Tumor Biol. DOI 10.1007/s13277-013-1575-4
RESEARCH ARTICLE
Clinical significance of RKIP mRNA expression in non-small cell lung cancer Qin Wang & Xiaodong Wu & Ting Wu & Gui-mei Li & Yi Shi
Received: 10 November 2013 / Accepted: 17 December 2013 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Raf-1 kinase inhibitor protein (RKIP) expression was associated with the onset, development, invasion, and metastasis of numerous tumor types including prostate cancer, melanoma, colorectal cancer, liver cancer, and breast cancer. However, RKIP mRNA expression and the clinical significance in non-small cell lung cancers (NSCLC) remain unresolved. Real-time PCR was performed to detect the expression of RKIP mRNA in 126 pairs of lung tumor tissues (TT) and surrounding normal tissues (sNT). Correlations between RKIP mRNA expression and clinicopathological features were evaluated by statistical analysis. In the 126 patients examined, RKIP mRNA expression was significantly lower in lung TT than the sNT (p0.05). The level of RKIP mRNA expression was found to be significantly downregulated in NSCLC, and the lower mRNA levels correlated with poorer differentiation, advanced pathologic TNM stage in patients with NSCLC. Keywords Raf-1 kinase inhibitor protein . Real-Time PCR . Non-small cell lung cancers Q. Wang : Y. Shi (*) Department of Respiratory and Critical Care Medicine, Jinling Hospital, Clinical School of Nanjing, Second Military Medical University, Nanjing 210002, China e-mail: [email protected] X. Wu : T. Wu Nanjing University School of Medicine, Nanjing, China G.5 cm far from the tumor edge were immediately snap frozen in liquid nitrogen and then stored at −80 °C until use. Necrotic or hemorrhagic tissues were excluded. None of the patients received any chemotherapy or radiotherapy before surgery. Clinic-pathological data were obtained by medical records located in the archives room. The records included the patient age, gender, size of tumor, histological differentiation, lymph nodal status, and pathological stage. The postoperative pathological staging was determined according to the 7th Edition of the TNM classification [21]. The research ethics committee of Jinling Hospital affiliated to Nanjing University School of Medicine approved this protocol and informed written consents were obtained from all participants. RNA extraction and cDNA synthesis Total RNA was isolated from frozen tissue with TRIzol (Invitrogen) following the manufacturer’s protocol. With random hexamer primers, 2 μg RNA was reverse transcribed to cDNA using the PrimeScript™ first-strand cDNA synthesis kit (Takara Inc.) and following the manufacturer’s protocol. Real-time quantitative reverse transcription-polymerase chain reaction Primers for RKIP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed and synthesized by Takara
The difference of the expression of RKIP mRNA in TT and sNT was performed using the paired t test. Associations between RKIP expression and the clinicopathologic characteristics were analyzed using the chisquare test or Fisher’s exact test. All statistical procedures were performed using SPSS (version 17.0 SPSS Inc, Chicago). Values of p< 0.05 were considered as statistically significant.
Results Characteristics of the NSCLC patients The characteristics of all included patients are listed in Table 1. The 126 patients with lung TT and sNT consisted of 95 males and 31 females, aged between 21 and 78 years old (mean 55.6 years). According to the classification of the World Health Organization, the specimens were classified into 63 (50 %) adenocarcinomas, 49 (38.9 %) squamous cell carcinomas, and 14 (11.1 %) others (large cell carcinomas, adenosquamous carcinomas, and carcinoid). The average tumor size was 4.18 ± 1.16 cm (range 1.1– 8.6 cm). Forty-six cases involved tumors ≤3 cm in size, whereas 80 cases involved tumors >3 cm in size. There were 58 negative cases and 68 positive cases for lymph node metastases. RKIP mRNA expression in lung cancer tissue We detected the levels of RKIP mRNA in lung TT and sNT. The relative mRNA level of the RKIP gene (RKIP per GAPDH, mean±SD) showed significant differences between lung cancer tissue (mRNA 1.601±0.072) and the surrounding normal lung tissue (mRNA 2.637±0.042). The paired t test
Tumor Biol. Table 1 RKIP mRNA expression and the association with the clinicopathological factors of non-small cell lung cancer Number of RKIP mRNA expression patients (%) Low High P All patients Gender Male Female Age =60 year old Size of tumor 3 cm Histology Squamous cell carcinoma Adenocarcinoma other Lymph node metastasis (pN) N0 N1-3 p-TNM stages I II III IV
126
67
59
95 (75.4) 31 (24.6)
50 17
45 14
59 (46.8) 67 (53.2)
34 33
25 34
46 (36.5) 80 (63.5)
24 43
22 37
49 (38.9) 63 (50) 14 (11.1)
33 32 2
16 31 12
0.238
0.347
0.381
58 (46.0) 68 (54.0)
25 42
33 26
52 (41.3)
25
27
26 (20.6) 45 (35.7) 3(2.4)
20 19 3
6 26 0
0.104
Fig. 2 The association between RKIP mRNA expression (compared to sNF) and the clinical characteristics of the patients. The results reveal a significant downregulation of RKIP in NSCLC with a poorer N-stage (p=0.019)
0.019*
Relationship between RKIP mRNA expression and clinicopathologic factors
0.015*
