WB and IP assays were performed as previously described 19 , 20 , 27 , 28 , 29 , Briefly, HEKT cells were seeded in 6-well plates, transfected, and treated, as described in the Results and Figures. After 38 to 48 hours, the cells were collected, and the cell lysates were immunoprecipitated with anti-Myc or anti-Flag antibodies. Cell lysates were immunoprecipitated with anti-Myc Ab and probed with the different antibodies indicated in the text and Figures.
A wound-healing migration assay was performed following previous protocols 27 , The cell monolayers were gently scratched and washed with culture medium. Cell images were captured after culturing at different time periods as indicated in each experiment. The Transwell invasion assay was performed following previous protocols 27 , Cells in the upper compartments were removed, washed with PBS, and fixed.
The invaded cells were stained with crystal violet Sigma-Aldrich, C and quantified by counting the number of fluorescent cells. Single cell migration assay was performed following the protocols provided by the Bio-protocol www. Data analysis was performed following protocols provided by the Bio-protocol www. Anchorage-independent soft agar colony formation assay was performed following previous protocols Growth medium 1. The ability of a single cell to grow into a colony was passed through a colony formation assay as previously described 26 , In the present study, we investigated whether HBx induces autophagy.
We next evaluated the interaction site of HBx to Vps Ctrl HepG2 with vehicle. The co-treatment of CQ, which blocks the binding of autophagosomes to lysosomes by altering the acidic environment of lysosomes and induces the accumulation of LC3-II in cells, enhanced the levels of LC3-II in both cells Fig. We next examined the functional role of HBx in cancer cell migration, invasion, and mobility.
Ctrl HepG2 treated with vehicle. In contrast, marked inhibitions were detected in both cells co-treated with autophagy inhibitors, 3-MA or CQ Fig. To further verify the ability of single-cell migration, time-lapse image microscope analysis was performed. On the contrast, after the co-treatment of 3-MA, the ability of migration was markedly attenuated in both cells Fig. Next, we examined HBx-induced cell proliferation using clonogenic assay Fig.
The number of colonies in the clonogenic assay was significantly increased in the HBx-HepG2 treated with vehicle, as compared with those of Ctrl HepG2 treated with a vehicle Fig. Ctrl HepG2 treated a vehicle. Consistently, an autophagy inhibitor, 3-MA or CQ, led to marked attenuation of the colony formation Fig. These results suggest that HBx positively regulates the liver cancer progression induced by TLR4 stimulation.
Ctrl SNU Ctrl SK-Hep-1 cells treated with vehicle. Moreover, the mobility was markedly enhanced with the LPS treatment, whereas marked attenuation was observed in the co-treatment of 3-MA Fig.
On the contrast, the co-treatment of autophagy inhibitors, 3-MA or CQ, resulted in a marked inhibition in both cells Fig. Taken together, these results suggest that HBx functionally promotes the migration, invasion, and mobility of liver cancer cells induced by TLR4 stimulation.
HBx protein plays a pivotal role in the pathogenesis of HBV-related liver diseases through the functional regulation of various host proteins that regulate hepatocyte differentiation and proliferation 1 , 2 , 3 , Previous reports have demonstrated that the interaction between HBx and different cellular proteins, including DNA repair proteins, damaged DNA-binding proteins, cell cycle-related proteins, and autophagy-related proteins is critically implicated in the pathogenesis of HCC following HBV infection 1 , 2 , 3 , 35 , To elucidate the pathogenic mechanism by which HBx is involved in the development of liver carcinogenesis, it is essential to understand the molecular and cellular mechanisms involved in liver cancer progression in response to the cellular stimulus.
Recent evidence has demonstrated that autophagy promotes carcinogenesis at the early stages, and tumor progression in HCC 37 , 38 , Importantly, previous studies have demonstrated that HBx is functionally implicated in the autophagy processes or induction 25 , 36 , 40 , 41 , 42 , HBx induced the formation of autophagosome, but inhibited autophagic degradation by impairing lysosomal maturation in Huh7 hepatoma cells HBx has also been shown to induce autophagy in HepG2 and primary liver cancer cells through various cellular signals, thereby promotes liver cancer migration and invasion 41 , 42 , These results suggest that HBx may have different roles in the regulation of autophagy in a context-dependent manner.
