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Abstract

BACKGROUND:

Despite the improvement in the diagnosis and the management of laryngeal squamous cell carcinoma (LSCC), many patients with advanced-stage have poor prognosis in the form of recurrence, metastasis or death. So, recognition of new molecular markers would facilitate the development of targeted therapies.

OBJECTIVES:

To investigate miR-221 expression in LSCC and its possible correlation to apoptotic protease activating factor-1 (Apaf-1). Also, we aimed to investigate the association between miR-221 and Apaf-1 expressions and the clinicopathological features of LSCC.

METHODS:

We investigated the expression of miR-221 (by qRT-PCR) and Apaf-1 (by qRT-PCR and immunohistochemistry) in primary LSCC and adjacent normal tissues.

RESULTS:

We found significant up-regulation of miR-221 and significant down-regulation of Apaf-1 expression in LSCC tissues compared to normal nearby laryngeal tissues. In addition, significant associations between up-regulated miR-221 and down-regulated Apaf-1 expressions and clinical stage and lymph node (LN) metastasis ( $P<$ 0.001 for each) were found. Furthermore, there was a negative correlation between miR-221 gene expression and Apaf-1 gene expression ( $r=-$ 0.73, $P<$ 0.001).

CONCLUSION:

miR-221 can be considered as a diagnostic marker in LSCC and Apaf-1 may be considered as a possible target of miR-221.

Keywords

Laryngeal squamous cell carcinoma miR-221 Apaf-1

Abbreviations

LSCC	Laryngeal squamous cell carcinoma
miRNAs	MicroRNAs
Apaf-1	Apoptotic protease activating factor-1

Ct	The cycle threshold
LOH	Loss of heterozygosity
LN	Lymph node

1. Introduction

Laryngeal cancer is considered one of the most prevalent head and neck malignancies. Squamous cell carcinoma represents about of 95% of diagnosed cases. Laryngeal squamous cell carcinoma (LSCC) is more common in males [1, 2]. Treatments for LSCC are surgery, chemotherapy, radiotherapy or combined regimen. In spite of advances in the diagnosis and the management of LSSC, the survival rates have not changed. Many patients with advanced LSCC suffer from recurrence, metastasis and eventually death [3]. So, recognition of new molecular markers responsible for LSCC initiation and progression can facilitate the development of targeted therapies.

MicroRNAs (miRNAs) are a class of short, non-coding, single-stranded RNAs with about 22- nucleotides. They are mainly transcribed by RNA Polymerase II and they are regulated by promoters and enhancers. In addition, they acquire a cap and polyadenylated tail of their initial product such as other RNA Polymerase II derived genes [4]. They have a post-transcriptional effect on gene expression as they bind to certain mRNAs at their 3’ untranslated region causing degradation or translational inhibition of the transcript [5].

In addition, miRNA dysregulation is the third mechanism, after the loss of heterozygosity (LOH) and DNA methylation, for inactivation of tumor suppressor genes such as apoptotic protease activating factor-1 (Apaf-1) [6]. Moreover, miRNAs were measured easily in different biological fluids without a change in stability. Thus, miRNAs expressions may be considered as possible diagnostic and prognostic biomarkers for cancers [7].

MiR-221 is up-regulated in many types of malignancies including glioblastoma, gastric, colorectal, bladder, kidney, ovarian, breast, lung and papillary tumors of the thyroid [8]. Lupini et al. proved the role of miR-221 in cancer promotion by various signals. They investigated some of the possible miR-221 target genes, including Apaf-1 in multiple cell lines by mRNA and protein analysis [9]. Apaf-1 was recognized as a target of miR-221 in bronchogenic carcinoma cell lines [10]. Moreover, Sun et al. correlated Apaf-1expression to miR-221 in LSCC cell line. They suggested that Apaf-1 may be a new target of miR-221. However, they didn’t investigate the expression levels in fresh tissue specimens or correlate them to clinicopathological features [11].

Carcinogenesis develops from the imbalance between apoptosis and proliferation due to the dysregulation of either of them. Apoptosis can occur by extrinsic and intrinsic (cytochrome C-dependent) pathways. In the intrinsic one, an apoptosome is formed from Apaf-1 interaction with caspase-9. So, Apaf-1 is considered as a tumor suppressor gene in different types of malignancies [12].

