Tissue Bank and used for the experiment. Normal mammary epithelial cells and cancer cells were purchased from 6 patients and used for the experiment. All regulations on the use of human tissues were conducted in compliance with the regulations of the Catholic University Institutional Ethics Committee.

2. Cell culture

DMEM culture medium containing 10% bovine serum was used for culturing all the cell lines used in the experiment. In a constant temperature incubator that maintained 37 ℃ 5% CO ₂ The cell lines were cultured in an environment where the concentration was maintained. In the intracellular injection of the outer genome, 35 mm ² Culture dish. In all other cases, the cell line was cultured and maintained using a 100 mm ² culture dish.

3. Fabrication of external dielectric carriers

A total of four plasmid forms and three microRNA-type outer genomes were used for the experiment through intracellular injection. The three types of microRNAs were purchased from Biona, and four plasmids were prepared by inserting the 3 'ends of p130Cas and p130Cas into the back of the fluorescent protein based on the pEGFP-C1 transporter purchased from BD Bioscience And two reporter plasmids inserted at the 3 'end of the mutant p130Cas were constructed (miR-362- 3p / 329 for miR-24-3p). A pEGFP-C1 transporter was used as a control for plasmid-shaped outer genome.

FIG. 1 shows a schematic diagram of a structure of the outer dielectric body for confirming the expected binding sites of microRNAs.

4. Intracellular injection of external genome

The injection of all external genomes used in the experiments was carried out using Lipofectamine 2000 from Invitrogen. Each outer dielectric was treated with Lipofectamine 2000 as a manufacturer's manual according to the experiment and then mixed with the cell culture solution to inject the outer genome into the cells.

5. p130Cas  Over-expressing cell line production

For the experiment to confirm whether the effect of microRNA expression on the cells was regulated by p130Cas, p130Cas overexpressed cell line and control cell line were prepared. 3'-terminal p130Cas overexpressed outer genomes were prepared, and cells were injected into the cells using lipofectamine 2000 and treated with G418 antibiotics. The colonies of surviving colonies were obtained by culturing in the culture medium containing the antibiotics for 2 weeks to establish a cell line having a single outer genome. FIG. 2 shows photographs of the p130Cas over-expressing cell line and the control group.

6. Analysis of gene expression changes in cells and tissues

Invitrogen's triazole solution was used for the analysis of gene expression and changes in the tissues purchased and the cells used in the experiments. After the tissue crushing and cell harvesting, the triose solution was treated to obtain mRNA, and cDNA was synthesized using Toyobo's cDNA synthesis kit. Real-time quantitative PCR method was used to quantify the gene expression and changes in the experimental and control groups.

7. Analysis of protein expression in cells

The obtained cells were subjected to RIPA to obtain intracellular proteins, and then the same total amount of proteins was separated by size in an SDS-PAGE gel through electrophoresis.

The specific size of the separated proteins was transferred to the PVDF membrane, and the antibody of the protein was attached to the PVDF membrane. After the secondary antibody, which can quantify the expression level of a specific protein, was treated, the light irradiated from the specific protein was recorded on the X-ray film to quantitatively analyze the change of the protein to be observed.

8. Cell activity analysis

To investigate the effects of microRNAs on the cancer cell line through p130Cas regulation, cell activity of the control and experimental groups was measured by MTT assay. The control and experimental cells were inoculated in 96-well culture dishes in the same amount and cultured for 2 days. Cell activity was then quantitated.

9. Analysis of drug reactivity in cells

We investigated the differences in drug reactivity between breast cancer cell lines treated with tamoxifen, which is an anticancer drug, and control group. Tamoxifen was diluted to a concentration of 10 μM in the culture medium, and then cultured for 2 days. After 2 days of culture, live cells were assayed by MTT assay to quantify the drug reactivity of the cells.

10. Cellular Colony Formability  analysis

Through the analysis of colony forming ability which can measure the degree of early cancer development of cancer cell line, it was confirmed that microRNA can affect early cancer development ability of cell line. The control and experimental groups were inoculated into a 6-well culture dish with 500 cells, and cultured for 3 weeks. After confirming colony formation on the culture dish, the number of colonies was quantified through crystal violet staining.

