Abstract
Objective
The objective of the study was to explore the inhibitive role of cyclin D1 gene silence in laryngeal squamous cell carcinoma by lentivirus-mediated RNA interference.
Materials and Methods
Cd1-RNAi-Lentivirus and the control lentivirus (GFP-Lentivirus) were transfected into Hep-2 cells. Reverse transcriptase polymerase chain reaction was performed to explore the cyclin D1 expression level in Cd1-RNAi-Lentivirus–transfected Hep 2 cells. The apoptosis and viability of Cd1-RNAi-Lentivirus–treated Hep-2 cells were measured with flow cytometry and methyl thiazolyl tetrazoliym assay. In an animal experiment, 10 mice bearing Hep-2 cell tumor were intratumorally injected with Cd1-RNAi-Lentivirus; and the other 10 mice were injected with GFP-Lentivirus. Terminal deoxytransferase–mediated dUTP nick end labeling stains and transmission electron microscope were used to observe the apoptosis in the xenografts.
Results
Cyclin D1 was knocked down after Cd1-RNAi-Lentivirus was transfected into Hep-2 cells. The proliferative ability of Hep-2 cells was significantly inhibited by Cd1-RNAi-Lentivirus, and a significant apoptosis of Hep-2 cells was also observed after Cd1-RNAi-Lentivirus transfection. The average weight and volume of tumors in the Cd1-RNAi-Lentivirus–treated group were significantly lower than those in the control group ( P < .01). The significant apoptosis was detected with terminal deoxytransferase–mediated dUTP nick end labeling stain and transmission electron microscope.
Conclusions
The present findings suggest that cyclin D1 gene silence by lentivirus-mediated RNA interference can inhibit growth and promote apoptosis of laryngeal squamous cell carcinoma.
1
Introduction
Laryngeal squamous cell carcinoma (LSCC) is frequently occurring in head and neck cancers, which comprise a diverse group of tumors arising from the epithelium of the upper respiratory tract, paranasal sinuses, and salivary and thyroid glands. In 2000, the global incidence of laryngeal cancer was 142 000 cases . Treatment methods of LSCC comprise radiotherapy, surgery, chemotherapy, or a combination of modalities. Despite refinement of multimodal therapies over the last 20 years, 5-year survival figures remain poor .
Cyclin-dependent kinases (CDKs) play key roles in cell cycle, and their activities are modulated by their binding to cyclins . Binding of cyclin D to CDK4 and CDK6 leads to the phosphorylation of the retinoblastoma protein. Phosphorylation of the retinoblastoma protein prevents it from repressing the E2F family of transcription factors and leads to the transcription of several genes required for the G1-to-S phase transition, thereby promoting cell proliferation . The human cyclin D1 gene was initially cloned as a breakpoint rearrangement within parathyroid adenoma . The cyclin D1 gene is amplified and overexpressed in human breast cancers, colon and prostate cancers, hematopoietic malignancies, and head and neck cancers . Because of its pivotal role in promoting cell cycle progression and, hence, cell division and proliferation, such overexpression might be expected to coincide with poor prognosis.
In this study, we used lentivirus to deliver a specially designed small interfering RNA (siRNA) for human cyclin D1 gene into the laryngeal cancer cell line Hep-2 to inhibit cyclin D1 protein expression. The results suggest that cyclin D1 gene silence can effectively suppress the growth and promote apoptosis of LSCC.
2
Methods
2.1
Lentivirus vectors for cyclin D1 siRNA
siRNA targeting human cyclin D1 was inserted into lentivirus gene transfer vector encoding green fluorescent protein (GFP), and the recombinant lentivirus was constructed by Genechem Co Ltd, Shanghai, China. The target sequence of the siRNA was 5′-GGAGAACAAACAGAT CATC-3′ (GenBank: NM_053056 ), which was verified by sequencing. The recombinant lentivirus of the SiRNA targeting cyclin D1 (Cd1-RNAi-Lentivirus) and the control lentivirus (GFP-Lentivirus) were prepared and titered to 10 9 transduction units per milliliter.
2.2
Cell line and lentivirus siRNA gene transfection
The Hep-2 cells of human LSCC were kindly provided by the Laboratory of Cell Pathology, Harbin Medical University. Cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (Gibco, Foster, Canada) and incubated in a humidified (37°C, 5% CO 2 :air) incubator. Hep-2 cells were plated in 24-well plates (2 × 10 4 cells per well) and cultured for 12 hours. Lentiviruses were diluted to 0.2 mL (10 8 transduction units per milliliter) with complete medium containing polybrene (8 mg/mL) and added to the cells for incubation for 1 hour at 37°C; 0.3 mL of fresh polybrene-DMEM was added to Hep-2 cells and incubated for 24 hours. After transfection, polybrene-DMEM was replaced with fresh DMEM medium; and the cells were cultured for another 48 hours.
