Detection of matrix metalloproteinase gene expression by reverse transcription-polymerase chain reaction in human hepatocellular carcinoma

International Hepatology Communications ELSEVIER

6 (1996)

36-42

Detection of matrix metalloproteinase gene expression by reverse transcription-polymerase chain reaction in human hepatocellular carcinoma Iwata Ozaki, Kyosuke Yamamoto*, Toshihiko Mizuta, Yoichi Setoguchi, Fumitaka Morito, Takahiro Sakai Division

of Hepatology

and Metabolism, Department of Internal Medicine, 5-1-I Nabeshima, Saga 849, Japan

Saga Medical

School.

Received 5 August 1996;accepted 14 August 1996

Abstract

Matrix metalloproteinase(MMP) has been reported to play an important role in the processof tumor developmentand metastasisthrough the alterations of cell-cell and/or cell-extracellular matrix interactions. We studied the gene expressionof matrix metalloproteinase MMP-2 and MMP-7, and also TIMP-1, one of the inhibitors of metalloproteinase,in the tissue of human hepatocellular carcinoma by using semi-quantitative reversetranscription-polymerasechain reaction (RT-PCR) method. MMP-2 and MMP-7 have been expressedin advanced hepatocellularcarcinomasas well as in non-cancerous, chronically diseasedliver tissue, and the expressionlevels of these MMPs in human hepatocellularcarcinoma(HCC) tissuesweregreater than that of non-canceroustissues.This data suggeststhat MMP-2 and MMP-7 may be involved in the processof the development, invasion and metastasisof HCCs.

Keywords: Gene expression;Hepatocellularcarcinoma;Metalloproteinase;RT-PCR

* Corresponding author.Tel.: + 81952316511; fax: + 81 952323026. 0928-4346/96/$12.00 Copyright0 1996ElsevierScience IrelandLtd. All rightsreserved PII

SO928-4346(96)00324-6

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1. Introduction The process of tumor development and metastasis appears to involve the alterations of cell-cell and/or cell-extracellular matrix (ECM) interactions. Recent study has indicated the matrix metalloproteinases (MMPs), which are the degrading enzyme of ECM, may play an important role in these processes [l]. There have been, so far, few studies on MMP in human hepatocellular carcinoma (HCC), probably because of the small mRNA expressions of MMPs in HCC tissues. We examined the gene expression of 72 kD gelatinase (MMP-2), pump-l (MMP7) and one of the inhibitors of MMPs (TIMP-1) in human HCC tissue by using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method [2].

2. Materials

and methods

2.1. Patients We studied 15 cases of HCC associated with surrounding non-cancerous liver tissue including four chronic hepatitis and eleven liver cirrhosis diagnosed by histopathological examination under light microscopy. We also examined three cases of normal controls without liver diseases. The clinical details of these cases are listed in Table 1. The histopathological grading of HCC was based on Edmondson’s grading system [3] and the clinical stage was based on TNM classification system by UICC [4]. 2.2. Liver tissues Liver tissue samples of HCC and surrounding non-cancerous lesions were obtained from each patients by surgical resection performed for the treatment of patients with hepatic neoplasms or by autopsy performed within 3 h after death at the Hospital of Saga Medical School. The portion of each human liver sample was used for histopathological diagnosis by light microscopy. The remaining liver tissue was immediately frozen in liquid nitrogen and stored at - 80°C until RNA extraction. 2.3. RNA extraction and semi-quahjication

by RT-PCR

Total RNA was extracted from liver tissue by using the guanidium method as previously described [5]. The sequence of oligonucleotide primers for RT-PCR was described as follows: MMP-2; nt 1937-1957, nt 2430-2450 [6]; MMP-7; nt 350-371, nt 601-620 [7]; TIMP-1; nt 310-320, nt 575-594 [S]; p-actin; nt 101-121, nt 1192-1211 [9].

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cDNA (complementary DNA) was synthesized from 0.2 to 2 pg of total RNA with reverse transcriptase (BRL, Gaithersburg, MD) with random primers (Takara, Kyoto). Subsequently reverse transcription reaction was used for PCR amplification with forward and reverse primers by using PCR kit with Taq polymerase (Ampli TaqTM, Takara). PCR reaction mixture was subjected to 15-40 successive cycles of amplification consisting of heat denaturation (92°C for 1 min), annealing (55°C for 1 min) and extension (72°C for 2 min). PCR products were size-fractionated in 2% agarose gels and visualized under UV light. Intensities of PCR products were evaluated by using nega-film of PCR products with densitometric scanning.

