MiRNA-145 induces apoptosis in a gallbladder carcinoma cell line by targeting DFF45

2.1 Cell culture and gallbladder carcinoma tissue

Normal gallbladder epithelial cells were extracted from healthy gallbladder tissue isolated from gallbladder polyps obtained from cholecystectomy patients. The isolated tissue was pathologically extracted from polyp sections that had no obvious inflammation.

A GBC-SD human gallbladder low-differentiation carcinoma cell line was purchased from Shanghai Institutes for Biological Sciences, CAS. The GBC-SD cell line and normal gallbladder tissue were cultured at 37°C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% glutamine. All fresh tissue samples used in this study were isolated at the Second Xiangya Hospital, the Third Xiangya Hospital and the Hunan People’s Hospital from specimens surgically resected between January 2011 and June 2013, with tissue sizes approximating 0.8 cm × 0.8 cm × 0.6 cm. Among the 15 gallbladder carcinoma tissue specimens, six were extracted from male patients and nine were extracted from female patients. All patients were between 40 and 67 years-of-age (mean 54.3 years). Prior to surgery, no gallbladder carcinoma patient underwent radiotherapy or chemotherapy, and each patient had complete medical records. After surgery, all patients were confirmed as having gallbladder carcinoma after pathological diagnoses. Controls were composed of the same 15 patients, with control tissues being derived from adjacent tissues that were ≥ 3 cm in distance from the tumor and showing no cancerous invasion on pathological diagnosis. Less than 0.5 h after isolation, tissue specimens were stored in Cryo Tubes with liquid nitrogen freezing until experiments.

2.2 Quantitative real-time RT-PCR

Total RNA was isolated using Trizol (Invitrogen, city, state). RNA extracts were subsequently reverse-transcribed into cDNA using a PrimeScript RT Master Mix (Takara, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using the SYBR Premix Ex TaqTM kit (Takara, Japan). The specific primer sequences used for quantitative real-time PCR were as follows: hsa-miRNA-145-F:

5’-AGTCGCTAGCCCACCCTGGCTGCTACAGATG-3’, and hsa-miRNA-145-R:

5’-ATCGGAATTCGGACAGCCTTCTTCTTGAACC-3‘. Primers were synthesized by Shanghai RiboBio Co. Ltd. (China). Expression of miRNA-145, relative to U6, was measured using the 2^–ΔΔCt method. Results were analyzed according to the manufacturer’s instructions. Experiments were performed independently, in triplicate, and data from a representative experiment are shown.

2.3 miRNA microarray and data analysis

Global miRNA expression data were obtained using Affymetrix miRNA 1.0 arrays (Affymetrix). Total RNA samples were labeled using an Affymetrix FlashTag Biotin HSR RNA Labeling Kit according to the manufacturer’s protocol. Briefly, 500 ng of each total RNA sample was poly(A)-tailed at 37°C for 15 min using poly (A) polymerase enzyme and ATP. The resultant mixture was biotinylated by ligating biotin-labeled fragments to the 3′-ends of RNA molecules. Labeled samples were hybridized on miRNA 1.0 arrays at 48°C and 60 rpm for 16 h. Arrays were subsequently washed and stained using the standard Affymetrix protocol with an Affymetrix Hybridization, Wash, and Stain Kit using a FS 450 fluidic station instrument. Arrays were scanned using an Affymetrix GeneChip Scanner 3000 instrument. An Affymetrix GeneChip Command Console 1.1 software package was used for data analysis.

2.4 Construction of miR-hsa-miRNA-145 expression vector, strains and plasmids

E. coli Top10 (Invitrogen) cells and plasmid pLL3.7 (Addgene) were used to construct eukaryotic miRNA expression vectors. First, DNA was extracted from miRNA-145 expressing cells and vector-transformed E. coli Top10 cells. The NCBI database was used to design primers that facilitated amplification of the full-length miRNA-145 molecule. PCR amplification of the template was subsequently performed and the amplicons were digested with restriction endonucleases compatible with the vector multi-cloning site. The vector was digested with compatible restriction enzymes, and both insert and plasmid were separated using 1% agarose gel electrophoresis. Gel extraction procedures were subsequently used to purify the vector and associated insert. The hsa-miRNA-145 fragments were ligated to the plasmid backbone and the ligated products were used to transform competent E. coli cells. Resultant transformants were screened for the presence of the hsa-miRNA-145 inserts.

