JP2013224860A - Post-transcriptional control of expression of mdr1/p-glycoprotein (p-gp) by micro-rna 145 (mir-145) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for determining an effect of medication discharged outside a cell by P-gp.SOLUTION: A biological sample collected from a subject is examined by a method that includes a step for measuring an amount of human miR-145 and determines an effect of medication discharged outside a cell by P-gp. It is preferable that the medication may be one selected from a group comprising anti-cancer drug, immunosuppressive drug, anti-HIV drug, colchicine, digoxin, quinidine, verapamil, and steroid. Further, it is preferable that the biological sample is blood.

Description

  The present invention relates to a mechanism related to post-transcriptional regulation of MDR1 / P-gp by miR-145, and relates to a kit for determining the effect of a drug excreted extracellularly by P-gp.

The P-glycoprotein encoded by the Multidrug resistance protein 1 (MDR1) gene is an ATP-binding cassette transporter that transports drugs out of the cell (Schwab et al., 2003: All non-patent literature is named at the end. ), Expressed in cancer cells in addition to various normal tissues (liver, small intestine, kidney, brain, placenta, etc.), and plays an important role in drug pharmacokinetics control and anticancer drug resistance (Cordon-Cardo et al., 1990; Gottesman et al., 2002). It is known that there are large individual differences in the expression level and function of P-gp (Masuda and Inui, 2006). In a previous report, we reported that the expression level of P-gp in the small intestine increased after hepatic ischemia reperfusion (Ikemura et al., 2009). However, the mechanism by which the amount of P-gp expression in the small intestine increases after hepatic ischemia reperfusion remains unclear. In recent years, it has been shown that the stability of MDR1 mRNA or translational regulation from mRNA may affect individual differences in the expression level and function of P-gp (Kimchi-Sarfaty et al., 2007; Wang and Sadee , 2006), elucidation of the post-transcriptional regulatory mechanism of P-gp is regarded as important.
Micro RNA (miRNA), a small functional RNA, has attracted attention as a new post-transcriptional regulator (E Eulalio et al., 2008). miRNA inhibits mRNA degradation and translation by binding to the 3′-untranslated region (3′-UTR) of the target mRNA (Filipowicz et al., 2008; Kim, 2005). In recent years, the role of miRNA has attracted attention not only in the cancer research area but also in the pharmacokinetic area, and it has been reported to be involved in post-transcriptional regulation of drug transporters, drug metabolizing enzymes, and nuclear receptors. (Dalmasso et al., 2011; Pan et al., 2009; Takagi et al., 2010; Takagi et al., 2008). It has also been reported that miR-27a and miR-451 act on some transcription factor and indirectly regulate P-gp expression (Li et al., 2010; Zhu et al., 2008). .

However, there is no data that miRNA controls the expression level and function of P-gp by direct action of MDR1 on 3′-UTR.
The present invention has been made in view of the above circumstances, and an object thereof is to provide a kit for determining the effect of a drug excreted extracellularly by P-gp.

The present inventor examined the miRNA expression change in the small intestine after hepatic ischemia-reperfusion and the role of miRNA in post-transcriptional regulation of P-gp in small intestinal epithelial cells. The inventors have found that the expression level and function of P-gp can be controlled through the suppression of translation from mRNA by direct action on the RNA, and the present invention has been completed.
Thus, the drug effect diagnostic kit according to the present invention comprises (A) a step of fixing a biological sample collected from a subject, (B) a step of measuring the amount of human miR-145, (C) the step ( And a step of detecting, measuring and determining a drug excreted extracellularly by P-gp based on B).
In the above invention, the drug excreted extracellularly by P-gp is one selected from the group consisting of anticancer drugs, immunosuppressive drugs, anti-HIV drugs, colchicine, digoxin, quinidine, verapamil, and steroids. Preferably there is.
The biological sample is preferably blood and urine.
The method for determining the effect of a drug excreted extracellularly by P-gp according to the invention for solving the above-mentioned problem is to measure the amount of human miR-145 in a biological sample collected from a subject. Including a process.
P-gp is a membrane protein having a molecular weight of about 180,000 encoded by the MDR1 gene. P-gp is a transporter that excretes extracellular compounds such as compounds that exist on the cell membrane and has cytotoxic properties. Various normal tissues (eg small intestine, lung, proximal tubule of kidney, blood brain barrier) Expressed in capillary endothelial cells). P-gp is also called ATP-binding Cassette Subfamily B 1 (ABCB1) or Multidrug resistance protein 1 (MDR1). P-gp is composed of 1280 amino acids and is a 12-transmembrane glycoprotein. Amino-terminal and carboxyl-terminal ends are both present in the cell, and there is an ATP-binding domain between the sixth and seventh of the 12 transmembrane domains and at the carboxyl-terminal end. doing.

