WO2010016638A1

WO2010016638A1 - Composition containing anti -microrna for treating or preventing solid cancers

Info

Publication number: WO2010016638A1
Application number: PCT/KR2008/005789
Authority: WO
Inventors: Duk-Soo Bae; Jeong-Won Lee; Young-Ae Park
Original assignee: Samsung Life Public Welfare Foundation Samsung Medical Center
Priority date: 2008-08-05
Filing date: 2008-10-01
Publication date: 2010-02-11
Also published as: KR20100016844A; KR101042052B1

Abstract

A technology for preventing or treating solid cancer, preferably, cervical cancer, which is characterized in that a synergistic anti-cancer effect is achieved by the combined administration of an anti-cancer agent and an anti-microRNA complementarily binding to a specific microRNA that increases in its production specifically in solid cancer, preferably, cervical cancer, is herewith provided.

Description

COMPOSITION CONTAININGANTI-MICRORNA FOR TREATING OR PREVENTING SOLID CANCERS

Technical Field A composition for preventing or treating solid cancer, preferably, cervical cancer, which contains an anti-microRNA complementarily binding to a specific microRNA the level of which increases specifically in cervical cancer, as well as an anti-cancer agent, for synergistic effect by the combined administration of the anti- microRNA and the anti-cancer agent. Background Art

The treatment of cancer has been conducted by surgery, chemical therapy, and radiation therapy, wherein the surgery would be the most effective on a perfect treatment. However, chemical therapy or radiation therapy is also supplementarily used because there are limitations in the application of surgery depending on locations, stages and types of cancers. Since 1940s, chemotherapy has received much attention because it is relatively easily applicable regardless of stages or types of cancer. So far, a variety of anti-cancer chemotherapic agents, i.e., anti-cancer agents, have been developed. Currently, there are about 40 kinds of anti-cancer agents which are generally used, and they can be classified into the types of anti- metabolites, antibiotics, microtuble inhibitors, steroid type drugs, alkylating agents, targeted therapeutic agents and the like. However, many of anti-cancer agents developed so far accompany restrictions in their applications due to decease in cancer cell sensitivity and side effects.

Among the above, alkylating agents are the most widely used type of anti- cancer agents. By introducing an alkyl-group into a cancer cell, and thereby binding to the Guanine N-7 position and other critical regions on DNAs, they interfere with formation of base pairs and cross-links, thereby causing single- or double-strand breaks.

Cisplatin, which is an alkylating agent type anti-cancer agent, is a platinum based heavy metal compound. In cisplatin, platinum is the central atom and is bonded to four ligands — two chlorine atoms and two amino groups — in cis position. Cisplatin binds with two neighboring guanines on the DNA strands and forms interstrand crosslinks, thereby interfering with DNA synthesis. In other words, cisplatin binds to the DNA double strands in the nucleus of a cancer cell. Interfering with DNA transcription, cisplatin suppress the growth and proliferation of the cancer cell, removing the cancer cell and exerting anti-cancer effect. Among the currently used anti-cancer agents, cisplatin is being used in priority order over other drugs, but problems are beginning to appear — such as development of tolerance to cisplatin in cancer cells.

Therefore, it is one of the major research subjects in the area of anti-cancer therapy, along with development of novel anti-drugs, to develop agents and methodologies that can improve the anti-cancer capacity of alkylating agents including cisplatin.

Meanwhile, MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNAs which regulate gene expression. Mature miRNAs are of 18 to 25 nucleotides in length and are processed from hairpin precursors. MircoRNAs complementarily binds to their target mRNAs and act as a post-transcriptional regulator. They are known to induce unstabilization, by inhibiting mRNA translation and thus down-regulating the gene expression, or by catalyzing the process of cleavage of the mRNA. Special cellular functions associated with miRNAs include cell proliferation, metabolism regulation, developmental time courses, apoptosis, hematosis, neurogenesis, human oncogenesis, DNA methylation, and chromatin modification.