3. Results

3.1. Smi-quantljkation

of matrix metalloproteinase

by RT-PCR

assay

For the semi-quantification of each mRNA by using RTPCR, we first determined the optimal amount of applied RNA or optimal cycles of PCR [2]. The PCR products of serially diluted RNA samples, 1- 10 - 5 pug, were visualized by ethidium bromide staining and UV light (Fig. 1) and evaluated by densitometry (Fig. 2). The results showed that the signal increased exponentially according to the total amount of RNA. Moreover, the PCR products by changing the PCR cycles from 15 to 40 cycles showed that the signals of the RT-PCR product increased exponentially as the number of PCR cycles increased (Figs. 1 and 2). Taking these together, we examined the mRNA levels of each matrix metalloproteinases in human liver tissue

gRNA

MYP-7 271 b.p.

MMP-2 514 b.p.

TIMP-1 296 b.p.

M: QX174/Hlncll

v

digest

Fig. 1. RT-PCR products of MMP-2, MMP-7, TIMP-I and b-actin Relationship between the PR-PCR products and amount of total number of PCR (right). M: size marker, OXI74/HincII digest.

in 2% agarose gel electrophoresis. RNA applied (left) and the cycle

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1. Ozaki et al. J International Hepatology Communications 6 (1996) 36-42

Total RNA

( wg )

Fig. 2. Densitometric semiquantification of the RT-PCR products showed association with the amount of RNA applied (A) and with the number of PCR cycle (B). MMP-7 (0), MMP-2 (a), TIMP-1 (W), /3-actin (A).

at the condition of reverse transcription from 2 ,ug of total RNA and subsequent 30 cycles of PCR for MMP-2, from 1 lug and 25 cycles of PCR for MMP-7 and TIMP-1, and from 0.2 pug and 25 cycles for /3-actin, respectively. 3.2. Expression of matrix metalloproteinases in HCC

We determined the mRNAs for matrix metalloproteinases in human liver tissues. As shown in Fig. 3, MMP-2 showed no expression in normal liver tissues, where as Non-cancerous llver :ontrol

-

HCC ’

MMP-7

241 b.p.

MMP-2

514 b.p.

TIMP-1

294 b.p.

pactin

540 b.p.

M: ~$X174/Hlncil

digest

Fig. 3. Gene expression of MMP-2, MMP-7, TIMP-1 and B-actin in human liver tissue detected by RT-PCR. The lane numbers shown are as follows; 1: Case 2, 2: Case 3, 3: Case 4, 4: Case 9, 5: Case 10, 6: Case 11. Case number is indicated in Table 1.

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MMP-7 showed weak but positive expressions. In the non-cancerous liver tissues, MMP-2 showed faint expression only in the part of the cases, whereas MMP-7 showed slightly increased expressions in all cases. In the tissue of HCC, MMP-2 showed fairly increased expressions in five cases with multiple HCC (Case 9 in Table 1; lane 4 in Fig. 3) or with extrahepatic metastasis (Case 4, 6, 10 and 11 in Table 1; lane 2, 5 and 6 in Fig. 3). MMP-7 showed significantly increased expressions in twelve cases among fifteen cases of HCC (Case 2- 13 in Table 1; lane l-6 in Fig. 3). When compared with the expression in non-cancerous liver tissue, MMP-2 and MMP-7 showed significantly increased expressions in HCC of the corresponding cases. Although TIMP-1 increased in the noncancerous and neoplastic liver tissue compared with normal control liver, there was no differences in expressions of TIMP-1 between the tissues from chronically diseased non-cancerous liver and HCCs. Expression of p-actin was well maintained among all the samples examined.

4. Discussion

HCC is the characteristic tumor which frequently shows invasion into the blood vessels from the early stage and that shows intrahepatic and extrahepatic metastasis [lo]. In the malignant neoplasms other than HCC, the increased expression of MMP-2 and MMP-9, or the decreased expression of TIMP-1 have been reported to suggest the involvement of these matrix metalloproteinase and/or imbalance between MMPs and TIMPs in the process of invasion and metastasis [l]. Recent studies using immunoassay for matrix metalloproteinase have showed that the serum levels of MMP-9 [l l] increased in subjects with HCC. These results imply the possibility of the increased production of MMPs in the whole body or the decreased catabolism in the subjects of HCC. We succeeded to determine mRNA levels of MMP-2 and MMP-7 in the human liver tissue including HCC by using semi-quantitative RT-PCR assay. The present study demonstrated that MMP-2 and MMP-7 have been expressed in many HCCs as compared with normal liver. This data suggests the involvement of these MMPs in the development of HCC and in the process of tumor invasion or metastasis of HCC.

References [I] Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis. An imbalance of positive and negative regulation. Cell 1991; 64: 327-336. [2] El-Deiry WS, Nelkin BD, Celano P, Yen R-WC, Falco JP, Hamilton SR, Baylin SB. High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer. Proc Nat1 Acad Sci USA 1991; 88: 3470-3474. [3] Edmondson HA, Steiner PE. Primary carcinoma of the liver: a study of 100 cases among 48 9000 necropsies. Cancer (Phila.) 1954; 7: 462-503. [4] Hermanek P, Sobin LH. editors. TNM Classification of Malignant Tumors. 4th Edn. UICC. Berlin: Spring Verlag, 1987.

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