The effect of tumor-associated protein RCAS1 gene silencing on blood pressure and urinary protein excretion in pregnant mouse: a pilot study




Objective


The level of tumor-associated receptor-binding cancer antigen that is expressed on SiSo cells (RCAS1) is decreased significantly in preeclamptic pregnancies. We hypothesized that RCAS1 protein gene silencing might affect blood pressure and proteinuria in pregnant mice.


Study Design


On postcoital day 7.5, pregnant imprinting control region mice were subjected to the transfer of small interfering RNA (siRNA) against RCAS1 protein into the uterine cavity with the use of a hemagglutinating virus Japan envelope. Scramble siRNA was used as a negative control. Blood pressure and urine albumin/creatinine measurements were performed. The effect of the transferred siRNA was examined in uterine samples on postcoital day 8.5 with the use of Western blotting and immunohistochemistry analyses.


Results


In the RCAS1 siRNA group, blood pressure significantly raised on postcoital days 9.5, 10.5, 11.5, and 15.5, whereas urine albumin/creatinine ratio was significantly increased on postcoital day 9.5


Conclusion


Our results suggest the importance of RCAS1 protein in the pathophysiologic condition of preeclampsia.


Preeclampsia is a potentially dangerous complication of the second half of pregnancy or the early period after delivery. With respect to systemic inflammation, there is substantial evidence to support the concept that preeclampsia develops when the normal inflammatory response of pregnancy exaggerates. Although decidualization of the endometrium is associated with a massive recruitment of NK cells after embryo implantation, dysfunctional NK-cell activation could result in the maternal syndrome of preeclampsia. On the other hand, the tumor-associated antigens that can be recognized by the immune system might have a crucial role in the development of preeclampsia. Recent studies have defined the expression of tumor-associated proteins in trophoblast tissues and shown that a significant imbalance in expression of tumor-associated proteins could lead to early trophoblast apoptosis in preeclampsia. Moreover, human T and NK cells express the receptor for receptor-binding cancer antigen expressed on SiSo cells (RCAS1) at a high level after being activated; RCAS1 inhibits proliferation and induces apoptotic cell death of receptor-positive immune cells. According to our recent investigation, the placental RCAS1 messenger RNA expression and maternal blood serum RCAS1 concentrations were decreased significantly in preeclamptic patients. These findings lead us to hypothesize that RCAS1 gene silencing might induce preeclampsia-like symptoms in mice.


The tumor-associated antigen RCAS1 was defined originally by its immunoreaction with the 22-1-1 monoclonal antibody, which was raised by immunization of mice with the human uterine cervical adenocarcinoma cell line SiSo. The gene product was then termed receptor-binding cancer antigen expressed on SiSo cells (RCAS1), which is identical with the estrogen-responsive protein, estrogen receptor-binding fragment–associated gene 9 (EBAG9). Incubation of NIH3T3 mouse fibroblastic cells with recombinant murine EBAG9 protein results in the suppression of cell growth. RCAS1 inhibits in vitro growth of receptor-expressing cells (T and NK cells) and induces apoptotic cell death, which suggests its involvement in immune escape of tumor cells. Moreover, knockdown of RCAS1 expression by RNA interference recovered T-cell growth and proliferation in vitro. RCAS1 protein is also involved in regulating apoptosis of erythroid progenitor cells and may have a critical role in erythropoesis.


RCAS1 protein is expressed in various human tissues and organs and may have a role in the process of placental detachment, in immune tolerance during pregnancy, in stillbirth, and in pregnancies that are complicated by gestational diabetes mellitus. According to our investigation, in normal pregnant mice, RCAS1 protein messenger RNA was increased significantly on postcoital day 7.5, thus pointing the importance of the postcoital day 7.5 for RCAS1 protein expression in connection with placentation as a possible target for in vivo studies. The aim of the present study was to transfer RCAS1 small interfering RNA (siRNA) to the pregnant mouse uterus on postcoital day 7.5 and to monitor its possible effects on blood pressure and urine albumin/creatinine ratio (ACR).


Materials and Methods


Animals


This study was conducted with female imprinting control region strain mice, 7-8 weeks old (SLC, Shizuoka, Japan). The mice were housed in an animal care facility with a controlled environment, and females were kept with males of the same strain overnight. When a vaginal plug was detected the next morning, that was considered postcoital day 0.5 of the pregnancy. All animal experiments were approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine.


