A total of 80 samples from patients with pathologically confirmed GBC were collected from Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University (Changsha, China) between 2008 and 2012 following cholecystectomy. None of the patients that participated in the present study were treated with radiotherapy, chemotherapy or other treatments prior to cholecystectomy. The patients included 30 males and 50 females, with an average age of 48.2±2.6 years. The pathological stage of GBC was classified according to 6th edition of the tumor-node-metastasis (TNM) classification of the American Joint Committee on Cancer (15). Poorly differentiated tumor tissues exhibit poor maturity and high degree of malignancy, while well-differentiated tumor tissues are similar in appearance to normal cells and exhibit a low degree of malignancy. The histological grade was evaluated by hematoxylin and eosin staining. Tumor invasion and lymph metastasis were assessed according to the standard criteria proposed by the Chinese Research Society for GBC (16). The clinicopathological features of all patients are presented in Table I. The overall survival time of each patient was measured from the date of initial surgical operation to mortality. The follow-up time ranged from 1 to 60 months.

This medicinal study was approved by the Research Ethics Committee of Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, and written informed consent was also obtained from all patients or their families. All patient specimens were made anonymous and handled according to the legal and ethical standards for protecting human rights.

Total RNA from 80 samples of fresh GBC tissues and adjacent normal gallbladder tissues or GBC cell lines were extracted using a miRNeasy mini kit (Qiagen, Inc., Valencia, CA, USA). Subsequently, for hsa-miR-372, the cDNA was synthesized by using One Step Prime script miRNA cDNA Synthesis kit (Qiagen, Inc.) according to the manufacturer's protocol. qPCR was performed on the Roche Light Cycler 480 II real-time PCR System using a SYBR Premix Ex Taq II kit (Takara Bio, Inc., Otsu, Japan). The qPCR conditions were: 95°C for 5 min then 40 cycles of denaturation at 95°C for 10 sec and for the annealing/elongation step, 60°C for 30 sec. Hsa-miR-372 was amplified by using forward primers with the DNA sequence of the mature miRNA (5′-ACTATTCTGATGTCCAAGTGGA-3′) and common reverse primers provided in the First Strand cDNA Synthesis kit. U6 small nuclear RNA was used as an internal control with the following primers: forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-ACGCTTCACGAATTTGCGT-3′. For CLIC1, the first-strand cDNA was synthesized by using a RevertAid H Minus First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. qPCR was also performed on the Roche Light Cycler 480 II real-time PCR System using a SYBR Premix Ex Taq II kit. The qPCR conditions were 98°C for 10 min and 40 cycles of denaturation at 98°C for 10 sec and for the annealing/elongation step, 65°C for 30 sec. GAPDH was used as an internal control. The primer sequences of CLIC1 and GAPDH were as follows: CLIC1 forward, 5′-TCTGAACCCTGAGTCCAAC-3′ and reverse, 5′-GAGTCCCTTCTCCAGATTGT-3′; and GAPDH forward, 5′-GAAGGTGAAGGTCGGAG-3′ and reverse, 5′-GAAGATGGTGATGGGAT-3′. All reactions were run in triplicate, the relative expression levels of hsa-miR-372 and CLIC1 were normalized to the expression of U6 and GAPDH, respectively using the 2^−ΔΔCq method (17).

Target genes of hsa-miR-372 were predicted by using TargetScanHuman (version 7.1; www.targetscan.org) and miRanda (2010 version; www.microrna.org) algorithms.

To generate a reporter plasmid for the dual-luciferase reporter assay, the full length 3′-untranslated region (UTR) of the CLIC1 gene was synthesized by PCR. The following primers were used: 5′-GCCCCTCCTGGGACTCCCTCAACCC-3′ (forward) and 5′-TTGCGTAAAAACACTTGATTTTTAT-3′ (reverse). Subsequently, PCR products were cloned into a psiCHECK-2 vector (Promega Corporation, Madison, WI, USA) to produce psiCHECK-2-CLIC1 wild type luciferase reporter plasmid. The correct clones were confirmed by Sanger DNA sequencing. The mutant CLIC1 3′-UTR sequence plasmid was synthesized by GeneCopoeia (GeneCopoeia, Inc., Rockville, MD, USA) and also inserted into the psiCHECK-2 vector to construct psiCHECK-2-CLIC1 mutant (MUT) luciferase reporter plasmid.

G-415, OCUG-1 and SGC-996 human GBC cell lines were purchased from the Japan Health Science Research Resources Bank (Osaka, Japan). All cells were cultured in Dulbecco's modified Eagle's medium/F12 (Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) in a humidified atmosphere with 5% CO₂ at 37°C.

GBC cells were seeded onto 24-well plates (4×10⁵/well) and cotransfected with 100 nM hsa-miR-372 mimics (5′-AAAGUGCUGCGACAUUUGAGCGU-3′) or corresponding mimics negative control (5′-CAGCUCAAGUGUCGCCAUGUTCU-3′) and WT or MUT luciferase reporter plasmids (100 ng) by using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After transfection for 24 h, the luciferase activities were analyzed using a dual luciferase reporter gene assay kit (Promega Corporation) in a TD-20/20 luminometer (Turner Designs, San Jose, CA, USA). Renilla luciferase activity was normalized to firefly luciferase activity.