Notably, TLR4 plays a vital role in the development and pathogenesis of HCC and the cancer progression by autophagy induction 14 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , Although several reports have suggested that HBx is functionally implicated in autophagy by regulating the mTOR pathway and the expression of BECN1 25 , 36 , the molecular and cellular mechanism by which HBx is involved in the TLR4-induced autophagy and thereby in liver cancer progression are still unclear.
Herein, we investigated the regulatory mechanism of HBx in the autophagy induction and examined whether HBx is implicated in the progression of liver cancer induced by TLR4. It has been reported that the expression of Bcl-2 is increased in various human cancers and is associated with resistance against apoptosis or autophagy during tumorigenesis and chemotherapy 44 , In addition, we found that HBx interacted with the C2 domain of Vps34 protein.
Given the molecular mechanism of HBx in the regulation of autophagy, we evaluated whether HBx is involved in liver cancer progression through autophagy induced by TLR4 stimulation. In contrast, the co-treatment of autophagy inhibitors, 3-MA or CQ, resulted in the attenuation of these activities. Together, these results strongly suggest that HBx positively regulates liver cancer progression induced by TLR4 stimulation. In summary, as illustrated in Fig. Accumulating evidence has demonstrated that autophagy and TLR4 are functionally implicated in HCC progression and tumorigenicity 14 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , Although the functional role of HBx in the regulation of autophagy induced by TLR4 in vivo is further required, our results will provide insight into the pathogenesis of HBV-induced carcinogenesis and tumor progression, and the development of new therapeutic agents against HBV-induced liver diseases.
Conflicts of Interest: The authors declare no potential conflicts of interest. National Center for Biotechnology Information , U. Journal List Immune Netw v.
Immune Netw. Published online Oct Find articles by Juhee Son. Find articles by Mi-Jeong Kim. Find articles by Ji Su Lee. Find articles by Ji Young Kim. Find articles by Eunyoung Chun. Find articles by Ki-Young Lee. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Correspondence to Ki-Young Lee. Correspondence to Eunyoung Chun. The Korean Association of Immunologists. Wound-healing migration assay A wound-healing migration assay was performed following previous protocols 27 , Transwell invasion assay The Transwell invasion assay was performed following previous protocols 27 , Single cell migration assay Single cell migration assay was performed following the protocols provided by the Bio-protocol www.
Anchorage-independent soft agar colony formation assay Anchorage-independent soft agar colony formation assay was performed following previous protocols Colony formation assay The ability of a single cell to grow into a colony was passed through a colony formation assay as previously described 26 , Open in a separate window. Figure 1. At 38 h post-transfection, the transfected cells were extracted and the cell lysates were subjected to IP with anti-Myc Ab followed by IB using anti-Flag or anti-Myc Ab.
IP assay was performed with anti-HA Ab. IP assay was performed with anti-Flag Ab. BECN1 interacts with Bcl-2 therefore inhibiting the autophagy induction left. Figure 2. C Truncated mutants of Vps34, were generated as described in Materials and Methods. A schematic model for the interaction between HBx and Vps34 protein is shown down. Figure 3. HBx-expressing HepG2 cells enhance cell migration, invasion, and mobility in response to TLR4 stimulation We next examined the functional role of HBx in cancer cell migration, invasion, and mobility.
Figure 4. Fixed cells were stained with crystal violet B. A representative experiment is shown D. Figure 5. Data analysis on the speed of cell mobility was performed following protocols provided by the Bio-protocol B www. After incubation for 18 days C , colonies were stained with 0. For visualization, the foci were stained with 0. Liver cancer has been shown to result from a series of mutations in specific oncogenes and tumor suppressor genes.
In a recent study, X protein stimulates ROS generation in the mitochondria due to collapse of the membrane potential and increases the mutation frequency, that evokes malignant transformation. In the past 10years, the possibility that several viral proteins directly engaged in the DNA damage has increased to some extent.
From an evolutionary viewpoint, it is noteworthy that several arrangement proteins have been found in viruses. Thus, there is some clue that a small amount of X protein acts as an arrangement protein for HBV replication dependent upon cellular DNA damage due to generated ROS as an amplified signal. Publication types Review.
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