Preparation of the hemagglutinating virus Japan envelope (HVJ-E) vector–mediated siRNA for in vivo transfer


Ten thousand hemagglutinating activity units of an inactivated column-purified HVJ-E vector, Genome-ONE NEO (Ishihara Sangyo Co Ltd, Fukuoka, Japan) initially were centrifuged at 13,500 g for 15 minutes at 4°C. The pellet was mixed with 20 μL of 2% Triton-X/TE and centrifuged at 13,500 g for 15 minutes at 4°C. After removal of the supernatant, the tube was tapped to collapse the pellet. The pellet was dissolved in 5 nm of siRNA solution for 5 mice and mixed well. Finally, human tubal medium (Nippon Medical and Chemical Instrument Co Ltd, Osaka, Japan) was added to yield a final volume of 250 μL. RCAS1 siRNA (5′-CAACUAUACGGAAAACUCA-3′; n = 21) and the scrambled siRNA (5′-ACCUAACUAAUCGGAAAAC-3′; n = 14) as a negative control were used (Sigma-Genosys Ltd, Hokkaido, Japan). SiRNA target sequence was defined with the use of a web-based online software system (siDirect sequences) for computing highly effective siRNA sequences with maximum target specificity for mammalian RNA interference ( http://genomics.jp/sidirect/index.php?type=fc ).


In vivo siRNA transfer experiments


Mice on postcoital day 7.5 were anesthetized with Nembutal (Dainippon Sumitomo Pharma, Osaka, Japan) that was diluted in phosphate-buffered saline solution at a concentration of 1.25 mg/mL (1.5 mL/mouse introduced intraperitoneally) and subjected to laparotomy to expose the pregnant uterus. Twenty-five microliters of vector-mediated siRNA suspension was injected into the upper (ovarian) side of the uterine cavity of each horn with a 30G needle. The needle was inserted into the uterine cavity between the amniotic sacs, outside the amniotic cavity. The cervix was clamped for 10 minutes. All the procedures were performed slowly, taking care not to rupture the amniotic membranes or not to damage the cervix. Finally, the incision was closed to allow the recovery of the mice.


Sample preparation for Western blotting and immunohistochemical analysis


We collected uteri that contained fetuses from pregnant mice on postcoital day 8.5 at 24 hours after siRNA transfer. Samples from 5 mice were obtained from each group at the designated day of gestation. A portion of the collected tissues was paraffin embedded; the remaining tissues were frozen in liquid nitrogen and stored at –80°C until use.


Western blotting analysis of whole uteri from pregnant mice on postcoital day 8.5 at 24 hours after siRNA transfer


The specimens of uteri with placentae and fetuses (each approximately 100 mg in weight, frozen at –80°C) were homogenized with 0.5 mL of lysis buffer that consisted of distilled water, 0.5 mol/L Tris-HCl (pH 6.8), 10% sodium dodecylsulfate, glycerol, 6% β-mercaptoethanol and 1% bromophenol blue. Homogenates were centrifuged at 4°C for 20 minutes at 1200 g . An Immunomini NJ-2300 apparatus (InterMed, Tokyo, Japan) was used to determine the protein concentration in the lysates. Fifty micrograms of protein was subjected to electrophoresis on 10% sodium dodecylsulfate-polyacrylamide gel with a Bio-Rad Power-Pac 200 apparatus (Bio-Rad Laboratories Inc, Hercules, CA) then were transferred to a nitrocellulose membrane (0.45 μm) with a Bio-Rad Mini-Transblotter apparatus (Bio-Rad Laboratories Inc). Commercially available mouse lung tissue extract was used as a positive control (Santa Cruz Biotechnology Inc, Santa Cruz, CA). The membrane was incubated with 5% dry milk (Amersham Biosciences, Arlington, IL) in Tris-tween buffered saline (TTBS) buffer (pH 8.0) that contained 0.005 mol/L Tris-HCl, 0.138 mol/L NaCl, 0.0027 mol/L KCl, and 0.1% Tween x 20, (Nacalai Tesque Inc, Kyoto, Japan) followed by incubation with the primary goat polyclonal antibody to RCAS1/EBAG9 (Abcam Inc, Cambridge, MA) in TTBS buffer at a dilution of 1:500. The primary polyclonal goat anti–β-actin antibody (Abcam Inc) at a dilution of 1:500 and a normal goat immunoglobulin G (Santa Cruz Biotechnology Inc) were used as loading and negative controls, respectively. After overnight incubation of the membrane at 4°C, we used anti-goat immunoglobulin, horseradish peroxidase–linked antibody (from donkey; Abcam Inc) in TTBS buffer at a dilution of 1:5000 as the secondary antibody. RCAS1 immunoreactivity was visualized with an enhanced chemiluminescence Western blotting detection kit (Amersham Biosciences). Western blotting experiment for each specimen from 5 mice was performed 3 times. Blot densitometry analysis was performed with the National Institutes of Health Image J software. The results were normalized by the expression of β-actin in each sample.