2.3
Detection of cyclin D1 expression by reverse transcriptase polymerase chain reaction
After transfection with Cd1-RNAi-Lentivirus for 72 hours, the total RNA was extracted from the transfectants of each group using Trizol (Invitrogen, Carlsbad, Canada). For reverse transcriptase polymerase chain reaction (RT-PCR) analysis, 2 μ g of total RNA was used to synthesize complementary DNA using a first-strand complementary DNA synthesis kit (Promega, Madison, WI). Two pairs of primer sequences were designed and synthesized by Shanghai Biological Engineering Corporation (Shanghai, China) as follows: human cyclin D1 forward primer was 5′-CTGGAGCCCGTGAAAAAGAGC-3′, reverse primer was 5′-CTGGAGA GGAAGCGTGTGAGG-3′, and product size was 433 base pairs. Human β -actin primer forward was 5′-CCCAGCACAATGAAGATCAAGATCAT-3, reverse primer was 5′-ATCTGCTGGAA GGTGGACAGCGA-3′, and product size was 107 base pairs. The conditions for the RT-PCR were 94°C for 5 minutes, 58°C for 30 seconds, and 72°C for 1 minute, for 35 cycles. The products were then electrophoresed on a 1.5% agarose gel containing ethidium bromide. β -Actin was served as the positive control; and RT-negative tubes, as the negative controls.
2.4
MTT assay
Hep-2 cells were incubated in 96-well plates at a density of 105 cells per well with RPMI 1640 medium supplemented with 10% fetal bovine serum for 24 hours. After Cd1-RNAi-Lentivirus transfection for 24, 48, 72, and 96 hours, 20 μ L of sterile methyl thiazolyl tetrazoliym (MTT) dye (5 mg/mL; Sigma-Aldrich Corp, St Louis, MO) was added and incubated for another 4 hours at 37°C. Afterward, 150 μ L of dimethyl sulfoxide was added to each well and thoroughly mixed for 10 minutes. Cells transfected with GFP-Lentivirus were used as controls. Spectrometric absorbance at a wavelength of 492 nm was measured on an enzyme immunoassay analyzer (model 680; Bio-Rad, Hercules, CA). This test was repeated at least 3 times. The cell growth rate was calculated by following formula:
Cell growth rate = (mean absorbance in 6 wells of the treatment group) / (mean absorbance in 6 wells of the cells control group) × 100% .
2.5
Cell apoptosis detection by flow cytometry
After transfection with Cd1-RNAi-Lentivirus for 72 hours, cells were washed twice with ice-cold phosphate-buffered saline and resuspended in binding buffer (BD Pharmingen, Franklin Lakes, NJ) to a final concentration of 1 × 10 6 cells per milliliter. Cell suspensions were fixed with 70% ethanol and washed with the APO-DIRECT Wash Buffer (BD Pharmingen). Cell apoptosis was assessed by annexin V-FITC/propidium iodide staining with the flow cytometric apoptosis detection kit (BD Pharmingen) and analyzed by the FACScan (BD, San Jose, Canada). Hep-2 cells without any treatment were used as internal control, and the experiments were repeated at least 3 times.
2.6
Animal experiments
All animal experiments were approved by the institutional Animal Welfare Committee. BALB/c-nu mice(5–6 weeks old; 20 g in weight), provided by the Central Animal Facility of Harbin Medical University, were bred in an aseptic condition according to standard guidelines. Twenty mice underwent subcutaneous injection of 100 μ L cell suspension of Hep-2 (1 million) in the dorsal scapula region. The size of tumors was blindly measured twice a week with calipers, and the volume of tumor was determined using the simplified formula of a rotational ellipsoid ( L × W 2 × 0.5). Once tumors reached approximately 0.5 to 0.6 cm 3 , 10 mice in the experimental group were treated by intratumoral injection of 200 μ L Cd1-RNAi-Lentivirus and the other 10 mice in the control group were treated with 200 μ L of GFP-Lentivirus once a week for 3 weeks. Tumors were harvested from mice 1 week after the end of treatment. (All animal in vivo experiments were repeated 3 times.)
2.7
Terminal deoxytransferase–mediated dUTP nick end labeling procedure
Apoptosis was detected using the terminal deoxytransferase–mediated dUTP nick end labeling (TUNEL) method in situ apoptosis detection kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. After deparaffinization, dehydration, and inactivation of intrinsic peroxidase activity, 20 paraffin sections of each tumor specimen were incubated with 2 μ g/mL proteinase K at 37°C for 15 minutes. Afterward, the sections were treated with terminal deoxynucleotidyl transferase and biotinylated dUTP. After stopping the reaction with TB buffer (30 mmol/L sodium chloride, 30 mmol/L sodium citrate), the samples were investigated by microscopy. Controls for the TUNEL procedure were treated in the same manner as the test samples except that the TdT enzyme was omitted from the reaction mixtures in both kits and was replaced with dH 2 O. No labeling was found in the controls.
