The current study consisted of 46 cesarean section puerperas with HDCP and 30 normal delivery puerperas. The peripheral blood was collected from patients from February 2013 to November 2014 in Laiwu City People's Hospital (Laiwu, China; n=14), Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Jinan, China; n=17) and Jining No. 1 People's Hospital (Jining, China; n=15). A single peripheral blood sample was collected from each patient or control 2 h before delivery. According to the blood pressure level and clinical classification standard in HDCP patients (16), the 46 patients are classified into grades as follows: 28 patients in grade I, 13 in grade II and 5 in grade III. The average age of patients in the grade III group was 28.6 years and the median age was 28 years (23.5–35 years). The average age of patients in the normal control group was 27.5 years old, and the median was 26 years old. The details of patients are presented in Table I. Prior written and informed consent were obtained from every patient and the study was approved by the Ethics Review Board of Laiwu City People's Hospital, Affiliated Hospital of Shandong University of Traditional Chinese Medicine and Jining No. 1 People's Hospital.

A total of 10 female Sprague Dawley rats aged 5–6 weeks, and weighing 150–200 g were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and were housed at 25°C with 30% humidity and a 12 h light/dark cycle, and with free access to water and food. After experiment, the animals were sacrificed by cervical dislocation. The cardiac apex was removed and washed with normal saline twice from 3-day-old newborn Sprague Dawley rats by cutting along the sternum midlines of the chest wall. The tissue was cut into sections ~1 mm³ using ophthalmic scissors, then washed twice in phosphate buffered saline (PBS) without calcium and magnesium ions. The tissue pieces were moved into 10 ml centrifuge tubes for digestion by trypsin and type II collagenase. The myocardial cells were collected by centrifugation at 500 × g for 5 min at room temperature. The collected cells were cultured for 2 days at 37°C in an atmosphere containing 5% CO₂ with High glucose-Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; BD Biosciences, Franklin Lakes, NJ, USA).

The supernatant was collected following centrifugation at 2,000 × g for 10 min at room temperature from a 5 ml anticoagulated peripheral blood sample from each patient. A total of 250 µl serum was mixed with 750 ml TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to extract total RNA based on the phenol-chloroform method (17). The miR cDNA was synthesized from total RNA by reverse transcription with an miRNA cDNA kit (Takara Biotechnology, Co., Ltd., Dalian, China). The reverse system of miRNA (miScript II RT kit; Qiagen, AB, Sollentuna, Sweden) included: 5 µl miRNA template, 10 µl 2X miRNA Reaction Buffer mix, 2 µl 0.1% bovine serum albumin, 2 µl miRNA PrimeScript RT Enzyme Mixture (Takara Biotechnology, Co., Ltd.,) and 1 µl H₂O. The reaction was completed at 37°C for 60 min with PolyA primer, then the reaction solution was added to RT RNAase Free H₂O to a total volume of 50 µl. Quantitative detection was completed according to the manufacturer's protocol as previously described (18) (miScript SYBR-Green PCR kit; Qiagen AB). The system included the following: 10 µl qRT-PCR-Mix, 0.5 µl forward primers and 0.5 µl reverse primers, 5 µl cDNA and 4 µl ddH₂O.

The miScript SYBR-Green PCR kit (Qiagen AB) was used to detect the expression of miR-181b in peripheral blood samples. U6 was used as internal reference. The primers were as follows: miR-181b forward, 5′-AACATTCATTGCTGTCGGTGGGT-3′ (reverse primer was provided in the kit without sequence information); U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; and Uni-miR primers were provided in the kit without sequence information. The miRNA reaction system included 10 µl RT-qPCR-Mix, 0.5 µl forward primer and 0.5 µl reverse primer, 5 µl cDNA and 13 µl ddH₂O, to make a total of 30 µl. PCR was performed using a thermocycler (StepOne Plus; Thermo Fisher Scientific, Inc.) as follows: 95°C for 10 min followed by 40 cycles of 95°C for 30 sec and 60°C for 30 sec. Each sample had 3 replicates and the average was used.

