KR101469917B1 - Composition for diagnosis of radio-resistance or radio-sensitive PSAP marker and use thereof - Google Patents

Abstract

The present invention relates to a radiation-sensitive or sensitive diagnostic composition comprising an agent for screening a marker for radiation resistance or sensitivity of a cancer and measuring the presence of the marker, a radiation-sensitive or sensitive diagnostic kit comprising the composition, Or susceptibility to radiation or radiation therapy in cancer patients. By using the radiation-sensitive or sensitive diagnostic composition of the present invention, it is possible to provide radiation therapy that is customized by judging the radiation resistance or sensitivity of cancer cells to be treated with radiation, and to improve the therapeutic effect and side effects Can be reduced. It can also be used to judge the prognosis of patients treated with radiotherapy (to predict the therapeutic effect).

Description

A radiation-sensitive or sensitive diagnostic composition comprising an agent for measuring PSAP and a use thereof.

The present invention relates to a radiation-sensitive or sensitive diagnostic composition comprising an agent for screening a marker for radiation resistance or sensitivity of a cancer and measuring the presence of the marker, a radiation-sensitive or sensitive diagnostic kit comprising the composition, Or susceptibility to radiation or radiation therapy in cancer patients.

Cancer is now the biggest factor in determining the health and death of modern people worldwide. Statistically, about 30-50% of cancer patients receive radiation therapy at one point in time. However, the effectiveness of radiation therapy varies with the nature of the cancer, the patient, and radiation as it is combined with other therapies. Cancers in which radiation therapy is widely practiced include head and neck cancer, laryngeal cancer, cervical cancer, parietal cancer, lung cancer, brain cancer, breast cancer, and colon cancer. Although these carcinomas may be the main targets of radiation therapy, strategies that improve treatment efficiency are needed because of poor response to radiation therapy. In addition, although the initial response is good due to good radiation therapy, recurrence frequently occurs, and it is important to take measures against recurrent cancer to improve the efficiency of radiation therapy. There is a difference in the sensitivity of radiation therapy to cancer cells. However, by developing a technique that can predict radiation susceptibility to individuals before radiation therapy, it is possible to treat the cancer by controlling individual radiation dose, can do.

Therefore, in order to examine the sensitivity of cancer cells to radiation before radiation therapy, a method of accurately and rapidly analyzing the degree of death of cancer cells after irradiation with radiation is necessary. The most direct and ideal method is to treat cells with trypan blue and then count the living cells using a microscope and a cell counting device. However, it takes too much time and effort, and can not be used when the number of samples is large There is a problem. Radiotherapy is very different in cancer patients with histologic findings or stage, and it is known that there are many factors influencing the radiation sensitivity. The most important reason for the tumor to have radiation resistance is the intrinsic radiosensitivity of tumor cells. When cancer cells are exposed to radiation, complex molecular biologic changes lead to death or recovery. Mechanisms that regulate the function of genes and proteins involved in signal transduction in these processes have been identified, and all of these have recently been shown to enhance the effectiveness of radiation therapy It could be a target to be known. Studies on radiation resistance have been carried out so far and some of them are based on the development of radiation sensitizers to overcome radiation resistance. However, the inherent or acquired radiation resistance of the tumor allows some cells to survive, limiting the effect of radiation therapy. This phenomenon represents a poor prognosis for laryngeal cancer patients after radiation therapy. Some studies have published tumor radiation resistance markers that can be used as diagnostic and therapeutic indications for radiation therapy. Cancer cells often change cellular components as a way to protect against chronic radiation damage. For example, growth regulatory proteins such as epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase / Akt and RAS are activated or overexpressed in radiation-resistant tumors. The status / expression of p53 or Bcl-2 / -xl Cell cycle and cell death, and is associated with tumor radiation resistance, and cellular antioxidant proteins such as peroxiredoxin-II and Mn-SOD (manganese superoxide dismutase) are frequently involved in the oxidative stress tolerance of cancer cells. Reported that radiation therapy can induce acquired radiation resistance and drug resistance by modulating proteins associated with P-glycoprotein and multidrug resistance. Although these results suggest that some of the radiation resistance and anticancer drug resistance of cancer cells Although it is understandable, the specific cancer types and detailed molecular mechanisms based on these phenomena The radiation resistance-related factors that have been identified so far include cyclooxygenase-2 (Terakado N, Shintani S, Yano J, Chunnan L, Mihara M, Nakashiro K, et al., Oral Oncol 2004; 40: 383-9), epithelial growth factor receptor (EGFR) (Milas L, Fan Z, Andratschke NH, Ang KK., Int J Radiat Oncol Biol Phys 2004; 58 (3) (IGFBP-1 receptor), manganese (IGFBP-1 receptor), and manganese (IGFBP-1) Superoxide dismutase, and survivin. However, the mechanism has been partially elucidated, and there are few markers that can be used as useful predictors of the results of radiation therapy and increase the radiation sensitivity.

Laryngeal cancer is the sixth most common cancer in the world, the largest subgroup of head and neck cancer. As smoking and alcohol consumption increase, it becomes a major risk factor for laryngeal carcinoma and human papillomavirus is another potential factor. One of the main treatment methods for the removal of malignant tumors is radiation therapy, which is commonly used to treat laryngeal cancer. Surgical surgery, chemotherapy and radiation therapy are commonly used to treat various types of cancer. . Therefore, a marker that can diagnose resistance in such laryngeal cancer is needed.

Therefore, the present inventors have made intensive efforts to find a marker capable of diagnosing radiation resistance of cancer, and as a result, they have constructed a radiation resistant cell line and analyzed radiation resistant cell lines and control cell lines to identify genes showing differences in expression, The present invention has been completed on a marker for radiation resistance diagnosis and its use.

One object of the present invention is to provide a composition for diagnosing the radiation resistance or sensitivity of cancer cells, which comprises an agent for measuring the level of mRNA of PSAP (Prosaposin, NCBI GenBank Gene ID: 5660) gene or a protein thereof.

Another object of the present invention is to provide a kit for the diagnosis of radiation resistance or sensitivity of a cancer cell comprising the composition.

Another object of the present invention is to provide a method of providing information for diagnosing radioactive resistance of a cancer patient using the composition.

Another object of the present invention is to provide a method for detecting a marker for radiation resistance or sensitivity in order to provide information necessary for predicting a radiation therapy prognosis of a cancer patient.

In order to achieve the above object, the present invention provides a composition for diagnosing radiation resistance or sensitivity of cancer cells, which comprises an agent for measuring the level of mRNA of PSAP (Prosaposin, NCBI GenBank Gene ID: 5660) gene or its protein do.

The term "PSAP (Prosaposin)" in the present invention refers to a precursor of saposins A, B, C, and 4 as a glycoprotein that separates lipid substrates around the membrane to facilitate access to degradative enzymes . However, the association of PSAP with radiation sensitivity and resistance is not known at present. The relationship between PSAP and radiation sensitivity and resistance was first described by the present inventors.

Preferably, the composition for radiation resistance or sensitivity diagnosis of cancer cells is selected from the group consisting of CDK11A (Cell division cycle 2-like 2, NCBI GenBank Gene ID: 728642), MRFAP1 (Mof4 family associated protein 1, NCBI GenBank Gene ID: 93621), HNRNPUL1 (Heterogeneous nuclear ribonucleoprotein U-like 1, NCBI GenBank Gene ID: 11100), WDFY3 (WD repeat and FYVE domain containing 3, NCBI GenBank Gene ID: 23001) and CDC27 homolog, NCBI GenBank Gene ID: 996), or an agent that measures the level of the mRNA of the at least one gene. The gene may be one or more, two or more, three or more, four or more, five or more, and may be used as a marker for radiation resistance or radiation sensitivity.