The aim of this study was to investigate the expression of miR-221 and Apaf-1 in LSCC tissues and to investigate the correlation between the two markers. We aimed also to analyze the relationship between miR-221 and Apaf-1 levels and the clinicopathological characteristics of LSCC patients.

2. Patients and methods

This study was carried out at Departments of Otorhinolaryngology and head and neck surgery, Pathology and Medical Biochemistry and Molecular Biology, Faculty of Medicine, Zagazig University between January 2015 and December 2016. A total of 50 patients with primary LSCC were enrolled in this study after exclusion of previous radiotherapy or chemotherapy before surgery and after obtaining written consents from all cases.

Patients were diagnosed by endoscopy and imaging (CT and/or MRI). All patients underwent full history taking, clinical examination. Under general anesthesia, direct laryngoscopy was performed and fresh laryngeal tissues (including cancer and the nearby non-malignant tissues) were obtained at surgery time and separated into two parts. One part was frozen and kept at $-$ 80 ${}^{\circ}$ C for RNA extraction. The other part was preserved in formalin solution for histopathological and immunohistochemistry studies.

We depended on The TNM criteria for clinical staging, and the World Health Organization criteria for histopathologic grading [13].

2.1 RNA extraction and real-time PCR

Total RNA with miRNAs from laryngeal tissues was extracted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) following the instructions of the manufacturer. Reverse transcription of RNA was performed using the Power cDNA synthesis Kit (iNtRON Biotechnology, Seongnam, Korea). Quantitative Real-time PCR was performed using Mx3005P™ (Stratagene, La Jolla, CA, USA) according to the following protocol: initial denaturation and polymerase activation for 15 min at 95 ${}^{\circ}$ C, followed by 40 cycles of denaturation for 15 sec at 94 ${}^{\circ}$ C; annealing for 30 sec at 55 ${}^{\circ}$ C; and finally extension for 30 sec at 70 ${}^{\circ}$ C. Twenty uL volume reactions were performed with 5 uL of the cDNA, 100 pmol/uL of each primer (0.5 uL each) (Biolegio, Netherlands) [4, 14], 10 uL of PCR EvaGreen Master Mix (Jena Bioscience GmbH, Jena, Germany) and 4 uL distilled water. The sequence of the primers utilized in the study was listed in Table 1.

Table 1
Primer sequence of the studied genes

Forward Reverse

GADPH TGAAGGTCGGAGTCAACGGATT CATGTGGGCCATGAGGTCCACCA

Apaf-1 CACGTTCAAAGGTGGCTGAT TGGTCAACTGCAAGGACCAT

miR-221 ACACTCCAGCTGGGAGCTACATTGTCTGCTGG CTCAACTGGTGTCGTGGA

U6 CTCGCTTCGGCAGCACA AACGCTTCACGAATTTGCGT

	Forward	Reverse
GADPH	TGAAGGTCGGAGTCAACGGATT	CATGTGGGCCATGAGGTCCACCA
Apaf-1	CACGTTCAAAGGTGGCTGAT	TGGTCAACTGCAAGGACCAT
miR-221	ACACTCCAGCTGGGAGCTACATTGTCTGCTGG	CTCAACTGGTGTCGTGGA
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT

MiR-221 expression was normalized to U6 while Apaf-1 expression was normalized to GADPH as housekeeping genes. The cycle threshold (Ct) values were calculated. The expression level of the studied genes was normalized by calculating the $\Delta$ Ct value. The amplitude of change of the expression (fold change) in cancerous tissues relative to the control adjacent tissues was analyzed by the 2 ${}^{-\Delta\Delta\text{Ct}}$ equation.

2.2 Immunohistochemical staining

For immunohistochemistry, 4 $\mu$ thicknesses of tumor tissues were obtained from 50 paraffin blocks of LSCC tissues. Laryngeal sections were subjected to dewaxing, re-hydration then blocking by the means of hydrogen peroxide, and antigen retrieval in 10 mM citrate buffer (pH 6.0) at a microwave. Sections were left for 60 min with the human Apaf-1 antibody (Rabbit monoclonal [E38] ab32372; Abcam, Cambridge, UK) at a dilution of 1:500. Visualization of binding sites of primary antibodies was done by Dako EnVision TM kit (Dako, Copenhagen, Denmark). Then, counterstaining of tissue sections was performed with Mayer’s hematoxylin. The omission of the primary antibody was performed to obtain negative controls. Positive control was gastric adenocarcinoma.