Evidence increasingly supports that miRNA gene expression is involved with oncogenesis and progress process of human cancers. Dinstinctive patterns of miRNA expressions are being reported in lung cancers, breast cancers, glioblastoma, hepatocellular carcinoma, thyroid papillary carcinoma, and recently, rectal cancers. Further, it is reported that miRNA expression signature is linked with clinical results of certain diseases. All this data suggest that miRNAs play a significant role in various types of human cancers.

However, molecular understanding of miRNA-mediated regulation of gene expression is still incomplete, and little has been known about the role played by miRNAs in oncognesis as well. DETAILED DESCRIPTION Technical Problem

The object is to identify specific miRNAs capable of improving anti-cancer effects of existing anti-cancer agents, and to provide a use thereof for preventing or preventing solid cancers.

To achieve said object, an embodiment provides a composition for preventing or treating solid cancers, which comprises an anti-cancer agent and a specific miRNA which is identified to increase in its production level specifically in solid cancers, preferably in cervical cancer. Technical Solution

The present invention relates to a technology using microRNA for suppressing proliferation of cancer cells. More precisely, the inventors found that the level of a certain microRNA is increased in a cancer call, and the proliferation of the cancer cell is inhibited when an anti-microRNA oligonucleotide complementary to a primary microRNA (pri-microRNA) of the microRNA is introduced into the cancer cell. Based on such findings, the inventors also found that, when the anti- microRNA oligonucleotide is used combined with an existing anti-cancer agent, a synergetic effect can be obtained, to complete the present invention. In particular, an embodiment suggests a result for miRNA regulations in early invasive squamous cell carcinomas (ISCCs) and normal cervical epithelial tissue sets, using an analysis by real-time quantitative PCR array method having high sensitivity and efficiency.

It was firstly identified herein that the expression levels of miRNAs are different between in early invasive squamous cell carcinomas and in normal cervical epithelial tissue, and that miR-199a, among the miRNAs, was observed to specifically increase in its production in the epithelial tissues of cervical cancer, suggesting miR-199a as a target for cervical cancer treatment.

Therefore, the present invention relates to a technology for treating and/or preventing cervical cancer which is of combined application of a miR-199a inhibitor and an anti-cancer agent. An embodiment provides a dosage form (administration formulation) or unit dosage form for treating and/or preventing cervical cancer, which contains a miR- 199 inhibitor and an anti-cancer agent. In the dosage for, a miR-199 inhibitor and an anti-cancer agent may be provided respectively in separate formulations, or provided together in a single formulation.

In another embodiment provides a composition for treating and/or preventing cervical cancer, which contains a miR-199 inhibitor and an anti-cancer agent as active ingredients.

In another embodiment provides a method of treating and/or preventing cervical cancer, which comprises the step of administrating a miR-199 inhibitor and an anti-cancer agent to a patient in need thereof. The method of treating and/or preventing cervical cancer can be characterized in that the miR-199 inhibitor and the anti-cancer agent can be provided respectively in separate formulations, or provided together in a single formulation.

In another embodiment provides a method of formulating a dosage form for treating and/or preventing cervical cancer containing a miR-199 inhibitor and an anti-cancer agent. The method of formulating a dosage form can be characterized in that a miR-199 inhibitor and an anti-cancer agent can be provided respectively in separate formulations, or provided together in a single formulation.

The above miR-199a may be originated from mammals, preferably from humans, and have nucleotide sequence of SEQ ID NO: 1 (5'- CCCAGUGUUCAGACUACCUGUUC-3'). Inhibition against miR-199a can be achieved by an anti-miR-199a which binds to miRNA-199a and inhibits its activity. For example, the miR-199a inhibitor may be a nucleotide complementary to at least 10, preferably at least 15 nucleotides, which are serially located within the nucleotide sequence of SEQ ID NO: 1. More preferably, the miR-199a inhibitor may be an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2 (5'- GAACAGGUAGUCUGAACACUGGG-S').

