A total of 443 patients (male, 134; female, 309; age range 17–81 years; male/female, 0.434) with PISs treated in the Southwest Hospital, Third Military Medical University (Chongqing, China) from December 2012 to May 2016 were included in the present study. Furthermore, 560 healthy individuals (male 245; female 315; age range 16–78 years; male/female, 0.778) were selected from the physical examination center of the same hospital as the control group. The inclusion criteria of the experimental group were confirmed diagnosis of PISs by imaging study; no prior liver resection, no history of chronic liver disease, no hepatitis A, B, or C virus or cytomegalovirus infection, no gallstones, and no tumors. The inclusion criteria of the control group were confirmed absence of PISs by imaging, no history of chronic liver disease, no hepatitis A, B, or C virus or cytomegalovirus infection, and no gallstones. Five milliliters of peripheral blood were collected from each of the above PIS patients and healthy participants, and the lymphocytes were immediately separated using human peripheral lymphocyte separation medium (Tianjin Hao Yang Biological Manufacturing Co., Ltd., Tianjin, China; http://www.tbdscience.com). The obtained samples were stored in a −80°C freezer.

Clinical data, including information on sex, age, height, body weight, indicators in the preoperative liver function test, preoperative serum jaundice, recurrent cholangitis, and stone type were collected (Tables I and II). Stone removal of the PIS patients was confirmed by abdominal ultrasound, abdominal CT, cholangiography and magnetic resonance imaging. Postoperatively, the patients were followed up every three months, and a stone found at follow-up was considered as a recurrence. The experiments were approved by the Ethics Committee of the Southwest Hospital of the Third Military Medical University of China. Written informed consent was obtained from all participants.

The Blood Genome DNA Extraction kit (Takara Bio, Inc., Otsu, Japan) was used to extract genomic DNA from the peripheral blood lymphocytes of patients and controls. In the 28 exon coding regions of the ABCB11 gene, the first exon does not participate in the coding of the protein, so Primer 6.0 primer design software (primerdesign.co.uk/home) was used to design 27 pairs of primers for the PCR amplification of the remaining exons (Table III). The PCR amplification was performed with the PrimeSTAR Max DNA Polymerase kit (Takara Bio, Inc.) and the cycling conditions were as follow: 30 cycles of 98°C for 10 sec, 55°C for 5 sec and 72°C for 30 sec. The amplified products were then sequenced using the ABI 3730XL Gene Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Total mRNA was extracted from the 200 mg freshly frozen liver tissue of the PIS patients and the control group using the Eastep Super Total RNA Extraction kit (Promega Corporation, Madison, WI, USA). PIS patient and control group liver tissue samples were cryopreserved in liquid nitrogen at the time of excision to facilitate RNA extraction and RT-qPCR analysis. The specific experimental procedure was conducted according to the manufacturer's protocol. DNA removal and reverse transcription were performed with the PrimeScript^™ RT Reagent kit with gDNA Eraser (Takara Bio, Inc.) at 42°C for 2 min. Finally, the mRNA expression of the ABCB11 gene in the PIS patients was detected by RT-qPCR using SYBR Premix Ex Taq II (Takara Bio, Inc.) and a quantitative fluorescence PCR analyzer.

Total RNA was extracted from 293 cells 48 h after transfection with wild-type and mutant A856V plasmids using the Eastep Super Total RNA Extraction kit (Promega Corporation), followed by DNA removal and reverse transcription using the PrimeScript^™ RT Reagent kit with gDNA Eraser (Takara Bio, Inc). The mRNA expression was detected by RT-qPCR using SYBR Premix Ex Taq II (Takara Bio, Inc.) and a quantitative fluorescence PCR analyzer.

The results of freshly frozen liver tissues and cells were normalized to the expression of β-actin. The primers for BSEP were forward primer, 5′-dTTGCCTTTGCCCAGTGCATCAT-3′ and reverse primer, 5′-dGGTTGTCGGTCCAGCAGTTGAA-3′ (BSEP), and those for β-actin were forward primer, 5′-dCCTGGCACCCAGCACAAT-3′ and reverse primer, 5′-dGGGCCGGACTCGTCATAC-3′. For the quantitative measurement of ABCB11 mRNA expression, RT-qPCR was performed using a CFX96 Real-Time system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with the following cycling conditions: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec (24).