11. Tumor of the cell Formability  analysis

To investigate the effect of microRNA on cancer development in the body, xenograft assay was performed to confirm the change of cancer development in the body. Tumor formation was observed after 2 × 10 ⁶ cells of control and experimental groups were injected subcutaneously into immunodeficient nude mice. Four weeks after the injection, tumors were harvested and the effect of microRNAs on cancer development was quantified by size comparison.

12. Cell Mobility Analysis

Three different experiments were performed to observe the changes in cell mobility.

Scratch wound healing assay was performed to confirm direct cell mobility. The migration assay, which can confirm the environment similar to the movement of cancer cells in the body, can be confirmed in the laboratory and the invasion change, which is one of the mechanisms of cell migration in the body Invasion assays were performed.

In the case of Scratch wound healing assay, the cells of the control and experimental groups were cultured in a 35-mm ² culture dish and then cultured at a density of 90% or more. When the cells were cultured using a 200 μl pipet tip, (1%) medium containing low serum concentration was added to the cells, and the cell mobility was quantified by measuring the decreasing interval after 48 hours.

The migration assay was performed using a migration chamber from BD Bioscience. After 3 x 10 ⁴ cells were inoculated on the top of the migration chamber, the number of cells moved to the bottom of the chamber after 24 hours was confirmed by staining.

In the case of the invasion assay, matrigel coating was applied before inoculation of cells in the same chamber top. After 1 × 10 ⁵ cells were inoculated on the top of the coated migration chamber, the number of cells moved to the bottom of the chamber after 24 hours was determined by staining and quantified.

Example  1: Micro RNA  After the treatment, p130Cas  Decreased expression

The microRNA was injected into the cell line of MCF7, a breast cancer cell line, using lipofectamine 2000 to induce overexpression. Cells in which overexpression of microRNAs were induced were cultured for 48 hours. The expression of p130Cas in the cells was determined by Western blotting, in which the MCF7 cell line cultured under the condition that the microRNA was overexpressed was separated and proteins were isolated from the cells to quantify the expression amount of a specific protein.

As a result, as shown in Fig. 3, it was confirmed that the expression of p130Cas was inhibited in all three experimental groups of MCF7 in which overexpression of microRNAs occurred. Protein expression of p130Cas decreased by 40% in all three experimental groups compared to the control group.

We also investigated the effect of microRNAs on p130Cas mRNA and inhibited p130Cas expression.

The effects of microRNAs are largely influenced by the two types of target mRNA expression. In order to confirm whether the target mRNA is directly destroyed, the expression of p130Cas mRNA in the test group induced by overexpression of microRNA into MCF7 Respectively.

As a result, as shown in Fig. 4, no significant difference was found in the experimental group and the control group in all three microRNAs. In the experimental group treated with three kinds of microRNAs, only differences within the range of error that could occur experimentally were shown.

Therefore, based on these results, it was confirmed that the role of microRNA is not involved in the direct destruction of mRNA.

Example  2: Micro RNA Direct p130Cas mRNA Through the combination of p130Cas Inhibition of the expression of

Reporters were constructed to confirm the exact binding sites of microRNA with p130Cas mRNA. In silico analysis, we estimated the point where bonding is expected. Reporters containing the 3'-end of p130Cas containing the predicted part were constructed to confirm the inhibition of expression and a reporter transformed with the nucleotide sequence of the predicted part was constructed to confirm whether the microRNAs could bind properly Respectively. The expression level of GFP, a reporter protein that is inhibited when the microRNA was bound using the prepared plasmid, was observed and quantified by Western experiment.

As a result, as shown in Fig. 5, it was confirmed that all three microRNAs directly inhibited the expression of the target protein by binding to a site predicted to bind p130Cas mRNA through In silico analysis, and in the modified reporter The effect was inhibited.

All of the three microRNAs showed a 40% reduction in expression and were statistically significant. No statistically significant changes were observed in modified reporters.

Example  3: p130Cas  Decreased cellular activity due to decreased expression

It is known that p130Cas plays a role as an intermediate messenger in various signal transduction steps in cells and also has a role of increasing cell activity.

Therefore, we investigated the effect of decreased p130Cas expression on cell activity in experimental group treated with microRNA, compared with the control group.

Three microRNAs were induced to overexpress in MCF7 cells using lipofectamine and cultured for 48 hours. The changes in cell activity were compared by MTT assay.

