Paclitaxel was purchased from Tianjin YiFang Science and Technology, Ltd. (Tianjin, China). 5-Fluorouracil injection, cisplatin and cytoxan were obtained from Zhejiang Chinese Medical University Second Clinical Medical College. Antibodies against Lin28 and β-actin, and horseradish peroxidase-conjugated secondary antibodies were purchased from Boster Biological Technology, Ltd. (Wuhan, China). Antibodies against caspase-3 and -9, BAX, cytochrome c, Bcl-2 and Bcl-xL were purchased from Hangzhou HuaAn Biotechnology Co., Ltd. (Hangzhou, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). HiFi-MMLV cDNA kits and UltraSYBR Mixture were obtained from Beijing Kang Century Biotechnology Co., Ltd. (Beijing, China).

The human hepatoma cell line Hep3B was obtained from Boster Biological Technology, Ltd., and was routinely cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37°C and 5% CO₂.

To develop a paclitaxel-resistant HCC cell line, Hep3B cells were exposed to gradually increasing concentrations of paclitaxel (0.01–0.2 μM) in complete medium. Briefly, Hep3B cells were seeded in culture flasks at a density of 4–5×10⁵ cells/ml and allowed to grow. After 24 h incubation, paclitaxel (0.01 μM) was added, and the cells were incubated for another 24 h. Then, the cells were washed 3 times with D-Hanks solution and the medium was changed to paclitaxel-free medium. The cells were incubated and allowed to grow until confluent. Then, the cells were subcultured and re-exposed to double the dose of drug. This process was repeated until the cells were resistant to 0.2 μM paclitaxel. After successful development, Hep3B/TAX cells were maintained in complete medium containing a low concentration of paclitaxel (0.01 μM).

Hep3B and Hep3B/TAX cells were seeded in 35 mm petri plates at a density of 1×10⁵ cells/ml. After 24 h incubation, cells were visualized and photographed under a Nikon Eclipse 80i microscope connected to a DS-5M-L1 camera.

A previously described 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake method (11) was used to determine the effect of drugs on the proliferation and viability of Hep3B and Hep3B/TAX cells. Experiments were repeated three times with 6 wells for each treatment to ensure the reproducibility of results. The IC₅₀ value was defined as the dose of the drug required to inhibit cell growth by 50% and was calculated using the improved Karber’s method.

To determine whether Hep3B/TAX cells were more resistant to paclitaxel than the parental Hep3B cells, Hep3B/TAX and Hep3B cells were seeded in 35 mm petri plates at a density of 1×10⁵ cells/ml. After 24 h incubation, cells were treated with paclitaxel for another 24 h, and untreated cells served as control. Next, the cells were washed twice with PBS and resuspended in 100 μl incubation buffer (PBS buffer containing 2% BSA and 2% FBS), and 100 μl of Guava Nexin reagent was added. After incubation for 20 min at RT in the dark, all the samples were filtered sequentially through 200 μm mesh sieves and analyzed using Guava EasyCyte 8 flow cytometer (EMD Millipore, USA). Annexin V-PE-negative and 7-AAD-negative cells were considered alive.

Total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions. cDNA was synthesized using a HiFi-MMLV cDNA kit. To synthesize let-7 family cDNAs, specific RT-primers were used that were based on the sequence of each family member, and the RT-primer for U6 was the same as the reverse primer (Table I). Real-time PCR was conducted using UltraSYBR Mixture. All primers were synthesized by GenScript Co., Ltd. (Nanjing, China). All samples were run in triplicate, and changes in gene expression were calculated using the ΔΔCt method.

Total protein was extracted from cells using Protein Extraction Reagent (Boster Bioengineering, Wuhan, China) containing 1 mM phenylmethanesulfonyl fluoride (PMSF) (Roche Molecular Biochemicals, Indianapolis, IN, USA). Protein concentrations were determined by the BCA protein assay (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China). The proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Pall Gelman Laboratory Corporation, Ann Arbor, MI, USA). Then, the western blot analyses were probed with antibodies against Lin28, caspase-3 and -9, BAX, cytochrome c, Bcl-2, Bcl-xL and β-actin. The protein bands were detected by enhanced chemiluminescence.

Data are expressed as means ± SD of experiments performed in triplicate. Statistical analysis was performed using one-way analysis of variance (ANOVA) for multiple comparisons and t-tests for comparisons between groups. A P-value of <0.05 was considered to indicate a statistically significant difference.

