【摘要】目的：探讨KAI－1mRNA在肾细胞癌中的表达及与浸润转移的关系。方法：采用RT－PCR方法检测35例肾癌及癌周正常肾组织KAI－1mRNA的表达。结果：肾癌组织中KAI－1表达率明显低于癌周正常组织（P＜0.05）;有肾周浸润或局部淋巴结转移的患者（Ⅲ、Ⅳ期）骨癌组织中KAI－1表达明显低于Ⅰ、Ⅱ期患者（P＜0.05）。结论：KAI－1基因的低表达可能与肾细胞癌的进展有关。

中图分类号：R737.11文献标识码：A

文章编号：1000－467X(2000)06－0554－04

Decreased KAI－1 expression in advanced renal cell carcinoma

KONG Xian－guo

Department of Urology,Affiliated Tielu Hospital, Shanghai Tiedao University,

Shanghai 200072,P.R.China

ZHANG Yuan－fang, DING Qiang, et al.

Department of Urology,Huashan Hospital,Shanghai Medical University,

Shanghai 200040,P.R.China

【 Abstract】 Objective： To investigate the expression of KAI－1 mRNA in renal cell carcinoma and the relationship with the metastasis. Method： RT－PCR was used to detect KAI－1 mRNA expression in specimens from 35 cases with renal cancer and adjacent normal kidney tissues. Results： KAI－1 expression rate in renal cancer was significantly lower than that in normal kidney tissues (P＜ 0.05); tumors of patients with extrarenal invasion or regional lymph node metastasis (stage Ⅲ orⅣ ) had significantly lower KAI－1 expression levels than tumors of patients with stage Ⅰ or Ⅱ disease (P＜ 0.05).Conclusion： Decreased expression of the KAI－1 gene may be related to disease progression in patients with renal cancer.

Key words： Kidney neoplasms; KAI－1; Metastasis

Metastasis, the main cause of death for most cancer patients, remains one of the most important but least understood aspects of cancer. Both positive and negative regulators of metastasis are likely to exist. A recently discovered antimetastatic gene that is present in prostate cancer has been named KAI- 1. When human prostate cancer samples were transplanted into nude mice, KAI- 1 was found to suppress the ability of prostate cancer cells to metastasize^[1]. KAI- 1 expression appears to be reduced in human cell lines that derive from metastatic prostate tumors, compared with its expression in normal prostate tissue^[1].

The KAI- 1 gene is located on human chromosome 11P11.2～ 13 and encodes a protein of 267 amino acids with a molecular mass of 29 610 daltons. KAI- 1 belongs to a structurally distinct family of membrane glycoproteins.They function in cell- cell and cell- extracellular matrix interactions, thereby potentially influencing the ability of cancer cells to invade tissue and to metastasize^[1]. The importance of KAI- 1 in cancers other than prostate cancer has been evaluated, KAI- 1 expression is up regulated in early pancreatic cancer and decreased in the presence of metastases^[2], and decreased mRNA expression of KAI- 1 correlates with poor prognosis in patients with non small cell lung cancer^[3].

Renal cell carcinomas (RCC) is known to meta- stasize early, and histological features such as cell type, architecture, and tumor grade are of limited prognostic value. Many of the renal cancer patients exhibit evidence for metastatic disease at the time of diagnosis. The reasons for the proclivity of renal cancer cells to metastasize are not known. The aim of the present study was to determine whether there are alterations in KAI- 1 expression in primary renal cancers and evaluate whether KAI- 1 mRNA expression correlates with clinical parameters of renal cancer patients.

1 MATERIALS AND METHODS

1.1 Patients

We studied 35 patients with RCC who underwent surgery between December 1995 and May 1998 at the Department of Urology of Shanghai Huashan Hospital. Their clinical records and histopathological diagnoses were fully documented. According to the Robsin classification of renal cancer, there were 7 patients with stageⅠ disease, 15 patients with stageⅡ ,7 patients with stageⅢ , and 6 patients with stageⅣ .

1.2 Tissue Sampling

Renal cancer tissue samples and adjacent normal kidney tissue samples were frozen in liquid nitrogen immediately after surgical removal and maintained at － 80℃ until use.

1.3 RT- PCR Analysis

Total cellular RNA was purified from frozen tumor or kidney tissues by the acid guanidinium thiocyanate procedure^[4]. On the basis of the nucleotide sequence of KAI- 1^[1],5＇- CAAGATCTATGGGCTCAGCCTGTAT- CAAAGTCACC- 3＇ was used as the sense primer and 5＇-CCAAGCTTTCAGTACTTGGGGACCTTGCTGTAGTC- 3＇as the antisense primer. This primer pair amplifies a 798bp fragment (nucleotides 168- 965). The reaction mixture was subjected to 30 PCR amplification cycles of 50s at 94℃ , 75s at 54℃ , and 90s at 72℃ . β- actin DNA amplification was used as the internal PCR control^[5];the sense primer was 5＇- CTATTGGCA- ACGAGCGGTTC- 3＇ , and the antisense primer was 5＇ CTTAGGAGTGGGGGTGGCTT- 3＇ .The same PCR conditions were used to amplify β-actin DNA, the amplified fragment was 776bp. Tubes containing all ingridents except templates were included in all runs and served as negative reaction controls. The amplified DNA samples were run on a 1.5％ agarose gel, and bands were visualized with ethidium bromide and photographed with a camera. Densitometric analysis of the photographic negatives was used for band quantification^[6].

1.4 Specimen Classification Based on RT- PCR Results

The value obtained for KAI- 1 by densitometry of the band of a given tissue sample was divided by that of β- actin and was referred to as the KAI- 1 expression ratio. When the ratio value of a given specimen was ≥ 0.2, it was considered to indicate positive, and if the value was ＜0.2, it was considered as denoting negative expression.

1.5 Statistical Analysis

The statistical significance of the difference between the incidence of KAI 1 positive expression and several clinical and pathological parameters was assessed by the exact text for 2× 2 table, P＜0.05 was considered to indicate statistical significance.

2 RESULTS

2.1 KAI- 1 Expression in Renal Cancer and Normal Kidney Tissues Analyzed by Quantitative RT- PCR

The KAI- 1 expression ratio ranged from 0 to 1.2 with a mean of 0.748. All the normal kidney tissues were positive. Of the 35 primary renal cancer cases studied, 25(71.4％ ) were positive and 10(28.6％ ) were negative(Fig.1). KAI- 1 expression rate in cancer was significantly lower than that in normal kidney tissues(P＜0.05).

2.2 Relationship between KAI- 1 Gene Expre- ssion and Known Prognostic Factors

Analysis of the 35 patients whose renal cancers were tested for KAI- 1 gene expression revealed that there were no statistically significant relationships (χ² test) between gene expression and the patients- age at surgery or sex. By contrast, KAI- 1 expression was significantly associated with tumor stage (P＜0.05; table 1). Tumors of patients with extrarenal invasion or regional lymph node metastasis (stageⅢ or Ⅳ ) had significantly lower KAI- 1 expression levels than tumors of patients with s