The above anti-cancer agent may be an alkylating agent type anti-cancer agent, for example, cisplatin. The alkylating agent is capable of introducing an alkyl-group (R-CH2) into a reacting compound and highly reactive. When contacting to a cell, it mostly reacts with the guanine N-7 position, distorts the DNA structure, and causes single- or double-strand breaks, thereby exhibiting anti-cancer and cytotoxic effects. The anti-cancer agents classified to this group may include cisplatin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, thiotepa, busulphan, DTIC (dacarbazine), procarbazine, and the like.

Therefore, the anti-cancer agent according to an embodiment may be one or more selected from the group consisting of cisplatin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, thiotepa, busulphan, DTIC (dacarbazine), procarbazine, and the likes. Preferably, the anti-cancer agent may be cisplatin.

The dose of the miR-199a inhibitor may be within the range from 0.001 to 500 mg/kg, preferably, from 0.01 to 100 mg/kg, based on the content thereof in the formulation, but is not limited thereto. It can be adjusted according to patient's condition and seriousness of the disease. There are no special limitations with regard to a dose of the anti-cancer agent.

It may be determined according to the dosage and administration regimes as directed for a given anti-cancer agents. In the case of cisplatin, for example, it can be applied within the range from 1 to 100 mg/m2(body surface area), preferably, 10 to 50 mg/ m2. In a desired embodiment, the administration of cisplatin may be generally conducted by one coure (meaing an administrative period unit of an anticancer agent including administering period of several consecutive days wherein the of anti-cancer agent is intravenously administered in a row, then followed by administration stopping period of several weeks) wherein the administration of cisplatin is continued in the amount of 15-20 mg/m2 once per a day for consecutive 5 days, and stopped for at least 2 weeks, and, depending on the patient's conditions, another coure wherein the administration of cisplatin is continued in the amount of 25-30 mg/m2 once per a day for consecutive 5 days, and stopped for at least one week; however it is not limited thereto, and it can be properly adjusted depending on the patient's conditions or the anti-cancer agent's efficacy. The anti-microRNA molecules described herein may be produced by a conventional DNA synthesizer and directly used, or cloned into an expression vector. The expression vector may be selected from the group consisting of a plasmid, a lentiviral vector, an adenoviral vector, and the likes, which are conventionally used for transcription and expression of mammalian cells and other types of target cells. The genes encoding the anti-microRNA described herein may be prepared and inserted into the expression vectors in conventional manners.

The composition according to the present invention may contain an anti- microRNA molecule produced as described above or cloned into an expression vector, as an active ingredient. The composition may contain the anti-microRNA molecule alone or along with pharmacologically acceptable carriers. The pharmacologically acceptable carriers may include solvents, dispersive media, coating agents, anti-microbial agents and anti-bacterial agents, isotonic agents, absorption retarders, and the like, which are suitable to pharmaceutical administration. Supplementary active materials may be further included. The anti-microRNA may be provided in a formulation suitable to a given administration route. The composition may be administered through any conventional oral or non- oral administration route, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, percutaneous (local) or rectal administration, or administration through inhalation or mucous membrane, but not be limited thereto. Administration regimes can follow conventional medical or pharmacological methods, safety and efficacy of which are proven. Toxicity and treatment efficacy of the composition of the present invention can be assessed by the standard pharmacological process applied in cell cultures and experiment animals, which measures LD50 (the lethal dose of a drug for 50% of the population) and ED50 (the minimum effective dose for 50% of the population). A therapeutic index is defined as the ratio between the toxicity and the efficacy, that is, LD50:ED50. For a given therapeutic agent, a high therapeutic index is preferable to a low one, and it is necessary to design a delivery system to target a site affected by the therapeutic agent, in order to minimize injuries to uninfected cells and reduce side effects.

A suitable dosage of the composition according to the present invention comprising anti-microRNA depends on the expression and activity of pri- microRNAs to be regulated. In a situation where anti-microRNAs are administered to a patient, the physician (a veterinarian or a researcher) may initially apply a low amount of dose and may increase the amount until desirable response is achieved. In addition, a dose specific to a patient is determined with many factors taken into account — such as the activity of substance to be applied, the patient's age, weight, general health condition, and infection with a venereal disease and eating habit, administration frequency and times, administration routes, excretion rates, other drugs administered along with, and extent of expression and activity to be controlled.

