Histidine-rich calcium binding protein promotes growth of hepatocellular carcinoma in vitro and in vivo

Abstract

We have recently shown that the histidine-rich calcium binding protein (HRC) promotes the invasion and metastasis of hepatocellular carcinoma (HCC). In the current study, we evaluated whether HRC may also affect the growth of HCC. We found that ectopic expression of HRC obviously enhanced proliferation and colony formation, while suppression of HRC exhibited inhibitory effects. Furthermore, we demonstrated that HRC promoted tumor growth in nude mice. These effects may result from the ability of HRC to upregulate cyclinD1 and cyclin-dependent kinase 2 (CDK2) expressions and promote G1/S transition. Further study showed that MEK/ERK signaling pathway was involved in HRC-induced cell proliferation. Interestingly, overexpression or depletion of HRC revealed its regulation on endoplasmic reticulum stress (ERS) and apoptosis, which was partially dependent on PERK/ATF4/CHOP signaling pathway. In addition, blocking ERS using 4-phenylbutyric acid (4-PBA) not only downregulated the expression of PERK, ATF4 and CHOP, but also significantly decreased apoptosis induced by HRC silence, whereas ERS inducer thapsigargin (TG) exerted the opposite effects. Our study thus demonstrates a role of HRC in promoting HCC growth, besides its role in inducing HCC metastasis, and highlights HRC as a promising intervention target for HCC.

Keywords: Apoptosis; endoplasmic reticulum stress; hepatocellular carcinoma; histidine-rich calcium binding protein; proliferation.

Figure 1

Histidine-rich calcium binding protein (HRC) promotes hepatocellular carcinoma (HCC) cell proliferation in vitro. (a) Relative expression of HRC in SMMC-7721 cells transfected with plasmids (pcDNA3.1-HRC and pcDNA3.1-vector) and Sk-hep-1 cells transfected with siRNA (si control and si HRC) were examined by RT-qPCR and western blotting. (b) Representative micrographs (left) and quantification (right) of crystal violet-stained cell colonies in treated SMMC-7721 and Sk-hep-1 cells. (c) Cell Counting Kit-8 (CCK-8) assay revealed that overexpression of HRC in SMMC-7721 cells enhanced cell proliferation, and HRC knockdown in Sk-hep-1 cells suppressed cell proliferation. (d) Representative micrographs (left) and quantification (right) of EdU-labeling cells in treated SMMC-7721 and Sk-hep-1 cells. Each bar represents the mean ± SD of three separate experiments. *P < 0.05, **P < 0.01.

Figure 2

Histidine-rich calcium binding protein (HRC) promotes tumor growth of hepatocellular carcinoma (HCC) in vivo. (a) Images of the tumors from the mice (left) and xenograft model of nude mice (right) in each group (n = 6 per group). (b) and (c) RT-qPCR and western blotting showed the level of HRC was actually higher in the SMMC-7721-HRC group than in the SMMC-7721-vector group.V:SMMC-7721-vector, H:SMMC-7721-HRC. (d) Growth curves of tumor resulting from injection of the SMMC-7721-HRC or SMMC-7721-vector cells into nude mice. The tumor volumes were estimated using calipers. (e) Mean tumor weights of nude mice in each group were measured on day 35. Each bar represents the mean ± SD of six mice per group. **P < 0.01.

Figure 3

Histidine-rich calcium binding protein (HRC) contributes to G1/S transition in hepatocellular carcinoma (HCC) cells. (a, b) Cell cycle analysis of treated SMMC-7721 and Sk-hep-1 cells. Left, representative image of flow cytometry analysis. Right, mean ± SD from three independent experiments of the percentages of cells in each cell cycle phase. (c, d) The levels of cyclinD1, cyclinE, CDK2 and CDK4 in treated SMMC-7721 and Sk-hep-1 cells were assessed by RT-qPCR and western blotting. GAPDH was used as a loading control. *P < 0.05, **P < 0.01.

Figure 4

Histidine-rich calcium binding protein (HRC) enhances cell proliferation by the MEK/ERK pathway. (a) The levels of phospho-ERK, phospho-MEK and phospho-Akt were determined by western blotting analysis. (b, c) The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) (b) and EdU assay (c) after SMMC-7721 cells were pretreated with U0126 (MEK/ERK inhibitor) or LY294002 (PI3K/Akt inhibitor). Each bar represents the mean ± SD of three separate experiments. *P < 0.05, **P < 0.01.

Figure 5

Effect of Histidine-rich calcium binding protein (HRC) on endoplasmic reticulum (ER) stress-mediated cell apoptosis. (a) FACS analysis of treated SMMC-7721 cells (left) and Sk-hep-1 cells (right) labeled with Annexin-V FITC and propidium iodide (PI) as markers for apoptosis. (b, c) Caspase 3 activity of treated SMMC-7721 and Sk-hep-1 cells. (d, e) The expression of Bax, Bcl-2 and the levels of ER stress-related proteins, including PERK, CHOP, ATF4 and Grp78 (BIP) in treated SMMC-7721 and Sk-hep-1 cells were determined by RT-qPCR and western blotting. Each bar represents the mean ± SD of three separate experiments. *P < 0.05, **P < 0.01.

Figure 6

Endoplasmic reticulum (ER) stress is involved in the induction of histidine-rich calcium binding protein (HRC)-suppressed apoptosis. (a) The expression of PERK, ATF4 and CHOP were assessed by RT-qPCR and western blotting after cells were pretreated with 1 mM thapsigargin (TG). (b) Sk-hep-1 cells were pretreated with 1 mM 4-phenylbutyrate acid (4-PBA). The mRNA and protein levels of PERK, ATF4 and CHOP were measured. (c) After treatment with 1 mM TG, SMMC-7721 cells apoptosis was determined by flow cytometry. 1. Vector+DMSO, 2. HRC+DMSO, 3. HRC+TG, 4.Vector+TG. (d) After pretreatment with 1 mM 4-PBA, Sk-hep-1 cells apoptosis was determined by flow cytometry. 1. si control+DMSO, 2. si HRC+DMSO, 3.si HRC+4-PBA, 4.si control+4-PBA. (e) Caspase 3 activity of hepatocellular carcinoma (HCC) cells pretreated with TG or 4-PBA were assessed. Each bar represents the mean ± SD of three separate experiments. *P < 0.05, **P < 0.01.