Interfered long non-coding RNA HELLPAR or up-regulated microRNA-448 inhibits nasopharyngeal carcinoma progression via suppression of ADAM10

Abstract

Objective: Nasopharyngeal carcinoma (NPC) is a highly invasive malignancy with poor prognosis, necessitating further exploration of its molecular mechanisms. While HELLP-associated long non-coding RNA (HELLPAR), microRNA-448 (miR-448), and a disintegrin and metalloprotease 10 (ADAM10) have been implicated in other malignancies, their regulatory interplay and functional roles in NPC remain unclear. This study aimed to investigate the role of HELLPAR in NPC progression through its interaction with miR-448 and ADAM10.

Methods: Cancerous and adjacent non-cancerous tissues were collected from 53 NPC patients admitted to our hospital between 1st January 2018 and 1st January 2020. Transcript levels of HELLPAR, miR-448, and ADAM10 were measured using quantitative real-time PCR (RT-qPCR), while the protein expression levels of ADAM10 were assessed by Western blotting. Long-term survival data were analyzed to assess the correlation between HELLPAR expression and patient prognosis. The binding interactions of HELLPAR/miR-448 and miR-448/ADAM10 were predicted and experimentally validated. Overexpression and knockdown constructs for HELLPAR, miR-448, and ADAM10 were transfected into NPC cells to assess their effects on proliferation, invasion, and apoptosis.

Results: HELLPAR and ADAM10 were significantly upregulated at both the RNA and protein levels in NPC tissues and cells, while miR-448 was notably downregulated. Suppression of HELLPAR inhibited NPC cell proliferation and invasion while promoting apoptosis. Mechanistically, HELLPAR functioned as a competitive endogenous RNA (ceRNA) by binding to miR-448, thereby downregulating its RNA expression. Overexpression of miR-448 counteracted the tumor-promoting effects of HELLPAR. Additionally, miR-448 directly targeted and suppressed ADAM10. Overexpression of ADAM10 reversed the inhibitory effects of miR-448 on NPC cell proliferation and invasion.

Conclusion: The HELLPAR/miR-448/ADAM10 axis plays a critical role in NPC progression. Suppressing HELLPAR expression enhances miR-448 activity, which in turn downregulates ADAM10 at both RNA and protein levels, leading to reduced NPC cell proliferation and invasion while promoting apoptosis.

Keywords: A disintegrin and metalloprotease 10; Apoptosis; Invasion; Long non-coding RNA HELLPAR; MicroRNA-448; Nasopharyngeal carcinoma; Proliferation.

Fig. 1

Differential expression of HELLPAR in NPC tissues and cells. A HELLPAR expression levels in paracancerous tissues and NPC tissues were tested by RT-qPCR; B HELLPAR expression levels in NP69, HK-1, TW03, C666-1, and HNE-3 cells were assessed; C The relationship of HELLPAR expression and the survival of NPC patients was analyzed by Kaplan–Meier analysis; * P < 0.05, ** P < 0.01; N = 53

Fig. 2

HELLPAR expression influences the biological functions of NPC cells. A. HELLPAR interference efficiency in NPC cells was assessed by RT-qPCR; B. The proliferation ability of cells after sh-HELLPAR treatment was tested by CCK-8 assay; C. The apoptosis rate after sh-HELLPAR treatment was assessed by flow cytometry; D. The invasive ability of cells after sh-HELLPAR treatment was measured by Transwell assay; * P < 0.05 vs sh-NC

Fig. 3

HELLPAR competitively binds miR-448 and regulates its expression. A. The subcellular localisation of HELLPAR was observed by nuclear and cytoplasmic separation assay, with GAPDH as a cytoplasmic marker and U6 as a nuclear marker; B. Starbase website predicted the binding site of HELLPAR to miR-448; C. The binding of HELLPAR to miR-448 was verified by dual-luciferase assay; D. The binding of HELLPAR to miR-448 was tested by RIP assay; E: The enrichment of miR-448 for HELLPARRNA was measured by RNA pull-down assay; F. miR-448 expression in NPC tissues and cells was assessed by RT-qPCR; G. miR-448 expression after overexpression of HELLPAR was tested by RT-qPCR; * P < 0.05

Fig. 4

HELLPAR sponges miR-448 to affect the biological functions of NPC cells. A. miR-448 expression after oe-HELLPAR + miR-448 mimic treatment was tested by RT-qPCR; B. The proliferation ability of cells after oe-HELLPAR + miR-448 mimic treatment was assessed by CCK-8 assay; C. The apoptosis rate after oe-HELLPAR + miR-448 mimic treatment was measured by flow cytometry; D. The invasive ability of cells after oe-HELLPAR + miR-448 mimic treatment was assessed by Transwell assay; * P < 0.05

Fig. 5

miR-448 affects the biological functions of NPC cells by regulating ADAM10. A. Starbase website predicted the target binding site between miR-448 and ADAM10; B. The targeting relationship between miR-448 and ADAM10 was verified by dual-luciferase assay; C. ADAM10 expression in NPC tissues and cells was assessed by RT-qPCR and western blotting; D. ADAM10 expression in cells treated with miR-448 mimic + oe-ADAM10 was measured by western blotting; E. The proliferation ability of cells treated with miR-448 mimic + oe-ADAM10 was tested by CCK-8 assay; F. The apoptosis rate after treatment of miR-448 mimic + oe-ADAM10 was assessed by flow cytometry; G. The invasive ability of cells treated with miR-448 mimic + oe-ADAM10 was determined by Transwell assay; * P < 0.05