The primary myocardial cells were cultured in an environment of 1% O₂, 5% CO₂ and 94% N₂. Following 2, 4, 8, 12 and 24 h, cells were collected to extract total RNA according to the phenol-chloroform extraction protocol. Following reverse transcription, the expression of miR-181b and TIMP3 was detected by RT-qPCR. The primers of TIMP3 were as follows: Forward 5′-GAAGAGAGTACCGGCATCGG-3′ and reverse 5′-CCATTCTCCCCCTGCCAAAT-3′. PCR was performed ysing the StepOne Plus thermocycler as follows: 95°C for 10 min and 40 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C 30 sec, followed by 1 min at 72°C for extension. The step for detecting miR-181b was completed as previously described.

The primary myocardial cells were divided into 4 groups based on transfection: Negative control, miR-181b mimic, miR-181b inhibitor, and TIMP3-short interfering RNA (siRNA). The cells were cultured in antibiotic-free H-DMEM medium with 10% FBS at 37°C and transfected at 70% confluence. A total of 25 pmol/µl negative controls and small interfering RNA and 1.25 or 1 µl Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was added into Eppendorf tubes containing 50 µl Opti Memi medium (Thermo Fisher Scientific, Inc.), respectively. The two tubes were mixed following 5 min of standing, followed by another 20 min standing at room temperature. The mixture was added to each well when the cell density reached 70–90%. The H-DMEM medium with 10% FBS was replaced following 6 h of transfection. Following 48 h of transfection, at which point the interfering effect was evident, TIMP3 protein expression was detected by western blot analysis.

Following 48 h transfection, the primary myocardial cells were washed twice using pre-cooled PBS, then lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) with 1% phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology) on ice for 5 min. Mixed with 5X SDS loading buffer and incubated at 100°C for 10 min, 20 µg of protein was loaded into SDS-PAGE at 100 V constant voltage. The proteins were transferred to a polyvinylidene fluoride membrane at 300 mA constant current for 1 h. Following blocking for 1 h using 5% skim milk at room temperature, the primary antibodies were added to incubate at 4°C overnight. The primary antibodies used were mouse anti-mouse GAPDH antibody (ab8245; 1:2,000) and rabbit anti-mouse TIMP3 (ab39184; 1:800). The secondary antibody was horseradish peroxidase-conjugated (HRP) goat anti-rabbit immunoglobulin G (ab6721; 1:10,000) and HRP goat anti-mouse immunoglobulin G (ab6789; 1:10,000). All antibodies were purchased from Abcam (Cambridge, MA, USA). The membrane was developed and exposed by ECL of the BeyoECL plus kit (Beyotime Institute of Biotechnology). Quantity One software version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to carry out the quantitative analysis. The experiment was performed in triplicate.

To investigate the role of miR-181b in myocardial injury, the present study analyzed the apoptosis rate of the negative control group, miR-181b mimic group and TIMP3 siRNA group, all of which were cultured in hypoxia for 24 h. Annexin V-FITC staining was performed according to the protocol of the FITC Annexin V Apoptosis Detection kit I (BD Biosciences). Briefly, cells were digested and rinsed with PBS. 1×10⁶ cells were seeded and incubated for 15 min at room temperature with 5 ul PI and FITC coupling Annexin V, and BD FACSVerse flow cytometry (BD Biosciences) was subsequently used to detect the apoptosis rate of cells in each group. Positive Annexin v indicates the apoptosis cells are in early stage, while propidium iodide (PI) positive means the cells are necrotic. Annexin V and PI double positive indicates that cells are in late stage apoptosis. The analysis was performed using the BD FACSuite Software v1.0.3 provided with the flow cytometer.