Immunohistochemical staining of RCAS1 in whole uteri from pregnant mice on postcoital day 8.5 at 24 hours after siRNA transfer


To determine the gene silencing efficiency of the transferred siRNA against RCAS1 protein in the uterus, whole uteri specimens that contained placenta and fetuses from 5 mice from each group on postcoital day 8.5 at 24 hours after siRNA transfer were examined immunohistochemically. Immunohistochemical staining was performed with paraffin-embedded sections. The sections were deparaffinized and rehydrated in Xylene, and 100%, 95%, 70% ethanol. A heat-induced antigene retrieval method with sodium citrate buffer (pH 6.0) was used, followed by incubation with 10% rabbit serum in PBS buffer (pH 7.4; Nacalai Tesque Inc, Kyoto, Japan) for 1 hour at room temperature. Next, the sections were incubated overnight with goat polyclonal antibody to RCAS1/EBAG9 in PBS buffer at a dilution of 1:50 (Abcam Inc). Normal goat immunoglobulin G at a dilution of 1:50 was used as a negative control (Santa Cruz Biotechnology Inc). After being washed, the sections were incubated with Alexa Fluor 555 rabbit anti-goat immunoglobulin G (H+L) secondary antibody in PBS buffer at a dilution of 1:1000 (Invitogen, Eugene, OR). The slides were counterstained with Bis-benzimides (Hoechst 33258; Nacalai Tesque Inc, Kyoto Japan), washed with water, and dehydrated.


For calculation of the positively stained placental cell number, the quantification system (field = 1 mm 2 at ×100 magnification) was used. Two examiners counted randomly selected 8 fields from each section. The number of RCAS1 positively stained placental cells was quantified as the percentage of the sum of unstained and stained cells in the placenta.


Urine ACR measurement


Urine was collected at designated gestational days in the morning at 8:00-9:00 before blood pressure measurements were taken. Urine samples were centrifuged at 11,100 g for 10 minutes at room temperature and stored at –20°C until used. Albumin and creatinine concentrations were evaluated with the Albuwell M ELISA kit (Exocell Inc, Philadelphia, PA) and Creatinine Companion chemical assay kit (Exocell Inc), respectively. Intra- and interassay precision of samples within the useful range have a coefficient of variation of <10% of the mean for both assays. The ACR was calculated in the following manner: albumin (mg/dL)/creatinine (mg/dL) × 1000 = ACR (mg/g). Urine ACR measurements were performed in the RCAS1 siRNA group (n = 16) and the scrambled siRNA group (n = 9). ACR was measured on postcoital days 3.5, 5.5, 7.5, 8.5, 9.5, 10.5, 11.5, 13.5, 15.5, 17.5, and 19.5 before labor.


Blood pressure measurement


Blood pressure was measured in awake mice with the use of an automated tail cuff system (Softron BP-98; Softron, Tokyo, Japan) according to the manufacturer’s instructions at designated days of pregnancy. Results from the first 5 measurements were discarded; the average value that was obtained from the next 5 measurements was counted. The mice were in the restrainer for 15-20 minutes. Measurements were performed at each designated gestational day in the morning from 11:00-12:30. Blood pressure was measured in RCAS1 siRNA group (n = 16) and the scramble siRNA (negative control) group (n = 9). Blood pressure measurements were performed on postcoital days 3.5, 5.5, 7.5, 8.5, 9.5, 10.5, 11.5, 13.5, 15.5, 17.5, and 19.5 before labor.


Evaluation of course of pregnancy after in vivo siRNA transfer


To evaluate the placental size and fetal weight after siRNA transfer, 5 mice from each group were killed on postcoital day 18.5; the remaining mice in each group were monitored until delivery.


Statistical analysis


Data that were obtained from study groups on each designated day of gestation were subjected to the Mann-Whitney U test; Western blotting and immunohistochemistry data were subjected to 1-way analysis of variance with the use of the Statview statistics package (Abacus Concepts, Inc, Berkeley, CA); a probability value of < .05 was considered to indicate significance.

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Jul 6, 2017 | Posted by in GYNECOLOGY | Comments Off on The effect of tumor-associated protein RCAS1 gene silencing on blood pressure and urinary protein excretion in pregnant mouse: a pilot study

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