LncRNA CCAT1 enhances chemoresistance in hepatocellular carcinoma by targeting QKI-5

Abstract

A major reason for treatment failure of cancer is acquisition of drug resistance. The specific mechanisms underlying hepatocellular carcinoma (HCC) chemoresistance need to be fully elucidated. lncRNAs involve in drug resistance in some cancers, however, the exact functions of lncRNA colon cancer-associated transcript 1 (CCAT1) in oxaliplatin resistance in HCC are still unknown. Our study indicated that CCAT1 promoted HCC proliferation and reduced the apoptosis induced by oxaliplatin. Knockout of CCAT1 could increased chemosensitivity in vitro and in vivo. Further study found that QKI-5 was an important mediator and blocking of QKI-5/p38 MAPK signaling pathway could enhance oxaliplatin sensitivity. In conclusions, CCAT1 promoted proliferation and oxaliplatin resistance via QKI-5/p38 MAPK signaling pathway in HCC. Targeting CCAT1 in combination with chemotherapeutics may be a promising alternative to reverse drug resistance in HCC treatment.

Figure 1

CCAT1 enhanced HCC resistance to oxaliplatin in vitro. (A) HCC cells received OXA treatment for 48 h, the expression of CCAT1 was determined by qRT-PCR. (B, C) HCCLM3 and HepG2 HCC cell lines were knocked out of CCAT1 and exposed to increasing concentrations of oxaliplatin from 0.1 to 1 mM for 48 h to determine the IC50 values by CCK-8 assay. (IC50 of HCCLM3 control and KO-CCAT1: 46.56 vs. 23.22 μM; HepG2 control and KO-CCAT1: IC50: 27.98 vs. 13.28 μM, p < 0.01 respectively). (D) The colony formation assay showed that the numbers of colonies were reduced when CCAT1 was knocked out. (E) Significantly increased proportion of apoptotic cells by in the KO-CCAT1 groups. (F) Significantly increased caspase-3 activation following knock out of CCAT1, indicated by red fluorescence staining (p < 0.01). (G) Caspase-3 and caspase-7 activities were measured by Caspase-Glo® 3/7 Assay (p < 0.05).

Figure 2

CCAT1 involved in HCC resistance to oxaliplatin in vivo. All groups received intraperitoneal injections of OXA (0.8 mg/kg/w). (A) Tumor growth curves of subcutaneous implantation models of HCC. (B) The average tumor volumes were calculated using the following formula: V(mm³) = (L × W²) × 0.5 (L: tumor length, W: width). (C) TUNEL assay was applied to detect HCC apoptosis in subcutaneous implantation tumor (Original magnification: × 200). (D) Immunohistochemistry staining for Bel-2 and active caspase-3 expressions in subcutaneous tumor (Original magnification: × 400). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

CCAT1 interacted with QKI-5. (A) Subcellular localization plots displayed by lncATLAS. (B) This nuclear location was confirmed by the cytoplasmic and nuclear extracts measured from qRT-PCR. (C) RNA-binding proteins of CCAT1 was predicted by RBPDB (http://rbpdb.ccbr.utoronto.ca/) database. (D) Immunohistochemistry staining indicated that QKI-5 was located in the nucleus (Original magnification: × 400). (E) CCAT1 was enriched with QKI-5-immunoprecipitation. RNA pull-down assay was applied to detect the QKI-5 antibody.

Figure 4

Overexpression of QKI-5 reversed oxaliplatin resistance induced by CCAT1. (A) Western blotting analysis of expression levels of QKI-5, JNK, p-JNK, ERK1/2, p-ERK1/2, p38 and p-p38 MAPK in control and pcDNA3.1-QKI-5 cells. (B) The phosphorylation and total levels of p38 MAPK in the CCAT1 and QKI-5 overexpression HCC cells. (C) IC50 of oxaliplatin was determined when QKI-5 was upregulated. (D) The colony formation assay showed that overexpressed QKI-5 significantly attenuated cell proliferation induced by CCAT1 both in HCCLM3 and HepG2 cells. (E, F) Fluorescence staining and Caspase-Glo® 3/7 Assay showed caspase-3 activity influenced by CCAT1 and QKI-5. Overexpressed QKI-5 could increase caspase-3 activity reduced by CCAT1. *P < 0.05, **P < 0.01, ***P < 0.001.