As a result, as shown in FIG. 6, in the experimental group in which miR-362-3p and 329 were overexpressed among the three microRNAs, the cell activity was decreased by about 20% as compared with the control group and statistically significant However, miR-24-3p among the microRNAs showed a slight inhibition of cell activity, but no statistically significant difference was observed. It was confirmed that there is a difference in the effect of each microRNA on cell activity.

Example  4: Micro RNA  By overexpression MCF  Cell line Colony Formative  Check for degradation

The effect of microRNA on colony formation of cells was examined through colony formation experiments of cells closely related to the early tumorigenic ability during cancer development.

The control and MCF7 cell lines with overexpression of three microRNAs were used. The control and experimental cells were inoculated into a 6-well culture dish and cultured for 3 weeks to observe the result of colony formation. The formed colonies were stained with a crystal violet solution to measure the number of colonies formed to quantify colony forming ability.

As a result, in the control MCF7 cell line, the colony forming ability of 50 ~ 30% was decreased when the microRNA was overexpressed (FIG. 7). Statistical significance was confirmed in all experimental groups.

In addition, in order to confirm whether the degradation of these cancer cells actually occurs due to the degradation of p130Cas, the same experiment is carried out using a cell line overexpressing p130Cas in which the 3'- saw.

As a result, MCF7 overexpressing p130Cas, which is not influenced by microRNA, was found to have no effect on colony forming ability even when microRNA was overexpressed. Thus, degradation of p130Cas in microRNA affected the degradation of colony forming ability (Fig. 7).

Example  5: p130Cas Of drug-induced changes

In addition, We investigated the effect of overexpression of microRNA on cell drug reactivity using tamoxifen, a kind of anticancer drug.

It is known that relatively strong expression of p130Cas in the MCF7 cell line with anticancer drug resistance is presumed to affect the drug reactivity of the cell when the expression of p130Cas is induced by microRNA treatment.

Experimental results showed that in the three experimental groups overestimating microRNA as expected, the cell death rate was about 10% higher when tamoxifen was treated compared to the control group (Figure 8)

When tamoxifen was added to the culture medium and treated with tamoxifen for 48 hours in the experimental group and the control group, the survival rate of tamoxifen-treated cell line was compared with that of the control group, whereas the control group showed a mortality rate of about 40% Respectively.

In order to confirm that this result is a direct cause of decreased expression of p130Cas due to overexpression of microRNAs, the same experiment was conducted using a cell line overexpressing p130Cas in which the 3'-terminal of the microRNA was removed.

As a result, MCF7 overexpressing p130Cas not affected by microRNA showed no significant difference in drug reactivity even when overexpression of microRNA was observed.

These results demonstrate that overexpression of three microRNAs directly inhibits the expression of p130Cas, resulting in a change in drug reactivity. These results indicate that the anticancer therapy used for the treatment of breast cancer overcomes the resistance of the cancer drug, Which can be applied to the method of increasing the height of the body.

Example  6: p130Cas  Changes in cell mobility due to decreased expression

One of the methods of cell mobility, Scratch wound healing assay, showed a change in cell mobility in cells overexpressing microRNA.

As a result, as shown in FIG. 9, it was confirmed that the cell mobility was lowered in the experimental group using over-expressed MCF7 through the three microRNA intracellular injections as compared with the control group.

In addition, control and experimental cells were inoculated in the same amount in the culture dish, and then cells having the same area were removed to form empty spaces. The distance of movement of adjacent cells moved to the empty space from which the cells were removed was measured and quantified, In the cell line in which microRNA was overexpressed, 30% ~ 40% of the three experimental groups showed decreased cell mobility compared with the control group, and it was confirmed through repeated experiment that there was a statistically significant difference in mobility.

To confirm whether these results are directly related to the decreased expression of p130Cas due to overexpression of microRNAs, we conducted the same experiment using a cell line overexpressing p130Cas in which the 3'-terminal region of the microRNA was removed.

As a result, MCF7 overexpressing p130Cas, which is not influenced by microRNA, showed no significant difference in cell mobility even when overexpressing microRNA.

These results indicate that overexpression of the three microRNAs in the cells directly inhibited the expression of p130Cas, resulting in changes in cell mobility.

In addition, migration and invasion assays were performed to investigate the effect of microRNA on cell mobility and invasiveness.