2.8
Transmission electron microscope examination
Tumor tissues were fixed (1 hour) with 2.5% glutaraldehyde using 0.1 mol/L Na cacodylate buffer (pH 7.3), washed in a buffer (1 hour), and then postfixed (1 hour) with 1% osmium tetroxide. Fixed specimens were rinsed and dehydrated in a graded ethanol series. Ultrathin sections were cut, mounted on copper grids, and stained with uranyl acetate and lead citrate by standard methods. Stained grids were examined and photographed in a JEM 1200EX transmission electron microscope (Tokyo, Japan).
2.9
Statistical methods
Results were expressed as mean ± SD. Student 2-sided t test was used to compare the values of the test and control samples. A value of P < .05 was considered as significant.
2
Methods
2.1
Lentivirus vectors for cyclin D1 siRNA
siRNA targeting human cyclin D1 was inserted into lentivirus gene transfer vector encoding green fluorescent protein (GFP), and the recombinant lentivirus was constructed by Genechem Co Ltd, Shanghai, China. The target sequence of the siRNA was 5′-GGAGAACAAACAGAT CATC-3′ (GenBank: NM_053056 ), which was verified by sequencing. The recombinant lentivirus of the SiRNA targeting cyclin D1 (Cd1-RNAi-Lentivirus) and the control lentivirus (GFP-Lentivirus) were prepared and titered to 10 9 transduction units per milliliter.
2.2
Cell line and lentivirus siRNA gene transfection
The Hep-2 cells of human LSCC were kindly provided by the Laboratory of Cell Pathology, Harbin Medical University. Cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (Gibco, Foster, Canada) and incubated in a humidified (37°C, 5% CO 2 :air) incubator. Hep-2 cells were plated in 24-well plates (2 × 10 4 cells per well) and cultured for 12 hours. Lentiviruses were diluted to 0.2 mL (10 8 transduction units per milliliter) with complete medium containing polybrene (8 mg/mL) and added to the cells for incubation for 1 hour at 37°C; 0.3 mL of fresh polybrene-DMEM was added to Hep-2 cells and incubated for 24 hours. After transfection, polybrene-DMEM was replaced with fresh DMEM medium; and the cells were cultured for another 48 hours.
2.3
Detection of cyclin D1 expression by reverse transcriptase polymerase chain reaction
After transfection with Cd1-RNAi-Lentivirus for 72 hours, the total RNA was extracted from the transfectants of each group using Trizol (Invitrogen, Carlsbad, Canada). For reverse transcriptase polymerase chain reaction (RT-PCR) analysis, 2 μ g of total RNA was used to synthesize complementary DNA using a first-strand complementary DNA synthesis kit (Promega, Madison, WI). Two pairs of primer sequences were designed and synthesized by Shanghai Biological Engineering Corporation (Shanghai, China) as follows: human cyclin D1 forward primer was 5′-CTGGAGCCCGTGAAAAAGAGC-3′, reverse primer was 5′-CTGGAGA GGAAGCGTGTGAGG-3′, and product size was 433 base pairs. Human β -actin primer forward was 5′-CCCAGCACAATGAAGATCAAGATCAT-3, reverse primer was 5′-ATCTGCTGGAA GGTGGACAGCGA-3′, and product size was 107 base pairs. The conditions for the RT-PCR were 94°C for 5 minutes, 58°C for 30 seconds, and 72°C for 1 minute, for 35 cycles. The products were then electrophoresed on a 1.5% agarose gel containing ethidium bromide. β -Actin was served as the positive control; and RT-negative tubes, as the negative controls.
2.4
MTT assay
Hep-2 cells were incubated in 96-well plates at a density of 105 cells per well with RPMI 1640 medium supplemented with 10% fetal bovine serum for 24 hours. After Cd1-RNAi-Lentivirus transfection for 24, 48, 72, and 96 hours, 20 μ L of sterile methyl thiazolyl tetrazoliym (MTT) dye (5 mg/mL; Sigma-Aldrich Corp, St Louis, MO) was added and incubated for another 4 hours at 37°C. Afterward, 150 μ L of dimethyl sulfoxide was added to each well and thoroughly mixed for 10 minutes. Cells transfected with GFP-Lentivirus were used as controls. Spectrometric absorbance at a wavelength of 492 nm was measured on an enzyme immunoassay analyzer (model 680; Bio-Rad, Hercules, CA). This test was repeated at least 3 times. The cell growth rate was calculated by following formula:
Cell growth rate = (mean absorbance in 6 wells of the treatment group) / (mean absorbance in 6 wells of the cells control group) × 100% .
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