2.3 Immunohistochemistry assessment

According to the intensity of staining, a score for Apaf-1 immune-expression assessment was utilized as follow: negative (0), weak (1), moderate (2), and strong (3). However, the extent of staining score depending on the percentage of positively stained cells was 0% (0), 1–25% (1), 26–50% (2), 51–75% (3), and 76–100% (4). The final staining score was calculated by adding the intensity and the extent scores. Four groups of final score were obtained, 0 $=$ negative; 1–3 $=$ weak; 4–5 $=$ moderate; and 6–7 $=$ strong [15].

2.4 Statistical analysis

Means $\pm$ standard deviations (SD) were used to express quantitative data while qualitative data were expressed as numbers and percents (%). Independent Student t-test was used to compare two groups of normally distributed data while Mann-Whitney U test was used for non-normally distributed data. Comparison between more than two groups of normally distributed data was performed by one-way ANOVA test while the comparison between non-normally distributed data was performed by Kraskall Wallis H test. Comparison between categorical variables percentages was performed using Chi-square test or Fisher’s exact test. The relation between gene expressions was calculated by Spearman’s rank correlation coefficient.

A statistically significant difference was detected at P values $<$ 0.05 while a highly significant difference was detected at P values $<$ 0.001. Data were analyzed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA).

3. Results

3.1 Patient characteristics

This study included 50 patients with primary LSCC; 40 males (80%) and 10 females (20%), with mean age $\pm$ SD (61.42 $\pm$ 9.4). The age ranged from 32 to 76 years. Thirty-five tumors (80%) were glottic while 15 (20%) were supraglottic. Thirteen cases (26%) were well differentiated; 21 cases (42%) were moderately differentiated and 16 cases (32%) were poorly differentiated. Twenty-seven patients (54%) were without lymph node (LN) metastasis while 23 patients (46%) were with LN metastasis (Table 2).

Table 2
Characteristics of the studied groups

Total Stages (I–II) N $=$ 27 Stages (III–IV) N $=$ 23 P

Age

$<$ 60 22 (44%) 18 (81.8%) 4 (18.2%) 0. 6

$\geqslant$ 60 28 (56%) 23 (82.1%) 5 (17.9%)

Gender

Male 40 (80%) 22 (55%) 18 (45%) 0. 78

Female 10 (20%) 5 (50%) 5 (50%)

Site

Glottic 35 (80%) 20 (57.1%) 15 (42.9%) 0. 26

Supraglottic 15 (20%) 7 (46.7%) 8 (53.3%)

Grade

Well 13 (26%) 13 (100%) 0 (0%) $<$ 0. 001 ${}^{}$

Moderate 21 (42%) 14 (66.7%) 7 (33.3%)

Poor 16 (32%) 0 (0%) 16 (100%)

Lymph node

Negative 27 (54%) 22 (81.4%) 5 (18.6%) $<$ 0. 001 ${}^{}$

Positive 23 (46%) 5 (21.7%) 18 (78.3%)

MiR221gene expression 3.86 $\pm$ 4.14 1.31 $\pm$ 0.89 6.85 $\pm$ 4.47 $<$ 0. 001 ${}^{}$

APF1gene expression 0.59 $\pm$ 0.35 0.82 $\pm$ 0.23 0.31 $\pm$ 0.25 $<$ 0. 001 ${}^{}$

APF1 immune expression

Negative 36 (72%) 14 (38.9%) 22 (61.1%) 0. 007 ${}^{*}$

Weak 6 (12%) 5 (83.3%) 1 (16.7%)

Moderate 6 (12%) 6 (100%) 0 (0%)

Strong 2 (4%) 2 (100%) 0 (0%)

${}^{*}$ Statistically highly significant difference ( $P<$ 0.05). ${}^{**}$ Statistically highly significant difference ( $P<$ 0.001).

All supraglottic cases showed lymph node involvement. No distant metastasis was detected by clinical examination and imaging in all cases. All patients with glottic lesions complained of hoarseness of voice. While all patients with supraglottic mass complained of discomfort on swallowing and 10 patients presented with stridor. After staging, the patients were further subdivided into two groups. Group 1 included 27 patients with early LSCC (T1-T2) and group 2 included 23 patients of late LSCC (T3-T4). No significant difference between the two groups concerning the age, the gender or tumor site was found ( $p=$ 0.6, 0.78 and 0.26) respectively but a significant difference between the two groups concerning pathological grading and lymph node involvement was found ( $p<$ 0.001 for each) (Table 2).