Membrane proteins were extracted from the liver tissues of PIS patients and 293 cells transfected with wild-type and mutant A856V ABCB11, using the Mem-PER^™ Plus Membrane Protein Extraction kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The Bicinchoninic Protein Quantification kit (CWBiotech, Beijing, China) was used for protein concentration determination. Equal amounts of protein (20 µg) were separated by 6% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA), which were blocked and incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study included the following: Anti-BSEP (1:500; cat. no. 155421; Abcam, Cambridge, UK) and anti-Na/K-ATPase (1:1,000; cat. no. 58475; Abcam). The membranes were washed with Tris-buffered saline/Tween-20 and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature. The SuperSignal West Femto Substrate Trial kit (Thermo Fisher Scientific, Inc.) was used for signal detection.

The wild-type plasmid pEZ-M02-BSEP contains all the open reading frames (GeneCopoeia Inc., Rockville, MD, USA) of the ABCB11 gene. This plasmid was used as a template to obtain the A856V mutation by site-directed mutagenesis (25). The ABCB11 coding sequences of the above plasmids were verified by sequencing.

A total of 293 cells and MDCK cells obtained from the Cell Bank of the Chinese Academy of Sciences were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) with 1% penicillin-streptomycin solution (Beyotime Institute of Biotechnology, Haimen, China) and 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO₂ humidified atmosphere. A total of 5×10⁵ 293 cells were seeded in 6-well plates and transiently transfected with 2,500 ng DNA and Lipofectamine^® (Invitrogen; Thermo Fisher Scientific, Inc.) per well after 24 h at 37°C humidified atmosphere containing 5% CO₂ when the cell confluence reached 80%. A total of 1×10⁵ MDCK cells were seeded in 24-well plates and transiently transfected with 500 ng of DNA and Lipofectamine^® (Invitrogen; Thermo Fisher Scientific, Inc.) per well after 24 h at 37°C humidified atmosphere containing 5% CO₂ when the cell confluence reached 70%. The transfected cells were incubated at 37°C humidified atmosphere containing 5% CO₂, and were harvested 48 h later for subsequent experiments.

The MDCK cells were seeded on slides 24 hour at room temperature prior to transfection. Forty-eight hours after transfection, the cells were fixed with 2% paraformaldehyde for 10 min at room temperature and infiltrated with 1% Triton X-100 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 5 min at room temperature, followed by blocking with 10% goat serum (Gibco; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Following rinsing with PBS, one group of 1×10⁵ cells was incubated with rabbit anti-BSEP polyclonal antibody (1:100; cat. no. 155421; Abcam) and chicken anti-Na/K-ATPase monoclonal antibody (1:200; cat. no. 353; Abcam) overnight at 4°C, then washed with PBS and incubated with goat anti-rabbit Alexa Fluor 488 (1:200; cat. no. 150077; Abcam) and goat anti-chicken Alexa Fluor 647 (1:200; cat. no. 150171; Abcam) at room temperature for 2 h. Another group of 1×10⁵ cells was incubated with rabbit anti-BSEP polyclonal antibody (1:100; cat. no. 155421) and mouse anti-calnexin polyclonal antibody (1:200; cat. no. 112995) overnight at 4°C, then washed with PBS and incubated with goat anti-rabbit Alexa Fluor 488 (1:200; cat. no. 150077) and goat anti-mouse Alexa Fluor 647 (1:200; cat. no. 150115; all Abcam) at room temperature for 2 h. Images of the cells were obtained using a Leica CTR 4000 laser scanning confocal microscope.

The genotype frequencies at all mutation loci were directly obtained by counting and the use of the Hardy-Weinberg equilibrium. The unconditional logistic regression model was used to evaluate the correlation between the ABCB11 gene mutations and PISs. The evaluation indicators included odds ratio (OR) and 95% confidence interval (CI); the odds ratio (OR) was corrected for sex, age and body mass index. The correlation between the genotype and the clinical data of the PIS patients was analyzed using the Chi-square test. All experimental data were analyzed by SPSS software (PASW Statistics 22, IBM Corp., Armonk, NY, USA) or Graphpad Prism (v6.0 for Windows; GraphPad Software, Inc., La Jolla, CA, USA). Data are presented as the mean ± standard deviation of four independent experiments. Statistical analyses shown in the figures were performed using paired Student's t-tests or one-way analysis of variance with least significant difference post hoc test. P<0.05 was considered to indicate a statistically significant result.