Examples of drugs excreted extracellularly by P-gp include anticancer drugs (irinotecan, doxorubicin, vinblastine, paclitaxel, etc.), immunosuppressive drugs (tacrolimus, cyclosporin A, etc.), anti-HIV drugs, colchicine, fexoso Examples include phenazine, digoxin, quinidine, verapamil, oseltamivir, loperamide, steroid and the like. Of these, anticancer drugs, immunosuppressive drugs, or digoxin are preferable.
MicroRNA (miRNA) is a single-stranded RNA of about 20 to 25 bases that is present in cells and is a noncoding RNA (ncRNA) that is not translated into protein and has the function of regulating the expression of other genes. It is considered to be. The DNA sequence that forms the mature miRNA is longer than the miRNA, and includes the miRNA sequence and a nearly complementary reverse sequence. When this DNA sequence is transcribed into single-stranded RNA, the miRNA sequence and the reverse complementary sequence are combined to form a double strand, and take a hairpin loop structure as a whole to become a primary miRNA (pri-miRNA). When part of the pri-miRNA molecule is cleaved by Drosha in the nucleus, it becomes a miRNA precursor (pre-miRNA), and then the pre-miRNA molecule is transported out of the nucleus by a carrier protein (Exportin-5), Dicer results in a 20-25 base miRNA sequence. A part of miRNA contains a sequence complementary to mRNA (mostly a sequence for 3'-UTR region), and binding of the mRNA and miRNA suppresses translation of mRNA or degrades mRNA. It is thought to cause. Thus, miRNA is thought to have a function of controlling protein translation.

miR-145 is a kind of miRNA. miR-145 has the sequence 5′-GUCCAGUUUUCCCAGGAAUCCCU-3 ′ (SEQ ID NO: 1). miR-145 is abundantly expressed in various tissues, and its expression decreases as cancer progresses, and it has been found to be associated with tumor cell development and suppression of metastasis. The present inventor's findings clarified that miR-145 regulates the expression and function of P-gp through repression of translation from mRNA by direct action of MDR1 on 3'-UTR. It was.
The biological sample means a sample collected from a subject outside the body, and includes a collected piece from each tissue and blood (serum, plasma, blood cells (including red blood cells and white blood cells)). miRNA is known to exist stably in tissues, saliva, urine, and human plasma. For this reason, in the present invention, blood (plasma) or urine that can be collected easily and repeatedly may be used as a biological sample.
As a step (method) for measuring the expression level of miR-145, methods generally used for miRNA measurement, such as Northern hybridization, microarray, RNase protection assay, and TaqMan ^R MicroRNA assay can be used. Of these, the TaqMan MicroRNA assay is preferably used from the viewpoint of sensitivity and the like.
According to the present invention, the amount of human miR-145 is measured for a biological sample collected from a subject. miR-145 regulates P-gp expression and function through translational repression from mRNA by direct action of MDR1 on 3′-UTR. Therefore, when the amount of miR-145 is low, the expression of P-gp is not suppressed, and the drug excretion activity to the outside by P-gp is high. You can take measures such as increasing it. On the other hand, when miR-145 is high, the expression of P-gp is suppressed, and the drug excretion activity by P-gp is low. You can take actions such as

  Thus, according to the present invention, a kit for determining the effect of a drug excreted extracellularly by P-gp is provided.

