| Japanese Journal of Clinical Oncology | Pages |
Messenger RNA Expression of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Human Hepatocellular Carcinoma
Introduction
Subjects And Methods
Tissue Samples
RT-PCR
Southern Blotting of PCR Products
Preparation of Standard Curves
Statistical Analysis
Results
Preparation of Standard Curves
Expression of MMPs and TIMPs mRNA in HCC
Expression of MMPs and TIMPs mRNA and Clinicopathological Features
Discussion
Acknowledgments
References
Messenger RNA Expression of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Human Hepatocellular Carcinoma
Methods: Messenger RNA expression of gelatinase A, gelatinase B, matrilysin and tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 were analyzed in 30 patients with hepatocellular carcinoma by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The results were contrasted with the clinicopathological data of the patients.
Results: Enhanced mRNA expression of gelatinase A, gelatinase B and matrilysin in tumor was observed in 20, 22 and 19 of 30 patients, respectively. Enhanced mRNA expression of gelatinase A or gelatinase B and of matrilysin showed trends toward presence of capsular invasion (P = 0.078) and intrahepatic metastasis (P = 0.064), respectively. Concomitant overexpression of gelatinase A and matrilysin was associated with portal invasion, intrahepatic metastasis and recurrence within the first postoperative year (P < 0.05). A modest increase of mRNA expression of TIMP-1 and TIMP-2 in tumor was observed in half of the patients, but did not correlate with any clinicopathological features.
Conclusion: Our results suggest that semiquantitative RT-PCR analysis of MMPs may be helpful in disease management of patients with hepatocellular carcinoma.
INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most common causes of cancer death worldwide. Efforts to attain long survival times have been weakened by the high incidence of intrahepatic recurrences (1,2). An understanding of the molecular mechanisms underlying the progression and recurrence of HCC will facilitate prognosis and therapeutic strategies.
A large body of evidence indicates that matrix metalloproteinases (MMPs) play a crucial role in tumor progression and enhanced MMP expression has been reported in various human malignant tumors (3-5). Several studies have described the expression of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs) in HCC (6-10). Overexpression of gelatinase B in tumor has been reported to associate with capsular invasion (7,8). Increased messenger RNA (mRNA) expression of TIMP-1 and TIMP-2 in tumor has also been reported (7,9). We have demonstrated an association between recurrence within the first postoperative year and enhanced secretion of active forms of gelatinase A and matrilysin (10). We believe that the role of this upregulation of MMPs is crucial for the recurrence of HCC. However, for a clearer picture, it is important to evaluate the relationship between recurrence and the expression of TIMPs in addition to MMPs. For instance, imbalance of the gelatinase A/TIMP-2 mRNA ratio has been correlated with lymph node metastasis in breast carcinoma (11).
In this study, we analyzed mRNA expression of MMPs (gelatinase A, gelatinase B and matrilysin) and TIMPs (TIMP-1 and TIMP-2) in 30 paired specimens of HCC and adjacent non-tumoral liver using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The results were contrasted with the clinicopathological data for the patients.
SUBJECTS AND METHODS
Tissue Samples
Thirty surgical specimen pairs of HCC and adjacent non-tumoral liver were obtained from patients undergoing surgical resection. Informed consent was obtained from each subject. A potentially curative resection was performed on 28 patients. After surgical resection, tissues were immediately frozen in liquid nitrogen and used for RNA extraction. Approximately 100 mg of samples were applied in the experiment. The degree of cell differentiation was classified according to the criteria of Edmondson and Steiner (12). The degree of TNM classification was decided according to the general rules for the clinical and pathological study of primary liver tumor (13). The data for the patients have been described previously (10).
