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Inhibition of Human Telomerase Reverse Transcriptase in Hep-2 Cells Using Short Hairpin RNA Expression Vectors
Shi-Ming Chen, PhD;
Ze-Zhang Tao, MD;
Qing-Quan Hua, PhD;
Dan Liu, MD;
Hua-Ming Chi, MD;
Qing Cai, PhD
Arch Otolaryngol Head Neck Surg. 2006;132:200-205.
ABSTRACT
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Objective Telomerase activity is mainly regulated by the human telomerase reverse transcriptase (hTERT) gene. Our objective was to investigate the effect of short hairpin RNA (shRNA) on hTERT expression and telomerase activity in laryngeal cancer cells.
Design Short hairpin RNA expression vectors targeting the messenger RNA of hTERT were constructed. Cells were treated with shRNA expression vectors directed against 2 different hTERT sites, control vectors that included mismatched shRNA and those without shRNA. The expression of hTERT was determined by reverse-transcriptase polymerase chain reaction and Western blotting. The activity of telomerase was measured by telomeric repeated amplification enzyme-linked immunosorbent assay. The cell viability was examined using the 3-(4,5-dimethyl thizol-2-yl) 2,5-diphenyl tetrazolium bromide assay.
Results We found that treatment of shRNA expression vectors induced a significant decrease in hTERT messenger RNA expression, the level of hTERT protein, telomerase activity, and cell viability. All of these effects were seen regardless of the target site, and the shRNA control showed none of these effects.
Conclusion Our results suggest that shRNA directed against hTERT inhibits telomerase activity through suppression of the hTERT expression in laryngeal cancer cells and that RNA interfering technology may be a promising strategy for the treatment of laryngeal cancers.
INTRODUCTION
Telomeres act as protective chromosomal end caps, and their length is maintained by a dynamic equilibrium between processes that shorten and lengthen telomeric DNA.1-5 Telomere shortening is a checkpoint for signaling the onset of cellular senescence. In healthy human somatic cells, telomere size is reduced with each division until a critical length is reached and cell death ensues. However, most tumor cells can overcome this cellular time bomb by expressing telomerase—an RNA protein complex that progressively elongates telomeric DNA.6 Most healthy cells lack this enzyme and thus have a finite doubling capacity.
Telomerase is a ribonucleoprotein complex that includes an RNA template molecule (TER) and a human telomerase reverse transcriptase (hTERT). Other subunits include a protein, dyskerin, which binds to TER. Through reverse transcribing of its RNA template moiety, telomerase synthesizes telomeric DNA, and the loss of telomeric sequences during DNA replication will be compensated.7 Telomerase activity is mainly regulated by the hTERT gene. Approximately 85% of all cancers are telomerase-positive. Most laryngeal cancer cells persistently express high levels of telomerase activity, whereas a healthy laryngeal epithelium rarely displays it.8-9
RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) targets homologous messenger RNA (mRNA) for degradation, thus effectively silencing gene expression at the posttranscriptional level. Introduction of a 19 to 21 nucleotide small interfering RNA (siRNA) duplex is sufficient to induce RNA interference in mammalian cells.10-11 DNA vectors constructed to mediate RNAi by expressing short hairpin RNA (shRNA) from RNA polymerase III promoters are a newly established technique that can produce long-term, stable, and highly specific gene silencing.12-13 These findings have opened a brand new avenue for the research of gene function and gene therapy.14 In this study, we tested whether the shRNA expression vectors can be employed to suppress the expression of the hTERT gene and telomerase activity in telomerase-positive laryngeal squamous carcinoma (Hep-2) cells, and we evaluated its effect on cell proliferation.