The composition according to the present invention can be administered to mammals, preferably to humans, while dosages can be adjusted according to a subject's age and seriousness of the disease. For example, a dose can be within the range of from 0.001 to 500 mg/kg, preferably, from 0.01 to 100 mg/kg, of the effective ingredient per kg of body weight. Administration of the composition according to the present invention can be conducted by any manner of administration as commonly used. For example, oral or rectal administration, or intravenous, intramuscular, subcutaneous or intrauterine injection can be applied. The composition according to the present invention may be prepared for oral administration in such forms as powders, granules, tablets, capsules, suspensions, emulsions, syrup, and aerosols; or for non-oral administration in such forms as transdermal patches, suppositories, or injections.

Tissue-specific pattern of miRNA expression has recently been reported and is viewed to reflect an aspect of embryologic development. According to a few reports, over- or under-expression of certain miRNAs is specifically observed in specific tumor types. The present inventors discovered that overexpression of specific miRNAs — rather than their underexpression — is predominantly found in ISCC (invasive squamous cell carcinoma) cases, compared to normal cervical epithelium (cervical epithelium) tissues. Overexpression of miRNAs in cervical cancer has not been reported. The results obtained by the present inventors can be explained by tissue-specificity of miRNA expression, as shown through large-scale profiling studies using a variety of tissue types of tumors.

Most of the human miRNAs are expressed from the introns of protein- coding genes, and approximately 1/3 of them are located within the introns of annotated mRNAs. Those intron mRNAs, generally having the same direction with the pre-mRNAs, can be regulated by the promoter that controls the mRNA precursors. Until now, more than 90 of intron miRNAs have been identified by biological informatic approaches, but for most of them, their functions remain not disclosed. Intron miRNAs generally have an identical manner of expression with their host gene's mRNAs. The present inventors found by biological informatics analysis that DNM2 intron 16 is the host gene of miR-199a intron 16 and that miRNAs of DNM2 is expressed along with the intron mRNAs (See Fig. 1).

Knock-down of over-expressed miRNAs or silent (silent) miRNA expression in cancer cells can lead to apoptosis of the cancer cells. Recently, 'antagomirs', a novel type of chemically synthesized oligonucelotides, are reported to can effectively silence endogenous miRNA in vivo. In an embodiment of the present invention, it was confirmed that anti-miR-199a reduces the growth of the cervical cancer cells (SiHa and ME-180), and increases cisplatin-induced cell toxicity (see Fig. 2). DNA injuries induced by cisplatin can increase the ability of anti-miR-199a to suppresss the growth. In summary, it was confirmed through the present invention that anti-miR-

199a inhibits cell growth and promotes chemotherapeutic reactions (in vitro), thus presenting that miR-199a can be a potential target of cervical cancer treatment.

MicroRNAs as described above can be used for cancer treatment, and an application for diagnosis of cancer cell proliferation is possible by measuring the extent of microRNA expression.

Brief Description of the Drawings

Fig. 1 shows a result of real-time quantitative PCR analysis for DNM2 intron 16, the host gene of miR-199, and indicates that mRNA level is significantly higher in ISCC (P <0.0001). Figs. 2 A to 2E show the suppression of cell growth by anti-miR-199a oligonucleotides in cervical squamous cell carcinoma. Fig. 2A shows the relative expressions of miR-199a in cervical squamous cell carcinoma tissues and normal cervical squamous epithelial cells, as measured by TaqMan real-time PCR (p 0.0001). Fig. 2B shows the suppression of the miR-199a expression by anti-miR- 199 in squamous cells in the cervix (ME- 180 and SiHa). Fig. 2C shows the cell growth inhibited by anti-miR-199a. Figs. 2D and 2E show the cell growth inhibited with increasing amounts of cisplatin when cisplatin is administered along with anti- miR-199a (Columns, the average value of three independent experiments; bars , SE (*, p <0.05; **, p <0.01)). Fig. 3 shows the inhibition of cell growth in the three kinds of cervical squamous epithelial cells (Caski, ME- 180, and SiHa) by the combined administration of the oligonucleotide of anti-miR-199a and cisplatin, an anti-cancer agent, in which it is confirmed that increase in cell growth effect by the combined administration of the oligonucleotide of anti-miR-199a and cisplatin, as shown in Figs 2D and 2E (ME-180 and SiHa), identically applies to different cervical squamous epithelial cells (Caski).