It is a figure which shows the result when the target binding site of miR-145 is estimated by the miRanda program in 3'-UTR of MDR1 and Mdr1a mRNA of human (A), rat (B) and mouse (C). It is a graph showing the results of confirming the expression level of miR-145, Mdr1a and Mdr1b in the upper small intestine (A, D, G), middle small intestine (B, E, H), and lower small intestine (C, F, I) . miR-145 expression level (A, B, C) was measured by RT-PCR and corrected based on U6 snRNA expression level. The mRNA levels of Mdr1a (D, E, F) and Mdr1b (G, H, I) were measured by RT-PCR and corrected based on the expression level of GAPDH. Each data is shown as an average value ± S.E. (N = 5). In the graph, “*” indicates that it was significant at a 5% risk rate compared to sham-treated rats (P <0.05). 3 is a graph showing the effect of miR-145 on 3′-UTR of MDR1 mRNA in HEK293 cells. Reporter plasmid (50 ng) with 3'-UTR sequence of MDR1 mRNA and miR-145 precursor (10, 30, 50 nM) or miR-control precursor (50 nM) or vehicle (Control) in HEK293 cells The luciferase activity was measured 24 hours later. Each data is shown as an average value ± S.E. Of three independent test results. In the graph, “**” was significant at 1% risk (P <0.01) compared to control, and “***” was 0.1% risk (P <0.01) compared to control. 0.001) is significant. It is a graph which shows the test result which confirmed the influence which the miR-145 inhibitor has with respect to the expression level of miR-145, MDR1 mRNA, and P-gp in Caco-2 cell. Data after 48 hours after transfection of Can-2 cells with 50 nM miR-145 inhibitor, 50 nM miR-control inhibitor or Vehicle (Control) are shown. (A) The amount of miR-145 was measured by the RT-PCT method and corrected based on the U6 snRNA expression level. (B) MDR1 mRNA level was measured by RT-PCR method and corrected based on GAPDH expression level. (C) The expression levels of P-gp and Villin in Caco-2 cells were determined by Western blot analysis. (D) The expression level of P-gp was corrected based on Villin data. Each data is shown as an average value ± S.E. Of three independent test results. In the graph, “*” indicates that it was significant at a risk rate of 5% compared to the control (P <0.05). This is a result showing Rho123 transport activity via P-gp in Caco-2 cells. Caco-2 cells were transfected with 50 nM miR-145 inhibitor or 50 nM miR-control inhibitor or Vehicle (Control). After 48 hours, the cells were incubated with 1 μM rhodamine 123 (Rho123) at 37 ° C. for 1 hour in the presence or absence of cyclosporin A (CsA; 10 μM). (A) Data showing the amount of Rho123 accumulated in Caco-2 cells in the presence or absence of CsA. (B) Data showing the transport activity of Rho123 via P-gp, calculated by subtracting the intracellular accumulation of Rho123 in the absence of CsA from the intracellular accumulation of Rho123 in the presence of CsA . Each data is shown as the mean ± SE of three independent test results. In the graph, “*” indicates that the risk rate was 5% (P <0.05) compared with the control. “ ^†† ” indicates a significant difference (P <0.01) with a 1% risk rate compared to the amount of Rho123 accumulated in Caco-2 cells in the absence of CsA.

Next, embodiments of the present invention will be described with reference to the drawings. However, the technical scope of the present invention is not limited by these embodiments, and various forms can be made without changing the gist of the invention. Can be implemented. Further, the technical scope of the present invention extends to an equivalent range.
Materials and methods <Test materials>
miR-145 precursor (Pre-miR ^TM miRNA precursor, AM17100, product ID PM11480), miR-145 inhibitor (Anti-miR ^TM inhibitor, AM17000, product ID AM11480), control oligo for miR-145 precursor (Pre- miR ^™ Negative control, AM17110) and control oligos for miR-145 inhibitors (Anti-miR ^™ Negative control, AM17010) were obtained from Ambion (Austin, TX, USA). Rho123 was obtained from Wako Pure Chemical. All other reagents were of the highest available purity.