The association of the six genes PSAP, CDK11A, MRFAP1, HNRNPUL1, WDFY3 and CDC27 with radiation sensitivity and resistance is currently unknown. According to one embodiment of the present invention, PSAP, CDK11A, MRFAP1, HNRNPUL1 or WDFY3 are overexpressed in a radiation-resistant cell line than in a normal cell line. According to one embodiment of the present invention, (Table 1, Fig. 1A), confirming that the above six genes are radiation-resistant or sensitive genes.

In addition, the radiation-sensitive or radiation-sensitive diagnostic composition may further comprise an agent for measuring the level of the mRNA of INPP4B (Inositol polyphosphate-4-phosphatase type II, NCBI GenBank Gene ID: 8821) gene or its protein. Preferably, the INPP4B gene may be a marker capable of further diagnosing cancer resistance. The anticancer agent includes, but is not limited to, an anticancer agent capable of diagnosing resistance to the marker gene of the present invention, for example, bleomycin, cisplatin, etoposide, doxorubicin. According to one embodiment of the present invention, it was confirmed that INPP4B is a marker gene resistant to an anticancer agent (FIG. 5).

 Information on the genes can be obtained from known databases, such as, but not limited to, NCBI GenBank.

The term "radio-resistance" in the present invention refers to a state that is not easily affected by irradiation and has little change in cell repair or proliferation upon exposure to radiation.

The term "radio-sensitive" in the present invention refers to a condition that is easily affected by irradiation and shows changes in cell repair or proliferation upon exposure to radiation.

The radiation-sensitive or sensitive diagnostic composition of the present invention is composed of a gene having a differential expression level in a radiation-resistant cancer cell versus a normal cancer cell, so that the radiation resistance of an individual or a cell can be judged. That is, it can be judged that the level of expression of the radiation resistance-detecting composition of the present invention is higher or lower than that of the control group, and that cancer cells have radiation resistance or sensitivity.

In the examples of the present invention, CDC27 expression level of the radiation sensitive diagnostic marker is lower than that of the control group in the radiation sensitive cancer cell line, while the radiation resistance diagnostic marker is PSAP, CDK11A, MRFAP1, HNRNPUL1, WDFY3, or INPP4B, the expression levels of the radiation resistance diagnostic marker were higher in the radiation-resistant cancer cell lines than in the control group.

If the radiation resistance or sensitivity of the cancer to be treated is judged and the level thereof is determined, the radiation dose of the radiation treatment can be determined in accordance with the determination, so that the efficiency and treatment result of the radiation treatment can be improved.

The term "marker or diagnosis marker" in the present invention means a substance which can be diagnosed by distinguishing a cancer cell or an individual having a cancer disease from a radiation-resistant cell or an individual, a radiation-sensitive cell or an individual, Such as polypeptides, proteins or nucleic acids (such as mRNA), lipids, glycolipids, glycoproteins or sugars (such as monosaccharides, disaccharides, oligosaccharides, etc.) which show an increase or decrease in cancer cells or individuals with radiation resistance .

Also, the cancer which can be measured by the composition of the present invention is not limited to cancer which can be distinguished by the expression difference of the marker gene of the present invention, but preferably includes cancer of head, neck, larynx, cervix, , Brain cancer, breast cancer, and colon cancer, and more preferably lung cancer, laryngeal cancer, colon cancer, or breast cancer.

The level of gene expression in a biological sample can be determined by identifying the amount of mRNA or protein.

The term " measurement of mRNA expression level " in the present invention is used to determine the presence or the mRNA level of the marker genes in a biological sample in order to diagnose radiation resistance and sensitivity. RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection (RPA), and reverse transcriptase-polymerase chain reaction assay, Northern blotting, DNA chip, and the like.

In the present invention, " measurement of protein expression level " is a process for determining the presence or the degree of expression of a protein expressed from the marker gene in a biological sample to diagnose radiation resistance or sensitivity. Preferably, The amount of the protein can be confirmed by using an antibody specifically binding to the protein. Methods for analysis include 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. .

The agent for measuring the level of the gene mRNA of the present invention can be prepared by a person skilled in the art using various programs based on the sequence of the gene known in known databases in a primer which specifically binds to the gene.

The term "primer " in the present invention means a short nucleic acid sequence capable of forming a base pair with a complementary template with a short, free 3 'terminal hydroxyl group and functioning as a starting point for template strand copying do. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The primer of the present invention is a sense and antisense nucleic acid having 7 to 50 nucleotide sequences for each marker gene-specific primer. Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis. The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate , Carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents. The nucleic acid sequences of the present invention can also be modified using a label that can provide a detectable signal directly or indirectly. Examples of labels include radioactive isotopes, fluorescent molecules, and biotin.

 And the agent for measuring the protein level of the gene comprises an antibody specific for the protein.

The term " antibody " in the present invention means a specific protein molecule directed to an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. As described above, since the radiation-resistant and sensitive marker proteins have been identified, the production of antibodies using the same can be easily carried out by using techniques well known in the art. Polyclonal antibodies can be produced by methods well known in the art for obtaining serum containing antibodies by injection of the above-mentioned radiation-sensitive or sensitive marker protein antigens into animals and blood collection from the animals. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like. Monoclonal antibodies may be obtained from the hybridoma method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519), or the phage antibody library (Clackson et al, Nature, 352: 624 Biol., 222: 58, 1-597, 1991) techniques. The antibody prepared by the above method can be separated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

The term "cancer cell" in the present invention refers to any cell that exhibits unregulated growth in the tissues or organs of a multicellular organism.

The radiation resistance diagnostic marker of the present invention was used to establish a radiation resistant cell line using a laryngeal cancer cell line and to quantify the gene expression level in comparison with a control group (normal laryngeal cancer cell: HEp-2 cell) ≪ / RTI >

According to one embodiment of the present invention, a radiation resistant cell line was constructed, and the established radiation resistant cell line was examined for radiation resistance. RT-PCR was used to confirm the expression of the gene in both the cell lines and the control cells. The gene that was found to show a lower expression level in the radiation-resistant cell line than the control was CDC27 as a result of the nucleotide sequence analysis. On the other hand, the gene confirmed to have a higher expression level in the radiation-resistant cell line than the control group, As a result of analysis, 6 were all of PSAP, CDK11A, MRFAP1, HNRNPUL1 or WDFY3. These genes have yet to be identified in the literature as novel radiolabel or sensitive diagnostic markers that have not been described as having radiation, particularly radiation-resistance and sensitivity-related functions.

The term "irradiation " in the present invention includes exposure to the action of electromagnetic radiation or alpha or beta particles including, for example, X-rays, ultraviolet rays, alpha particles and gamma rays.

The term "gray (Gy) " in the present invention is the amount of radiation that releases 1J in 1 kg of representative biological tissue.

In another aspect, the present invention provides a radiation-sensitive or sensitive diagnostic kit comprising the radiation-sensitive or sensitive diagnostic composition. The kit may be an RT-PCR kit, a DNA chip kit, a microarray, or a protein chip kit. The diagnostic kit according to the present invention can be prepared according to a conventional method used in the art using the above radiation-sensitive or sensitive diagnostic composition. In addition, the expression of the radiation-sensitive or sensitive gene may be measured using a kit containing a radiation-sensitive or radiation-sensitive diagnostic marker according to the present invention.

