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Chinese Journal of CancerResearch 16(2): 90 --92, 2004

CORRELATION ANALYSIS BETWEEN STK15 GENE AND LARYNGEAL CARCINOMA ZHAO Xu ~z~Jl~ 1, SUN Kai-lai ~ ] x ~

LI Fu-cai ~ : T J 1.

1,

LI Ying-hui ~ : ~

1,

XU Zhen-ming ~,~,~Y]

2,

1Department of Medical Genetics, China Medical University, Shenyang 110001," 2Department of Otolaryngology, 463 Hospital of PLA, Shenyang 110001 C L C number: R739.65

D o c u m e n t code: A

Article ID: 1000-9604(2004)-02-0090-03

ABSTRACT Objective: To explore the relationship between STK15 gene abnormal expression and laryngeal carcinoma. Methods: Tumor tissues and matched normal tissues were taken from 55 LSCC patients. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect STK15 expression in 110 specimens. Results: In 38 of the 55 cases (69.1%), the STKI5 expression at the mRNA levels was higher than that of the paired normal tissue. The ratio of ADV (average density value) of STKI5 gene to ADV of 13-actin gene was 1.22x~0.49 in the cancer tissue, and 0.99i'0.54 in the paired normal tissue with a significant difference (t=4.539, P<0.01). Conclusion: There was obvious association between the STK15 overexpression and laryngeal carcinoma. It may serve as an alternative mechanism of activating the pathogenesis of human laryngeal squamous cell carcinoma. Key words: Laryngeal carcinoma; STK15; Centrosome abnormality; RT-PCR

STK15 (also known as BTAK, Aurora-A) is a human serine/threonine kinase and essential for chromosome segregation and centrosome functions[~]. The STK15 gene is amplified, and its transcript is also highly expressed in various cancers. Overexpression of STK15 protein induces increased numbers of centrosome, aneuploidy, and transformation of the cells [2]. Received date: Oct. 9, 2003; Accepted date: Feb. 19, 2004. Foundation item: This work was supported by the National

Natural Science Foundation of China (No.30171008) and the Natural Science Foundation of Liaoning Province (No.2001101039). "Author to whom correspondence should be addressed. Phone: (0086-24)-23256666 ext. 5324; E-mail: .X.u [email protected]; Biography: ZHAO Xu(1977-), Female, master of medicine, China Medical University, majors in medical genetics.

In order to explore expression of STK15 gene in the development of human LSCC, we tested both laryngeal squamous cell carcinoma tissues and paired normal tissues from 55 northeastern patients for STK15 ezpression by RT-PCR.

MATERIALS AND METHODS Samples

55 LSCC tissues and matched normal tissues were obtained from the No. 463 Hospital of Air-Force. All cases were first affected patients of laryngeal carcinoma in northeast of China from November 2000 to April 2003, and did not receive radiotherapy and chemotherapy before surgery. All cases were confirmed by clinical and pathological data and all tumor tissue fragments examined by molecular analysis were bordered by another fragment that was processed for histological diagnosis and confirmed as being malignant. Normal specimens were taken from the distal resection margin, which were at least 2 cm apart from tumor tissues at the time of surgery. All tissues were quickly frozen at -70°C until analysis. R N A Isolation and R e l a t i v e R e v e r s e TranscriptaseP o l y m e r a s e Chain Reaction ( R T - P C R ) Analysis

Total RNA was extracted from the tissues using TRIZOL Reagent (GIBCO-BRL) according to the manufacturer's instructions, and the RNA samples were resuspended in 100% formamide and quantified spectrophotometrically at 260 nm. All RNA isolates had an OD260:OD2s0 between 1.8 and 2.0, indicating clean RNA isolates. RT-PCR was performed according to the protocol provided with the SuperScript one-step RT-PCR system (Takara). Oligo-(dT)z0 (Takara) was used as primer in cDNA synthesis. Total RNA (2 gtg) was combined with 0.1 ~tg oligo-dT, 10 mmol/L dNTPs, 40 mmol/L MgCI2,