A human hepatoma paclitaxel-resistant model system was established, as described in the Materials and methods section, to study the molecular mechanisms of drug resistance. After 9 months of development, we obtained Hep3B/TAX cells that grew stably in DMEM containing 0.2 μM paclitaxel. Microscopic observation showed that the Hep3B/TAX cells were elongated compared with their parental cells, especially at low cell density (Fig. 1A, a-1 and a-2), and the number of black particles in cytoplasm of Hep3B/TAX cells increased (Fig. 1A, b-1 and b-2). The doubling time for Hep3B was 44.21±1.47 h and that of Hep3B/TAX cells was 49.16±1.89 h, therefore the growth rate of paclitaxel-resistant cells was significantly lower than that of the parental cells (Fig. 1B, P<0.01). Next, to determine the IC₅₀ value of paclitaxel, Hep3B and Hep3B/TAX cells were treated with various doses of paclitaxel for 48 h and then cell viability was assessed using the MTT assay. As shown in Fig. 2A, we found that the survival rate of Hep3B/TAX cells was much higher than that of their parental cells after treatment with the same dose of paclitaxel. The IC₅₀ values were 0.2 μM for Hep3B cells and 5.65 μM for Hep3B/TAX cells at 48 h, and the drug resistance index was 28.25 (Table II). These data suggest that Hep3B cells are more sensitive to paclitaxel than Hep3B/TAX cells. We also assessed the response of Hep3B and Hep3B/TAX cells to other chemical drugs besides paclitaxel, and we found that Hep3B/TAX cells exhibited cross-resistance to cisplatin, 5-fluorouracil and cytoxan (Fig. 2B–D and Table II).

After 24 h exposure to paclitaxel in DMEM, Hep3B and Hep3B/TAX cells were harvested, and their apoptosis rates were determined. As shown in Fig. 3, we observed that untreated Hep3B and Hep3B/TAX cells both showed very low apoptosis rates (1.55±0.55 and 2.41±0.69%, respectively; Table III). After treatment with paclitaxel, the percentage of apoptotic Hep3B cells markedly increased to 21.65±2.28% (0.1 μM) and 30.57±4.74% (0.4 μM), whereas the apoptosis rate in Hep3B/TAX cells was still very low, only 2.63±0.87, 3.82±0.99 and 3.55±0.57% following treatment with 0.1, 0.2 and 0.4 μM paclitaxel. Therefore, compared with Hep3B cells, Hep3B/TAX cells were much less sensitive to paclitaxel.

Activation of caspases is a hallmark of apoptosis; to measure this, Hep3B and Hep3B/TAX cells were treated with 0.2 μM paclitaxel for 24 h, and whole cell extracts were prepared and caspase-9 and -3 activation was assessed. Western blot analysis showed that cleavage of caspase-9 and -3 in Hep3B/TAX was reduced (Fig. 4A). Furthermore, we found that in Hep3B/TAX cells, the expression of Bax was reduced whereas the expression of anti-apoptosis protein Bcl-2 was enhanced (Fig. 4A), and real-time PCR analysis confirmed these findings (Fig. 4B). These results indicated that Hep3B/TAX cells are resistant to paclitaxel-induced apoptosis. Although this phenotype may be related to drug resistance, it nevertheless confirmed the successful development of a drug-resistant cell line.

To determine whether Lin28 expression is associated with the drug resistance observed in HCC, we examined the expression of Lin28 in Hep3B and paclitaxel-resistant Hep3B/TAX cells by qPCR. We found that the mRNA level of Lin28 in the paclitaxel-resistant cell line was 75-fold higher than that in the parental Hep3B cells (Fig. 5A) and the Lin28 protein level in Hep3B/TAX cells was also much higher than that in Hep3B cells (Fig. 5B). Then, we measured the expression of let-7 family miRNAs, which are regulated by Lin28. The results showed that the expression of all the let-7 family members tested was reduced except let-7b (Fig. 5C). The anti-apoptotic protein Bcl-xL plays a transcendental role in chemoresistance in tumor cells, and Bcl-xL is regulated by let-7. Therefore, we examined the expression of Bcl-xL both in Hep3B and Hep3B/TAX cells by western blotting. We observed that the level of Bcl-xL protein was much higher in Hep3B/TAX cells than in Hep3B cells. All the data suggest that the Lin28/let-7/Bcl-xL pathway is associated with drug resistance in Hep3B cells.