HLA-DQB1-AS1 Promotes Cell Proliferation, Inhibits Apoptosis, and Binds with ZRANB2 Protein in Hepatocellular Carcinoma

Abstract

Major histocompatibility complex, class II, DQ beta 1 antisense RNA 1 (HLA-DQB1-AS1) conferred the susceptibility to hepatocellular carcinoma. Sustaining cell growth and resisting apoptosis are two hallmarks of hepatocellular carcinoma. The present study explored the role of HLA-DQB1-AS1 in the proliferation and apoptosis of hepatocellular carcinoma cells and investigated its downstream pathway. Colony formation assay was performed to assess cell proliferation. Cell apoptosis was assessed with the TdT-mediated dUTP nick end labeling method. HLA-DQB1-AS1 deficiency exerts antiproliferative and proapoptotic effects on hepatocellular carcinoma cells. Moreover, based on bioinformatic analysis combined with the results of RNA immunoprecipitation assay, HLA-DQB1-AS1 was revealed to bind with zinc finger RANBP2-type containing 2 (ZRANB2) protein. ZRANB2 was upregulated in hepatocellular carcinoma at a clinical and cellular level. HLA-DQB1-AS1 caused no significant effects on ZRANB2 mRNA and protein expression. ZRANB2 knockdown suppressed cell proliferation and enhanced cell apoptosis of hepatocellular carcinoma. Moreover, ZRANB2 overexpression rescued the anticancer effect of silenced HLA-DQB1-AS1 in hepatocellular carcinoma cells. In conclusion, HLA-DQB1-AS1 promotes cell proliferation and inhibits apoptosis in hepatocellular carcinoma by the interaction with ZRANB2 protein.

Figure 1

HLA-DQB1-AS1 is upregulated in hepatocellular carcinoma. (a) HLA-DQB1-AS1 expression, log2 (FPKM + 0.01) transformed, in 374 liver hepatocellular carcinoma (LIHC) tissues and 50 normal samples on EOCORI database. (b) RT-qPCR analysis of HLA-DQB1-AS1 expression in hepatocellular carcinoma cell lines and normal liver cell line LO2. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 2

HLA-DQB1-AS1 deficiency exerts antiproliferative and proapoptotic effects on hepatocellular carcinoma cells. (a) RT-qPCR was performed to confirm the knockdown efficiency of HLA-DQB1-AS1. (b, c) Colony formation and TUNEL assays were performed to assess the proliferation and apoptosis of MHCC97-H and Huh-7 cells after sh-HLA-DQB1-AS1#1/2 transfection. ^∗∗∗P < 0.001.

Figure 3

HLA-DQB1-AS1 binds with ZRANB2 protein. (a) RT-qPCR analysis of ZRANB2 expression in hepatocellular carcinoma cell lines and normal liver cell line LO2. (b, c) Effects of sh-HLA-DQB1-AS1#1/2 on ZRANB2 mRNA and protein expression, as assessed by RT-qPCR and western blotting. (d) RIP assay was performed to explore the interaction between HLA-DQB1-AS1 and ZRANB2 protein in MHCC97-H and Huh-7 cells after sh-HLA-DQB1-AS1 transfection. ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 4

Bioinformatic information of ZRANB2. (a) ZRANB2 expression, log2 (FPKM + 0.01) transformed, in 374 LIHC tissues and 50 normal samples on EOCORI database. (b) Overall survival and disease-free survival curves of liver hepatocellular carcinoma patients with high or low expression of ZRANB2 were obtained from GEPIA database. (c) ZRANB2 expression in different stages of liver hepatocellular carcinoma was obtained from GEPIA database. (d) Expression correlation of HLA-DQB1-AS1 and ZRANB2 in liver hepatocellular carcinoma tissues and adjacent normal tissues was obtained from GEPIA database.

Figure 5

ZRANB2 knockdown suppressed proliferation and enhanced apoptosis of hepatocellular carcinoma. (a) RT-qPCR was performed to confirm the knockdown efficiency of ZRANB2. (b, c) Colony formation and TUNEL assays were performed to detect the proliferation and apoptosis in MHCC97-H and Huh-7 cells after sh-ZRANB2#1/2 transfection. ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 6

ZRANB2 overexpression rescued the anticancer effect of silenced HLA-DQB1-AS1. (b, c) Colony formation and TUNEL assays were performed in MHCC97-H and Huh-7 cells after transfection of sh-NC and sh-HLA-DQB1-AS1 and cotransfection of sh-HLA-DQB1-AS1+pIRES-hrGFP-1a-ZRANB2. ^∗∗P < 0.01; ^∗∗∗P < 0.001.