Figure 1. Correlation between band intensities and amounts of serially diluted cDNAs. Standard curves for gelatinase A (a), gelatinase B (b), matrilysin (c), TIMP-1 (d), TIMP-2 (e) and GAPDH (f) cDNA. Vertical and horizontal axes: band intensity (mm) and amount of serially diluted cDNA (µg), respectively. Total RNA was extracted from specimens using the acid guanidinium thiocyanate-phenol-chloroform extraction method and treated with DNase I. The cDNA was synthesized from 1 µg of total RNA using MMLV reverse transcriptase (Perkin-Elmer/Cetus, Norwalk, CT) with random hexamers. The cDNA was amplified with the following PCR oligonucleotides (14): sense 5[prime]-CCA CGT GAC AAG CCC ATG GGG CCC C-3[prime] and antisense 5[prime]-GCA GCC TAG CCA GTC GGA TTT GAT G-3[prime] for gelatinase A; sense 5[prime]-GGT CCC CCC ACT GCT GGC CCT TCT ACG GCC-3[prime] and antisense 5[prime]-GTC CTC AGG GCA CTG CAG GAT GTC ATA GGT-3[prime] for gelatinase B; sense 5[prime]-AGA TGT GGA GTG CCA GAT GT-3[prime] and antisense 5[prime]-TAG ACT GCT ACC ATC CGT CC-3[prime] for matrilysin; sense 5[prime]-TGC ACC TGT GTC CCA CCC CAC CCA CAG ACG-3[prime] and antisense 5[prime]-GGC TAT CTG GGA CCG CAG GGA CTG CCA GGT-3[prime] for TIMP-1; sense CCG AAT TCT GCA GCT GCT CCC CGG TGC ACC CG-3[prime] and 5[prime]-GGA AGC TTT TAT GGG TCC TCG ATG TCG AG-3[prime] for TIMP-2; and 5[prime]-CAG CCG AGC CAC ATC G-3[prime] and 5[prime]-TGA GGC TGT TGT CAT ACT TCT C-3[prime] for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH served as an internal control of the reaction. PCR was carried out for 1 cycle of 94°C for 4 min followed by 20-30 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 30 s. All reactions were controlled without reverse transcriptase. The PCR products were electrophoresed in a 3% Nusieve agarose gel (FMC Bioproducts, Rockland, ME) and transferred to nylon membranes in 0.4 N NaOH. The membranes were hybridized with cDNA probes labeled using the random primer method in 50% formamide-5× Denhardt's solution-3× SSC-100 µg/ml salmon sperm DNA-1% SDS at 42°C. Probes were partial cDNAs obtained using RT-PCR, each of which was revealed to be identical with the respective original gelatinase A, gelatinase B, matrilysin, TIMP-1, TIMP-2 and GAPDH cDNA with DNA sequencing. After overnight hybridization, followed by washing, the membranes were exposed to X-ray films at -70°C. Hybridization signals on the films were quantified with a densitometer (Bio-Rad, Richmond, CA). Standard curves for PCR were prepared as described previously (15). Plasmid (1 µg) containing amplified regions of gelatinase A, gelatinase B, matrilysin, TIMP-1, TIMP-2 or GAPDH was diluted serially and logarithmically. Diluted samples were amplified with PCR using the same intervals and cycles as those used for the clinical samples. Southern blotting of amplified products was performed with radiolabeled gelatinase A, gelatinase B, matrilysin, TIMP-1, TIMP-2 or GAPDH cDNA. The resulting band intensities of the autoradiograms were measured with a densitometer. The band intensity of each sample was expressed as the peak height (mm) of the curve drawn with the densitometer. The correlation between the quantity of cDNA before PCR and band intensities was analyzed using a semilogarithmic scale. The relative amount of mRNA in tumor tissue versus adjacent non-tumoral tissue from the same patient calculated by the standard curve was defined as the tumor to non-tumor expression ratio. Each expression ratio of gelatinase A, gelatinase B, matrilysin, TIMP-1 and TIMP-2 was corrected by the GAPDH expression ratio. The gelatinase A/TIMP-1 and gelatinase B/TIMP-2 expression ratio was calculated as described previously (11). Figure 2. RT-PCR analysis of