METHODS
CELL LINE AND CULTURE
Hep-2 cells were obtained from the American Type Culture Collection and maintained in medium (Gibco-BRL, Carlsbad, Calif) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah), 100 U/mL penicillin G sodium, and 100 µg/mL streptomycin sulfate in 5% carbon dioxide at 37°C. Cells were divided into 5 different treatment groups: 1, shRNA1 (plasmid involved in enhanced green fluorescent protein gene [pEGFP], can express shRNA targeted to the hTERT mRNA site 1); 2, shRNA2 (pEGFP, can express shRNA targeted to the hTERT mRNA site 2); 3, shRNA3 (pEGFP, can express shRNA targeted to no human gene); 4, pEGFP; and 5, medium-only as a negative control.
shRNA DESIGN AND PLASMID CONSTRUCTION
Short hairpin RNA 1 and 2 were designed according to the complementary DNA sequence of the hTERT (GenBank accession No. AB085628); shRNA3, used as a control (it did not target any specific human gene), was also designed. Short hairpin RNAs encoding the DNA template were designed as follows: a 19-nucleotide target sequence as a sense strand followed by a spacer and complementary antisense strand and then 4 continuous thymines as terminate signal (Figure 1A and B). The shRNAs were subcloned into the pEGFP-C1 (Figure 1C) with human U6 promoter between the BamHI and HindIII restriction sites. Plasmids were constructed by Wuhan Genesil Biotechnology Company (Wuhan, China). All of the constructs used in this study were verified by DNA sequencing.
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Figure 1. The structures of plasmid that can express short hairpin RNA (shRNA). A, Predicted structures of shRNAs. B, Design of the shRNA template. hTERT indicates human telomerase reverse transcriptase; pshRNA1–hTERT+EGFP, plasmid involved in enhanced green fluorescent protein (EGFP) gene, can express shRNA-targeted hTERT messenger RNA (mRNA) site 1; pshRNA2–hTERT+EGFP, plasmid involved in EGFP gene, can express shRNA-targeted hTERT mRNA site 2; pshRNA3+EGFP, plasmid involved in EGFP gene, can express shRNA-targeted to no human gene. C, Schematic of the pEGFP vectors. An shRNA encoding template was inserted between BamHI and HindIII restriction sites downstream of the U6 promoter. Transcripts of the template in B will form a 19-nucleotide double-stranded stem with a 9-nucleotide loop hairpin that targets hTERT mRNA or shRNA3 that targets no specific human gene mRNA. HSV TK indicates herpes simplex virus thymidine kinase; Kan/Neo, kanamycin sulfate/neomycin sulfate; MCS, multiple cloning site; PCMV IE, immediate early promoter of cytomegalovirus; pEGFP-C1, the plasmid involved in the EGFP gene and the customer is C-1; PSV40 E, SV40 plasmids; pUC ori, pUC origin of replication; SV40 ori, simian virus 40 origin of replication; SV40 polyA, simian virus 40 polyadenylation.
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TRANSFECTION AND DETECTION OF TRANSFECTION EFFICIENCY
Twenty-four hours before transfection, cells were seeded onto 6-well plates with an antibiotic-free growth medium at a density of 2 x 104 cells per well, so that the confluence would reach approximately 75% at the time of transfection. Cells were transfected with 2 µg per well of shRNA plasmid-targeting hTERT, another gene, or an empty vector using 4 µL of transfection reagent (Metafectene; Biontex, Munich, Germany) following the protocol provided by the manufacturer. After 24 hours of administration, the fluorescence was detected using confocal microscopy (model TCS SP2 MP; Leica, Solms, Germany).
DETECTION OF hTERT mRNA EXPRESSION BY RT-PCR
At 12, 24, 48, and 72 hours, total RNA was extracted from the cancer cells after treatment with the different plasmids using Trizo, a chemical reagent to isolate total RNA (Invitrogen, Carlsbad, Calif). The total RNA (2 µg) was reverse transcribed using a first-strand complementary DNA synthesis kit (Promega, Madison, Wis) in a 25-µL reaction containing 1 x reverse transcriptase (RT) reaction buffer (Promega), 200 µM deoxyribonucleoside triphosphates, 10 ng random hexamer primer (Promega), 20 U RNAsin, and 400 U Moloney murin leukemia virus (Promega) RT for 90 minutes at 42°C, and then heated for 10 minutes at 72°C. After heat inactivation of the RT at 94°C for 2 minutes, 2 µL of the RT reaction mixture and 48 µL of the polymerase chain reaction (PCR) mixture were mixed and then amplified with PCR. The hTERT upstream primer sequence is 5'-ACT TTG TCA AGG TGG ATG TGA CGG-3'; hTERT downstream primer sequence, 5'-ACG GCT GGA GGT CTG TCA AGG TAG-3'. The  -actin upstream primer sequence is 5'-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3'; -actin downstream primer sequence, 5'-CGA GCG GGA AAT CGT GCG TGA CAT TAA GGA GA-3' (primers from Shanghai Institute of Biochemistry, Shanghai, China). The conditions for the PCR were 94°C for 10 seconds; 60°C for 20 seconds; and 72°C for 1 minute, for 38 cycles. The RT-PCR products were then electrophoresed on a 1.5% agarose gel containing ethidium bromide. -Actin served as the positive control, and RT-negative tubes served as the negative control.
