Approaching TLR3Agonist DsRNA Combined With Sorafenib To Suppress HCC In Vitro And Ex Vivo

Objective Toll-like receptor3(TLR3) is a member of Toll-like receptors which recognize pathogen-associated molecular patterns (PAMPs) leading to the activation of the innate immune response. Recent studies demonstrated that synthetic dsRNAs may produce therapeutic effects in a target-independent manner through the stimulation of the toll-like receptor-3(TLR3)/interferon pathway, suppression of angiogenesis and inhibition the proliferation of tumor cells, suggesting that activation of this pathway may have therapeutic potential. In this study, we approach that TLR3agonist dsRNA (BM-06) combined with sorafenib to study its effect on HCC in vivo and in vitro.Methods First, we detected the expression of TLR3in100samples of human hepatocellular carcinoma tissue. Then we screened the most effect dsRNA (BM-06) from four designed dsRNA which can activate TLR3/interferon pathway. Then after treating HepG2.2.15cells with BM-06, NF-κB activity was checked by subcellular fractionation, which represent the activity of TLR3in response to BM-06. HCC cellline HepG2.2.15cells were divided into five groups-BM-06group, sorafenib group, BM-06+sorafenib group, poly(I:C) group and control group. After BM-06treated cells alone or combined with sorafenib, cell apoptosis was identified by Annexin V and PI staining through FACS, migration ability was measured by Transwell migration assay and cell proliferation assay was performed by CCK-8method. The expression of HBsAg and HBcAg of HepG2.2.15cells were tested by qRT-PCR and Western blot. By constructing orthotopic HCC SD rats model with2-AAF, after treatment with BM-06alone or combination with sorafenib, obtain tumor tissue, detect the expression of Laminin, PCNA, survivin and bcl-2.Results The positive expression rates of TLR3and NF-κB in human HCC tissue were64%and57%. TLR3was maily located in membranous (55%) not plasma (45%). NF-κB was maily located in plasma (76%) not nucleus (24%). After BM-06treatment, compared with the control group NF-κB activity increased, indicating that BM-06can activate TLR3. BM-06and sorafenib can significantly trigger apoptosis in HepG2.2.15cells(F=1.106、1.040、1.811、1.800each group) and inhibit migration (F=1.160、1.800、1.400、1.800each group) and proliferation (72h F=4.025、1.088、2.966、2.102) of them, especially the combination group (P<0.05). The secration of HBsAg and HBcAg of HepG2.2.15cells was suppressed after treated with BM-06(0.30±0.02,0.41±0.03respectively), lower than the control group (0.76±0.07,0.73±0.05respectively)(HBsAg:F=2.069, HBcAg:F=2.203, P<0.05). In orthotopic HCC SD rats model, the liver/body weight of BM-06group, sorafenib group, BM-06+sorafenib group and poly(I:C) group were (3.82±0.25、4.40±0.12、4.16±0.13and4.51±0.21), lower than control group (5.31±0.28)(F=2.626、1.103、2.944、2.860, P<0.05). In rat liver tumor, node average diameter of four groups (0.81±0.02mm2、1.21±0.03mm2、1.02±0.04mm2and1.63±0.04mm2respectively) were reduced than control group(3.2±0.033mm2)(F=2.308、2.681、2.160、2.160, P<0.05). The expression of PCNA of four groups (7.43%±1.31%,16.81%±2.27%,12.50%±1.19%and18.13%±1.39%) were lower than control group (24.64%±2.28%). The expression of bcl-2of four groups (30.13%±2.42%,49.82%±3.53%,38.55%±3.47%and65.14%±4.58%) were lower than control group (81.83%±5.45%). The expression of survivin of four groups (22.11%±2.29%,30.54%±3.31%,29.73%±2.43%and42.15%±3.38%) were lower than control group (71.82%±4.44%)(PCNA:F=2.536,1.550、1.715、2.579, bcl-2:F=1.399、1.293、1.161、1.412, survivin: F=1.128、2.243、1.964、1.276, all P<0.05). The was obviously lower combination therapy inhibited tumor growth and induced apoptosis of HCC cells more efficient than single agent alone. In addition, the expression of Laminin, bcl-2and survivin of BM-06group were significantly lower than poly(I:C) group (Laminin:F=2.050, bcl-2: F=2.010, survivin:F=1.014, P<0.05), BM-06is superior to poly(I:C) in inhibiting HCC growth, promoting apoptosis of HCC cells.Conclusions1. TLR3was expressed in HCC tissue.2. In this study, we found the TLR3agonist dsRNA (BM-06) can effectively activate TLR3in HepG2.2.15cells as weii as in ilver of HCC rat and then activate NF-κB.3. BM-06effectively inhibits the proliferation and invasion of HCC cells in vivo and vitro and promotes the apoptosis of HCC cells through TLR3pathway.BM-06can inhibit the secretion of HBsAg and HBcAg of HepG2.2.15cells.4. BM-06can significantly enhance the anti-tumor effect of sorafenib, the combination of sorafenib and a TLR3agonist dsRNA like BM-06is a viable combination.5. BM-06is superior to poly(I:C) in suppressing HCC and shows a sequence specific.