MMPs and TIMPs mRNA expression in HCC. Representative cases are shown. GAPDH served as an internal control. C and N, matched RNA samples isolated from carcinoma and adjacent non-tumoral liver, respectively. All three cases (cases 2, 7 and 17) with concomitant overexpression of gelatinase A and matrilysin developed recurrence within the first postoperative year, whereas the remaining three cases did not. Statistical analysis was performed using Fisher's exact test or the Mann-Whitney non-parametric test. A p value of <0.05 was considered to be statistically significant and a p value of <0.1 was considered suggestively significant. For semiquantitative detection using PCR, standard curves were made as shown in Fig. 1. The correlation between the quantity of cDNA before PCR (Q) and the band intensities (I) was analyzed using a semilogarithmic scale. The linear relationships, determined by the least-squares approximation, were as follows: I(gelatinase A) = -25.4logQ + 103; I(gelatinase B) = -24.4logQ + 100; I(matrilysin) = -27.5logQ + 112; I(TIMP-1) = -25.0logQ + 108; I(TIMP-2) = -29.7logQ + 117; and I(GAPDH) = -25.6logQ + 108.5. The relationships between band intensities and plasmid concentrations were almost linear within the ranges corresponding to 10-4-10-7 µg of plasmids containing gelatinase A (correlation coefficient r = -0.957, P < 0.05; paired t-test), 10-4-10-7 µg of gelatinase B plasmids (r = -0.979, P < 0.05), 10-4-10-7 µg of matrilysin plasmids (r = -0.964, P < 0.05), 10-4-10-7 µg of TIMP-1 plasmids (r = -0.983, P < 0.05), 10-4-10-7 µg of TIMP-2 plasmids (r = -0.966, P < 0.05) and 10-3-10-6 µg of GAPDH plasmids (r = -0.989, P < 0.05). These results indicate that the amount of transcripts could be evaluated semiquantitatively within these ranges. Semiquantitative RT-PCR was performed in 30 paired specimens of HCC and adjacent non-tumoral liver, each pair from the same patient. When RNA samples had been incubated in RT reactions without reverse transcriptase, no gene products of interest were amplified using PCR (data not shown). This indicates that a trace amount of residual genomic DNA did not show any band even after hybridization with the cDNA probe. Representative data are shown in Fig. 2. Tumors that exhibited a tumor to non-tumor expression ratio of [ge]10 were considered to have enhanced expression of each transcript in tumor compared with adjacent non-tumoral liver. Enhanced mRNA expression of gelatinase A, gelatinase B and matrilysin in tumor was observed in 20, 22 and 19 of 30 patients, respectively. A modest increase, namely a ratio of >5 and <10, in TIMP-1 and TIMP-2 mRNA was observed in 16 and 14 of 30 patients, respectively. The results are summarized in Table 1. There were no significant differences with respect to tumor size, tumor grade, capsular formation, portal invasion, venous invasion or recurrence within the first postoperative year. Enhanced mRNA expression of gelatinase A or gelatinase B and of matrilysin showed trends toward presence of capsular invasion (P = 0.078) and intrahepatic metastasis (P = 0.064), respectively. Among various combinations of overexpression, concomitant overexpression of gelatinase A and matrilysin was found to associate with portal invasion (P = 0.035), intrahepatic metastasis (P = 0.033) and recurrence within the first postoperative year (P = 0.040). A modest increase of TIMP-1 and TIMP-2 mRNA and gelatinase A/TIMP-2 and gelatinase B/TIMP-1 mRNA ratios did not correlate with any clinicopathological features (data not shown). Using semiquantitative RT-PCR, we examined the expression of MMPs and TIMPs in liver tissues from patients with HCC. It should be noted that sampling variations may exist in this kind of