WESTERN BLOTTING
Cells were harvested at the indicated time after transfection, washed with cold phosphate-buffered saline solution, and total proteins were extracted in the extraction buffer (150 mM sodium chloride; 50 mM Tris hydrochloride, pH 7.5; 1% glycerol; and 1% Nonidetp-40 substitute solution [Sigma-Aldrich Corp, St Louis, Mo]). Equal amounts of protein (15 µg per lane) from the treated cells were loaded and electrophoresed on an 8% sodium dodecyl sulfate polyacrylamide gel and then transferred onto an enhanced chemiluminescence system of nitrocellulose membranes (Hyband, Buckinghamshire, England). The blotted membrane was incubated with the antibodies to hTERT (1:750; Santa Cruz Biotechnology, Santa Cruz, Calif) and -actin (1:1000; Cell Signaling Technology Inc, Beverly, Mass), followed by treatment with secondary antibody conjugated to horseradish peroxidase (1:5000). The proteins were detected by the enhanced chemiluminescence system and exposed to x-ray film. Protein expressions were semiquantitatively analyzed with the Villber Lourmat Scanning System (Marne-la-Vallée, France).
MEASUREMENT OF TELOMERASE ACTIVITY
Telomerase activities were measured according to the protocol of a telomerase PCR enzyme-linked immunosorbent assay kit (Roche, Mannheim, Germany). The experiments were performed in triplicate. Cell extract was heated to 65°C for 10 minutes as a negative control, and an extract of 293 cell lines with telomerase activity (from the kit) was used as a positive control. Briefly, a telomeric repeated amplification protocol reaction involves the following: (1) primer elongation; (2) telomerase inactivation: perform 1 cycle at 94°C for 5 minutes; and (3) amplification: perform 30 cycles for amplification at 94°C for 30 seconds for denaturation, at 50°C for 30 seconds for annealing, at 72°C for 90 seconds for polymerization, and then at 72°C for 10 minutes for balance. Twenty-microliter PCR products were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then detected using a silver nitrate staining assay. Five-microliter RT-PCR products were used to make the enzyme-linked immunosorbent assay reaction. The absorbance (A) value of wavelength of 450 to 655 nm to calculate (A = A450–A655) was determined. The value of A was presented as telomerase activity.
IN SITU CELL APOPTOSIS DETECTION
The apoptotic cell was identified using a modified end-labeling technique originally described by Zhang et al.15 All procedures were performed following the manufacturer's instructions (in situ cell apoptosis detection kit; Wuhan Boster Biological Technology Co, Wuhan, China). Two controls per assay were performed: incubating sections with DNase I served as a positive control, and omission of the terminal transferase from the reaction mixture served as a negative control.
CELL VIABILITY ANALYSIS BY THE MTT ASSAY
The 3-(4,5-dimethyl thizol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay was performed to assess the effect of different shRNAs on cell proliferation. Cells (3 x 103cells per well) were incubated in 96-well plates and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. At 24 hours after seeding, the culture medium was replaced by new medium. Then cells were transfected with 45 µL of fetal bovine serum–free medium per well, containing 0.1 µg of plasmid DNA and 0.4 µL of transfection reagent (Biontex). After 20, 44, 68, and 92 hours, 10 µL of sterile MTT dye (5 mg/mL; Sigma-Aldrich Corp) was added to the cells and incubated for another 4 hours at 37°C, and then 200 µL of dimethyl sulfoxide was added and thoroughly mixed for 30 minutes. Spectrometric absorbance at a wavelength of 492 nm was measured on an enzyme immunoassay analyzer (model 550; Bio-Rad, Hercules, Calif).
