j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 5 1 5 e5 2 0
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Prognostic value of microRNA-100 in esophageal squamous cell carcinoma Shu Zhou, BS, Benqiang Yang, MD,* Yanjuan Zhao, BS, Sen Xu, BS, Hongwei Zhang, BS, and Zhao Li, BS Department of Radiodiagnosis, the General Hospital of Shenyang Military Region, Shenyang, China
article info
abstract
Article history:
Background: MicroRNA-100 (miR-100) has been demonstrated to be implicated in tumori-
Received 11 May 2014
genesis and tumor progression of human esophageal squamous cell carcinoma (ESCC).
Received in revised form
However, its expression patterns in ESCC are controversial and its prognostic value in this
7 June 2014
malignancy has not been fully elucidated. The aim of this study was to investigate the
Accepted 1 July 2014
expression and clinical significance of miR-100 in ESCC.
Available online 6 July 2014
Materials and methods: Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to detect expression levels of miR-100 in 120 self-paired
Keywords:
specimens of ESCC and adjacent normal esophageal tissues. The associations of miR-100
MicroRNA-100
expression with clinicopathologic features, locoregional progression-free survival (LPFS),
Esophageal squamous cell carcinoma
distant progression-free survival (DPFS), and overall survival (OS) of patients with ESCC
Real-time quantitative PCR
were statistically analyzed.
Locoregional
Results: Compared with adjacent normal esophageal tissues, the expression levels of miR-100
progression-free survival
in ESCC were significantly decreased (normal versus ESCC: 3.53 1.22 versus 1.89 0.38,
Distant progression-free survival
P 6 cm 68 (56.67) 6 cm 52 (43.33) Clinical stage I 20 (16.67) II 50 (41.67) III 30 (25.00) IV 20 (16.67) Differentiation Well 44 (36.67) Moderately 56 (46.67) Poorly 20 (16.67) TNM clinical classification Depth of invasion T1 20 (16.67) T2 45 (37.50) T3 25 (20.83) T4 30 (25.00) Regional lymph nodes N0 55 (45.83) N1 65 (54.17) Distant metastasis M0 70 (47.9) M1 50 (52.1)
MiR-100 expression (n, %)
P
Low
High
46 (54.12) 20 (57.14)
39 (45.88) 15 (42.86)
NS
40 (61.54) 26 (47.27)
26 (38.46) 29 (52.73)
NS
30 (53.57) 26 (65.00) 10 (41.67)
26 (46.43) 14 (35.00) 14 (58.33)
NS
36 (52.94) 30 (57.69)
32 (47.06) 22 (42.31)
NS
20 26 20 0
0 24 10 20
0.008
(100.00) (52.00) (66.67) (0)
(0) (48.00) (33.33) (100.0)
25 (56.82) 30 (53.57) 10 (50.00)
19 (43.18) 26 (46.43) 10 (50.00)
NS
6 20 15 25
14 25 10 5
0.02
(30.00) (44.44) (60.00) (83.33)
(70.00) (55.56) (40.00) (16.67)
31 (56.36) 35 (53.85)
24 (43.67) 30 (46.15)
NS
26 (37.14) 40 (80.00)
44 (62.86) 10 (20.00)
0.008
Bold P value ¼ Differences with statistical significance. NS ¼ not significant; TNM ¼ tumor node metastasis.
confirmed to have ESCC and were treated with radiotherapy alone between 1999 and 2008, with a total dose of 34e80 Gy (median 60 Gy). A total of 56, 40, and 24 lesions were located in the upper-, middle-, and lower-thoracic esophagus, respectively. The length of the lesions varied from 1.2 to 16 cm (median 6 cm). Table 1 summarized the clinicopathologic characteristics of all 120 ESCC patients enrolled in this study. All 120 ESCC patients were followed up for at least 5 y or until death. The patients were followed with office visits and physical examinations every 3 mo for the first year and then every 6 mo for the next 2 y, and finally annually. The diagnostic examinations consisted of esophagography, computed tomography, chest x-ray, abdominal ultrasonography, and bone scan when necessary to detect recurrence and/or metastasis. The overall survival (OS) was defined as the time from the start date of follow-up to the date of death or the end date of follow-up. The locoregional progression-free survival (LPFS) was defined as the time from diagnosis to tumor locoregional progression (the first end point) or cancer-
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 5 1 5 e5 2 0
517
relative death (the second end point). The distant progressionfree survival (DPFS) was defined as the time from diagnosis to tumor distant progression.
2.2.