<Animals>
9-week-old Wistar male rats were used in the experiment (obtained from SLC Japan Co., Ltd.). Rats were fasted for 12 hours prior to the experiment and water ad libitum. This experiment was approved by the Mie University Review Board for Animal Experiments and was performed according to the National Institutes of Health (NIH) guidelines (Institute of Laboratory Animal Resources, 1996).
Details of hepatic ischemia-reperfusion model rats are as reported in our report (Ikemura et al., 2009). Briefly, after intraperitoneal administration of 50 mg / kg pentobarbital to rats, blood flow to the left and middle lobes of the liver was blocked for 60 minutes using a vascular clip, resulting in 70% hepatic partial imagination. Raised blood. The mock-treated rats were treated in the same way without blocking the blood flow to the left and middle lobes of the liver. Twelve hours after reperfusion, small intestine (upper, middle, and lower) tissues were collected.

<Total RAN extraction and RT-PCR>
Total RNA was isolated using the mirVana ^™ miRNA isolation kit (Ambion, Austin, TX). For miRNA, use TaqMan ^R MicroRNA Reverse Transcription Kit and TaqMan ^R MicroRNA Assay for miR-145 and U6 (both Applied Biosystems, Foster, CA, USA), and extract only the desired miRNA from total RNA by stem-loop hybridization. Reverse transcribed. For the expression level of MDR1 mRNA, cDNA was synthesized from total RNA (100 ng) using PrimeScript ^™ RT reagent kit (Takara Bio Inc.) according to the attached manual. Thereafter, cDNA was subjected to RT-PCR using SYBR ^R Premix Ex Taq ^TM (Takara Bio Inc.). The primers used for PCR were as follows. Human MDR1 sense 5'-CCCATCATTGCAATAGCAGG-3 '(SEQ ID NO: 2), human MDR1 antisense 5'-TGTTCAAACTTCTGCTCCTGA-3' (SEQ ID NO: 3), rat Mdr1a sense 5'-GACGGAATTGATAATGTGGACA-3 '(SEQ ID NO: 4), rat Mdr1a antisense 5'-GTACGTCGTCATCCAGAGC-3 '(SEQ ID NO: 5), rat Mdr1b sense 5'-GAAATAATGCTTATGAATCCCAAAG-3' (SEQ ID NO: 6), rat Mdr1b antisense 5'-GGTTTCATGGTCGTCGTCTCTTGA-3 '(SEQ ID NO: 7), human And rat GAPDH sense 5′-CCTTCATTGACCTCAACTA-3 ′ (SEQ ID NO: 8), human and rat GAPDH antisense 5′-GGAAGGCCATGCCAGTGAG-3 ′ (SEQ ID NO: 9). Amplification and detection of specific DNA products was performed using the ABI7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The miR-145 expression level was corrected based on the U6 sn RNA expression level. The MDR1 mRNA expression level was corrected based on the GAPDH expression level.

<RNA labeling and hybridization using miRXplore ^TM microarray>
The quality and stability of the isolated RNA samples were evaluated using the Agilent 2100 bioanalyzer platform (Agilent Technologies, Boeblingen, Germany). RNA samples were labeled with Hy3 and Hy5 according to the protocol of miRXplore ^™ (Miltenyi Biotec, Bergisch-Gladbach, Germany), respectively. The labeled samples were then hybridized overnight with the miRXplore ^™ microarray using the Hyb ^™ hybridization station (Miltenyi Biotec, Bergisch-Gladbach, Germany).