Values are expressed as mean ± SD of multiple experiments. Analysis of covariance with the Dunnett or Tukey-Kramer posttests was used for multiple groups, and the t test was used for comparison between 2 groups using SPSS statistical software (version 11.0; SPSS Inc, Chicago, Ill).
RESULTS
FLUORESCENT EXPRESSION AFTER TRANSFECTION BY DIFFERENT PLASMIDS
In this study, we constructed recombinant plasmids that would express shRNAs. To detect whether the plasmids could be successfully transfected into Hep-2 cells, green fluorescent protein (GFP) was used as a marker for all of the plasmids. As shown in Figure 2, the fluorescence of GFP illustrated the efficiency of the transfection. Treatment with shRNA1 or shRNA2 induced the death of many cells, whereas treatment with shRNA3 and pEGFP did not.
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Figure 2. Short hairpin RNA (shRNA) expression vectors, which contained the enhanced green fluorescent protein (EGFP) marker gene, expressed fluorescence in Hep-2 cells after 24 hours of exposure. A, shRNA1-treated cells; B, shRNA2-treated cells; C, shRNA3-treated cells; D, pEGFP-treated cells; E, control cells without treatment by any vector. After treatment with shRNA1 or shRNA2, many cells showed green fluorescence and died. The dead cells were small and round (arrows) (original magnification x400).
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mRNA EXPRESSIONS OF hTERT
We next determined, using RT-PCR, whether hTERT mRNA expression was altered in shRNA-treated Hep-2 cells. The data presented in this study demonstrate that shRNA-induced mRNA expression decreased in a time-dependent manner. As shown in Figure 3, the hTERT mRNA expressions were decreased after 12 hours of exposing the Hep-2 cells to shRNA1 or shRNA2. As time went by, the effect was more and more explicit. After administration of shRNA1 for 72 hours, the maximum suppressive level of hTERT expression was observed.
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Figure 3. Reverse-transcriptase polymerase chain reaction assay for human telomerase reverse transcriptase (hTERT) and -actin messenger RNA (mRNA) in short hairpin RNA (shRNA)-treated and untreated Hep-2 cells. Lane 1, shRNA1-treated cells; lane 2, shRNA2-treated cells; lane 3, shRNA3-treated cells; lane 4, pEGFP (plasmid involved in enhanced green fluorescent protein gene)–treated cells; and lane 5, control cells without treatment. The mRNA expressions of hTERT were significantly decreased by 48 and 72 hours of exposure to shRNA1 and shRNA2, respectively, compared with the control. Arrows show (A) hTERT, 215 base pairs (bp), and (B) -actin, 480 bp.
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PROTEIN EXPRESSIONS OF hTERT
Protein expressions of hTERT were analyzed by Western blotting (Figure 4A). Strong expression of hTERT protein was observed in the control cells (lane 5), in pEGFP-treated cells (lane 4), and in shRNA3-treated cells (lane 3). However, the levels of hTERT proteins were obviously decreased by treatment with shRNA1 or shRNA2 (lanes 1 and 2). As shown in Figure 2B, hTERT protein in control cells was 100%. Following exposure to shRNA1 for 24 and 48 hours, hTERT protein in Hep-2 cells progressively declined to 45.1% and 9.9%, respectively. Following exposure to shRNA2, it declined to 51.2% and 18.2%, respectively.
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Figure 4. The vectors that express short hairpin RNA (shRNA) suppressed the human telomerase reverse transcriptase (hTERT) proteins in Hep-2 cells. A, Lanes 1 through 5 show the expression of the hTERT protein treated with shRNA1, shRNA2, shRNA3, pEGFP (plasmid involved in enhanced green fluorescent protein gene), and medium only, respectively. B, The level of hTERT protein was decreased significantly by the treatment of Hep-2 cells with shRNA1 or shRNA2. Results are expressed as mean ± SD for 3 independent experiments. Asterisk indicates P<.05; dagger, P<.01 compared with the control.
