| Aims Primary liver cancer is a common type of malignant tumor. The incidence of this cancer has increased annually in recent years, and approximately half of all cases are found in China. Most liver cancers are hepatocellular carcinomas (HCC). Hepatitis B virus (HBV) infection is internationally recognized as a major cause of HCC and a contributing factor to metastasis in HCC. Metastasis usually has occurred during the early stage of HCC development. For patients with HCC, prognosis including risk of death, metastasis and recurrence following surgery is worse with higher serum HBV DNA levels. During the metastasis processes of malignant tumor cells, unattached tumor cells can avoid anoikis. This is an early event and precondition for metastasis. It is an essential characteristic of metastatic tumor cells. Previous studies showed that special AT-rich binding Protein-1 (SATB1) can regulate gene expression by anchoring specific DNA sequences and recruiting chromatin remodeling complexes to promote tumor growth and metastasis. After SATB1 expression is silenced, the anchorage-independent growth is inhibited and anoikis is induced in metastatic breast cancer cell lines. This study aimed to confirm the relationship between HBV, SATB1 and hepatocellular carcinoma cell anoikis and their specific regulatory mechanisms.Methods Forty pairs of liver cancer and their adjacent normal liver tissues (including twenty pairs of HBV-related HCC and their adjacent normal liver tissues, and twenty pairs of HBV-uninfected HCC and their adjacent tissues) were was obtained from the patients with histoically confirmed HCC. Real-time quantitative PCR (RT-qPCR) and western blot were used to assay the SATB1 expression in liver cancer and their adjacent normal liver tissues, and five liver cancer cell lines with different metastatic potential (including Hep3B and HepG2.2.15 cells that were positive for HBV DNA, and SK-HEP-1, HepG2 and SMMC-7721 cells that were negative for HBV) to confirm the relationship between SATB1 and HCC metastasis, HBV infection and so on. In order to further study the relationship between HBV, SATB1 and anoikis, the pBlue-HBV plasmid containing whole-genome of HBV that can encode various HBV proteins was transfected into liver cancer cell line with low SATB1 expression, and the expression of SATB1, cell anoikis and soft agarose single-colony formation were analyzed in these cells, we transfected the expression vectors containing individual seven viral structural genes of HBV into HepG2 cells and examined the expression of SATB1 to screen the special HBV-encoded protein that can regulate SATB1 expression. We stably transfected the expression vectors containing this viral gene into HepG2 and Hep3B cells, and analyzed the expression of SATB1, cell anoikis and soft agarose single-colony formation to clarify the role of HBV-encoded protein regulation of SATB1 expression and anoikis resistance. In order to investigate the special signal transduction pathway that HBV-encoded protein regulated SATB1 expression and anoikis resistance, We used inhibitors of the PI-3K, JNK, ERKs and P38 MAPK signaling molecules to treat of HepG2-HBx cells respectively. CaspGLOWTM Rhodamine Active Caspase Staining Kit was used to analyze the activation of caspase-3, caspase-8 and caspase-9 in apoptotic cells. The hallmark protein of death receptor apoptotic pathway FADD were analyzed to investigated the special apoptotic pathway that SATB1 inhibited anoikis.Results We assayed the expression of SATB1 in 40 pairs of human HCC and corresponding adjacent normal tissues. Twenty-two pairs of samples were high expression (55%); i.e., mRNA and protein levels of SATB1 in the hepatoma tissue were significantly higher (by≥2.0-fold) than those in corresponding adjacent noncancerous tissue. And there were certain correlations between the high expression of SATB 1 and the HBV infection (p<0.05), tumor size (p<0.05), differentiation degree (p<0.05), portal invasion (p<0.05) and lymph node metastases (p<0.05). Both SATB1 mRNA and protein were high expression in liver cancer cell lines HepG2.2.15 containing whole-genome of HBV and SK-HEP-1 with high metastatic