<Analysis with miRXplore ^TM microarray>
The fluorescent signal of the hybridized miRXplore ^™ microarray was detected using a laser scanner system (Agilent Technologies, Boeblingen, Germany). ImaGene ^R software (Biodiscovery, El Segundo) was used to measure the average signal and average background intensity of each spot in miRXplore ^™ microarray images. The PIQOR ^™ analyzer software (Miltenyi Biotec, Bergisch-Gladbach, Germany) was used to subtract the background and then normalize the net signal intensity data to calculate the Hy5 / Hy3 ratio of miRNA. The cut-off value was set to 1.5 times.

<Cell culture>
Cultured human intestinal epithelial cell carcinoma Caco-2 and cell line HEK293 derived from human fetal kidney were obtained from American Type Culture Collection (Rockville, MD, USA). Caco-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% non-essential amino acids without the addition of antibiotics and used between passages 40-60. HEK293 cells were cultured in DMEM containing 10% fetal bovine serum without adding antibiotics and used between passages 60-70. These cells were cultured at 37 ° C. in a humidified atmosphere under 5% carbon dioxide.

<Transfection of miR-145 inhibitors into Caco 2 cells>
For Caco-2 cells cultured on plastic plates using SiPORT ^™ NeoFX ^™ transfection reagent (Ambion, Austin, TX, USA) and Opti-MEMI medium (Invitrogen, Carlsbad, CA, USA) , 50 nM miR-145 inhibitor, 50 nM miR control inhibitor, or Vehicle. After 48 hours, Rho123 transport activity was measured or total RNA and protein were extracted from the cells.

<Luciferase assay>
A 683 base pair fragment of MDR13'-UTR was inserted downstream of the luciferase gene of the pLightSwitch reporter vector (Swithgear Genomics, Menlo Park, CA, USA). For HEK293 cells cultured on 96-well plates, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), add 50 ng MDR1 3'-UTR reporter plasmid and 50 nM miR-145 precursor or control precursor. Co-transfected. After 24 hours, cellular luciferase activity was measured using a LightSwtch luciferase assay system ^™ (Swithgear Genomics, Heidelberg, Germany) and an Appliskan ^™ luminometer (Thermo scientific, USA).

<Western blotting for P-gp>
Protein isolated from Caco-2 cells (12.5 μg) was separated by 7.5% SDS-PAGE. Western blotting for P-gp using the C219 monoclonal antibody (Calbiochem, San Diego, CA, USA) was performed as previously reported (Shimomura et al., 2002). A polyclonal antibody against Villin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used according to the attached manual. The relative intensity of the band in each lane was measured using image processing software: Image J 1.38 (National Institutes of Health, Bethesda, MD, USA) to determine the intensity ratio of P-gp and Villin (Omae et al ., 2005).

<Rho123 transport test using Caco 2 cells>
The intracellular accumulation of Rho123 was examined using Caco-2 cells. The composition of the incubation solution used was 145 mM NaCl, 3 mM KCl, 1 mM CaCl ₂ , 0.5 mM MgCl ₂ , 5 mM D-glucose, 5 mM HEPES (pH 7.4). After removing the culture medium, the cells were washed once with the above incubation solution and then preincubated for 30 minutes at 37 ° C in the presence or absence of the P-gp inhibitor cyclosporin A (10 µM). did. After preincubation, cells were incubated with 1 μM Rho123 in the presence or absence of 10 μM cyclosporin A for 1 hour at 37 ° C. Thereafter, the drug solution was removed and washed once with an ice-cooled incubation medium containing 3% bovine serum albumin and twice with an ice-cold incubation medium. Cells were solubilized with 1N NaOH, and the amount of cellular protein was measured by the Bradford method (Bradford, 1976) using Coomassie Brilliant Blue Protein Assay Kit (Nacalai Tesque). At this time, bovine gamma globulin was used as a standard product. In order to confirm the intracellular accumulation of Rho123, elution was performed with 1 mL of extraction solution (incubation solution: methanol = 50: 50), and Rho123 was measured by HPLC. For HPLC analysis, an HPLC system (LC-20AB, Shimadzu Corp.) was used. Reversed-phase Chemcobond 5-ODS-H column (4.0 × 150 mm, Chemco Corp.) and fluorescence detector (RF-10AXL, Shimadzu Corp.) Was used. The column temperature was set to 40 ° C. Rho123 was eluted with 20 mM sodium acetate (pH 4.0): methanol = 20: 80 at a flow rate of 1 ml / min. The wavelength of the fluorescence detector was an excitation wavelength of 488 nm and a measurement wavelength of 535 nm.