The term "kit" in the present invention refers to the expression level or protein expression level of at least one gene selected from the group consisting of PSAP alone or further CDK11A, MRFAP1, HNRNPUL1, WDFY3, CDC27 and INPP4B, Means a tool capable of diagnosing radiation resistance or sensitivity. The kit for diagnosing whether or not radiation resistance of the cancer cells of the present invention includes a PSAP alone or a primer for measuring one or more gene expression levels selected from the group consisting of CDK11A, MRFAP1, HNRNPUL1, WDFY3, CDC27 and INPP4B as a radiation resistance diagnostic marker, Or optionally one or more other component compositions, solutions, or devices suitable for the assay, as well as antibodies that recognize the marker.

In another aspect, the present invention provides a method for detecting cancer in a cancer patient, comprising: measuring mRNA level from a biological sample of a cancer patient using a primer that specifically binds to a PSAP gene; And comparing the change in the mRNA level with the mRNA level of the normal control sample. The present invention also provides a method for providing information for diagnosing radioactive resistance or sensitivity of a cancer patient.

Further comprising measuring and comparing mRNA levels from a biological sample of a cancer patient using a primer that specifically binds to one or more genes selected from the group consisting of CDK11A, MRFAP1, HNRNPUL1, WDFY3, CDC27 and INPP4B It is preferable to include a method of providing information for diagnosing radiation resistance or sensitivity of a cancer patient.

In another embodiment, the present invention relates to a method for detecting a protein level by contacting an antibody specific to a protein of a PSAP gene with a biological sample of a cancer patient to form an antigen-antibody complex, And comparing the change of the amount of protein formation with a protein level of a normal control sample. The present invention also provides a method for providing information for diagnosing radiation resistance or sensitivity of a cancer patient.

And further comprising measuring and comparing the protein levels of one or more genes selected from the group consisting of CDK11A, MRFAP1, HNRNPUL1, WDFY3, CDC27 and INPP4B to provide information for diagnosing radiation resistance or sensitivity in cancer patients .

For the purpose of the present invention, low levels of mRNA and protein expression of CDC27 gene in cancer cells can be judged as cancer cells exhibiting radiation resistance, while cancer cells exhibiting radiation sensitivity when the expression level of the gene is high. In addition, high expression levels of mRNA and protein of PSAP, CDK11A, MRFAP1, HNRNPUL1, WDFY3, or INPP4B genes can be judged as radiation-resistant cancer cells, while low levels can be judged as radiation-sensitive cancer cells.

In another aspect, the present invention provides a method for predicting radiation therapy prognosis of a cancer patient, which comprises comparing PSAP gene mRNA or a protein level thereof with a normal cell expression level from a sample of a patient who has received radiation therapy There is provided a method for detecting a marker for diagnostic of radiation resistance. Also provided is a method for detecting a radiation-sensitive or sensitive diagnostic marker comprising the further step of measuring and comparing the mRNA or protein level of one or more genes selected from the group consisting of CDK11A, MRFAP1, HNRNPUL1, WDFY3, CDC27 and INPP4B . In the method of the present invention, the cancer cells may be, but are not limited to, head and neck cancer, laryngeal cancer, cervical cancer, parietal cancer, lung cancer, brain cancer, breast cancer and colon cancer. Preferably, the cancer cells are derived from lung cancer, laryngeal cancer, colon cancer or breast cancer.

The term "prognosis prediction" in the present invention is to predict radiation-resistant or sensitive cancer cells through a gene showing a difference in expression in a radiation-resistant cancer cell line as compared to a normal control group after irradiation.

The term "differential expression" in the present invention refers to the level of expression of one or more identical biomarkers of the invention in a sample, and by measuring the amount or level of mRNA of a biomarker in one sample, Quot; refers to the difference in the expression level of the mRNA of the biomarker. "Differentially expressed" may also include a measure of a protein encoded by a biomarker of the invention in a sample or sample population in comparison to the amount or level of protein expression in the population of samples. Differential expression can be measured as described herein and as understood by those skilled in the art.

The term "gene expression pattern" or "gene expression profile" or "gene profile" in the present invention refers to the relative expression of radiation resistance and sensitivity related genes of cancer cells, , 4, 5, 6, 7, or both of the expression levels of one or more biomarkers of the invention. The gene expression pattern or gene expression profile or gene characteristic can be obtained from the measurement of the expression of the mRNA and / or the protein expressed by the gene corresponding to the biomarker of the present invention.

In the embodiment of the present invention, the function and the role of INPP4B were examined. More specifically, it was confirmed that induction of INPP4B expression in HEp-2 and RR-HEp-2 cells was not limited to radiation stimulation or specific cell types. In addition, INPP4B inhibited the radiation resistance of HEp-2 cells induced by ERK- Promoted phenotype, confirming that INPP4B increased radiation resistance and could selectively protect HEp-2 cells against radiation-induced cell death. In addition, INPP4B was found to be a positive biomarker for the development of anticancer resistance. Furthermore, INPP4B induces INPP4B induction by stresses such as radiation or anticancer drugs, promoting the resistance phenotype by Akt survival pathway in cancer cells.

By using the radiation-sensitive or sensitive diagnostic composition of the present invention, it is possible to provide radiation therapy that is customized by judging the radiation resistance or sensitivity of cancer cells to be treated with radiation, and to improve the therapeutic effect and side effects Can be reduced. It can also be used to judge the prognosis of patients treated with radiotherapy (to predict the therapeutic effect).