Chinese Journal o f Cancer Research 16(2): 90 --92, 2004

2 p.1 10xRT Buffer and 10 U AMV Reverse Transcriptase (Takara), and RNase free H20 was added to a total volume of 20 I-d. After incubation at 30°C for 10 min, the RT mix was preheated at 42°C for 60 min to denature secondary structures and then stopped by heating at 99°C for 5 min. The mixture was then cooled rapidly to 5 °C for 5 min. The cDNA stock was stored at -70°C. The resultant cDNA was used as template for PCR reactions with STK15 gene and 13-actin primers. To insure that no false positive PCR fragments would be generated from contaminating genomic DNA, primer sequences were designed to span intron regions. The primers correspond to STK15 gene exon 8 - 9 (Genebank accession #AF011468, 553 base-pair product) or 13-actin (Genebank accession #NM001101, 300 base-pair product). Primer sequences were as follows: STK15: 5'-GAC TAC CTG CCC CCT GAA AT - 3 ' and 5'-GCA CAA TTC TCG TGG CTA CTT -3'; 13-actin: 5'-AGA GCT ACG AGC TGC CTG AC - 3 ' and 5'-AGT ACT TGC GCT CAG GAG GA -3'. Polymerase chain reactions were performed on a GeneAmp PCR system 2400 (Perkin-Elmer) using-100 ng of cDNA, 10 pmoles of STK15 and 13-actin primers (synthesized by Sangon biotech, Shanghai, China), 2 mmol/L of dNTPs, 1 unit of Taq Polymerase (Promega) and 10xTaq polymerase buffer in a 25 lal volume. The PCR program initially started with a 94°C denaturation for 8 min, followed by 30 cycles of 94°C/30s, 60°C/45s, 72°C/1 rain. The PCR samples were electrophoresed on 1% agarose gels in TAE buffer [40 mmol/L Tris-acetic acid pH 8.0, 1 mmol/L EDTA]. The gels were photographed on top of a 280 nm UV light box and digitally captured with GDS8000 imaging system (Bio-Rad, US). The quantity and base pair size of the PCR generated DNA fragments were estimated relative to DNA ladder standards and densitometry values were measured with the Fluor Chem V2.0 Stand Alone program. RT-PCR values were presented as a ratio of STK15's signal divided by the 13-actin signal in the same selected linear (Figure 1). M

T r

N

1" r"

N

T

N

71"

N

I

Fig. 1. A representative RT-PCR gel of STK15 expression from 4 cases and a negative control. N: normal tissue, T: tumor tissue, M: marker, ¢ X174/Hae III marker. The product size of STK15 gene was 553bp and 13-actin was 300bp.

91

Statistical Analysis Data were analyzed using the SPSS software. RT-PCR signals were averaged from 2 replicates using cDNA from 55 LSCC and 55 Control samples. The differences between tumor group and paired normal group on the RT-PCR ratio values were analyzed using Student's t-test. Two-sided P-values below 0.05 were considered to be statistically significant.

RESULTS In 38 of the 55 cases (69.1%), the STK15 gene expression at mRNA level in tumor tissue was higher than that in the paired normal tissue. A representative RT-PCR gel was shown in Figure 1. The ratio of ADV (average density value) o f STK15 gene to ADV o f [3-actin gene was 1.22+0.49 in the cancer tissue, and 0.99+0.54 in the paired normal tissue with a significant difference (t=-4.539, P<0.01) (Figure 2). There was obvious association between the STK15 gene overexpression and laryngeal carcinoma. Tumor tissues

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Fig. 2. A summary of data for the 55 cases of STK15 gene expression.

DISCUSSION The centrosome is the major imcrotubule organizing center (MTOC) of human cells [3'4]. It maintains genomic stability by establishing the bipolar spindles during cell division and executes accurate segregation of chromosomes during mitosis [5"7]. Aberrations in the centrosome duplication cycle result in the formation o f monopolar or multipolar or multipolar spindles, and it has been long proposed that such aberrations may cause aneuploidy and contribute to cancer development [2'7]. Many types of tumors feature a high proportion of cells with centrosomal abnormalities and centrosome anomalies have been reported to arise at early stages of tumor formation and to expand concomitantly with tumor progression ts-~°3. Moreover, centrosome amplification in tumors and cell lines has been linked to numerous genetic aberrations, including overexpression of STK15 [11l. STK15 gene encodes a putative member of protein

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Chinese Journal of Cancer Research 16(2): 90--92, 2004

serine/threonine kinase family localized on chromosome 20q13 t12'13]. In proliferating cells, STK15 protein was found to be localized to the spindle pole during mitosis, especially from prophase through anaphase t1,14,ls]. From its localization data, STK15 was supposed to be required for a centrosome function(s), such as sister chromosome segregation and spindle formation. STK15 gene and its related kinases have been implicated in regulating equal segregation o f chromosomes during mitotic cell division cycle [~6]. The fact that overexpression of STK15 protein leads

to extra copies of centrosomes, aneuploidy, and transformation of the cells has shown its critical role in tumorigenesis [1'17]. As a possible pathway, we studied the expression of STK15 gene in LSCC tissues and the paired normal tissues. Our results led us to conclude that STK15 gene overexpression may lead to STK15 protein overexpression, serving as an alternative mechanism of activating the pathogenesis of human laryngeal carcinoma through centrosome amplification. In summary, there may be an important pathway of centrosome amplification in laryngeal carcinoma:

STK15 gene overexpression---> overexpressing STK15 protein Centrosome amplification--->Genomic instability~Tumor Although STK15 gene overexpression may not constitute a major primary cause for centrosome amplification in tumors, the hypothesis that STK15 gene overexpression contributes to tumor development remains attractive and is worthy of further investigation.

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