3.2 Reverse transcription-polymerase chain reaction (RT-PCR) results

In LSCC, the mean relative expression (fold change) of miR-221 and Apaf-1 compared to control were (3.86 $\pm$ 4.14 & 0.59 $\pm$ 0.35) respectively. There was a significant up-regulation of miR-221 expression and a significant down-regulation of Apaf-1 expression in LSCC specimens as compared to control adjacent tissue specimens. miR-221 was significantly up-regulated and Apaf-1 was significantly down-regulated in late stage LSCC compared to early stage ( $p<$ 0.001), as well (Table 2).

Table 3
Association between miR-221 and Apaf-1 gene expression and clinicopathological characteristics

Character No. of cases miR-221 expression p Apaf-1 expression p

Age

$<$ 60 22 2.8 $\pm$ 0.03 0.9 0.56 $\pm$ 0.3 0.7

$\geqslant$ 60 28 2.7 $\pm$ 0.1 0.6 $\pm$ 0.3

Gender

Male 40 3.62 $\pm$ 3.96 0.41 0.58 $\pm$ 0.34 0.71

Female 10 4.84 $\pm$ 4.94 0.62 $\pm$ 0.42

Site

Glottic 35 4.11 $\pm$ 4.04 0.38 0.57 $\pm$ 0.35 0.56

Supraglottic 15 2.88 $\pm$ 4.61 0.64 $\pm$ 0.37

Stage

I 10 1.09 $\pm$ 1.02 $<$ 0.001 ${}^{}$ 0.94 $\pm$ 0.17 $<$ 0.001 ${}^{}$

II 17 1.69 $\pm$ 0.45 0.76 $\pm$ 0.23

III 14 4.9 $\pm$ 2.11 0.43 $\pm$ 0.22

IV 9 9.89 $\pm$ 5.54 0.11 $\pm$ 0.01

Grade

Well 13 4.53 $\pm$ 5.83 0.79 0.94 $\pm$ 0.17 $<$ 0.001 ${}^{}$

Moderate 21 3.47 $\pm$ 3.86 0.68 $\pm$ 0.21

Poor 16 3.48 $\pm$ 2.92 0.17 $\pm$ 0.12

Lymph node

Negative 27 1.3 $\pm$ 0.3 $<$ 0.001 ${}^{}$ 0.82 $\pm$ 0.23 $<$ 0.001 ${}^{**}$

Positive 23 4.3 $\pm$ 0.9 0.31 $\pm$ 0.25

Character	No. of cases	miR-221 expression	p	Apaf-1 expression	p
Age
$<$ 60	22	2.8 $\pm$ 0.03	0.9	0.56 $\pm$ 0.3	0.7
$\geqslant$ 60	28	2.7 $\pm$ 0.1		0.6 $\pm$ 0.3
Gender
Male	40	3.62 $\pm$ 3.96	0.41	0.58 $\pm$ 0.34	0.71
Female	10	4.84 $\pm$ 4.94		0.62 $\pm$ 0.42
Site
Glottic	35	4.11 $\pm$ 4.04	0.38	0.57 $\pm$ 0.35	0.56
Supraglottic	15	2.88 $\pm$ 4.61		0.64 $\pm$ 0.37
Stage
I	10	1.09 $\pm$ 1.02	$<$ 0.001 ${}^{**}$	0.94 $\pm$ 0.17	$<$ 0.001 ${}^{**}$
II	17	1.69 $\pm$ 0.45		0.76 $\pm$ 0.23
III	14	4.9 $\pm$ 2.11		0.43 $\pm$ 0.22
IV	9	9.89 $\pm$ 5.54		0.11 $\pm$ 0.01
Grade
Well	13	4.53 $\pm$ 5.83	0.79	0.94 $\pm$ 0.17	$<$ 0.001 ${}^{**}$
Moderate	21	3.47 $\pm$ 3.86		0.68 $\pm$ 0.21
Poor	16	3.48 $\pm$ 2.92		0.17 $\pm$ 0.12
Lymph node
Negative	27	1.3 $\pm$ 0.3	$<$ 0.001 ${}^{**}$	0.82 $\pm$ 0.23	$<$ 0.001 ${}^{**}$
Positive	23	4.3 $\pm$ 0.9		0.31 $\pm$ 0.25

${}^{*}$ Statistically highly significant difference ( $P<$ 0.05). ${}^{**}$ Statistically highly significant difference ( $P<$ 0.001).

