HN1 was highly expressed in HCC and associated with poor prognosis of HCC patients.
First, the protein level of HN1 was analyzed by IHC in 43 HCC tissues and para-carcinoma tissues, and the protein level of Ki67 was also analyzed in HCC tissues to assess cell proliferation of malignant cells (Fig. 1a). We found that HN1 protein level was higher and positively correlated with Ki67 protein level in HCC tissues with Pearson's correlation of 0.6498 (Fig. 1b). The overexpression of HN1 in HCC tissues compared to para-carcinoma tissues was verified by western blot (Fig. 1c). Moreover, the protein and mRNA expression level of HN1 in human HCC cell lines were remarkably higher than that in the normal immortalized hepatic cell line LO2 (Fig. 1d, e). Notedly, Hep3B and Huh7, which have been proved to be sensitive to oxaliplatin , had lower level of HN1 than other cell lines in our study. The clinical data indicated that aberrant expression of HN1 was closely associated with the tumor size and the incidence of portal vein tumor thrombus (PVTT) of HCC (see additional file 1). Further analysis of the overall survival and progression free survival of the patients showed that patients with higher expression of HN1 had worse prognosis than those who had lower expression of HN1 (Fig. 1f, g). Overall, above data implied that HN1 was overexpressed in HCC tissues and closely associated with poor prognosis of patients.
HN1 knockdown inhibited HCC proliferation and metastasis in vitro.
To investigate the tumor-promoting mechanism of HN1 in HCC, two different siRNAs (siRNA#1 and siRNA#2) were transfected into HepG2 and MHCC-97L cell lines. The cells transfected with siHN1#2 had a lower HN1 mRNA expression and protein level than siHN1#1 (Fig. 2a, b). The MTT assay showed that downregulation of HN1 significantly inhibited HCC cell growth (Fig. 2c), and HN1 knockdown inhibited clonogenicity of HCC cells (Fig. 2d). In addition, c-Myc and cyclin D1 which reflect potency of cell proliferation also downregulated in HN1 knockdown cells (Fig. 2f). However, downregulation of HN1 had no effect on cell apoptosis (see additional file 2).
Given that higher expression of HN1 was related with PVTT patients, HN1 possibly promoted metastasis of HCC. As expected, the result of transwell assay showed that downregulation of HN1 decreased the migration and invasion of HCC cells (Fig. 2e). In addition, N-cadherin, E-cadherin and ZEB1 which reflect metastasis potency of HCC cells also altered following HN1 knockdown (Fig. 2g). Next, we overexpressed HN1 in Hep3B and Huh7 cell lines through pCMV-HN1 plasmid, and measured their potency of cell proliferation, migration and invasion. We found that these malignant phenotypes of HCC cells were consistently reinforced after HN1 overexpression (see additional file 3). In conclusion, gain and loss of function experiments indicated that HN1 could promote proliferation, migration and invasion of HCC cells.
HN1 knockdown increased sensitivity to oxaliplatin and aggravated DNA damage in HCC cells.
We established HN1 knockdown cells lines of HCC (HepG2 and MHCC-97L) through shRNA lentivirus vector and knockdown efficiency was confirmed by qRT-PCR and western blot (Fig. 3a, b). Taking the observation into account that patients with higher HN1 expression had worse prognosis after oxaliplatin-based chemotherapy treatment, we explored whether HN1 desensitized HCC to oxaliplatin. The CCK8 assay demonstrated that HepG2 and MHCC-97L cells with HN1 knockdown were more sensitive to oxaliplatin and had lower IC50 compared to those with negative control HCC cells (Fig. 3c). Alkaline comet assay suggested that HCC cells with HN1 knockdown had higher tail-positive proportion compared with negative control group, which indicated that there were more DNA lesions in HN1 knockdown HCC cells after oxaliplatin treatment (Fig. 3d). In addition, HN1 knockdown also reduced the survival of HCC cells in low dose of oxaliplatin (1µM and 5µM, Fig. 3e). Next, we treated HCC cells with gradient concentration of oxaliplatin (1µM, 5µm and 10µM) for 1 hour or 24 hours (Fig. 3f, g). We measured the histone modification related to DNA damage (γ-H2AX) and chromatin accessibility (acetylation of H3 and H4) [26–28]. We found that after oxaliplatin treatment, γ-H2AX level was higher in HN1 knockdown HCC cells while acetylation of H3 and H4 were higher in negative control HCC cells. Then time-dependence assay (cells treated with 5µM oxaliplatin for 0h, 6h, 12h and 24h) were performed to verify that HN1 could affect DNA damage repair and chromatin modification (Fig. 3h). We also confirmed that immunofluorescence intensity of γ-H2AX foci is higher in HN1 knockdown HCC cells after oxaliplatin treatment (5µM and 10µM, Fig. 3i). Finally, flow cytometry analysis demonstrated that HN1 knockdown HCC cells had a significantly higher apoptosis rate compared to negative control cells after oxaliplatin treatment (5µM and 10µM, Fig. 3j). All these results indicated that HN1 knockdown substantially sensitized HCC cells to oxaliplatin treatment.