potential (compared to Hep3B cells with low metastatic potential;ΔCT: 7.36±0.08 vs 10.27±0.06 and 7.46±0.09 vs 10.27±0.06, respectively;**p<0.05). After the HepG2 cells were transfected with pBlue-HBV, SATB1 expression was enhanced (ΔCT: 7.01±0.03 vs 9.59±0.02;**p<0.01), rate of anoikis was inhibited (22.68±1.25% vs 35.51±.86%;**p<0.01) and rate of single-colony formation was increased (24.89±2.23% vs 2.11±1.02%;**p<0.01). The ability of unattached liver cancer cells resistance to anoikis was correspondingly promoted (anoikis rate:21.20±0.81% vs 31.79±0.88%; cell colony rate:22.09±1.84% vs 10.67±1.14%;**p<0.01) or inhibited (anoikis rate:40.51±1.81% vs 27.48±0.79%; cell colony rate:4.91±0.76% vs 17.98±1.57%;**p<0.01) after the expression of SATB1 was enhanced or silenced in liver cancer cell lines. The expression vectors containing individual seven viral structural genes of HBV and the empty pEGFP-N1 vector were transfected into HepG2 cells respectively, and the results indicated that HBV-encoded HBx can significantly upregulate SATB1 expression (ΔCT:7.69±0.07 vs 10.02±0.03;**p<0.01). HepG2 and Hep3B cells were transfected with pEGFP-N1-HBx vector, and the results indicated that, in HepG2-HBx and Hep3B-HBx cells, SATB1 expression was increased notably alongside increased HBx expression (ΔCT:7.59±0.05 vs 9.86±0.02; 7.79±0.06 vs 10.39±0.02;**p<0.01), anoikis was reduced (21.86±1.79% vs 34.12±1.32%; 19.87±1.98% vs 35.07±1.56%;**p<0.01) and soft agarose single-colony formation was increased (22.32±1.82% vs 2.24±0.95%; 26.57±1.94% vs 5.04±0.99%; **p<0.01). Then HepG2-HBx cells were transfected with SATB1-siRNA1. In HepG2-HBx cells with that high HBx expression, following inhibition of SATB1 expression caused by SATB1-siRNA1, the ability of anoikis resistance was significantly suppressed, apoptosis was increased (35.34±1.59% vs 23.37±2.05%;**p<0.01), and single-colony formation was decreased (6.27±1% vs 23.18±1.83%;**p<0.01). We used inhibitors of the signaling molecules including PI-3K, JNK, ERK and P38 MAPK to treat of HepG2-HBx cells. After a 24 hour incubation with individual signaling molecule inhibitors, ERK and p38 MAPK inhibitors significantly suppressed SATB1 expression (ΔCT:9.92±0.03 vs 7.59±0.06; 10.17±0.04 vs 7.59±0.06;**p<0.01). The ability of anoikis resistance in these cells was lower than in DMSO-treated HepG2-HBx cells, rate of anoikis was increased (36.80±1.61% vs 25.21±1.54%; 38.51±2.19% vs 25.21±1.54%;**p<0.01) and rate of single-colony formation was inhibited (8.32±1.05% vs 22.45±2.26%; 6.36±1.37% vs 22.45±2.26%;**p<0.01). The activation of caspase-3, caspase-8 and caspase-9 in apoptotic cells was analyzed, and the results indicated that, as HBx and SATB1 expression increased, caspase-8 and caspase-3 activation decreased significantly (**p<0.01). Caspase-9 activation showed no correlation to HBx and SATB1 expression levels. We also investigated the expression of FADD, the upstream activator of caspase-8, and discovered an inverse relationship between SATB1 and FADD. SATB1 downregulated the expression of FADD (ΔCT:9.02±0.02 vs 6.84±0.03; 6.88±0.03 vs 8.41±0.06;**p<0.01). FADD expression in HepG2-HBx was lower than in HepG2 cells (ΔCT:7.75±0.03 vs 6.31±0.05; **p<0.01) but it was higher in HepG2-HBx-SATBl-siRNA1 cells than in HepG2-HBx cells (ΔCT:5.77±0.04 vs 7.69±0.02;**p<0.01).Conclusion Our study confirmed that HBV can induce SATB1 expression and suppress anoikis of unattached liver cancer cells, and in the seven HBV-encoded proteins (HBp, HBx, HBs, preS2, preSl, HBc and HBe), HBx can upregulate SATB1 expression by activating ERK and p38MAPK signal transduction pathways, inhibite the expression of Fas-associated protein with death domain (FADD) and the activation of caspase-8 and caspase-3 through SATB1, and further induce the resistance to anoikis in liver cancer cells. In conclusion, the study proves the effects of HBV-encoded HBx on SATB1 expression and anoikis resistance and outlines the specific mechanism by which it regulates these processes. We provide a new theoretical basis for research into the mechanism of HBV-related HCC metastasis. |
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