Each error bar in panels (ACD) and (F) represents the mean SD of three indie experiments. mice injected intracardially with HCCLM3\P/luc cells and cultured, and re\injected intracardially into mice. This procedure was repeated for four cycles. B) Volcano plot analysis of dysregulated proteins comparing HCCLM3\BM4 cells with HCCLM3\P cells. C) Representative images (left) and quantification (right) of RNF219 expression in normal liver tissue (= 23), HCC tissues without bone\metastasis (= 437), main HCC tissues with bone\metastasis (= 38), and HCC bone\metastasis tissues (= 6) (left panel). Scale CR2 bar, 50?m. D) Upper: BLI (left) and CT images (middle and right) of bone lesions from representative mice. Arrowheads: fractured bone site. Lower: KaplanCMeier bone metastasis\free survival curve and quantification of the osteolytic sites, BMD and fracture frequency from representative mice (= 8/group). E) CT images of trabecular section (upper) and quantification (lower) of bone parameters from representative mice (= 8/group). BV/TV, bone/tissue volume ratio; BS/TV, bone surface/ tissue volume ratio; Tb. = 8/group). Level bar, 50?m. Each error bar in panels (C?F) represents the mean SD of three independent experiments. Significant Isorhynchophylline differences were determined by one\way ANOVA with Tukey’s multiple comparison test (CCF). * ?0.05, ** ?0.01, *** ?0.001. 2.2. RNF219 Overexpression Promotes Bone Metastasis and SREs in HCC To determine whether RNF219 overexpression induces HCC\BM, we monitored the progression of BM after the intracardiac injection of control\ and RNF219\transduced HCC cells, which stably expressed firefly luciferase reporter. Prominently, the mice intracardially injected with RNF219\transduced HCC cells displayed earlier systemic bone metastatic onsets and a larger bone metastatic tumor\burden (Physique S1D,E, and Table S6, Supporting Information and Figure?1D). Micro\CT (CT) analysis showed that RNF219 also contributed to bone remodeling, as indicated by increased systematic severe osteolytic bone lesions, a reduced systemic bone mineral density (BMD), and a higher frequency of SREs, such as pathological fracture (Physique?1D). In the mean time, the relative trabecular volume, trabecular number, and trabecular thickness were significantly increased while the trabecular separation and trabecular bone pattern factor were decreased in HCC/RNF219 cells\injected mice compared to control mice (Physique?1E). Histological analysis revealed a larger osteolytic area and increased TRAP+\osteoclasts, but no alteration of alkaline phosphatase (ALP)+\osteoblasts, along the bone\tumor interface in RNF219/mice (Physique?1F). All these results suggested that RNF219 contributes to HCC\BM and SREs development. Consistently, compared with control mice, the RNF219\silenced HCC cells\injected mice exhibited delayed bone metastases, reduced BM lesions/osteolytic areas, less BMD reduction, and fewer SREs frequency (Physique S2ACG and Table S6, Supporting Information). Histological TRAP staining showed that RNF219\silenced HCC cells suppressed osteoclasts activation (Physique S2H, Supporting Information). Taken together, our results implicate RNF219 contributes to skeletal complications of HCC. 2.3. RNF219\induced LGALS3 Promotes Osteoclastogenesis Treatment with conditioned media (CM) Isorhynchophylline from HCC/RNF219 cells significantly increased the number of TRAP+\multinuclear osteoclasts and TRAP enzymatic activity, suggesting that RNF219 upregulation might enhance the capability of HCC cells in creating a bone tumor microenvironment (Physique? 2A). However, it experienced no effect on differentiation of pre\osteoblast MC3T3\E1 cells, as ALP+\osteoblasts and the relative RANKL/OPG ratio were unaltered (Physique S3A, Supporting Information), suggesting that RNF219\induced secretome promotes osteoclastogenesis. Open in a separate Isorhynchophylline window Physique 2 RNF219 induced\LGALS3 promotes osteoclastogenesis in vitro. A) Left: Osteoclast differentiation assays by TRAP staining (upper) or osteoblast differentiation assay by ALP staining (lower) in the presence of CM from indicated cells. Right: Quantification of the number of TRAP+\multinuclear osteoclasts, TRAP activity and ALP activity from your experiment in the left panel. B) Scatter diagram generated from dysregulated proteins in CM\HCCLM3/vector compared with CM\HCCLM3/RNF219 and in osteoclast (OC) compared with osteoclast precursor (OP). A full list is available in Table S7, Supporting Information. C) ELISA analysis of secreted LGALS3 protein expression in CM from indicated cells. D) Osteoclast differentiation assays in the presence of the indicated CM, or BSA, or purified LGALS3 from CM\HCCLM3/Flag\tagged LGALS3 cells. E) Osteoclast precursor Natural 264.7 cells were treated with BSA, or purified LGALS3 from CM\HCCLM3/flag\tagged LGALS3 cells, and then IF staining of LGALS3, Flag\LGALS3, CD98 and integrin ?0.01, *** ?0.001 and N.S.: not significant ( ?0.05). Analysis of the protein profiling in CM collected from HCC/vector and HCC/RNF219 cells and the proteomics data of mature osteoclast (OC) and osteoclast precursor (OP), [ 15 ] indicated that LGALS3 was one.