HN1 inhibited the degradation of HMGB1 through autophagy-lysosome pathway by interacting with TRIM28.
Given that the depletion of HN1 in HCC cells enhanced sensitivity to oxaliplatin-induced DNA damage, we hypothesized that deregulation of proteins in DNA repair pathway could be crucial to this sensitivity transformation. HMGB1, which can bind to damaged DNA, recruit other components and enhance DNA repair, was tested as a potential downstream effector of HN1. The result of qRT-PCR showed that mRNA expression level of HMGB1 was impervious followed by HN1 knockdown (Fig. 4a). The protein expression level of intracellular HMGB1, however, was obviously decreased both in nucleus and cytoplasm (Fig. 4b, c). This result was verified by immunofluorescence staining (Fig. 4d). These results indicated that HN1 could regulate the expression of HMGB1 in post-transcriptional level and affect its degradation. To verify this speculation, after cells were transfected into pCMV-HMGB1 expression plasmid for 72 hours, the protein synthesis inhibitor, cycloheximide (CHX) was added at a concentration of 2µg/mL for another 24 hours, 48 hours, 72 hours, and 96 hours respectively. The result of western blot showed that degradation of HMGB1 is prominently faster in HN1 knockdown cells than negative control cells (Fig. 4e). Next, we aimed to investigate which pathway was responsible for HN1-related HMGB1 degradation. The autophagy inhibitor, chloroquine (10µM) or proteasome inhibitor, MG132 (1µM) was individually added into cells for 24 hours in the presence of CHX (2µg/mL). The result of western blot showed that autophagy inhibitor chloroquine could slower evidently the degradation rate of HMGB1 in HN1 knockdown cells (Fig. 4f). Ubiquitination is a common post-translational modification for substrate protein ready to be degraded . Thus, we detected the endogenous ubiquitination of HMGB1. The result of IP showed that ubiquitination of HMGB1 was substantially increased followed by HN1 knockdown (Fig. 4g). These results indicated that HN1 prevent HMGB1 from autophagy-lysosome-depend degradation.
We next investigate how HN1 regulated the ubiquitination of HMGB1. Previous studies reported that TRIM28 could be essential to the ubiquitination and degradation of HMGB1 . The result of co-immunoprecipitation assay revealed that HN1 interacted with TRIM28 (Fig. 4h), which is also supported by STRING database (https://string-db.org). In addition, from analysis of Immunofluorescence co-localization assay, we found the high degree of co-localization of HN1 and TRIM28 protein (Fig. 4i). Our pre-test indicated that TRIM28 knockdown could slightly affect the protein level of HMGB1 in the normal or HN1 knockdown cells (see additional file 4).
It has been well-established that MAGE proteins can bind to RING domain of E3 ligase like TRIM28 and form cancer-specific ubiquitin ligase complexes which regulate ubiquitination and degradation of various tumor suppressors like p53 [30–32]. It's worth noting that MAGE-C2 and MAGE-A3/6 was specially upregulated in HCC [33–35]. Therefore, we hypothesized that MAGE-C2 or (and) MAGE-A3/6 could be involved in regulation of HMGB1 ubiquitination through TRIM28. We then knockdown mRNA expression of TRIM28, MAGE-A3/6 or MAGE-C2 with small interfering RNAs (siRNAs), and detected the protein level of HMGB1. We found that knockdown of TRIM28 and MAGE-A3/6 significantly restored the protein level of HMGB1, while knockdown of MAGE-C2 had no effect on HMGB1 protein level (Fig. 4j). As expected, knockdown of TRIM28 or MAGE-A3/6 could inhibit the ubiquitination of HMGB1 and restored its protein level in HCC cells with HN1 downregulation (Fig. 4k). Based on these observations, we drawn the conclusion that HN1 could inhibit the degradation of HMGB1 through autophagy-lysosome pathway via interacting with TRIM28-MAGE-A3/6 complexes.