Figure 1 shows INPP4B as a protein related to radiation resistance in HEp-2 cells. (A) HEp-2 maternal cells, and radiation resistant RR-HEp-2 and RR- # 6 clone variants were not treated or treated with 10 Gy radiation for 24 hours. The gene transcript was measured by performing a typical RT-PCR. ERBB2 and Bcl-xl were used as positive controls; P21 was used as a negative control; GAPDH was also used as a loading control. (B) HEp-2 parent cells, and RR-HEp-2 and RR- # 6 variants were treated with the indicated radiation dose. After 14 days, the survival fraction was measured by colony formation survival assay; Results are expressed as survival curves (top). INPP4B protein levels were measured by Western blot using β-actin as a loading control (bottom). (C) HEp-2 maternal cells (CON), radiation sensitivity (RS- # 7, RS- # 9 and RS- # 18) and radiation resistance (RR- # 3, RR- # 6 and RR- # 13) HEp- 2 cells did not (+) or treat 4Gy radiation. After 14 days, the colony forming survival fraction was quantified by automated colony counter; Results are expressed as survival curves (top). INPP4B protein levels were measured by Western blot using β-actin as a loading control (bottom). The data were expressed as formal results or mean ± standard deviation (performed 3 times).
Fig. 2 shows the induction of INPP4B-expressing cancer cells by treating with radiation or an anticancer agent. (A) HEp-2 parent cells (top), or HEp-2 parent cells and RR-HEp-2, and RR- # 6 variants (bottom) were irradiated with 10 Gy of radiation for the indicated time. (B) A549, H460, HCT116 and MCF7 cells were not irradiated (+) or irradiated (-) with 10 Gy of radiation for 24 hours. INPP4B transcription levels were measured by quantitative PCR using GAPDH as an internal control. (C) HEp-2 cells were exposed to radiation (10 Gy), bleomycin (Bleo; 10 μM), cisplatin (Cis; 10 μM), etoposide (Eto; 5 μM), or doxorubicin (Doxo; ) Or not (CON). INPP4A and INPP4B transcript levels were measured by quantitative PCR using GAPDH as an internal control. (D) HEp-2 and RR-HEp-2, and RR- # 6 variants were irradiated with 10 Gy of radiation for 24 hours. INPP4A, INPP4B and PTEN transcript levels were measured by either quantitative PCR (top) or typical PCR (bottom). GAPDH was used as an internal control or a loading control. The data were expressed as formal results or mean ± SD (4 runs).
FIG. 3 shows ERK-dependent induction of INPP4B expression by radiation in HEp-2 cells. (A) MAPK protein expression and phosphorylation levels were determined by western blotting. (B) HEp-2 cells were irradiated (+) or irradiated with 10 Gy of radiation in the presence or absence of 10 μM PD98059 (PD), 10 μM SB203580 (SB), or SP600125 (-), and cultured for 24 hours. INPP4B protein levels were measured by Western blot using β-actin as a loading control (top). INPP4B transcript levels were measured by performing a typical PCR (intermediate) or quantitative PCR (bottom). GAPDH was used as an internal control or a loading control. (C) HEp-2 cells were transfected with control siRNA (siCON) or 100 nM ERK-1/2 siRNA (siERK) for 48 hours and then irradiated (+) or irradiated with 10 Gy for 24 hours Did not do it(-). INPP4B and ERK protein levels were measured by western blotting using β-actin as a loading control. INPP4B transcript levels were measured by performing a typical PCR (intermediate) or quantitative PCR (bottom). GAPDH was used as an internal control or a loading control. The data were expressed as formal results or mean ± standard deviation (performed 3 times).
Figure 4 shows INPP4B-dependent modulation of radiation sensitivity in HEp-2 cells. (A and B) HEp-2 cells were transfected with empty vector (CON) or MYC-tagged INPP4B expression vector (INPP4B) and cultured for 24 hours. The cells were irradiated with the radiation dose indicated by radiation (A) or 10 Gy radiation (B). After 14 days, colony formation was quantified using an automated colony counter; The results are shown in the survival curves (A). After 48 hours, cell viability was measured using a FACScan flow cytometer; Data were expressed as the ratio of PI-positive cells (B, medium). The cleaved PARP and INPP4B protein levels were determined by Western blotting with β-actin as a loading control (B, right). (C and D) RR-HEp-2 cells were transfected with control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP4B) and cultured for 48 hours. The cells were irradiated with the indicated amount of radiation (C) or 10 Gy of radiation. (D) Cell viability (C), cell viability (D, left), cell morphology (D, medium) and western blot (D, right) experiments were performed similar to A and B. The data were expressed as formal results or mean ± SD (4 runs).
Figure 5 shows INPP4B-dependent modulation of anticancer drug sensitivity in stably transfected INPP4B-HEp-2 cells. (A and B) HEp-2 (CON) and stable INPP4B-HEp-2 (INPP4B) were exposed to vehicle, 10 μM bleomycin, 5 μM etoposide, or 1 μM doxorubicin for 24 h. The truncated PARP and Myc-tagged INPP4B expression levels were measured by Western blotting using β-actin as a loading control (A, top). Cell death was measured using a FACScan flow cytometer; The data are expressed as the ratio of PI-positive cells (A, bottom). (C and D) INPP4B-HEp-2 cells were transfected with either control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP 4B) for 48 h and then treated with vehicle or 1 μM doxorubicin for an additional 24 h. Western blotting (C, top), apoptosis (C, bottom) and cell morphology (D) experiments were performed as in A and B. (INPP4B- # 2, INPP4B- # 4 and INPP4B- # 6) were transfected with control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP4B) for 48 hours Were treated with vehicle or 1 μM doxorubicin for 24 h. Cleaved PARP and Myc-tagged INPP4B were measured by western blotting with beta-actin as a loading control. The data were expressed as formal results or mean ± SD (4 runs).
6 is a graph showing the induction of apoptosis by the deprivation of INPP4B in lung cancer cells. (A) A549 and H1299 cells were transfected with either control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP4B) for 48 hours. INPP4B protein levels were measured by Western blot using β-actin as a loading control (top). The level of INPP4B mRNA was measured by quantitative PCR using GAPDH as an internal control (bottom). (B and C) A549 (B) and H1299 (C) cells were transfected with control siRNA (-) or 100 nM INPP4B siRNA (+) for 48 h and then incubated with 1 μM doxorubicin for 24 hours or 48 hours 10 Gy of radiation was not irradiated (+) or irradiated (-). The cleaved PARP levels were determined by western blotting with β-actin as a loading control (top and middle). The morphology of the cells was observed under an optical microscope (bottom). The data were expressed as formal results or mean ± SD (4 runs).
7 is a graph showing changes in Akt activity due to the expression of INPP4B in laryngeal cancer and lung cancer cells. (A) HEp-2 cells and radiation-resistant RR-HEp-2 cells were irradiated with 10 Gy of radiation for the indicated time. Akt and GSK3 [alpha] / [beta] expression and phosphorylation levels were measured by Western blotting using [beta] -actin as a loading control. (B) RR-HEp-2 cells were transfected with control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP4B) for 48 hours and then irradiated or not irradiated with 10 Gy radiation for an additional 6 hours. Akt and GSK3 [alpha] / [beta] expression and phosphorylation levels were measured by Western blotting using [beta] -actin as a loading control. (C and D) Control HEp-2 (CON) and stably transfected INPP4B-HEp-2 (INPP4B) cells (C) or RR-HEp-2, A549 and H1299 cells ) Or 100 nM INPP4B siRNA (siINPP4B) for 48 hours. Akt and GSK3 [alpha] / [beta] expression and phosphorylation levels were measured by Western blotting using [beta] -actin as a loading control. (E and F) RR-HEp-2 cells were transfected with control siRNA (siCON) or 100 nM INPP4B siRNA siINPP4B) for 48 hours and then irradiated or not irradiated with 10 Gy of radiation for an additional 6 hours. Akt (E) or PP2A (F) were immunoprecipitated from each sample and immunocomplexes were used to measure PP2A phosphorylase activity. The PP2A inhibitor okadaic acid was used as a positive control. The data were expressed as formal results or mean ± SD (4 runs).

Hereinafter, the present invention will be described in more detail by way of examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  1: Cell preparation and establishment of radiation resistant cell lines

Lung cancer cell lines A549, H1299 and H460, laryngeal cancer cell line HEp-2, colon cancer cell line HCT116 and breast cancer cell line MCF7 were purchased from ATCC (American Type Culture Collection, Manassas, VA). Colon cancer cell line HCT116 and breast cancer cell line MCF7 were cultured in DMEM (Dulbecco's modified Eagle's medium, HEp-2) medium containing 10% FBS (fetal bovine serum), 50 μg / mL streptomycin and 50 units / mL penicillin, A549, H1299 and H460 were cultured in RPMIM 1640 (Roswell Park Memorial Institute medium 1640) medium containing 10% FBS (fetal bovine serum), 50 μg / mL streptomycin and 50 units / mL penicillin.

The radiation resistant cell line RR-HEp-2 and its mutants were established by the method disclosed in Proteomics (Kim JS et al. Chloride intracellular channel 1 identified using proteomic analysis of the radiosensitivity of HEp-2 cells via reactive oxygen species production. Proteomics 2010; 10: 2589-604). Several subclones (# 3, # 6, # 7, # 9, # 13, and # 18) isolated from the RR-HEp-2 cell colonies had their own clonogenic survival assay after 4 GY of radiation treatment. Were selected for radiation-resistant or radiation-sensitive HEp-2 variants. HEp-2 cells were maintained as control cells.