Real-time quantitative RT-PCR for miRNA
Real-time quantitative RT-PCR was performed to detect expression levels of miR-100 in 120 self-paired specimens of ESCC and adjacent normal esophageal tissues. Total RNA was isolated using the RNeasy Maxi Kit (Qiagen, Germany), according to the manufacturer’s protocol. The stem-loop RT primers were designed as follows: miR-100 50 -GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACC ACA AGT -30 and RNU6B 50 -CGC TTC ACG AAT TTG CGT GTC A-30 . Real-time quantitative RT-PCR was performed using the miScript Reverse Transcription and miScript SYBR Green PCR Kit, according to the manufacturer’s protocol (Qiagen, Germany). The PCR primers were designed as follows: miR-100 sense, 50 -GCG GCA ACC CGT AGA TCC GAA-30 and antisense, 50 -GTG CAG GGT CCG AGG T-30 ; RNU6B sense, 50 -CGC TTC GGC AGC ACA TAT ACT A-30 , and antisense, 50 -CGC TTC ACG AAT TTG CGT GTC A-30 . The 1 mg total RNA was reverse transcribed into complementary DNA using the miScript Reverse Transcription Kit. The 20 mL PCR system consisted of 10 mL of 2 QuantiTect SYBR Green PCR Master Mix, 2 mL of 10 miScript Universal Primer, 300 nM of forward primers, 1 mL of complementary DNA template, and RNase-free water. The reactions were incubated in 96-well optical plates at 95 C for 6 min and then followed by 30 cycles of 95 C for 20 s and 56 C for 10 min. The deltaedelta Ct method was used to calculate the fold change. For each sample, all experiments were done in triplicate.
2.3.
Statistical analysis
Statistical analysis in the present study was performed using SPSS software (SPSS Standard version 12.0; SPSS Inc Chicago, IL). The experimental data were representative of three independent experiments. The differences in miR-100 expression levels in ESCC and adjacent normal esophageal tissues were analyzed by two-way analysis of variance. The relationships between miR-100 expression and various clinicopathologic data of patients with ESCC were evaluated by the chi-square test. KaplaneMeier survival curves were constructed with tumor locoregional progression, distant progression, and death as the end points. Univariate and multivariate analyses were performed using Cox proportional hazard model to evaluate the correlation between miR-100 expression and patients’ prognosis. Differences were considered statistically significant when P was
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Prognostic value of microRNA-100 in esophageal squamous cell carcinoma Shu Zhou, BS, Benqiang Yang, MD,* Yanjuan Zhao, BS, Sen Xu, BS, Hongwei Zhang, BS, and Zhao Li, BS Department of Radiodiagnosis, the General Hospital of Shenyang Military Region, Shenyang, China
article info
abstract
Article history:
Background: MicroRNA-100 (miR-100) has been demonstrated to be implicated in tumori-
Received 11 May 2014
genesis and tumor progression of human esophageal squamous cell carcinoma (ESCC).
Received in revised form
However, its expression patterns in ESCC are controversial and its prognostic value in this
7 June 2014
malignancy has not been fully elucidated. The aim of this study was to investigate the
Accepted 1 July 2014
expression and clinical significance of miR-100 in ESCC.
Available online 6 July 2014
Materials and methods: Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to detect expression levels of miR-100 in 120 self-paired
Keywords:
specimens of ESCC and adjacent normal esophageal tissues. The associations of miR-100
MicroRNA-100
expression with clinicopathologic features, locoregional progression-free survival (LPFS),
Esophageal squamous cell carcinoma
distant progression-free survival (DPFS), and overall survival (OS) of patients with ESCC
Real-time quantitative PCR
were statistically analyzed.