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TELOMERASE ACTIVITY
Telomerase reverse transcriptase is the key component for the control of telomerase activity. We postulated that the inhibition of hTERT expression would decrease the telomerase activity. To verify this hypothesis, the effect of shRNAs on telomerase activity was examined. The results are summarized in Figure 5. The telomerase activity was distinctly decreased by shRNA1 or shRNA2 treatment but remained unaltered in the other treatments. As shown in Figure 5B, absorbance level of cells treated with shRNA1 or shRN2 was much lower than that of control cells after 24 to 72 hours of exposure. Telomerase activity in untreated Hep-2 cells was 100%. Following exposure to shRNA1 for 24, 48, and 72 hours, telomerase activity of Hep-2 cells progressively declined to 30.3%, 10.5%, and 9.3%, respectively. After exposure to shRNA2, it declined to 30.9%, 10.8%, and 10.2%, respectively.
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Figure 5. Summary of the results of the examination of the effect of short hairpin RNAs (shRNAs) on telomerase activity. A, Effect of shRNA vectors targeted against the human telomerase reverse transcriptase gene on telomerase activity after 48 hours of treatment. Lanes 1 to 7 represent negative control (cell extract was heated to 65°C); control (untreated Hep-2 cells); protein treated with pEGFP (plasmid involved in enhanced green fluorescent protein gene); shRNA3; shRNA2; shRNA1; and positive control (from the telomerase polymerase chain reaction enzyme-linked immunosorbent assay kit [Roche, Mannheim, Germany]), respectively. The telomerase activity was significantly decreased after treatment with shRNA1 or plasmids that can express shRNA2 (pshRNA2). B, Absorbance level of shRNA1 or shRNA2 transfection was much lower than that of control cells after 24 to 72 hours. bp Indicates base pair. Asterisk indicates P<.01; dagger, P<.001 compared with the control.
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IN SITU CELL APOPTOSIS DETECTION
After administration of shRNA1 or shRNA2, we observed many dead cells and wondered whether this effect was induced by the programmed cell death. Here, we used a terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL) reaction to detect the 3'-hydroxyl termini of DNA fragmentation in Hep-2 cells. As shown in Figure 6A, following incubation of Hep-2 cells with shRNA1 or shRNA2, most cells were TUNEL-positive, indicating that they were undergoing apoptosis (panels 1 and 2). In contrast, uninfected control cells had a negative appearance (panel 5). In addition, shRNA3-infected or pEGFP-infected cells also had a negative appearance, indicating that the effect was specific to special shRNAs (panels 3 and 4).
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Figure 6. TUNEL staining of Hep-2 cells after treatment with different short hairpin RNAs (shRNAs). A, Sections are from (1) shRNA1 treatment; (2) shRNA2 treatment; (3) shRNA3 treatment; (4) pEGFP (plasmid involved in enhanced green fluorescent protein gene) treatment; and (5) control. The arrows point to TUNEL-positive cells, which indicates the transfer of fragmented DNA from nuclei to the neuronal fiber terminals. B, The rate of Hep-2 cell apoptosis increased after treatment with special shRNA. Asterisk indicates P<.01 compared with the control (original magnification x400).
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To measure the apoptotic rate of shRNA-infected cells, 8 fields of vision at different time points, after 24 and 48 hours of exposure, were observed. As shown in Figure 6B, the apoptotic rate increased with time of infection. After exposure to shRNA1 for 24 and 48 hours, the rate of apoptosis for Hep-2 cells progressively increased to 32.3% and 34.5%, respectively. After exposure to shRNA2, it progressively increased to 29.2% and 30.1%, respectively.
CELL VIABILITY AFTER TREATMENT WITH shRNA EXPRESSION PLASMIDS
We report herein that suppressing both telomerase RNA and telomerase RT mRNA can reduce cell viability by down-regulating telomerase activity. The effect of shRNA expression plasmids on Hep-2 cell proliferation was examined using an MTT assay. As shown in Figure 7, shRNA1 and shRNA2 exhibited potent cytotoxic activity against Hep-2 cells. Following incubation with shRNA1 for 12, 24, 48, 72, and 96 hours, cell viability progressively decreased to 58%, 65%, 52%, 31%, and 10%, respectively. After incubation with shRNA2, it decreased to 68%, 69%, 68%, 36%, and 18%. Time-course inhibition by the special shRNAs in Hep-2 cells is also shown in Figure 7. Over time, the potent cytotoxic activity of special shRNAs was more obvious.