<Statistical analysis>
All data were expressed as mean ± SE. Statistical analysis was performed using GraphPad Prism 5.02 (GraphPad Software Inc., San Diego, CA, USA), followed by Dunnett's test followed by one-way analysis of variance. Differences between the two groups were performed by unpaired t-test. The significance level was p <0.05.

Test results <MiRNA expression changes and Mdr1a and Mdr1b mRNA expression levels in the small intestine after hepatic ischemia reperfusion>
RNA samples isolated from the upper small intestine of sham-treated rats and hepatic ischemia-reperfusion treated rats were screened against 475 types of mature miRNA using miRExplore ^TM microarray according to miRbase version 16.0. Table 1 shows a list of miRNAs that were increased 1.5 times or more or decreased to 1 / 1.5 or less.

  Ten miRNAs decreased and four miRNAs increased in the upper small intestine after hepatic ischemia-reperfusion. Of these miRNAs, only miR-145, which was found to decrease in humans, rats, and mice by target prediction search using the miRanda program, has a sequence complementary to the 3'-UTR of MDR1 and Mdr1a. The possibility of being a factor that regulates the expression of the MDR1 and Mdr1a genes was shown (FIG. 1). On the other hand, other miRNAs detected from the microarray analysis did not have complementary sequences to the 3′-UTR of MDR1 and Mdr1a. Furthermore, the miR-145 expression level in the small intestine was measured by RT-PCR analysis using total RNA extracted from mock-treated rats and rats after hepatic ischemia-reperfusion. 2A, 2B and 2C show the miR-145 expression level corrected based on the U6 snRNA expression level in the small intestine of each group of rats. The amount of miR-145 in the upper small intestine (2A), middle small intestine (2B), and lower small intestine (2C) after hepatic ischemia-reperfusion was approximately 33% compared to the miR-145 expression in the mock-treated rats, It decreased to 47, 48%. This data was almost the same as the result of microarray analysis (Table 1). Mdr1a mRNA expression level and Mdr1b mRNA expression level in mock-treated rats and rats after hepatic ischemia-reperfusion were evaluated by RT-PCR analysis (FIG. 2D-2I). There were no significant differences in the expression levels of Mdr1a and Mdr1b mRNA in the small intestine in both the mock-treated rats and the rats after hepatic ischemia-reperfusion.

<Inhibitory effect of miR-145 on 3'-UTR of MDR1 in HEK293 cells>
In order to confirm whether miR-145 acts directly on the 3'-UTR of MDR1 mRNA, the MDR1 3'-UTR sequence containing the miR-145 predicted target binding site was introduced downstream of the luciferase reporter gene. The reporter plasmid and miR-145 precursor or miR-control precursor were cotransfected into HEK293 cells. The inhibitory effect of miR-145 on 3'-UTR of MDR1 was evaluated by measuring luciferase activity in HEK293 cells. As shown in FIG. 3, luciferase activity was significantly reduced in a dose-dependent manner by miR-145 overexpression. On the other hand, miR-control precursors did not have a significant effect on luciferase activity.