Locoregional
Results: Compared with adjacent normal esophageal tissues, the expression levels of miR-100
progression-free survival
in ESCC were significantly decreased (normal versus ESCC: 3.53 1.22 versus 1.89 0.38,
Distant progression-free survival
P 6 cm 68 (56.67) 6 cm 52 (43.33) Clinical stage I 20 (16.67) II 50 (41.67) III 30 (25.00) IV 20 (16.67) Differentiation Well 44 (36.67) Moderately 56 (46.67) Poorly 20 (16.67) TNM clinical classification Depth of invasion T1 20 (16.67) T2 45 (37.50) T3 25 (20.83) T4 30 (25.00) Regional lymph nodes N0 55 (45.83) N1 65 (54.17) Distant metastasis M0 70 (47.9) M1 50 (52.1)
MiR-100 expression (n, %)
P
Low
High
46 (54.12) 20 (57.14)
39 (45.88) 15 (42.86)
NS
40 (61.54) 26 (47.27)
26 (38.46) 29 (52.73)
NS
30 (53.57) 26 (65.00) 10 (41.67)
26 (46.43) 14 (35.00) 14 (58.33)
NS
36 (52.94) 30 (57.69)
32 (47.06) 22 (42.31)
NS
20 26 20 0
0 24 10 20
0.008
(100.00) (52.00) (66.67) (0)
(0) (48.00) (33.33) (100.0)
25 (56.82) 30 (53.57) 10 (50.00)
19 (43.18) 26 (46.43) 10 (50.00)
NS
6 20 15 25
14 25 10 5
0.02
(30.00) (44.44) (60.00) (83.33)
(70.00) (55.56) (40.00) (16.67)
31 (56.36) 35 (53.85)
24 (43.67) 30 (46.15)
NS
26 (37.14) 40 (80.00)
44 (62.86) 10 (20.00)
0.008
Bold P value ¼ Differences with statistical significance. NS ¼ not significant; TNM ¼ tumor node metastasis.
confirmed to have ESCC and were treated with radiotherapy alone between 1999 and 2008, with a total dose of 34e80 Gy (median 60 Gy). A total of 56, 40, and 24 lesions were located in the upper-, middle-, and lower-thoracic esophagus, respectively. The length of the lesions varied from 1.2 to 16 cm (median 6 cm). Table 1 summarized the clinicopathologic characteristics of all 120 ESCC patients enrolled in this study. All 120 ESCC patients were followed up for at least 5 y or until death. The patients were followed with office visits and physical examinations every 3 mo for the first year and then every 6 mo for the next 2 y, and finally annually. The diagnostic examinations consisted of esophagography, computed tomography, chest x-ray, abdominal ultrasonography, and bone scan when necessary to detect recurrence and/or metastasis. The overall survival (OS) was defined as the time from the start date of follow-up to the date of death or the end date of follow-up. The locoregional progression-free survival (LPFS) was defined as the time from diagnosis to tumor locoregional progression (the first end point) or cancer-
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 5 1 5 e5 2 0
517
relative death (the second end point). The distant progressionfree survival (DPFS) was defined as the time from diagnosis to tumor distant progression.
2.2.
Real-time quantitative RT-PCR for miRNA
Real-time quantitative RT-PCR was performed to detect expression levels of miR-100 in 120 self-paired specimens of ESCC and adjacent normal esophageal tissues. Total RNA was isolated using the RNeasy Maxi Kit (Qiagen, Germany), according to the manufacturer’s protocol. The stem-loop RT primers were designed as follows: miR-100 50 -GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACC ACA AGT -30 and RNU6B 50 -CGC TTC ACG AAT TTG CGT GTC A-30 . Real-time quantitative RT-PCR was performed using the miScript Reverse Transcription and miScript SYBR Green PCR Kit, according to the manufacturer’s protocol (Qiagen, Germany). The PCR primers were designed as follows: miR-100 sense, 50 -GCG GCA ACC CGT AGA TCC GAA-30 and antisense, 50 -GTG CAG GGT CCG AGG T-30 ; RNU6B sense, 50 -CGC TTC GGC AGC ACA TAT ACT A-30 , and antisense, 50 -CGC TTC ACG AAT TTG CGT GTC A-30 . The 1 mg total RNA was reverse transcribed into complementary DNA using the miScript Reverse Transcription Kit. The 20 mL PCR system consisted of 10 mL of 2 QuantiTect SYBR Green PCR Master Mix, 2 mL of 10 miScript Universal Primer, 300 nM of forward primers, 1 mL of complementary DNA template, and RNase-free water. The reactions were incubated in 96-well optical plates at 95 C for 6 min and then followed by 30 cycles of 95 C for 20 s and 56 C for 10 min. The deltaedelta Ct method was used to calculate the fold change. For each sample, all experiments were done in triplicate.
2.3.
Statistical analysis
Statistical analysis in the present study was performed using SPSS software (SPSS Standard version 12.0; SPSS Inc Chicago, IL). The experimental data were representative of three independent experiments. The differences in miR-100 expression levels in ESCC and adjacent normal esophageal tissues were analyzed by two-way analysis of variance. The relationships between miR-100 expression and various clinicopathologic data of patients with ESCC were evaluated by the chi-square test. KaplaneMeier survival curves were constructed with tumor locoregional progression, distant progression, and death as the end points. Univariate and multivariate analyses were performed using Cox proportional hazard model to evaluate the correlation between miR-100 expression and patients’ prognosis. Differences were considered statistically significant when P was