The cells were irradiated at a dose of 3.81 Gy / min using a ¹³⁷ Cs line (Atomic Energy of Canada, Mssissauga, Canada) or 10 μM bleomycin (Sigma), 10 μM cisplatin (Sigma), 5 μM etoposide , Or 10 μM doxorubicin (Sigma). PD98059, SB203580 and SP600125 (Calbiochem) were used to inhibit ERK, p38 and JNK, respectively.

Example  2: Radiation resistance or sensitivity Marker  Gene selection

Genes differentially expressed between HEp-2 and RR-HEp-2 cell lines (RR-HEp-2 and RR- # 6) were screened to identify new radiation resistant or fecal marker genes.

<1-1> Typical PCR  Analysis and quantitative RT - PCR

Total RNA isolated using RNA STAT-60 (Tel-Test B Inc. USA) was reverse transcribed using ImProm-IITM reverse transcriptase (Promega). Quantitative RT-PCR reactions were performed three times using Chromo-4 cycler (Bio-Rad) and SYBR Premix Ex Taq (Takara Bio). In a typical RT-PCR experiment, the amplified signal from the target gene was normalized to the cycle threshold values (Ct values) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same reaction.

5'-CGG GCA TTC AGT GAC CTG AC-3 '(SEQ ID NO: 1) and Bac-xl (151-bp product, binding temperature 55 ° C, 32 cycles) Antisense primer-5'-TCA GGA ACC AGC GGT TGA AG-3 '(SEQ ID NO: 2); 5'-CAT ATG TCT CCC GCC TTC TG-3 '(SEQ ID NO: 3) and antisense primer-5' -ERBB2 (402-bp product, -CCC ACA CAG TCA CAC CAT AAC-3 '(SEQ ID NO: 4); 3 '(SEQ ID NO: 5) and 5'-GGA TGA CCT TGC CCA CAG (SEQ ID NO: 5) and GAPDH (305-bp product, binding temperature 55 ° C, 24 cycles), sense primer-5'- CAT CTC TGC CCC CTC TGC TGA- CCT-3 '(SEQ ID NO: 6); 5'-GGC TGC CAG TCC ATA ATC-3 '(SEQ ID NO: 7) and antisense primer-5 '-CAC ACT TTC TCC CAC TCC-3' (SEQ ID NO: 8); (440-bp product, binding temperature 55 ° C, 31 cycles), sense primer 5'-AAA GAA TGC AGG TAC ACA G-3 '(SEQ ID NO: 9) and antisense primer 5'- CTC TGT GCT GCT CTT AGG-3 (SEQ ID NO: 10); (580-bp product, binding temperature 55 ° C, 31 cycles), sense primer 5'-GAA AGA CATAT GAC ACC G-3 '(SEQ ID NO: 11) and antisense primer 5'- TTA GCA TCT TGT TCT GTT TG- 3 ' (SEQ ID NO: 12); 3 '(SEQ ID NO: 13) and antisense primer 5'-GGA GTG GTA GAA ATC TGT CAT G- 3 ' (SEQ ID NO: 14).

In addition, primer sequences and expression patterns used for identification of radiation resistant or sensitive marker genes are shown in Table 1 below.

gene NCBIID primer SEQ ID NO: Expression pattern INPP4B
8821
F: GAAACAGAAAGAAGAAATACC 15 Overexpression
R: CCCACTCTTCCTCATCATAG 16 PSAP
5660
F: GTGGTGCCAGAATGTGAAG 17 Overexpression
R: CTGAGGGTAGAGGAGGAGAG 18 CDK11A
728642
F: GTCTGCACATCACCGAAC 19 Overexpression
R: CATTTCTTCCTCACTTACTTC 20 GLUT1
6513
F: CTCATGGGCTTCTCGAAAC 21 Overexpression
R: CCGACTCTCTTCCTTCATCTC 22 JTB
10899
F: CCACCTCTGCTGGTTGCTC 23 Overexpression
R: CTATATGGACTCGATTTGCTTCC 24 ABCC5
10057
F: AAACAGGATCAGTAAAGAAG 25 Overexpression
R: AAGAACACCAGGATAACG 26 MRFAP1
93621
F: ATTATATATGCCGCTCCTAC 27 Overexpression
R: TGGCCTTCACTACCTGTTC 28 HNRNPUL1
11100
F: ACTCGTGAGGAAACTGCC 29 Overexpression
R: CTTGGAACAGGATCAGGG 30 WDFY3
23001
F: TTAAATGGTGCAACTCTG 31 Overexpression
R: TGGATGGTACTAAGAAGAAC 32 NDRG1
10397
F: AGATCTCAGGATGGACCCAAG 33 Low expression
R: CATGCCCTGCACGAAGTAC 34 CDC27
996
F: ACAAATCTTATCTGGTGGAG 35 Low expression
R: GTATCAGGTGAAATTACAGC 36

As a result, ERBB and Bcl-xl (positive control) were increased in RR-HEp-2 and p21 (negative control) were decreased compared to HEp-2, Top). The mRNA levels of the nine genes in normal RT-PCR (reverse transcription-polymerase chain reaction) were compared with untreated controls and HEp-2 in HEp-2 or RR-HEp2 (FIG. 1A, middle) while the two genes were down-regulated (FIG. 1A, bottom).

Inositol polyphosphate-4-phosphatase type II (INPP4B), and PSAP (RR-HEp-2) were detected in the radiation-resistant cell lines RR-Hep2 and RR- Prosaposin, Cell division cycle 2-like 2, GLUT1 (Glucose transport type 1), Jumping transport type 1 (JTB), ABCC5 (ATP-binding cassette, CFTR / MRP) (NF-I) and NF-κB (NF-κB), and NF-κB (NF-κB) 27 homolog) was underexpressed (Table 1 and Figures 1A-1C). Among these genes, 6 genes, specifically PSAP, CDK11A, MRFAP1, HNRNPUL1, WDFY3 and CDC27, were confirmed to be novel radiation resistant or sensitive markers with unknown radiation resistance.

These results suggest that the gene specifically increases or decreases the expression in radiation resistant or sensitive cell lines than in normal cancer cells and that the expression of the gene can be analyzed to specifically diagnose radiation resistance or sensitivity .

Example  3: INPP4B  Function analysis

Since most of the functions of INPP4B (inositol polyphosphate 4-phosphatase type II, hereinafter referred to as 'INPP4B') selected in Example 2 were not disclosed in relation to tumor radiation resistance, in the present invention, After confirming the role of INPP4B and examining the radiation, its function was analyzed.

<3-1> Bulk fluoride  analysis( Clonogenic assay )

Cell viability was measured by colony formation assay. More specifically, the cells were irradiated with a single dose of radiation, immediately treated with trypsin, and then diluted. Thereafter, a 60 mm tissue culture dish was coated with various cell densities (0, 2, 4 and 6 Gy of radiation 200, 400, 1500 and 3000 cells for irradiation). The colonies were irradiated for 14 days, fixed with methanol, stained with trypan blue solution and observed. The number of living cells was measured using a colony only colony counter (Imaging Products) composed of 50 or more cells.

As a result, the RR-HEp-2 and RR- # 6 cell lines showed a distinct radiophenotype by clonogenic survival assays (FIG. 1B, top) INPP4B was expressed (Fig. 1B, bottom). In the present invention, the relationship between the radiation resistance phenotype and INPP4B was confirmed by using radiation-sensitive and radiation-resistant HEp-2 clones. The radiation sensitivity of each clone was confirmed by analysis of colony formation after treatment with 4 Gy radiation (Fig. 1C, top).