2.5 Transient transfection of expression plasmid, stable transfection of cells, and clone selection

Cells were subcultured in a 6-well plate and grown until they reached 60–80% confluence and then culture media was changed to resistance-free 10% FBS DMEM complete medium. One to two hours after the media was changed, Lipofectamine 2000 (Invitrogen) was used to facilitate transfection. A total of 1 μl of the expression vector (final concentration of 2 μg/μl), 100 μl of serum-free medium and 5 μl of the transfection reagent, lip2000, were incubated at room temperature for 5 min. This reaction mixture was thoroughly mixed prior to the 5 min incubation step. Opti-MEM (Invitrogen) medium containing Lipofectamine 2000 was added, drop-wise, to an Eppendorf tube containing the requisite plasmids, and the reaction was allowed to proceed for 30 min. Finally, the reaction mixture was added to cultured cells (drop-wise) and shaken to re-suspend the associated suspension. The media was changed every 5 h.

For GBC-SD cells transfected after 48 h, complete medium containing 0.5 μg/ml G418 was used to screen for transfected clones. Media was changed every other day. Resultant cell cultures were monitored for two to three weeks, and clones visible to the naked eye were grown. Clones were then isolated to generate amplification cultures.

2.6 Apoptotic

Cells were harvested and rinsed twice with PBS and samples were diluted with 150 μl of 1 × annexin-binding buffer. Next, 5 μl of FITC-labeled enhanced-annexin V and 5 μl (20 μg/ml) of propidium iodide were added into the cell suspension. Cells were subsequently incubated in the dark for 15 min at room temperature. Flow cytometry was conducted using a FAC-SCalibur instrument (FACSVerse). Data were analyzed according to the manufacturer’s instructions. We repeated the experiment three times to confirm results.

2.7 Western blot

Total protein was extracted using RIPA buffer and a protease inhibitor cocktail (Pierce, Rockford, IL). Extracted protein was quantified using a BCA protein Assay Kit (Pierce) and separated by 9% SDS-PAGE. Gels were subsequently transferred onto PVDF membranes (Millipore, Bedford MA). Next, membranes were incubated with rabbit mono-clonal anti-caspase-3 antibody (1:5,000; Epitomics, Burlingame, CA) or mouse mono-clonal anti-GAPDH antibody (1:5,000, Abcam, Cambridge, MA). Membranes were subsequently incubated with secondary antibody. Signals were measured using ECL.

2.8 Dual-Luciferase reporter assay

We plated the GBC-SD cells in 24-well plates the day before transfection. Wild-type or seed-region-mutated DFF-45 3′-untranslated region (UTR) sequences were inserted into pGL4 plasmids following double restriction enzyme digestion. Reporter plasmids were co-transfected with miRNA-145 or the control into GBC-SD cells using Lipofectamine 2000 (Invitrogen). Cells were harvested 48 h later, and relative luciferase activities were measured using a Dual-Luciferase Assay System (Promega, Madison, WI).

3.1 Expression of miRNA-145 was down-regulated in GBC-SD cells and gallbladder carcinoma tissue

Micro-array hybridization of RNA specimens and scanning analyses showed dot-shaped florescent signals and data indicate that in GBC-SD cells miRNA-145 was down-regulated compared to normal gallbladder epithelial cells. Figure 1 shows miRNA-145 expression in gallbladder carcinoma tissues was significantly decreased compared with adjacent gallbladder tissues. These data agree with GBC-SD cell chip assay results.

Figure 1

miRNA-145 expression in adjacent gallbladder tissues (A) and gallbladder carcinoma tissues (B).

3.2 Successful construction of hsa-miRNA-145 over-expression vector and hsa-miRNA-145 over-expression gallbladder carcinoma stable cell line

We constructed an hsa-miRNA-145 over-expressing vector and after comparing DNA sequencing results with the original sequence from the NCBI reference genome, we observed that the GV143-hsa-miRNA-145 sequence was identical to the original sequence and had no single base mutations.

We subsequently generated GBC-SD cell lines that stably over-expressed hsa-miRNA-145. After G418 resistance screening, we obtained GBC-SD cell lines containing stably transfected empty plasmids and GBC-SD cell lines stably over-expressing the hsa-miRNA-145 plasmid. Total RNA was extracted from transfected and untransfected cells lines prior to real-time PCR analysis (Fig. 2). Data show that hsa-miRNA-145 in cells stably over-expressing hsa-miRNA-145 was significantly elevated compared with the normal control group (untransfected GBC-SD cell group) and the empty plasmid-transfected group (p < 0.05). Compared with the empty plasmid group, the normal control group had no significant changes in hsa-miRNA-145 expression (p > 0.05).

Figure 2

GBC-SD cells stably transfected with hsa-miRNA-145 have more hsa-miRNA-145 expression. A: hsa-miRNA-145 expression in normal non-transfected GBC-SD cells; B: GBC-SD cells transfected with empty plasmid C: cells over-expressing hsa-miRNA-145 (ns: non-significant differences; ^*: p<0.05, significant difference).