Examples

The present invention is further explained in more detail with reference to the following examples. These examples, however, should not be interpreted as limiting the scope of the present invention in any manner.

Example 1 : Tissue specimens

Fresh frozen tumor biopsy specimens (n=10) from patients with primary ISCC (International Federation of Gynecology and Obstetrics stage of IB to HA) were obtained at the time of surgery. A radical hysterectomy with pelvic lymph node dissection was done at the Department of Obstetrics and Gynecology, Samsung Medical Center, between January 2002 and October 2003. Tumor specimens were immediately snap-frozen at -80 ^°C . Only specimens containing >90% tumor cells, which were examined by a single gynecologic pathologist using H&E staining, were used in the analysis. Table 1 summarizes the patient characteristics.

* Lymph node metastasis

+ Parametrial invasion

J Resection margin involvement

As a control, normal cervical tissues (n=10) were obtained from patients undergoing hysterectomy for benign gynecologic disease. Fresh cervical biopsies (5-8 mm3) were obtained before undertaking any surgical procedures. Dispase II (2.4 units/mL; Roche) was used to obtain the normal epithelial tissues alone from the entire cervical tissues including the stroma. These biopsies were washed in sterile PBS for a few minutes and incubated for 1 hour in dispase II at 37°C, with the stromal side down. The epithelial sheets were then gently removed from the stromal layers and then washed twice in sterile PBS before extracting the total RNA.

Example 2: RNA extraction and reverse transcription Total RNA was extracted from the ISCC and normal epithelial tissues using an easy-spin (genomic DNA-free) Total RNA Extraction Kit (iNtRON Biotechnology). The concentration was quantified using the NanoDrop ND- 1000 Spectrophotometer (Nano-Drop Technologies). cDNA was synthesized from total RNA using stem-loop reverse transcription primers (provided from ABI) according to the TaqMan MicroRNA Assay protocol (PE Applied Biosystems; ref. 38).

Example 3: miRNA expression profiling using TaqMan MicroRNA assay

Rea-ltime PCR for miRNA expression profiling was done using a 7900HT Sequence Detection system (Applied Biosystems). The expression levels of the 157 human mature miRNAs were measured using the Human Panel Early Access Kit (PN 4365381; Applied Biosystems). Data normalizations were done using let-7a as the endogenous control according to the manufacturer's suggestions, miR-16 as a positive control, or cel-lin-4, ath-miR159a, and cel-miR-2 as the negative controls: mature miRNA hsa-let-7a (UGAGGUAGUAGGUUGUAUAGUU, SEQ IS NO: 3) mature miRNA hsa-miR- 16 (UAGCAGCACGUAAAUAUUGGCG, SEQ IS NO: 4) mature miRNA cel-lin-4 (UCCCUGAGACCUCAAGUGUGA, SEQ IS NO: 5) mature miRNA ath-miR159a (UUUGGAUUGAAGGGAGCUCUA, SEQ IS NO: 6) mature miRNA cel-miR-2 (UAUCACAGCCAGCUUUGAUGUGC, SEQ IS NO: 7)

Relative quantification of miRNA expression was calculated by the 2-ΔΔCT method (Livak KJ, SchmittgenTD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 2001;25:402-8). The relative expression values were multiplied by 106 in order to simplify the presentation of the data.

