Expression and alteration of insulin-like growth factor II-messenger RNA in hepatoma tissues and peripheral blood of patients with hepatocellular carcinoma

Abstract

Aim: To investigate the clinical values of serum free insulin-like growth factor II (IGF-II) levels and IGF-II mRNA in hepatocellular carcinoma (HCC) tissues and peripheral blood for diagnosis of HCC and monitoring of extrahepatic metastasis.

Methods: Total RNAs were extracted from HCC tissues or peripheral blood mononuclear cells from patients with HCC, liver diseases devoid of cancer, non-hepatic tumors, and healthy controls, respectively. IGF-II cDNAs were synthesized through random primers and reverse-transcriptase, amplified by polymerase chain reaction (PCR), and confirmed by DNA sequencing analysis. Serum free IGF-II levels in patients with different liver diseases were analyzed by an enzyme-linked immunosorbent assay.

Results: The amplified fragments of IGF-II mRNA by RT-PCR were identical to originally designed ones with a size of 170 bp and confirmed by sequencing analysis. The dilution experiments revealed that the lowest sensitivity of our system was 2 ng/L of total RNA. The positive frequencies of IGF-II mRNA were 100% in HCC tissues, 53.3% in para-cancerous tissues, and 0% in non-cancerous tissues, respectively. The serum free IGF-II levels were significantly higher in HCC than those in chronic hepatitis or liver cirrhosis. The positive frequency of circulating IGF-II mRNA was 34.2% in HCC, no amplified fragment was found in other liver diseases, extrahepatic tumors, and normal controls, respectively. The circulating IGF-II mRNA correlated with the stage of HCC, and its positive rate was 100% in HCC with extrahepatic metastasis and 35.5% in HCC with AFP-negative. No significant correlation was found between tumor sizes and circulating IGF-II mRNA fragment.

Conclusion: The abnormal expressions of free IGF-II and IGF-II mRNA are useful tumor markers for HCC diagnosis, differentiation of extrahepatic metastasis and monitoring postoperative recurrence.

Figure 1

Amplification of IGF-II genomes from human hepatoma tissues or circulating blood samples of patients with hepatocellular carcinoma. IGF-II mRNAs were synthesized according to IGF-II cDNA with random hexamers and moloney murine leukemia virus reverse-transcriptase, and detected with different primer pairs by nested PCR (170 bp). The positive fragments of IGF-II genome were found distinctly in hepatoma tissues or in peripheral blood of patients with hepatocellular carcinoma. A: the sensitive limitation of our detection system (2 ng/L), using total RNA with 10^-2-10^-8 fold dilution and then amplified by nested PCR; B: the amplified fragments (452 bp) of glyceraldehyde-3-phosphate dehydrogenase genome from liver tissues or peripheral blood as controls; C: the amplification of IGF-II genomes in liver tissues (No. 1-4) or circulating blood (No. 5-6). No. 1-2, the positively amplified fragments of IGF-II mRNA from cancerous tissues of patients with hepatocellular carcinoma; No. 3, the positively amplified fragments of IGF-II mRNA from para-cancerous tissue of patients with hepatocellular carcinoma; No. 4, no positively amplified fragment from non-cancerous tissue of patients with hepatocellular carcinoma; No. 5, no positively amplified fragment from circulating blood of patients with liver cirrhosis, and No. 6, the positively amplified fragment from peripheral blood of patients with hepatocellular carcinoma. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. M: DNA molecular weight marker.

Figure 2

Alignment of nucleotide sequences of the amplified fragments of IGF-II genome from cancerous tissue or circulating blood in patients with HCC by sequence analysis. Origin: the cited sequence (170 bp, nt 311-480) of human