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Figure 7. Cell viability of Hep-2 cells after exposure to the different short hairpin RNAs (shRNAs). The value of the cell viability is the mean ± SD of 5 independent experiments. The most prominent inhibition was found in cells treated with shRNA1 or shRNA2. Increased inhibition by the special shRNAs in Hep-2 cells over time was observed. pEGFP indicates plasmid involved in enhanced green fluorescent protein gene.
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COMMENT
RNA interference is now established as a general method to silence the specific genes in a variety of organisms. When transfected into cells, the dsRNA will interfere with the expression of homologous genes, subsequently disrupting their normal function. This suppression is mediated by 19-nucleotide to 23-nucleotide siRNAs, which induce degradation of mRNA based on complementary base pairing. In mammalian cells, transient delivery of synthetic siRNA that resembles the processed form of standard dsRNAi is effective in silencing specific gene expression. However, the duration of the silencing and the issues related to the transferring efficiency restrict the field of the applications of siRNA in mammals.16 The cellular expression of shRNA from vectors will solve all of these shortcomings of siRNA. The shRNAs are expressed from mammalian promoters on DNA vectors, which are introduced to cells by transfection or infection and possess double-stranded stems that are about 19 to 23 nucleotides in length and that serve as substrates for the enzyme dicer, which allows for the creation of stable, silenced cell lines.17-18
It is currently unclear how telomerase is activated or regulated in human tumor cells. The human telomerase complex comprises multiple components, but hTERT is the key component for the control of telomerase activity. Specifically, healthy human somatic cells can acquire the ability to maintain telomeres and replicate well beyond the Hayflick limit (the limit to the number of times a human cell can divide; the number is around 50) by stable, enforced expression of the hTERT gene.
In the research described herein, we have used shRNAs to inhibit the expression of hTERT to influence telomerase activity. The results show that we have succeeded in down-regulating or inhibiting hTERT gene expression to decrease the activity of telomerase in Hep-2 cells. After treatment with shRNA1 or shRNA2, absorbance of Hep-2 cells was distinctly reduced compared with the control, suggesting that some Hep-2 cells had died and some cells stopped growing. The result of the TUNEL assay indicated that the shRNAs produced this decrease in cell viability through apoptosis.
It was reported19 that the most effective short hairpin siRNA vectors decrease expression of target proteins or synthetic luciferase targets by more than 10-fold, but not all shRNA vectors inhibit this well. Some differences in effectiveness probably arise from differences in accessibility of the RNA target sequences for RNA secondary structure or bound proteins, as has been suggested for in vitro–synthesized siRNA duplexes.12, 20-21 However, differences in processing, stability, or synthesis also are likely to contribute to the differences in effectiveness among shRNAs. It was suggested12, 22 that varying the length of the duplex region or varying the sequence of the loop between the shRNA antisense and sense sequences could alter the effectiveness of shRNAs. Paddison et al22 suggested that siRNA duplexes of 25 to 29 nucleotides are generally more effective than shorter duplexes. Our observations indicate that shRNAs with duplex lengths of 19 nucleotides can be effective in reducing the expression of target genes. Moreover, there is no distinctive difference in effectiveness between shRNA1 and shRNA2, which target different sites of the hTERT gene. In our study, the effectiveness of RNAi emerged after 12 hours of treatment. With additional time, the effect becomes more explicit.
Research has proved that suppressing telomerase RNA and telomerase RT mRNA can reduce telomerase activity, restraining the growth of cancer cells.23-25 Although hTERT is more specific to malignant neoplasms than telomerase,26 it is unclear whether there is another telomerase activator and whether merely suppressing hTERT gene expression can suppress telomerase activity in the long run. Therefore, to effectively treat telomerase-positive tumors, further investigation is needed on the mechanisms by which telomerase can be optimally suppressed.
AUTHOR INFORMATION
Correspondence: Ze-Zhang Tao, MD, Department of Otolaryngology–Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, China (shimingchen0468{at}hotmail.com).
Submitted for Publication: May 27, 2005; final revision received July 22, 2005; accepted September 7, 2005.
Financial Disclosure: None.
Funding/Support: This study was supported by grant No. 30471873 from the National Natural Science Foundation of China, Beijing.
Acknowledgment: We thank Guo-Hua Ding, PhD (Renmin Hospital, Wuhan University, Wuhan, China), for helpful advice and critical reading of the manuscript.
Author Affiliations: Department of Otolaryngology–Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, China.
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