Example 4: Real-time quantitative reverse transcription-PCR analysis for DNM2 mRNA expression

DNM2, as an overlapping transcript of miR-199, and glyceraldehyde-3- phosphate dehydrogenase (GAPDH), as an endogenous control gene, were used in the same PCR reaction for real-time quantitative reverse transcription-PCR. In order to avoid amplification of the genomic DNA, the primers and probe for amplifying DNM2 and GAPDH were chosen to hybridize at the junction between the two exons as follows:

DNM2 (Hs00191900, Applied Biosystems; NM_001005362, exon boundary 13-14, probe 5^I-CATCCCCAATCAGGTGATCCGCAGG-3^I, SEQ ID NO: 8), and

GAPDH (4310884E, Applied Biosystems).

[PCR Conditions]

Thermal Cycling Parameters

The gene expression ΔCT value of DNM2 from each sample was calculated by normalization with GAPDH and relative quantification values were plotted ((XiY,

Shalgi R, FodstadO, PilpelY, JuJ. Differentially regulated microRNAs and actively translated messenger RNA transcripts by tumor suppressor p53 in colon cancer. Clin

Cancer Res 2006; 12:2014-24)).

Example 5: Cell lines and transfection of anti-miR-199a

All cell culture reagents were purchased from Invitrogen Life Technologies.

The human cervical cancer cell lines, SiHa and ME-180, were obtained from the

American Type Culture Collection. SiHa cells were grown at 37 ^°C in 5% CO2 in MEM supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 Ag/mL). ME-180 cells were maintained in McCoy's 5A and

RPMI 1640.

Cells were transfected with 100 nmol/L of anti -miR-199a (Ambion) or negative control using siPort Neo-FX (Ambion). Three days later, total RNA from the cells was isolated to examine the expression level of miR-199a using the RNA- spin total extraction Kit (Intron Biotech).

Example 6: Cell viability determined by [3-(4,5-dimethylthiazol-2-yl^*)-2,5- diphenyl-tetrazoliumbromide] assay Cervical cancer cells (SiHa and ME-180) were plated in a 96-well plate at

4,000 cells/well and then were allowed to grow for 3 days before the MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] assay. For the MTT assay, 1 mg/mL of MTT solution (1 niL of 10 mg/niL MTT in PBS added to 9 mL of serum-free medium) was added to each well. Then, the cells were incubated for 3 h in the dark. The formazan grain was then dissolved in DMSO (Sigma), and absorbance was read at 570 run using an ELISA plate reader (Bio-Rad). To further assess the effect of anti-miR-199a on cell growth, the transfected cells were treated with 1.5 and 3μg/mL of cisplatin for 2 days.

SPSS software (version 10.0, SPSS Inc.) was used to perform statistical analysis. The Mann- Whitney U test was used to evaluate the significance between the gene expression of tumor and nonmalignant tissue samples. Additionally, unsupervised hierarchical clustering was done on the PCR data to investigate the relationships among genes and among samples. Each miRNA raw data CT were median-centered for all samples before clustering. Hierarchical average-linkage clustering was done by means of the GeneSpring GX software (version 7.3.1, Agilent Technologies), using log-transformed, median-centered gene expression values and the Pearson correlation as similarity metrics.

Experimental Example 1 : Comparison of miRNA expression in ISCCs and normal cervical epithelial tissues

Expression profiles of the 157 miRNAs analyzed in the ISCCs and normal epithelial tissues was compared. As the results, there was a significant difference in the expression of 70 miRNAs in comparisons between the ISCCs and normal epithelial tissues (P < 0.05), 68 were up-regulated and 2 were down-regulated (Supplementary Table Sl). Among these, 10 miRNAs that were the most significantly overexpressed in ISCCs with fold changes of nearly >100 and a P < 0.0001, were as follows: miR-199-s, miR-9, miR-199a*, miR-199a, miR-199b, miR- 145, miR-133a, miR-133b, miR-214, and miR-127. By contrast, only two of the miRNAs, miR-149 (2.974-fold change) and miR-203 (3.704-fold change), showed significant down-regulation.