Based on the bioinformatics prediction, the mutant 3′ untranslated region (UTR) of TIMP3 and the wild-type 3′UTR were synthesized in vitro (Hanbio Biotechnology Co., Ltd., Shanghai, China) and cloned into the downstream region of the pMIR-REPORT luciferase vector (Hanbio Biotechnology Co., Ltd.) using HindIII and Spe1 restriction enzymes. HEK293T cells were co-transfected with miR-181b mimic and wild type TIMP3 3′UTR or the mutant 3′UTR. Following 24 h transfection, cells were lysed and luciferase intensity was measured using the GloMax^® 20/20 luminometer (Promega Corporation, Madison, WI, USA). The intensity of Renilla was used as control, and the protocol of the Dual Luciferase Reporter Gene Assay kit (Beyotime Institute of Biotechnology) was followed.

SPSS 17.0 (SPSS Inc., Chicago, IL, USA) software was applied for statistical analysis. All data is presented as the mean ± standard deviation and differences were determined by two-tailed Student's t-test. P<0.05 was considered to represent statistically significant differences.

To investigate the roles of miR-181b in HDCP, RT-qPCR was used to detect miR-181b expression in peripheral blood samples obtained from HDCP patients and the control. As Fig. 1A demonstrates, miR-181b expression was significantly lower in peripheral blood of patients with HDCP compared with the control puerpera (P<0.05). miR-181b expression was increased in patients in stage II/III hypertension compared with those in stage I (Fig. 1B). For detecting the correlation of miR-181b expression with TIMP3 expression, another 15 patients with HDCP and 15 healthy individuals were selected from the aforementioned patients to estimate the expression in blood. As Fig. 1C demonstrates, TIMP3 was significantly increased in patients with HDCP, which was negatively associated with the expression of miR-181b.

To assess the participation of miR-181b in the processes of hypoxia-injured myocardial cells, RT-qPCR was used to detect miR-181b expression and TIMP3 expression at different time points for hypoxia culturing. It was demonstrated that miR-181b expression was significantly decreased along with the duration of hypoxia in culturing cells (P<0.05). The expression of TIMP3 was significantly increased with the hypoxia time extension (P<0.05). miR-181b expression was negatively associated with TIMP3 expression, as presented in Fig. 2.

To investigate whether miR-181b has an influence on the expression of TIMP3 in myocardial cells, western blot analysis was completed, in order to detect the expression of TIMP3 protein in different groups of cells. The proteins were isolated in different groups of cells transfected with miR-181b mimics, miR-181b inhibitor and TIMP3 siRNA. It was indicated that TIMP3 protein expression was downregulated when miR-181b was upregulated (miR-181b mimic group), however the expression of TIMP3 increased when miR-181b was significantly downregulated (miR-181b inhibitor group; P<0.05; Fig. 3A). The expression of TIMP3 significantly decreased in the TIMP3 siRNA group (P<0.05; Fig. 3B).

To investigate the influence on apoptosis by miR-181b and TIMP3, flow cytome-try was applied to detect the rate of apoptotic cells using Annexin V and PI double staining. As Fig. 4 demonstrates, following culture in a hypoxic environment for 24 h, the apoptosis rate of myocardial cells in the miR-181b mimic group was significantly lower than in the negative control group (P<0.05). The rate of apoptosis also significantly decreased in the TIMP3 siRNA group compared with the negative control group (P<0.05). Combined with the bioinformatics prediction, it was indicated that miR-181b may inhibit the apoptosis of myocardial cells through regulating TIMP3 expression.

To determine whether TIMP3 was directly regulated by miR-181b, the luciferase intensity was detected using a dual luciferase assay. As presented in Fig. 5A, the binding sites of miR-181b seed region on the TIMP3 3′UTR were mutated in the in vitro synthesized sequence. The present study identified that the luciferase intensity was significantly decreased when co-transfected with miR-181b mimics and TIMP3 3′UTR luciferase vector compared with the negative control (P<0.05), while no significant difference between the negative control and the mutant TIMP3 3′UTR was observed (P>0.05; Fig. 5B). The results of the current study indicate that miR-181b may directly bind to the ‘seed region’ in 3′UTR of TIMP3 mRNA to regulate TIMP3 expression.