In the present study, peripheral blood lymphocytes were collected from 443 PIS patients and 560 healthy individuals for ABCB11 exon sequencing. Six mutations were detected, including three missense mutations and three synonymous mutations (Table IV). The missense mutation rs118109635 and the synonymous mutation rs497692 were detected within exons 21 and 24, of the ABCB11 gene, respectively. The gene frequency distributions of the two mutational loci in the PIS patients and the healthy control group differed significantly (P=0.025 and P=0.017, respectively). In addition, the missense mutations rs2287617 and rs2287622 and the synonymous mutations rs3815675 and rs2287616 were located in exons 9, 13, 4 and 9 of the ABCB11 gene, respectively. However, these four mutations showed no significant differences between PIS patients and healthy controls (P>0.05). These results indicated that there are two significant ABCB11 gene mutations in the PIS patients (rs118109635 and rs497692), consistent with previous findings (5).

The analysis of the ABCB11 gene mutations and the relevant clinical data for the PIS patients in the present study (Table II) showed that the genotype of the rs118109635 mutation (CT genotype) of the ABCB11 gene was correlated with preoperative jaundice (P=0.026) and that the homozygous genotype (GG) and the heterozygous genotype (GA) caused by mutations at the rs497692 locus in the PIS patients in the present study were associated with preoperative jaundice (P=0.011). In addition, the mutation at the rs2287617 locus of the ABCB11 gene was associated with the recurrence of PISs (P=0.009). Therefore, the rs118109635 and rs497692 mutations of the ABCB11 gene are closely associated with the clinical characteristics of PISs.

The expression of ABCB11 mRNA in the liver tissues of PIS patients with different genotypes was measured by RT-qPCR. The results showed that whereas the mRNA expression levels of the mutant rs497692 were decreased, the rs118109635 mutation had no significant impact on ABCB11 mRNA expression levels (Fig. 1C). The expression of BSEP in the liver tissue of PIS patients with different genotypes was detected by western blot analysis; BSEP was detected as a 140–150 kDa band (Fig. 1A). The results revealed that the expression of BSEP was significantly decreased in patients with the ABCB11 gene mutations rs118109635 and rs497692 (Fig. 1A and B). Thus, the ABCB11 gene mutations (rs118109635 and rs497692) may affect the transcription and translation of the ABCB11 gene.

The aim of this experiment was to investigate the effect of ABCB11 gene mutation on BSEP expression and its subcellular localization. Therefore, 293 and MDCK cell lines were used, albeit neither express BSEP protein, as experimental cells, and investigated the effect of ABCB11 gene mutations on protein expression and subcellular localization by transfection of ABCB11 mutant plasmid and wild-type plasmid. Due to the high expression of BSEP protein in liver cell lines, it may interfere with the observation of cell transfection experiment results. The 293 and MDCK cells are polarized cells commonly used to study trafficking of membrane proteins (17,23). The transfection efficiency in MDCK cells was quite low (25). Thus, to determine the effects of ABCB11 mutations on BSEP expression, 293 cells were used. Plasmids containing wild-type and mutated BSEP cDNA were constructed and transfected into 293 cells (25) to determine the effects of the A865V mutation on BSEP expression. The expression of BSEP in the A865V mutant measured by western blot analysis was significantly lower than that in wild-type 293 cells (Fig. 2A and B). Although the A865V mutation had no apparent effect on BSEP mRNA expression levels (Fig. 2C), the decrease in BSEP protein expression levels may be associated with the degradation of BSEP due to its retention in the endoplasmic reticulum as a result of a change in its three-dimensional structure (17). Thus, the above results indicated that the A865V mutation downregulated the translocation of the BSEP protein.

The cells used in this experiment needed to undergo multiple antibody incubation and PBS washing steps. The 293 cells have poor adhesion and can easily be detached from the slide during multiple incubation and washing (25). Thus, the impact of ABCB11 mutations against BSEP subcellular localization was assessed in MDCK cells. MDCK cells were transiently transfected with wild-type and mutant plasmids, and the expressed proteins were labeled and stained with antibodies to BSEP, calnexin and Na/K-ATPase to determine the subcellular location of BSEP by immunofluorescence confocal microscopy. Calnexin and Na/K-ATPase were used as markers for the endoplasmic reticulum and the cell membrane, respectively. Results demonstrated that wild-type BSEP was distributed inside the cells and in the cell membrane, whereas the A856V mutant BSEP was mainly distributed inside the cells and showed significantly reduced distribution in the cell membrane (Fig. 3). Thus, the A856V mutation affected the subcellular localization of BSEP.