As a result, the clones displaying the radiation resistant phenotype (RR- # 3, RR- # 13 and RR- # 6) showed pronounced upregulation of the INPP4B protein in contrast to HEp-2, while the INPP4B level was a radiation sensitive clone - # 7, RS- # 9 and RS- # 18) (Fig. 1C, bottom). These results suggest that increased levels of INPP4B may be associated with the development of radiation resistance in HEp-2 cells.

<3-2> Cell death analysis Apoptosis 분석 )

2 x 10 &lt; ⁵ &gt; cells were dispensed into 60 mm dishes and processed according to the indicated experimental conditions. Cell death was measured by western blot analysis of cleaved poly (ADP-ribose) polymerase (PARP) and cell morphology was observed under an optical microscope. The cells were analyzed with a FACScan flow cytometer (Becton Dickson) to quantify cell death.

<3-3> Western Blat  analysis( Western blot 분석 )

Cells were lysed with buffer containing 50 mM TRIS-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 and 0.1% SDS (sodium dodecyl sulfate) containing phosphatase inhibitors and phosphatase inhibitors Respectively. The following antibodies were used to detect the proteins: Rabbit polyclonal anti-phospho-Akt (Ser473), anti-Akt, antiphospho-p38, anti-p38, anti-phospho-JNK, anti- Truncated-PARP (Asp214) (Cell Signaling Technology); Rabbit monoclonal anti-phospho-GSK3? /? (Ser21 / 9) (Cell Signaling Technology); Mouse monoclonal anti-ERK (BD Transduction Laboratories); Mouse monoclonal anti-phospho-ERK and anti-myc (Santa Cruz Biotechnology Inc.); Mouse monoclonal anti-beta-actin (Sigma); Goto-polyclonal anti-INF4B (Santa Cruz Biotechnology, Inc.).

<3-4> siRNA On by INPP4B And ERKd Analysis of Knockdown of INPP4B and  ERK by siRNA )

Human ERK-1 siRNA 5'-CUC UCU AAC CGG CCC AUC U-3 '(SEQ ID NO: 38) and human ERK-1 siRNA 5'-CAG AUC UUU ACA AGC UCU U-3 '(SEQ ID NO: 39) was synthesized. Scrambled siRNAs that did not show any particular homology to the identified gene sequences and that did not regulate gene expression were used as negative controls.

The cells were transiently transfected by treatment with 100 nM siRNA in serum-free culture medium for 5 hours using Metafectene reagent (Biontex) according to the manufacturer's manufacturing method. The cells were further maintained for 48 hours, and target protein defects were determined by Western blot and quantitative RT-PCR analysis.

<3-5> INPP4B  Structures and Transfection

CDNA for human INPP4B was prepared by PCR using specific primers (sense 5'-GGG GTA CCG AGC CAC CAT GGA AAT TA AGA GGA AGG G-3 ': SEQ ID NO: 40, and antisense 5'-CCG CTC GAG CGG GGT GTC AGC TTT TCC ATA AG-3 ': SEQ ID NO: 41) and pCMV-Sport6-INPP4B (Open Biosystems). The CDNA was cloned into pcDNA3.1 (+) Myc / His vector (Invitrogen). HEp-2 cells were transfected with an expression vector using Metafectene reagent (Biontex). To establish a stable cell line, HEp-2 cells were infected with the INPP4B cDNA vector and cultured in a basic culture medium for 24 hours. Transfected cells were selected by adding 1 mg / mL G418 to the cultured cells. Stable HEp-2 cells expressing INPP4B were identified by Western blot analysis.

<3-6> Phosphatase  analysis( Phosphatase assay )

Cells were lysed in buffer solution containing 50 mM TRIS-HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40, 7.5% glycerol, 1 mM EDTA, 1 mM Na3VO4 and complete protease inhibitors. The cell lysate was precipitated with anti-Akt or anti-PP2A antibody (Santa Cruz Biotechnology, Inc.). Immune mixtures were collected using protein A sepharose beads; A quarter of the washed beads were used to perform Western blot analysis to confirm the immunoprecipitation efficiency of Akt and PP2A. The remaining 3/4 beads were analyzed for PP2A activity using the RediPlat96 EnzChek Serine / Threonine Phosphatase assay kit [Invitrogen (Molecular Probes)] according to the manufacturer's method. The fluorescence was measured using a Victor2 fluorescence microplate reader (Perkin Elmer Corp.). Okadaic acid (Sigma), which inhibits PP2A activity, was used as a positive control.

Experimental Example  One: INPP4B  Ensure induction is not limited to radiation stimulation or specific cell types

To investigate the INPP4B response to radiation, HEp-2 cells and RR-HEp-2 were treated with 10 Gy. As a result, INPP4B protein levels for radiation increased in a time-dependent manner in HEp-2 cells (Fig. 2A, top); This induction was even more severe in RR-HEp-2 cells (Fig. 2A, bottom). These results suggest that INPP4B is a radiolabeled protein and that the overaccumulation of this protein can be a mechanism to obtain the radiation resistance phenotype in HEp-2 cells. Other human cancer cells, including A549, H460, HCT116 and MCF7 cells, were treated with 10 Gy radiation to investigate whether the radiation-mediated induction of INPP4B expression was restricted to HEp-2 cells. Quantitative RT-PCR showed that the radiation treatment induced an increase in INPP4B transcription level in all cancer cell lines (FIG. 2B). These results suggest that the above phenomenon occurs in all human cancer cells.

In addition, since the regulation of radiation resistance genes often plays a role in the development of resistance to anticancer drugs in various types of cancer, INPP4B has been shown to inhibit bleomycin, cisplatin, and etoposide in HEp- etoposide, and doxorubicin. Surprisingly, quantitative RT-PCR showed that all drugs induced increased levels of INPP4B mRNA, while INPP4A, a homologue of the INPP4B gene, was not induced with these drugs (FIG. 2C).

We also investigated whether the radiation-induced up-regulation is extended to other lipid phosphatases, or to INPP4B, which regulates the 3-phosphoinositide lipid network. As a result, it was confirmed that lipid phosphatase INPP4A and PTEN were not induced by radiation treatment, unlike INPP4B, by quantitative PCR (FIG. 2D, top) and general PCR (FIG. 2D, bottom) analysis.

These results suggest that INPP4B is the only lipid phosphatase that controls the development of radiation resistant phenotype of HEp-2 cells.

Experimental Example  2: INPP4B of ERK - dependency Judo HEp -2 cell to confirm the radiation resistance phenotype promotion confirmation

Since radiation activates mitogen-activated protein kinase (MAPK) protein, we investigated whether induction of INPP4B expression is involved in MAPK activity in HEp-2. As a result, 10Gy radiation increased the active forms of ERK, JNK (c-Jun N-terminal kinase) and p38 kinase without changing the level of the corresponding protein (Fig. 3A).

Then, specific inhibitors of each MAPK were pretreated with the cells prior to treatment with radiation. As a result, as shown in Fig. 3B, ERK inhibition by PD98059 blocks radiation-induced increase at the INPP4B protein (upper) and mRNA (intermediate) levels, while JNK inhibition by SP600125 or p38 kinase inhibition by SB203580 is at the INPP4B level It had no effect. Quantitative PCR showed that INPP4B transcription level was 3.2-fold increased in radiation-treated HEp-2 cells compared to untreated control cells, and that this induction decreased 1.3-fold with ERK inhibition (Fig. 3B, bottom ).