Unsupervised hierarchical clustering was used to classify the samples without using any information on the identity of the samples (Fig. 1). Fig. 1 shows the results of Real-time quantitative PCR analysis of the mRNA level of DNM2 intron 16, which is the host gene of miR-199a, wherein the number of X axis means random serial number of the samples. The expression level of mRNA isolated from fresh frozen tumor biopsy specimens (n=10) from patients with primary ISCC (International Federation of Gynecology and Obstetrics stage of IB to HA) (see Table 1) and normal cervical tissues (n=10) from patients undergoing hysterectomy for benign gynecologic disease as a control, was analyzed. The mRNA level of DMM2 intron 16 was significantly higher in ISCCs (P < 0.0001). This procedure resulted in the classification of cancer samples into two major classes based on similarities in miRNA expression. Of interest was the observation of a difference in the LN metastasis between the two clusters (P = 0.052 using Pearson x2 test).

Experimental Example 2: Investigation of host gene of miR-199a In this experimental example, it was confirmed that miR-199a is an intronic miRNA located in host gene, DNM2 intron 16. The nucleotide sequence of the overlapping transcripts of the significantly expressed 70 miRNAs in ISCC was analyzed using Sanger miRNA registry (http://microrna.sanger.ac.uk). The chromosomal locations of overlapping transcripts were divided into 40 introns, 22 intergenic, 6 3 '-untranslated regions, and 2 exons. The gene lists of overlapping transcripts for the top 10 miRNAs are DNM, Clorfόl, C20orfl66, RP11-771D21.2, and RTLl.

DNM2 mRNA was identified as an overlapping transcript of miR-199-s, miR-199a*, and miR-199a. It was found that the nucleotide sequence at DNM2 intron 16 was complementary to the sequences of miR-199a* and miR-199a (see Table 2). Because intronic miRNAs are coordinately expressed with their host gene's mRNA, the mRNA level of DNM2 in the same tissues was evaluated using real-time quantitative PCR. It was found that the mRNA level was significantly increased in the ISCCs compared with the normal tissues (P < 0.0001; Fig. 1). Therefore, these findings suggest that miR-199a* and miR-199a are intronic miRNAs from the host mRNA, DNM2 intron 16.

[Table 2] Sequence of Mature hsa-mir-199

Experimental Example 3: Inhibition of cervical cancer cell growth by anti- mJR-199a

In order to evaluate the role of specific miRNAs in cervical carcinogenesis, miR-199a, which is one of the most up-regulated ISCCs was selected (Fig. 2A). The TaqMan real-time PCR revealed that anti-miR-199a significantly reduced the expression of miR-199a in cervical cancer cells, suggesting that anti-miR-199a is efficiently introduced into the cells and acts to knock down miR-199a (Fig. 2B). In addition, it was found that this inhibitor reduced cell growth (Fig. 2C). Furthermore, anti-miR-199a -mediated cell growth inhibition was increased in cisplatin-treated cells in a dose-dependent fashion (Fig. 2D and 2E).

In addition, the cell growth inhibition effect by a combination administration of cisplatin and anti-miR-199a, the effect of which was confirmed in cervical cancer cells, SiHa and ME- 180, as above, was further examined in a cervical cancer cell, Cask. As the results, it was revealed that the cell growth was equally inhibited by a combined administration of cisplatin 3ug/ml and anti-miR-199a in all the three types of cervical cancer cells (Fig. 3)

Claims

WHAT IS CALIMED IS:

1. An administration formulation for treating or preventing cervical cancer, comprising an oligonucleotide having the nucleotide sequence complementary to at least 15 nucleotides which are serially located within miR-199 having the nucleotide sequence of SEQ ID NO: 1 and an alkylating agent type anticancer agent, wherein the oligonucleotide and the alkylating agent type anti-cancer agent are provided respectively in seperate formulations, or provided together in a single formulation.

2. The administration formulation for treating or preventing cervical cancer according to Claim 1, wherein the oligonucleotide has the nucleotide sequence of SEQ ID NO: 2.

3. The administration formulation for treating or preventing cervical cancer according to Claim 1 , wherein the alkylating agent type anti-cancer agent is one or more selected from the group consisting of cisplatin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, thiotepa, busulphan, DTIC (dacarbazine), and procarbazine.