Total protein from G-415, OCUG-1 and SGC-996 cells was extracted by radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.) at 65°C and quantified using a bicinchoninic assay kit (Thermo Fisher Scientific, Inc.). Subsequently, the quantified proteins (50 µg) were denatured and subjected to 15% SDS-PAGE, and blotted onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were then blocked with 5% non-fat milk for 2 h at 37°C. Following washing with TBS-Tween-20 (0.1%) three times, the membranes were incubated with mouse anti-human CLIC1 (1:1,000; cat. no. ab77214; Abcam, Cambridge, MA, USA) and anti-GAPDH (1:2,000; cat. no. ab8245; Abcam) antibodies overnight at 4°C. Then, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG (1:2,000; cat. no. 7076; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at 37°C and visualized using an enhanced chemiluminescence western blot detection kit (GE Healthcare, Pittsburgh, PA, USA) according to the manufacturer's protocols.

Statistical analysis was performed using SPSS 19.0 (IBM Corp., Armonk, NY, USA). Each experiment was repeated at least three times. Data are presented as the mean ± standard deviation. A paired Student's t-test was performed to compare hsa-miR-372 expression in GBC tissues with adjacent normal gallbladder tissues. The χ² test was used to detect the association between hsa-miR-372 expression and clinicopathological features. The expression of hsa-miR-372 between the three GBC cell lines was compared using one-way analysis of variance and Dunnett's post-hoc test. Overall survival curves were evaluated by the Kaplan-Meier method and logrank test. Univariate Cox regression analysis was performed for prognostic parameters, and the importance of multiple parameters for survival was assessed by the Cox's proportional hazards model for multivariate analysis. P<0.05 was considered to indicate a statistically significant difference.

The expression of hsa-miR-372 was determined in 80 GBC tissue samples and adjacent normal gallbladder tissues by RT-qPCR. Following normalization to U6, the hsa-miR-372 expression in GBC tissues was significantly lower compared with adjacent normal gallbladder tissues (P=0.002; Fig. 1A). Additionally, hsa-miR-372 expression was also downregulated in GBC tissues with low differentiation compared with highly differentiated tissues (P=0.042; Fig. 1B).

RT-qPCR results indicated that hsa-miR-372 expression was significantly downregulated in patients with GBC. In order to further determine the potential associations between hsa-miR-372 expression and the clinicopathological features, 80 samples of GBC tissues were divided into low and high expression groups based on the average expression of hsa-miR-372 in GBC tissues. Patients with GBC that exhibited expression of hsa-miR-372 at levels lower than the average expression level (2.41) were assigned to the low expression group (n=55), and those samples with expression equal to or above the average value were assigned to the high expression group (n=25).

As summarized in Table I, low hsa-miR-372 expression was significantly associated with the presence of GBC histological grade (P<0.001), TNM stage (P<0.001), lymph node metastasis (P=0.014) and distant metastasis (P<0.001). However, there were no significant differences in the gender of patients with GBC (P=0.071), patient age (P=0.105), tumor size (P=0.191) or the presence of gallbladder stones (P=0.228) between the high and low hsa-miR-372 expression groups. Collectively, these results indicate that decreased expression of hsa-miR-372 may be relevant to tumor differentiation and metastasis, which are involved in tumorigenesis and the progression of GBC.

The association between hsa-miR-372 expression and the overall survival of GBC patients was evaluated by Kaplan-Meier analysis. As demonstrated in Fig. 2, low hsa-miR-372 expression was associated with a shorter overall survival time compared with patients with high hsa-miR-372 expression (P=0.002). Furthermore, multivariate Cox regression analyses indicated that hsa-miR-372 expression (P=0.004), histological grade (P=0.024) and lymph node metastasis (P=0.001) maintained an independent prognostic influence on the overall survival of patients with GBC (Table II).

The endogenous expression level of hsa-miR-372 in OCUG-1 and SGC-996 cells were relatively higher compared with G-415 cell, as determined by RT-qPCR (Fig. 3A). The expression levels of hsa-miR-372 in G-415, OCUG-1 and SGC-996 cells transfected with hsa-miR-372 mimics were significantly upregulated by 22.33-, 16.38- and 28.45-fold, respectively, when compared with levels in the mimics negative control transfection group (Fig. 3B), indicating that the transfection efficiency is suitable for further cell experiments.

As miRNAs primarily function by inhibiting their target genes, the targets of hsa-miR-372 in GBC were predicted by using bioinformatics analysis. Previous research demonstrated that CLIC1 expression was closely associated with GBC progression and may be regarded as an effective biomarker for predicting the prognosis of GBC (18,19). Complementary sequences were identified between hsa-miR-372 and the 3′UTR of CLIC1 (Fig. 4A), which indicated CLIC1 may be a target gene of hsa-miR-372. To further investigate whether hsa-miR-372 inhibits CLIC1 expression in GBC cells, G-415, OCUG-1 and SGC-996 cells were treated with hsa-miR-372 mimics or mimics negative control. RT-qPCR and western blot analysis demonstrated that transfection with hsa-miR-372 mimics markedly decreased the expression of CLIC1 mRNA and protein in all three GBC cell lines (Fig. 4B). In addition, a dual-luciferase reporter assay was performed to confirm the direct interaction between hsa-miR-372 and the 3′UTR of CLIC1. Notably, the results demonstrated that the relative luciferase activity of the wild-type CLIC1 3′UTR plasmid decreased in response to treatment with hsa-miR-372 mimics compared with negative control mimics (Fig. 4C). By contrast, hsa-miR-372 mimics exhibited no inhibitory effect on luciferase activity when transfected with the mutant CLIC1 3′UTR plasmid (Fig. 4C).