Endogenous ERKs were knocked down using siRNA (small interfering RNA) in HEp-2 cells to identify endogenous ERKs that regulate INPP4B expression. As a result, siRNA treatment effectively knocked ERK expression as shown in Fig. 3C. In particular, Western blot (FIG. 3C, top) and general RT-PCR (FIG. 3C, middle) were performed to confirm that endogenous ERK deletion significantly inhibited INPP4B induction by radiation (FIG. 3C). Quantitative PCR analysis confirmed that siRNA-regulated ERK knockdown reduced radiation-induced INPP4B mRNA levels by 38% compared to cells treated with radiation only (Fig. 3C, bottom). These results suggest that stress induced induction of INPP4B is dependent on ERK signal but not on JNK or p38 signal.

Overexpression of INPP4B tagged with wild-type Myc in HEp-2 cells was performed to confirm that INPP4B was essential for cancer radiation resistance. As a result of the colony formation survival analysis, overexpression of INPP4B increased cell survival after exposure to radiation (FIG. 4A). As a result of flow cytometry analysis, it was confirmed that cell death induced by treatment with 10 Gy radiation was markedly reduced at 48 hours in HEp-2 cells overexpressing INPP4B compared to HEp-2 cells (below 33%) , left). The results were confirmed by observing the cell morphology (Fig. 4B, middle).

In addition, it was confirmed that the cut-off level of PARP, which is a marker of apoptosis, is also reduced in HEp-2 cells in which INPP4B is overexpressed (FIG. 4B, right) compared with radiation treated HEp-2 cells, The results are consistent. Conversely, it was confirmed that siRNA mediated knockdown of endogenous INPP4B in RR-HEp-2 cells reduced survival after radiation treatment (Fig. 4C).

As measured by flow cytometry, endogenous INPP4B deletion increased radiation-induced RR-HEp-2 cell death from 16% (radiation alone) to 28% (radiation and INPP4B knockdown treatment) (FIG. 4D, left) The results were confirmed again by observing the morphology of the cells (Fig. 4D, middle). SiRNA targeting INPP4B effectively knocked INPP4B and found that the -PARP level cleaved in the substantially irradiated RR-HEp-2 cells was increased (Fig. 4D, right). These results suggest that INPP4B can selectively protect HEp-2 cells against radiation-induced cell death by increasing radiation resistance.

Experimental Example  3: INPP4B of HEp -2 Cells with Anti-Cancer Resistance

HEp-2 cells (INPP4B-HEp-2 cells) stably expressing Myc-tagged INPP4B were established in order to confirm whether INPP4B can regulate resistance to anticancer agents. As a result, surprisingly, INPP4B-HEp-2 cells showed markedly increased levels of basal INPP4B expression induced by the anti-cancer agents bleomycin, cisplatin, etoposide and doxorubicin. The cells were found to be resistant to anti-cancer drug-induced apoptosis by Western blot (FIG. 5A, top) and flow cytometry analysis of truncated-PARP (FIG. 5A, bottom). Although the degree of INPP4B induced by individual drugs was different, the anti-apoptotic effect of INPP4B was confirmed by morphological changes in all anticancer-treated cells (Fig. 5B).

In addition, INPP4B was inhibited by siRNA in order to confirm INPP4B dependence on anti-apoptotic phenotype in INPP4B-HEp-2 cells. As a result, INPP4B siRNA effectively blocked INPP4B expression, and remarkably re-sensitized to anti-cancer drug-induced apoptosis was confirmed by Western blot (Fig. 5C, top). As a result of the flow cytometry analysis, INPP4B deletion promoted apoptosis, and up to 60% (INPP4B-HEp-2 cells treated with doxorubicin infected with siRNA for INPP4B) in 33% (INPP4B-HEp-2 cells treated with doxorubicin) (Fig. 5C, bottom). &Lt; tb &gt;&lt; TABLE &gt; Similar results were confirmed by observation of morphological changes (Fig. 5D). This sensitization effect was further confirmed using other stable INPP4B-HEp-2 clones (# 2, # 4 and # 6). Further, the anticancer-resistant mutants were confirmed by western blotting on truncated- After INPP4B knockdown, it was confirmed that doxorubicin-induced cell death was increased, and it was confirmed that INPP4B was a positive biomarker in generation of anticancer resistance (Fig. 5E).

Experimental Example  4: Resistant phenotype INPP4B  In the role of lung cancer cells Akt  Check for changes in activity

In order to confirm that the resistance function of INPP4B is restricted to HEp-2 laryngeal cancer cells, A549 and H1299 lung cancer cells having resistance to radiation and anticancer drugs were investigated. As a result, it was confirmed that INPP4B siRNA efficiently knockdown INPP4B of A549 and H1299 cells with Western blotting (top) and quantitative PCR analysis (bottom), as shown in Fig. 6A. Similar to the results obtained in HEp-2 cells, the INPP4B silent phenomenon was detected in A549 cells by doxorubicin and radiation (Fig. 6B, top and middle) Lt; RTI ID = 0.0 &gt; cell death &lt; / RTI &gt; Under the same conditions as in the above experiment, this sensitization effect was greater in H1299 cells than in A549 cells (Fig. 6C). Because p53 differs between H1299 cells (p53 deficient) and A549 cells (wild type p53), the results suggest that the role of INPP4B for resistance is independent of the p53 signal.

Thus, whether or not the radiation can regulate Akt activity was examined. In order to confirm that INPP4B positively regulates Akt activity, RR-HEp-2 cells were transfected with siRNA and knockdown of endogenous INPP4B. Akt phosphorylation was highly induced in RR-HEp-2 cells exposed to radiation compared to HEp-2 cells expressing Akt activity; Consistent with this, GSK3? /? Phosphorylation, a known major target of Akt, was also increased (FIG. 7A). Although the RR-HEp-2 cells contained high phosphorylated Akt levels after irradiation, such Akt activity was largely inhibited by INPP4B deletion without altering Akt basal levels (FIG. 7B). In addition, phosphorylated Akt levels were very high in INPP4B-HEp-2 cells, but were dramatically down-regulated in INPP4B knockdown, which shows a close relationship between INPP4B expression and Akt activity levels (FIG. 7C). Similar to the results obtained in RR-HEp-2 cells, Akt phosphorylation levels were markedly reduced with INPP4B deletion in A549 and H1299, with a concomitant decrease in phosphorylated GSK3a / beta levels (Fig. 7D). Since phosphatase PP2A is known to directly inhibit Akt activity, it was determined in this invention whether INPP4B regulates PP2A activity. PP2A activity was greatly inhibited in RR-HEp-2 cells treated with okadaic acid used as a positive control (Fig. 7E and Fig. 7F). However, PP2A activity was associated with INPP4B mediated Akt activity as measured by Akt and PP2A immunoprecipitation assays (Figure 7E and Figure 7F), indicating that radiation treatment or INPP4B siRNA infection did not affect PP2A activity in RR-HEp-2 cells Respectively.