<Role of miR-145 on the expression level of MDR1 mRNA and P-gp in Caco-2 cells>
In order to confirm the regulation of P-gp expression via miR-145, Caco-2 cells were transfected with miR-145 inhibitor, miR-control inhibitor, or vehicle (control). Transfection with miR-control inhibitor had no effect on miR-145 expression. When miR-145 inhibitor was transfected, after 48 hours, the amount of miR-145 in Caco-2 cells decreased to about 16% compared to the control (FIG. 4). This confirmed that miR-145 expression level was knocked down by miR-145 inhibitor transfection.
48 hours after transfection into Caco-2 cells, mRNA concentration of MDR1 was measured by RT-PCR using total RNA. FIG. 4B shows the MDR1 mRNA expression level corrected based on the GAPDH expression level. The expression level of MDR1 mRNA was not changed by transfection of miR-145 inhibitor or miR-145 control inhibitor (FIG. 4B). For samples obtained from Caco-2 cells 48 hours after transfection, the expression levels of P-gp and Villin were compared by Western blotting (FIG. 4C). FIG. 4D shows the result of the expression level of P-gp corrected based on the signal of Villin. The expression level of P-gp increased to about 160% by transfection of the miR-145 inhibitor compared to the control. On the other hand, P-gp expression level did not change by transfection of miR-145 control inhibitor.

<Influence of miR-145 on P-gp function in Caco-2 cells>
To investigate the role of miR-145 on the excretory transport activity of P-gp, P-gp when Caco-2 cells were transfected with miR-145 inhibitor, miR-control inhibitor, or vehicle (control) Mediated Rho123 transport function was examined using cyclosporin A (CsA, P-gp inhibitor). As shown in Fig. 5A, in Caco-2 cells transfected with miR-145 inhibitors, Rho123 intracellular accumulation in the absence of CsA tends to decrease, although not significant compared to Vehicle (control). there were. In Caco-2 cells transfected with miR-145 inhibitor or miR-control inhibitor, Rho123 intracellular accumulation in the presence of CsA was significantly increased compared to intracellular accumulation in the absence of CsA. FIG. 5B shows the transport activity of Rho123 via P-gp calculated by subtracting the intracellular accumulation of Rho123 in the absence of CsA from the intracellular accumulation of Rho123 in the presence of CsA. . In Caco-2 cells transfected with miR-145 inhibitors, the transport activity of Rho123 via P-gp increased to about 280% of the control. On the other hand, Caco-2 cells transfected with miR-145 control inhibitor showed no effect on P-gp function.

<Discussion>
P-gp (MDR1) is an excretion transporter that regulates drug pharmacokinetics and drug resistance of cancer cells. Although it has been elucidated that the amount of P-gp expression in the small intestine increases after hepatic ischemia reperfusion, the detailed mechanism for this phenomenon remains unclear. miRNA plays an important role in drug pharmacokinetics and acquisition of drug resistance through post-transcriptional regulation of drug transporters and drug metabolizing enzymes. The purpose of this study was to investigate the changes in miRNA expression in the small intestine after hepatic ischemia reperfusion and the role of miRNA in post-transcriptional regulation of P-gp in small intestinal epithelial cells.
First, in order to investigate the regulation mechanism of the increase in P-gp expression level in the small intestine after hepatic ischemia reperfusion, we examined the expression level of Mdr1a and Mdr1b mRNA in each part of the small intestine (FIG. 2). However, there was no significant difference between the two groups in the Mdr1a and Mdr1b mRNA expression levels at all sites, so the increase in P-gp expression levels after hepatic ischemia-reperfusion was not transcriptional regulation but transcription. The possible involvement of post-regulation was shown. Next, in order to investigate the post-transcriptional regulation mechanism of P-gp mediated by miRNA, we compared miRNA expression fluctuations in the upper small intestine using mock-treated rats and rats after hepatic ischemia-reperfusion. As shown in Table 1, in the rat after hepatic ischemia / reperfusion, various miRNA expression levels were changed in the upper small intestine. Of these, miR-145, which was reduced by analysis using the miRanda program, has a complementary sequence to the 3'-UTR of MDR1 and Mdr1a mRNA, and thus focused on miR-145 expression reduction ( Figure 1). When the expression levels of miR-145 in the upper, middle, and lower parts of the small intestine were measured by RT-PCR, they were reduced to about 33%, 47%, and 48%, respectively (FIG. 2).