* Statistically significant difference (p
RESEARCH ARTICLE
Clinical significance of RKIP mRNA expression in non-small cell lung cancer Qin Wang & Xiaodong Wu & Ting Wu & Gui-mei Li & Yi Shi
Received: 10 November 2013 / Accepted: 17 December 2013 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Raf-1 kinase inhibitor protein (RKIP) expression was associated with the onset, development, invasion, and metastasis of numerous tumor types including prostate cancer, melanoma, colorectal cancer, liver cancer, and breast cancer. However, RKIP mRNA expression and the clinical significance in non-small cell lung cancers (NSCLC) remain unresolved. Real-time PCR was performed to detect the expression of RKIP mRNA in 126 pairs of lung tumor tissues (TT) and surrounding normal tissues (sNT). Correlations between RKIP mRNA expression and clinicopathological features were evaluated by statistical analysis. In the 126 patients examined, RKIP mRNA expression was significantly lower in lung TT than the sNT (p0.05). The level of RKIP mRNA expression was found to be significantly downregulated in NSCLC, and the lower mRNA levels correlated with poorer differentiation, advanced pathologic TNM stage in patients with NSCLC. Keywords Raf-1 kinase inhibitor protein . Real-Time PCR . Non-small cell lung cancers Q. Wang : Y. Shi (*) Department of Respiratory and Critical Care Medicine, Jinling Hospital, Clinical School of Nanjing, Second Military Medical University, Nanjing 210002, China e-mail: [email protected] X. Wu : T. Wu Nanjing University School of Medicine, Nanjing, China G.5 cm far from the tumor edge were immediately snap frozen in liquid nitrogen and then stored at −80 °C until use. Necrotic or hemorrhagic tissues were excluded. None of the patients received any chemotherapy or radiotherapy before surgery. Clinic-pathological data were obtained by medical records located in the archives room. The records included the patient age, gender, size of tumor, histological differentiation, lymph nodal status, and pathological stage. The postoperative pathological staging was determined according to the 7th Edition of the TNM classification [21]. The research ethics committee of Jinling Hospital affiliated to Nanjing University School of Medicine approved this protocol and informed written consents were obtained from all participants. RNA extraction and cDNA synthesis Total RNA was isolated from frozen tissue with TRIzol (Invitrogen) following the manufacturer’s protocol. With random hexamer primers, 2 μg RNA was reverse transcribed to cDNA using the PrimeScript™ first-strand cDNA synthesis kit (Takara Inc.) and following the manufacturer’s protocol. Real-time quantitative reverse transcription-polymerase chain reaction Primers for RKIP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed and synthesized by Takara
The difference of the expression of RKIP mRNA in TT and sNT was performed using the paired t test. Associations between RKIP expression and the clinicopathologic characteristics were analyzed using the chisquare test or Fisher’s exact test. All statistical procedures were performed using SPSS (version 17.0 SPSS Inc, Chicago). Values of p< 0.05 were considered as statistically significant.
Results Characteristics of the NSCLC patients The characteristics of all included patients are listed in Table 1. The 126 patients with lung TT and sNT consisted of 95 males and 31 females, aged between 21 and 78 years old (mean 55.6 years). According to the classification of the World Health Organization, the specimens were classified into 63 (50 %) adenocarcinomas, 49 (38.9 %) squamous cell carcinomas, and 14 (11.1 %) others (large cell carcinomas, adenosquamous carcinomas, and carcinoid). The average tumor size was 4.18 ± 1.16 cm (range 1.1– 8.6 cm). Forty-six cases involved tumors ≤3 cm in size, whereas 80 cases involved tumors >3 cm in size. There were 58 negative cases and 68 positive cases for lymph node metastases. RKIP mRNA expression in lung cancer tissue We detected the levels of RKIP mRNA in lung TT and sNT. The relative mRNA level of the RKIP gene (RKIP per GAPDH, mean±SD) showed significant differences between lung cancer tissue (mRNA 1.601±0.072) and the surrounding normal lung tissue (mRNA 2.637±0.042). The paired t test
Tumor Biol. Table 1 RKIP mRNA expression and the association with the clinicopathological factors of non-small cell lung cancer Number of RKIP mRNA expression patients (%) Low High P All patients Gender Male Female Age =60 year old Size of tumor 3 cm Histology Squamous cell carcinoma Adenocarcinoma other Lymph node metastasis (pN) N0 N1-3 p-TNM stages I II III IV
126
67
59
95 (75.4) 31 (24.6)
50 17
45 14
59 (46.8) 67 (53.2)
34 33
25 34
46 (36.5) 80 (63.5)
24 43
22 37
49 (38.9) 63 (50) 14 (11.1)
33 32 2
16 31 12
0.238
0.347
0.381
58 (46.0) 68 (54.0)
25 42
33 26
52 (41.3)
25
27
26 (20.6) 45 (35.7) 3(2.4)
20 19 3
6 26 0
0.104
Fig. 2 The association between RKIP mRNA expression (compared to sNF) and the clinical characteristics of the patients. The results reveal a significant downregulation of RKIP in NSCLC with a poorer N-stage (p=0.019)
0.019*
Relationship between RKIP mRNA expression and clinicopathologic factors
0.015*
* Statistically significant difference (p