Table 4

Association between Apaf-1 immune expression and clinicopathological characteristics

	Negative		Weak		Moderate		Strong		p
Age
$<$ 60	16	(72.7%)	2	(9.1%)	3	(13.6%)	1	(4.5%)	0.	9
$\geqslant$ 60	20	(71.4%)	4	(14.3%)	3	(10.7%)	1	(3.6%)
Gender
Male	29	(72.5%)	4	(10%)	5	(12.5%)	2	(5%)	0.	75
Female	7	(70%)	2	(20%)	1	(10%)	0	(0%)
Site
Glottic	30	(85.7%)	2	(5.7%)	2	(5.7%)	1	(2.8%)	0.	56
Supraglottic	9	(60%)	1	(6.7%)	3	(20%)	2	(13.4%)
Stage
I	1	(10%)	2	(20%)	5	(50%)	2	(20%)	$<$ 0.	001 ${}^{**}$
II	13	(76.5%)	3	(17.6%)	1	(5.9%)	0	(0%)
III	13	(92.8%)	1	(7.2%)	0	(0%)	0	(0%)
IV	9	(100%)	0	(0%)	0	(0%)	0	(0%)
Grade
Well	3	(23.1%)	3	(23.1%)	5	(38.5%)	2	(15.4%)	$<$ 0.	001 ${}^{**}$
Moderate	18	(85.7%)	2	(9.5%)	1	(4.8%)	0	(0%)
Poor	15	(93.7%)	1	(6.3%)	0	(0%)	0	(0%)
Lymph node
Negative	14	(51.8%)	5	(18.5%)	6	(22.2%)	2	(7.4%)	0.	007 ${}^{*}$
Positive	22	(95.6%)	1	(4.4%)	0	(0%)	0	(0%)

${}^{*}$ Statistically highly significant difference ( $P<$ 0.05). ${}^{**}$ Statistically highly significant difference ( $P<$ 0.001).

Table 5

Roc curve of MiR-221 in laryngeal squamous cell carcinoma

	AUC	Cut-off	p	CI	SNS	SPS	PVP	PVN	Accuracy
MiR-221	0.8	1.7	$<$ 0.001 ${}^{**}$	(0.7–0.96)	94%	82%	81%	95.6%	88%

AUC: area under curve; 95% CI: 95% confidence interval; SN: sensitivity, SP: specificity, PPV: positive predictive value, NPV: negative predictive value.

Concerning clinicopathological features, up- regulated miR-221 and down-regulated Apaf-1 were of high significant association with tumor stage and lymph node involvement ( $p<$ 0.001) (Table 3). There was a significant negative correlation between miR-221 and Apaf-1 mRNA expressions ( $r=-$ 0.73, $P<$ 0.001) (Fig. 1).

Figure 1.

Scatter plot with line chart for the correlation between Apaf-1 gene expression and miR-221 expression.

Receiver operating characteristic (ROC) curve analysis revealed that miR-221 could differentiate the late from early tumors with an AUC of 0.86, $P<$ 0.001. The optimal specificity and sensitivity to recognize early from late LSSC were (82% and 91% with a cutoff level $>$ 1.7) (Table 5, Fig. 2).

Figure 2.

ROC curve for miR-221gene expression in prediction of late laryngeal carcinoma. ROC curve: receiver operating characteristic curve.

Univariate Cox hazard regression analysis for diagnostic factors in LSCC showed significant differences in LN involvement, pathological grade and miR221 expression in laryngeal carcinoma (Table 6), suggesting that these factors are indicators of LSCC progression.

Table 6

Univariate Cox hazard regression analysis for prognostic factors in LSCC

	B	SE	p		Hazard ratio odds (CI 95%)
Age	0.4	0.5	0.	4	0.6	(0.2–1.9)
Grade	4.1	1.1	$<$ 0.	001 ${}^{**}$	5.9	(6.6–28.8)
LN	5.6	1.2	$<$ 0.	001 ${}^{**}$	27	(23–32)
Apaf-1 gene	0.2	0.5	0.	7	0.8	(0.2–2.5)
MiR-221	1.8	0.6	0.	003 ${}^{*}$	6.5	(1.8–22.5)

$P<$ 0.05 is significant. B: Regression coefficient; SE: Standard error; CI: Confidence interval.