Overexpression of HMGB1 restored the phenotypes resulted from HN1 knockdown in HCC cells.
To further investigate the essential roles of HMGB1 as a key mediator of HN1 in phenotypic regulation of HCC cells, we transfected pCMV-HMGB1 overexpression plasmid into HN1 knockdown cell lines and assessed their phenotype restoration. We confirmed the transfection efficiency of plasmid through qRT-PCR and western blot analysis (Fig. 5a, e). The results of MTT assay and clone formation assay showed that the potency of cell growth could be partly rescued by overexpression of HMGB1 in HN1 knockdown cells and so was potency of migration and invasion tested by Transwell assay (Fig. 5b-d). In addition, results of western blot showed that HMGB1 overexpression could partly reverse the protein expression level of the biomarkers of cell proliferation and metastasis in HepG2 and MHCC-97L cells (Fig. 5e). Finally, we observed that overexpression of HMGB1 could abolish the increased drug sensitivity induced by HN1 knockdown in HCC cells (Fig. 5f). The results of western blot revealed that DNA damage alleviated and chromatin accessibility increased followed by HMGB1 overexpression under oxaliplatin treatment (Fig. 5g).
Moreover, increasing evidences indicated that autophagy plays an essential role in regulation of malignant phenotypes of tumor and cancer resistance to chemotherapy agents. Given that HMGB1 is an acknowledged autophagy activator , we conjectured that HN1 could regulate autophagy via HMGB1 and further affect phenotypes of HCC cells. In contrast to shNC cells, the recognized autophagy stimuli HBSS employed in shHN1 cells failed to upregulate the expression of LC3-Ⅱ, which indicated that HN1 was essential to cell autophagy (Fig. 5h). To investigate the role of HMGB1 in this process, we transfected pCMV-HMGB1 overexpression plasmid into shNC and shHN1 HCC cells and results of western blot indicated that the overexpression of HMGB1 boosted the protein level of LC3-Ⅱand lowered the level of p62 in shHN1 cells (Fig. 5i). In addition, cells were transfected by exogenous GFP-mRFP-LC3 adenovirus to monitor and measure cell autophagy. We found that the formation of GFP-mRFP-LC3 puncta markedly increased in shHN1 cells with HMGB1 overexpression compared with single shHN1 cells (Fig. 5j). Finally, transmission electron microscope analysis provided potent testimony of autophagy. The overexpression of HMGB1 in shHN1 cells exhibited significantly more autophagosomes compared with single HN1 knockdown cells (Fig. 5k). Above evidences supported a pivotal role of HMGB1 in process of autophagy regulated by HN1.
The above results verified that HMGB1 acted as a key downstream effector of HN1 in regulation of malignant phenotypes of HCC cells such like cell proliferation, metastasis and chemosensitivity.
HN1 knockdown inhibited malignant phenotypes and enhanced the antitumor effect of oxaliplatin in vivo.
MHCC-97L cells stably transfected with shNC or shHN1 were subcutaneously injected into male nude mice. We found that knockdown of HN1 dramatically inhibited tumor growth compared with negative control group (Fig. 6a-c). From the result of IHC, we also found the protein level of Ki67 and HMGB1 were also decreased after HN1 knockdown (Fig. 6d). Additionally, the role of HN1 on HCC tumor metastasis was evaluated through caudal intravenous pulmonary metastasis model. The result of HE staining showed that HN1 knockdown reduced the number and size of visible pulmonary metastatic nodules and the Ki67 protein level in metastatic niche of lung (Fig. 6e, f). Briefly, HN1 knockdown inhibited tumor proliferation and metastasis in vivo. Furthermore, to investigate the effect of HN1 knockdown on HCC sensitivity to oxaliplatin treatment in vivo, the nude mice were treated with oxaliplatin or normal saline (NS) when some of tumors derived from HN1 knockdown or negative control MHCC-97l cells grew roughly to 100mm3. The growth curve, image and weight of tumor demonstrated that HN1 knockdown group was more sensitive to oxaliplatin treatment than the control group (Fig. 6g-i).