  This result was almost the same as the result of microarray analysis as shown in Table 1. From the above, P-gp expression level and miR-145 expression level showed a negative correlation. On the other hand, other miRNAs detected by microarray analysis did not have complementary sequences to MDR1 and Mdr1a 3′-UTR, but other miRNAs act on some P-gp transcription factors, The possibility of indirectly regulating the translation of P-gp cannot be denied. Furthermore, miR-21 has been reported to increase the expression of P-gp in lung cancer cells via the regulation of programmed cell death 4 (PDCD4), a tumor suppressor protein (Bourguignon et al., 2009). In this study, as shown in the results of microarray analysis in Table 1, miR-21 expression increased after hepatic ischemia reperfusion. Although not shown in the data, the results of RT-PCR analysis showed that there was no significant difference in miR-21 expression between mock-treated rats and rats after hepatic ischemia reperfusion. The subsequent increase in the expression level of P-gp in the small intestine was thought to be less involved in miR-21-mediated expression regulation.

  In order to confirm whether MDR1 is directly controlled by miR-145, luciferase assay using a plasmid containing MDR1 3′-UTR was performed using HEK293 cells (FIG. 3). As a result, it was shown that MDR1 is controlled by miR-145. Caco-2 cells were then used to examine how miR-145 affects protein levels, mRNA levels, and function with respect to P-gp by a knockdown approach. When the miR-145 inhibitor was transfected, it was found that the MDR1 mRNA expression level was not changed, but the P-gp expression level was increased and the Rho123 transport activity was increased. (Fig. 4B, 4C, 4D, 5B). These results indicate that P-gp is regulated by miR-145, and that the regulation mechanism is to suppress translation from mRNA by direct action of MDR1 on 3'-UTR. . These findings are the first to show that miRNA regulates P-gp expression by direct action of MDR1 on 3′-UTR.

  miR-145 is ubiquitous and abundant in normal human tissues, including small intestine (7942 copies per cell), kidney (2130 copies per cell), brain (991 copies per cell), and liver (606 copies per cell) (Lee et al., 2008). According to many studies, miR-145 expression levels are determined in colon cancer (Michael et al., 2003), breast cancer ((Iorio et al., 2005; Kim et al., 2011), and lung cancer (Yanaihara). et al., 2006) are known to have decreased expression levels in various cancer cells, including extensive studies on the role of tumor suppressor miRNAs involved in tumor cell development and metastasis suppression. Cancer Shi et al. Also report that miR-145 overexpression reduces the resistance to anticancer drugs caused by glucocorticoids and increases the sensitivity of P-gp to mitomycin. (Shi et al., 2012) Thus, miR-145 plays an important role in the mechanism of cancer development and drug resistance of cancer cells.

As a result of this study, it was clarified that miR-145 regulates the expression and function of P-gp through translational suppression from mRNA by direct action of MDR1 on 3'-UTR. It was also thought to be deeply involved in the expression and function enhancement of P-gp expressed in small intestinal epithelial cells after hepatic ischemia reperfusion. Furthermore, this study was thought to provide data to deepen our understanding of drug-resistance of cancer cells and novel mechanisms regarding individual differences in P-gp expression and function.
Thus, according to the present embodiment, a kit and a method for determining the effect of a drug excreted extracellularly by MDR1 can be provided.

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Claims (4)

(A) fixing a biological sample collected from a subject;
(B) measuring the amount of human miR-145;
(C) based on the step (B), detecting, measuring and determining a drug excreted extracellularly by P-gp;
A drug effect diagnostic kit comprising: The drug excreted extracellularly by P-gp is one selected from the group consisting of anticancer drugs, immunosuppressive drugs, anti-HIV drugs, colchicine, digoxin, quinidine, verapamil, and steroids A drug effect diagnostic kit. The kit for diagnosis of drug effect according to claim 1 or 2, wherein the biological sample is blood or urine. A method for measuring using the drug effect diagnostic kit according to any one of claims 1 to 3, comprising a step of measuring the amount of human miR-145 in a biological sample collected from a subject. A method for determining the effect of a drug excreted extracellularly by P-gp.