3.3 Apaf-1 immunohistochemistry results

Normal stratified squamous epithelium adjacent to LSCC showed cytoplasmic immunoreactivity of Apaf-1. Polymorphonuclear leucocytes in the submucosa showed positive stain, as well. Concerning LSCC; 36 cases (72%) were completely negative and 14 tumors (28%) showed positive expression of Apaf-1. Strong expression was detected only in 2 cases (4%) of stage I, moderate and weak expressions were revealed in 6 cases for each (12%). Decreased expression of Apaf-1 was significantly associated with tumor stage, tumor grade and lymph node involvement ( $P<$ 0.001, 0.001, 0.007 respectively).

Figure 3.

A: Normal stratified squamous epithelium of larynx shows strong expression of Apaf1 (IHC x400). B: Well differentiated LSCC (H&E x400). C: Well differentiated LSCC shows strong expression of Apaf1 (IHC x200). D: Poorly differentiated LSCC (H&E x400). E: Poorly differentiated LSCC shows weak expression of Apaf1 (IHC x200).

4. Discussion

In the last few years, many studies recognized many miRNAs with different expression patterns in LSCC and were proved to regulate LSCC progression [16, 17, 18]. MiR-221 acts as an oncogene in tumor initiation and progression by targeting tumor suppressor genes including p27, P57, ARH1, BIM, PUMA, TIMP3, phosphatase and tensin homolog and PTEN. By inhibition of these genes, activation of the AKT pathway occurs, initiating the cell cycle, and inhibiting apoptotic proteins. These effects lead to tumor proliferation [8].

Several transcription factors can regulate miR-221 expression through attachment to a promoter. For example, c-Jun was proved to be responsible for up-regulation in miR-221/222 transcription in extrahepatic cholangiocarcinoma [4]. In addition, both nuclear factor-kappa B and c-Jun, factors involved in carcinogenesis, were shown to be affected by miR-221 [19]. Furthermore, in vivo and in-vitro studies on estrogen receptor-negative breast cancer showed increased levels of miR-221 and were shown to directly target estrogen receptors. Estrogen-independent growth of breast cancer could result from up-regulated miR-221 which up-regulates the inhibitors of the $\beta$ -catenin pathway and down-regulates the inhibitors of Wnt pathway [20].

However, very few studies focused on miR-221 expression in LSCC and its role in LSCC is still unclear. And, its correlation with Apaf-1 and other apoptotic proteins still need further investigation. So, this study focused on the expression of miR-221 in LSCC and the correlation between miR-221 and its possible target, Apaf-1.

Previous studies demonstrated that Apaf-1 is a target of different miRNAs such as miR-17, miR-27a, miR-155, miR-23a and miR-24a [21, 22, 23, 24, 25]. A single study on LSCC cell line correlated Apaf-1expression to miR-221 [11].

In our study, miR-221 levels were significantly up-regulated in LSCC compared to normal nearby tissues. This result was the same as found by The Cancer Genome Atlas in 448 head and neck squamous cancers [26]. Also, Yilmaz et al. found significantly up-regulated miR-221 levels in the plasma of preoperative laryngeal cancer patients in comparison with healthy individuals. Furthermore, they found a significant reduction to normal values in miR-221 levels after surgery [27].

Also, miR-221 levels were significantly up-regulated in late stage compared to the early stage which was found in many tumors [28, 29, 30, 31]. In addition, miR-221 expression was significantly increased with advancing tumor stage and LN involvement.

Concerning Apaf-1 expression, we found that it was down-regulated in LSCC. This result was similar to that found in previous studies on LSCC [11] and other malignancies [23, 24]. Moreover, a negative correlation between miR-221 and Apaf-1 mRNA expressions was found. This finding was consistent with that found by Sun et al. [11] in Hep-2 cell lines. In addition, they found that inhibition of miR-221 in Hep-2 cells induced apoptosis in vitro and in vivo. Their immunohistochemical analysis revealed higher expression of the effector proteins in Apaf-1 apoptotic pathway including caspase-3, -8 and -9 with downregulation of miR-221 [11].

