Unraveling the oncogenic role of LINC00504 and its interaction with miR-545-3p and ARIHI in hepatocellular carcinoma: novel insights for molecular therapy

Abstract

Objectives: Hepatocellular carcinoma (HCC) is a serious global health threat associated with high morbidity and mortality. The importance of long non-coding RNAs (lncRNAs) in tumor progression is growing. The aim of this study was to explore the expression, functional properties and molecular mechanisms of LINC00504 in HCC.

Materials and methods: Tumor tissue samples from HCC patients were collected to analyze the expression of LINC00504, miR-545-3p, and ARIH1 mRNA using RT-qPCR, and compared with various HCC cell lines (PLC/PRF/5, SNU-182, Hep3B, HuH-7) and a human normal liver epithelial cell line (THLE-2). Cell proliferation, apoptosis, and invasion were assessed using transfection vectors, CCK8 assay, flow cytometry, and Transwell. Interactions among LINC00504, miR-545-3p, and ARIH1 were confirmed through database predictions and luciferase reporter gene assays.

Results: LINC00504 was underexpressed in HCC tissues and cell lines. Upregulation of LINC00504 inhibited cell proliferation, induced apoptosis, increased Bax and Caspase-3, decreased Bcl-2 mRNA, and suppressed invasion. miR-545-3p was overexpressed in HCC cells and was negatively regulated by LINC00504. Overexpression of miR-545-3p counteracted the effects of LINC00504 upregulation. ARIH1 was underexpressed in HCC tissues and had a negative correlation with miR-545-3p. miR-545-3p negatively regulated ARIH1 expression, and ARIH1 overexpression overturned the promotional effects of miR-545-3p on HCC cells.

Conclusion: This study uncovers the significant tumor-suppressing role of LINC00504 in HCC, potentially through a mechanism involving the targeting of miR-545-3p, which in turn inhibits the ARIH1. These findings offer new potential targets for HCC molecular treatment.

Keywords: ARIH1; Cellular processes; HCC; LINC00504; MiR-545-3p.

Fig. 1

A LINC00504 downregulation noted in HCC via LncBook. B LINC00504 expression reduced in HCC tissues. C LINC00504 significantly downregulated in HCC cell lines compared to THLE-2. *** P < 0.001, vs. Normal or THLE-2

Fig. 2

A SNU-182 and HuH-7 cell lines were transfected with the oe-LINC00504 plasmid to induce LINC00504 upregulation. B, C LINC00504 upregulation inhibits cell proliferation in SNU-182 and HuH-7 cells. D LINC00504 upregulation induces apoptosis in cancer cells. E, F LINC00504 upregulation promotes Bax/Caspase-3 and inhibits Bcl-2 in SNU-182 and HuH-7. G LINC00504 upregulation inhibits cancer cell invasion. *** P < 0.001, vs. CK

Fig. 3

A GEDS database shows miR-545-3p upregulation in hepatocellular carcinoma (LIHC) tissues. B miR-545-3p upregulated in HCC tissues. C miR-545-3p negatively correlates with LINC00504 in HCC. D In HCC cell lines in comparison to THLE-2, miR-545-3p upregulated. ***P < 0.001, vs. Normal or THLE-2

Fig. 4

A The binding sites between miR-545-3p and LINC00504, with primer sequences shown for WT and MT. B, C miR-545-3p negatively affects luciferase activity of WT-LINC00504 in SNU-182 and HuH-7 cells, not MT. D miR-545-3p overexpression does not alter LINC00504 expression in HCC cell lines. E LINC00504 overexpression significantly inhibits miR-545-3p expression in HCC cell lines. *** P < 0.001, vs. CK; ## P < 0.001, vs. oe- LINC00504

Fig. 5

A, B miR-545-3p overexpression counters LINC00504-mediated inhibition of SNU-182 and HuH-7 cell proliferation. C miR-545-3p reverses LINC00504-induced apoptosis. D, E miR-545-3p alters LINC00504 effects on Bax, Caspase-3, Bcl-2. F miR-545-3p reverses the inhibition of LINC00504 on cancer cell invasion. *** P < 0.001, vs. CK; ## P < 0.001, vs. oe- LINC00504

Fig. 6

A Binding sites between ARIH1 and miR-545-3p identified. B, C miR-545-3p reduces luciferase activity of WT-ARIH1. D miR-545-3p levels are unaffected by ARIH1 overexpression. E miR-545-3p inhibits ARIH1 expression. *** P < 0.001, vs. miR-NC; ## P < 0.001, vs. miR-mimic

Fig. 7

A, B ARIH1 reverses miR-545-3p promotion of SNU-182, and HuH-7 cell proliferation. C ARIH1 reverses the inhibition of apoptosis by miR-545-3p. D, E ARIH1 alters miR-545-3p effects on Bax, Caspase-3, Bcl-2 in SNU-182 and HuH-7. F ARIH1 reverses miR-545-3p promotion of cancer cell invasion. *** P < 0.001, vs. miR-NC; ## P < 0.001, vs. miR-mimic