experiment and this issue needs further investigation. The mRNA expression of gelatinase A, gelatinase B and matrilysin was increased in about two-thirds of tumor samples. In contrast, a modest increase of TIMP-1 and TIMP-2 mRNA was observed in half of the tumor samples. These results suggest that the balance of MMP/TIMP expression favors MMP in the majority of HCC. Although neither TIMP alone nor the MMP/TIMP mRNA ratio correlated with any clinicopathological features, enhanced mRNA expression of gelatinase A or gelatinase B and of matrilysin showed trends towards the presence of capsular invasion and intrahepatic metastasis, respectively. Because these and some other relationships reached statistical significance in our previous zymographic analysis (10), RT-PCR analysis in this study may be less informative than zymographic analysis. Nevertheless, the present study revealed that concomitant overexpression of gelatinase A and matrilysin was associated with portal invasion, intrahepatic metastasis and recurrence within the first year after surgery. Some of these intrahepatic recurrences were accompanied by distant metastasis. It is well known that portal invasion and intrahepatic metastasis are the most determinant pathological factors for recurrence. We suggest that patients with concomitant overexpression of gelatinase A and matrilysin could represent a high-risk group for developing early recurrences. Consequently, early postoperative screening for recurrence should be performed on patients in this group. On the basis of these results, longer follow-up studies are under way. Although zymographic analysis appears to have an advantage in MMP analyses (10,16), this is a somewhat cumbersome method and has not come into wide use. On the other hand, RT-PCR can be carried out with relative ease and with a much smaller amount of tissue. A relevant internal control can be used in RT-PCR and the quantification of results is easier than in zymographic analysis. RT-PCR has become widespread even in clinical laboratories. Furthermore, based on our results, multiplex RT-PCR can be developed and will be a more powerful method for rapid screening. Our results suggest that semiquantitative RT-PCR analysis of MMPs may be helpful in disease management of patients with hepatocellular carcinoma. Table 1. We thank Mr Daniel Day, Sapporo Medical University, for linguistic assistance. This work was supported by grants-in-aid from the Ministry of Education, Science and Culture (F.I., Y.H., K.I.) and from the Ministry of Health and Welfare (F.I., K.I.), Japan.
RT-PCR
Southern Blotting of PCR Products
Preparation of Standard Curves
Statistical Analysis
RESULTS
Preparation of Standard Curves
Expression of MMPs and TIMPs mRNA in HCC
Expression of MMPs and TIMPs mRNA and Clinicopathological Features
DISCUSSION
Gelatinase A
Gelatinase B
Matrilysin
Gelatinase A and matrilysin
With
Without
P
With
Without
P
With
Without
P
With
Without
P
Tumor size
NS
NS
NS
NS
<3 cm
7/20
2/10
6/22
3/8
7/19
2/11
5/14
4/16
>3 cm
13/20
8/10
16/22
5/8
12/19
9/11
9/14
12/16
Tumor grade
NS
NS
NS
NS
I-II
16/20
9/10
19/22
6/8
16/19
9/11
12/14
13/16
III-IV
4/20
1/10
3/22
2/8
3/19
2/11
2/14
3/16
Capsular formation
13/20
7/10
NS
13/22
7/8
NS
12/19
8/11
NS
9/14
11/16
NS
Capsular invasion
11/13
3/7
0.078
11/13
3/7
0.078
10/12
4/8
NS
8/9
6/11
NS
Portal invasion
9/20
2/10
NS
8/22
3/8
NS
9/19
2/11
NS
8/14
3/16
0.035
Venous invasion
4/20
1/10
NS
3/22
2/8
NS
3/19
2/11
NS
3/14
2/16
NS
Intrahepatic metastasis
12/18
3/10
NS
12/22
3/8
NS
12/19
3/11
0.064
10/14
5/16
0.033
Recurrence within
first postoperative year7/19
1/9
NS
6/19
2/9
NS
7/19
1/9
NS
6/12
2/16
0.040
Acknowledgments
References
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