3.3 GBC-SD cell lines stably transfected with hsa-miRNA-145 exhibited reduced growth

We studied three groups at different time points with an MTT assay, and plotted the GBC-SD growth curve (Fig. 3). Data show that on the fourth day of culture, GBC-SD cells stably transfected with hsa-miRNA-145 were inhibited 30% (p < 0.05), compared with the normal control group and empty plasmid group.

Figure 3

Effect of hsa-miRNA-145 on gallbladder carcinoma cell growth according to MTT assay. normal control : normal non-transfected GBC-SD cells; vect: GBC-SD cells transfected with empty plasmids; miR-145: GBC-SD cells transfected with hsa-miRNA-145 over-expression plasmids; p<0.05: significant difference.

3.4 Over-expression of hsa-miRNA-145 has no significant effect on the cell cycle

Flow cytometry data for GBC-SD cells transfected with hsa-miRNA-145 and normal control group cells appear in Fig 4. Cells over-expressing hsa-miRNA-145 in the S phase were slightly increased compared with the normal control group and the empty plasmid-transfected group; however, the difference was not significant (p > 0.05).

Figure 4

Cell cycle distribution for all phases of GBC-SD cells according to flow cytometry. A:normal non-transfected GBC-SD cells, B: GBC-SD cells transfected with hsa-miRNA-145 over-expression plasmids.

3.5 Hsa-miRNA-145 induces apoptosis of GBC-SD cells

Flow cytometry data showed that GBC-SD cells transfected with hsa-miRNA-145 were double-positive (Annexin V and PI) and more apoptotic than the normal control and this difference was significant (p < 0.05; Fig. 5).

Figure 5

Flow cytometry indicating apoptosis of GBC-SD cells (Annexin V-FITC). A:represents normal non-transfected GBC-SD cells; B: represents hsa-miRNA-145 transfection group.

We measured expression of caspase-3 with Western blot and (Fig. 6) show that relative expression of the caspase-3 protein in GBC-SD cells transfected with hsa-miRNA-145 was higher than in normal control and the empty plasmid transfection group. These differences were significant (p < 0.01) and show that caspase-3 expression was up-regulated after hsa-miRNA-145 over-expression.

Figure 6

Expression of caspase-3 protein in GBC-SD cells according to Western blot.

3.6 Hsa-miRNA-145 inhibits GBC-SD cell clone formation

Clone formation assays showed that clones formed from GBC-SD cells transfected with hsa-miRNA-145 were fewer than for controls (p<0.01; Fig. 7). However, compared with the empty plasmid group, GBC-SD cells not transfected were not different with respect to clones formed (p < 0.05).

Figure 7

Hsa-miRNA-145 inhibits GBC-SD clone formation. A: cells over-expressing hsa-miRNA-145; B: GBC-SD cells transfected with empty plasmids; C: normal non-transfected GBC-SD cells.

3.7 Over-expressed miRNA-145 down-regulates expression of DFF45 mRNA and protein

Sequence analysis showed that the DFF45 3’-UTR contains a putative binding site (854 bp–876 bp, Fig. 9A) for miR-145. After transfection of GC-SD cells with miRNA-145, we measured DFF45 mRNA and protein expression. Real-time PCR and Western blot revealed that compared with controls and transfected empty plasmids, over-expressed miRNA-145 cell lines had less DFF45 transcription (p < 0.05) and protein expression (p < 0.05; Fig. 8).

Figure 8

Western blot quantification of DFF45 protein expression in controls, transfected empty plasmid group and transfected miRNA-145 cell lines. Normal control: normal GBC-SDE cells not transfected; Vect: GBC-SD cells transfected with empty plasmids; MiRNA-145 GBC-SD cells transfected with over-expressed mlRNA-145 plasmids.

Figure 9

hsa-miRNA-145 targets DFF45-854 to regulate transcriptional activity. A, bioinformatics predicted target region and sequence of hsa-miRNA-145 in DFF45-854 and hsa-miRNA-145 target sequence mutants. B, hsa-miRNA-145 suppressed activity of normal DFF45-854; C, hsa-miRNA-145 had no suppressive effects related to activity of DFF45-854 mutants.

We next assayed relevant reporter plasmids and miRNA for fluorescent activity and observed that cells transfected with NC and DFF45-854, cells transfected with hsa-miRNA-145 and DFF45-854 had less fluorescent activity; the associated difference was significant (P = 0.005281; Fig. 9B). Compared with cells transfected with NC and DFF45-854 mutation, cells transfected with hsa-miRNA-145 and the DFF45-854 mutation had less fluorescent activity; however, the difference was not significant (P = 0.4784; Fig. 9C). With EZH2, a known target gene of miR-101 as a positive control, we observed that compared with cells transfected with NC and EZH2-CDS854, cells transfected with hsa-miR-101 and EZH2-CDS854 had less fluorescent activity. Thus, DFF45 is a direct target for miRNA-145 in gallbladder cancer cells.