Recent studies showed that the HH pathway is Seliciclib frequently activated in HCC (1-3). The increased expression of GLI1 protein in breast tumor and hepatoblastoma is also reportedly correlated with significantly poor prognosis (24-26). The HH pathway mediates the progression of breast cancer from non-invasive to invasive and serves as a significant independent prognostic indicator in gastric and bladder cancers (27,28). In our previous studies, we found an association with poor clinical prognosis of the HH pathway in human HCC (29) as well as the simultaneous expression of GLI1 in HCC and liver tissues adjacent to the tumor. These findings prompted us to expand our sample size to 46 HCC patients and extend their follow ups to confirm whether the expression of HH pathway components is associated with HCC progression and clinical outcome.

Materials and methods Patients and tumor samples This study included 46 HCC patients consisting of 40 (86.96%) males and 6 (13.04%) females with ages ranging from 35 years to 79 years (median age =49 years). All patients underwent surgical treatment from April 2002 to July 2005 in Beijing Cancer Hospital. The clinical and pathologic data included patients�� demographics (age, gender), tumor size, degree of histological differentiation, and complete follow-up record. The exclusion criterion was preoperative therapy. The study was approved by the Ethics Committee of Human Experimentation of Beijing Cancer Hospital. All samples of tumor and adjacent normal liver tissues were freshly obtained immediately after surgery.

The samples of tumor tissue were collected from the luminal aspect of the malignancy, whereas those of paired adjacent normal tissue were from the luminal aspect of the liver tissues 2 cm away from the tumor margin. All tissue samples were snap-frozen in liquid nitrogen for 30 min after resection and stored at �C80 ��C. RNA extraction and complementary DNA (cDNA) preparation The total RNA was isolated from 100 mg of each tissue sample using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer��s instructions, and stored at -80 ��C for further use. The reverse transcription (RT) of the total RNA (1 ��L) was performed using SuperScript First-Strand Synthesis System (Invitrogen). The internal control was glyceraldehyde-3-phosphate dehydrogenase (GAPDH; forward primer: 5′-TCA ACG GAT TTG GTC GTA TT-3′, reverse primer: 5′-AGT CTT CTG GGT GGG AGT GAT-3′, 540 bp).

Polymerase chain reaction (PCR) amplification To analyze the expression of individual HH gene, 1 ��L of cDNA was amplified with 6.25 units of AmpliTaq Gold (Roche, Basel, Switzerland) in 25 ��L reaction solution containing 0.5 mmol/L dNTPs and 1.5 mmol/L MgCl2. Anacetrapib The primer sequences for PCR of each gene were designed according to a previous study (9) or using Genbank sequences (Table 1).