Tumor grade was associated with Apaf-1 gene expression. This result was in the same line with that previously found in colorectal carcinoma, renal cancer, malignant melanoma, transitional cell carcinoma of the bladder, and breast cancer. Moreover, these studies explained Apaf-1 contribution to cancer progression by inactivation by DNA methylation [32, 33, 34, 35, 36]. On the other hand, no significant association between miR-221 expression and tumor grade was found. This finding was similar to that found by previous studies on hepatocellular carcinoma [37, 38].

5. Conclusion

Up-regulation of miR-221 and down-regulation of Apaf-1 were observed in LSSC. In addition, miR-221 expression was significantly increased with advancing tumor stage and LN involvement. So, miR-221 expression can be considered as a possible diagnostic marker in LSCC.

Footnotes

Acknowledgments

We thank Dr. Mohamed Abdelgawad-Department of Clinical Oncology, Faculty of Medicine, Zagazig University, Egypt- for his efforts in this study.

Conflict of interest

None.

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	Total		Stages (I–II) N $=$ 27		Stages (III–IV) N $=$ 23		P
Age
$<$ 60	22	(44%)	18	(81.8%)	4	(18.2%)	0.	6
$\geqslant$ 60	28	(56%)	23	(82.1%)	5	(17.9%)
Gender
Male	40	(80%)	22	(55%)	18	(45%)	0.	78
Female	10	(20%)	5	(50%)	5	(50%)
Site
Glottic	35	(80%)	20	(57.1%)	15	(42.9%)	0.	26
Supraglottic	15	(20%)	7	(46.7%)	8	(53.3%)
Grade
Well	13	(26%)	13	(100%)	0	(0%)	$<$ 0.	001 ${}^{**}$
Moderate	21	(42%)	14	(66.7%)	7	(33.3%)
Poor	16	(32%)	0	(0%)	16	(100%)
Lymph node
Negative	27	(54%)	22	(81.4%)	5	(18.6%)	$<$ 0.	001 ${}^{**}$
Positive	23	(46%)	5	(21.7%)	18	(78.3%)
MiR221gene expression	3.86 $\pm$ 4.14		1.31 $\pm$ 0.89		6.85 $\pm$ 4.47		$<$ 0.	001 ${}^{**}$
APF1gene expression	0.59 $\pm$ 0.35		0.82 $\pm$ 0.23		0.31 $\pm$ 0.25		$<$ 0.	001 ${}^{**}$
APF1 immune expression
Negative	36	(72%)	14	(38.9%)	22	(61.1%)	0.	007 ${}^{*}$
Weak	6	(12%)	5	(83.3%)	1	(16.7%)
Moderate	6	(12%)	6	(100%)	0	(0%)
Strong	2	(4%)	2	(100%)	0	(0%)

Up-regulated miR-221 expression as a molecular diagnostic marker in laryngeal squamous cell carcinoma and its correlation with Apaf-1 expression

Abstract

BACKGROUND:

OBJECTIVES:

METHODS:

RESULTS:

CONCLUSION:

Keywords

Abbreviations

1. Introduction

2. Patients and methods

2.1 RNA extraction and real-time PCR

Table 1 Primer sequence of the studied genes Forward Reverse GADPH TGAAGGTCGGAGTCAACGGATT CATGTGGGCCATGAGGTCCACCA Apaf-1 CACGTTCAAAGGTGGCTGAT TGGTCAACTGCAAGGACCAT miR-221 ACACTCCAGCTGGGAGCTACATTGTCTGCTGG CTCAACTGGTGTCGTGGA U6 CTCGCTTCGGCAGCACA AACGCTTCACGAATTTGCGT

2.3 Immunohistochemistry assessment

2.4 Statistical analysis

3. Results

3.1 Patient characteristics

5. Conclusion

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Primer sequence of the studied genes

Forward Reverse

GADPH TGAAGGTCGGAGTCAACGGATT CATGTGGGCCATGAGGTCCACCA

Apaf-1 CACGTTCAAAGGTGGCTGAT TGGTCAACTGCAAGGACCAT

miR-221 ACACTCCAGCTGGGAGCTACATTGTCTGCTGG CTCAACTGGTGTCGTGGA

U6 CTCGCTTCGGCAGCACA AACGCTTCACGAATTTGCGT