These results support that the induction of INPP4B by stress (radiation, anticancer agent) promotes the resistance phenotype by the Akt survival pathway in cancer cells.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> KOREA INSTITUTE OF RADIOLOGICAL & MEDICAL SCIENCES <120> Composition for diagnosis of radio-resistance or radio-sensitive          PSAP marker and use thereof <130> PA120672 / KR <160> 41 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bcl-xI Primer <400> 1 cgggcattca gtgacctgac 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bcl-xI Primer <400> 2 tcaggaacca gcggttgaag 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 Primer <400> 3 catatgtctc ccgccttctg 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ERBB2 Primer <400> 4 cccacacagt cacaccataa c 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Primer <400> 5 catctctgcc ccctctgctg a 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Primer <400> 6 ggatgacctt gcccacagcc t 21 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> INPP4A Primer <400> 7 ggctgccagt ccataatc 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> INPP4A Primer <400> 8 cacactttct cccactcc 18 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> INPP4B Primer <400> 9 aaagaatgca ggtacacag 19 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> INPP4B Primer <400> 10 ctctgtgctg ctcttagg 18 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PTEN Primer <400> 11 gaaagacatt atgacaccg 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTEN Primer <400> 12 ttagcatctt gttctgtttg 20 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> p21 Primer <400> 13 tgtccgtcag aacccatg 18 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> p21 Primer <400> 14 ggagtggtag aaatctgtca tg 22 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> INPP4B Primer <400> 15 gaaacagaaa gaagaaatac c 21 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> INPP4B Primer <400> 16 cccactcttc ctcatcatag 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PSAP Primer <400> 17 gtggtgccag aatgtgaag 19 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PSAP Primer <400> 18 ctgagggtag aggaggagag 20 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CDK11A Primer <400> 19 gtctgcacat caccgaac 18 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CDK11A Primer <400> 20 catttcttcc tcacttactt c 21 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GLUT1 Primer <400> 21 ctcatgggct tctcgaaac 19 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GLUT1 Primer <400> 22 ccgactctct tccttcatct c 21 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> JTB Primer <400> 23 ccacctctgc tggttgctc 19 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> JTB Primer <400> 24 ctatatggac tcgatttgct tcc 23 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ABCC5 Primer <400> 25 aaacaggatc agtaaagaag 20 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ABCC5 Primer <400> 26 aagaacacca ggataacg 18 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MRFAP1 Primer <400> 27 attatatatg ccgctcctac 20 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MRFAP1 Primer <400> 28 tggccttcac tacctgttc 19 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HNRNPUL1 Primer <400> 29 actcgtgagg aaactgcc 18 <210> 30 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HNRNPUL1 Primer <400> 30 cttggaacag gatcaggg 18 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> WDFY3 Primer <400> 31 ttaaatggtg caactctg 18 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WDFY3 Primer <400> 32 tggatggtac taagaagaac 20 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NDRG1 Primer <400> 33 agatctcagg atggacccaa g 21 <210> 34 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NDRG1 Primer <400> 34 catgccctgc acgaagtac 19 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CDC27 Primer <400> 35 acaaatctta tctggtggag 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CDC27 Primer <400> 36 gtatcaggtg aaattacagc 20 <210> 37 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> INPP4B siRNA <400> 37 cagaauguuu gagucacua 19 <210> 38 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> ERK-1 siRNA <400> 38 cucucuaacc ggcccancer 19 <210> 39 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> ERK-2 siRNA <400> 39 caggiuuua caagcucuu 19 <210> 40 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> INPP4B Primer <400> 40 ggggtaccga gccaccatgg aaattaaaga ggaaggg 37 <210> 41 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> INPP4B Primer <400> 41 ccgctcgagc ggggtgtcag cttttccata ag 32

Claims (15)

A composition for diagnosing radiation resistance of a laryngeal cancer cell, comprising an agent for measuring the level of mRNA of PSAP (Prosaposin, NCBI GenBank Gene ID: 5660) gene or a protein thereof.
The composition of claim 1, wherein the composition is selected from the group consisting of CDK11A (Cell division cycle 2-like 2, NCBI GenBank Gene ID: 728642), MRFAP1 (Mof4 family associated protein 1, NCBI GenBank Gene ID: 93621), HNRNPUL1 1, NCBI GenBank Gene ID: 11100), WDFY3 (WD repeat and FYVE domain containing 3, NCBI GenBank Gene ID: 23001), CDC27 (Cell division cycle 27 homolog, NCBI GenBank Gene ID: 996) and INPP4B (Inositol polyphosphate- -phosphatase type II, NCBI GenBank Gene ID: 8821). The composition for diagnosing radiation resistance of a laryngeal cancer cell according to claim 1, wherein the mRNA or the level of the mRNA of the at least one gene is selected from the group consisting of:
3. The composition for diagnosing radiation resistance of laryngeal cancer cells according to claim 2, wherein the composition further comprising an agent for measuring the level of the mRNA of the INPP4B gene or a protein thereof is capable of further diagnosing cancer resistance.
delete The composition for diagnosing radiation resistance of a laryngeal cancer cell according to claim 1 or 2, wherein the agent for measuring the level of the gene mRNA comprises a primer that specifically binds to the gene.
The composition for diagnosing radiation resistance of a laryngeal cancer cell according to claim 1 or 2, wherein the agent for measuring the protein level of the gene comprises an antibody specific to the protein.
A kit for the diagnosis of radiation resistance of a laryngeal cancer cell comprising the composition of claim 1 or 2.
8. The kit for diagnosing radiation resistance of a laryngeal cancer cell according to claim 7, wherein the kit is an RT-PCR kit, a DNA chip kit, a microarray or a protein chip kit.
Measuring the mRNA level from the separated biological sample using a primer that specifically binds to the PSAP gene;
Comparing the mRNA level with a normal control sample mRNA level; And
And judging that the mRNA level of the biological sample is higher than that of the normal control sample, and determining that the mRNA level of the biological sample is radioactive resistance.
10. The method of claim 9, further comprising measuring and comparing mRNA levels of one or more genes selected from the group consisting of CDK11A, MRFAP1, HNRNPUL1, WDFY3, CDC27 and INPP4B. And
If the mRNA level of the CDK11A, MRFAP1, HNRNPUL1, WDFY3 or INPP4B gene is higher than the control sample; Or judging that the level of mRNA of the CDC27 gene is lower than that of the control sample, it is judged to be radioactive resistance.
Contacting an antibody specific for the protein of the PSAP gene with an isolated biological sample to identify protein levels by antigen-antibody complex formation;
Comparing the change in protein level with a protein level in a normal control sample; And
And judging that the protein level of the biological sample is higher than that of the normal control sample, and judging the protein level to be radioactive resistance.
12. The method of claim 11, further comprising measuring and comparing protein levels of one or more genes selected from the group consisting of CDK11A, MRFAP1, HNRNPUL1, WDFY3, CDC27, and INPP4B; And
If the protein level of the CDK11A, MRFAP1, HNRNPUL1, WDFY3 or INPP4B gene is higher than the control sample; Or determining that the protein level of the CDC27 gene is lower than that of the control sample, the radioactive resistance is judged to be radioactive resistance.
Comparing the level of mRNA or a protein of the PSAP gene to the level of expression of normal cells from a separate sample of a patient who has undergone radiation therapy; And
And judging that the level of mRNA or protein of the separated sample of the patient is higher than that of a normal cell, the radiation resistance of the sample is determined to be radiation resistance.
14. The method of claim 13, further comprising measuring and comparing the mRNA or protein level of one or more genes selected from the group consisting of CDK11A, MRFAP1, HNRNPUL1, WDFY3, CDC27 and INPP4B. And
If the mRNA or protein level of the CDK11A, MRFAP1, HNRNPUL1, WDFY3, or INPP4B gene is higher than normal cells; Or judging that the CDC27 gene mRNA or its protein level is lower than that of a normal cell, it is judged to be radioactive resistance, and a method for providing information necessary for predicting the radiation treatment prognosis of a laryngeal cancer patient. delete

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