Experiments were carried out using a migration chamber capable of testing the mobility of three-dimensional cells. The cell migration was quantified by measuring the number of migrated cells by staining only the cells that passed through the pores of the membrane structure after the inoculation of the same amount of cells of the experimental group and the control group on the top of the porous membrane structure.

migration assay showed 30-50% reduced cell mobility in the three experimental groups compared to the control group (Fig. 10).

In the invasion assay, a matrigel matrix is formed on the upper side of the same migration chamber to form an environment similar to that of the cell-binding tissue in the body. Then, the cells are transferred to the bottom of the membrane structure And counted and quantified.

As a result, cell migration was reduced by 30 ~ 50% in invasion assay as well as migration assay.

To confirm whether these results are directly related to the decreased expression of p130Cas due to overexpression of microRNAs, we conducted the same experiment using a cell line overexpressing p130Cas in which the 3'-terminal region of the microRNA was removed.

As a result, MCF7 overexpressing p130Cas, which is not influenced by microRNA, showed no significant difference in cell mobility even when overexpressing microRNA. These results indicate that overexpression of the three microRNAs in the cell directly inhibits the expression of p130Cas, resulting in changes in cell mobility and invasiveness.

Example  7: p130Cas  In the body due to decreased expression Tumor formation ability  change

A xenograft assay was performed to determine whether microRNAs actually affect cancer development in the body. The control group and the same amount (2 x 10 ⁶ ) of the experimental group cells overexpressing the three microRNAs were injected subcutaneously in the nude mice, and then tumor formation was observed for 4 weeks. Four weeks after the tumor was identified, the tumor was collected from the experimental animals and the difference between the experimental and control groups was quantitated by weighing the tumors.

As a result, as shown in FIG. 11, it was confirmed that the sizes of the tumors formed by the three experimental cell groups were reduced by 85 ~ 70% compared to the control group, and it was confirmed that there was a significant difference through the statistical processing.

These results indicate that the overexpression of microRNAs can induce a decrease in p130Cas in cancer cells.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry Academic Cooperation Foundation of Catholic University <120> A Use of microRNA for Ddiagnosing and Treating Brest Cancer <130> PN1408-259 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> RNA <213> miR24 sequence <400> 1 gacaaggacg acuugacucg gu 22 <210> 2 <211> 22 <212> RNA <213> miR362 sequence <400> 2 acuuaggaac uuauccacac aa 22 <210> 3 <211> 22 <212> RNA <213> miR329 sequence <400> 3 uuucuccaau ugguccacac aa 22

Claims (14)

1. An anticancer pharmaceutical composition for p130Cas-activated cancer comprising microRNA-24 (miR24) or microRNA-362 (miR362) as an active ingredient, wherein the microRNA- 24 (miR24) or the microRNA-362 (miR362) represented by SEQ ID NO: 2 inhibits the expression of p130Cas. delete The method according to claim 1,
The p130Cas-activated cancer may be selected from the group consisting of breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, non-small cell lung cancer, brain cancer, larynx cancer, gallbladder cancer, pancreatic cancer, rectal cancer, pituitary cancer, thyroid cancer, , Gastric cancer, and bronchial cancer. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; The method of claim 3,
Wherein the cancer to which p130Cas is activated is a breast cancer. The method according to claim 1,
Wherein said microRNA is contained in said composition in the form of a vector introduced to express microRNA-24 (miR24) or microRNA-362 (miR362). delete delete delete delete (MiR24) or microRNA-362 (miR362) as an active ingredient, wherein said microRNA-24 (miR24) or microRNA- 362 &lt; / RTI &gt; (miR362) are each composed of the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. 11. The method of claim 10,
Wherein said microRNA is contained in said composition in the form of a vector introduced to express microRNA-24 (miR24) or microRNA-362 (miR362). delete A screening method of a substance for treating breast cancer comprising the steps of:
(a) treating an animal cell expressing microRNA-24 (miR24) or microRNA-362 (miR362) with an anticancer candidate substance; And
(b) selecting a substance for breast cancer treatment as a substance in which the expression of microRNA-24 (miR24) or microRNA-362 (miR362) is promoted as compared with a control group not treated with an anticancer candidate substance in the animal cell Wherein the method further comprises the step of identifying the expression of p130Cas according to the presence or absence of the anticancer candidate substance and selecting the substance for inhibiting the expression of p130Cas as a substance for treating breast cancer. delete

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