NEAT1_2-SFPQ axis mediates cisplatin resistance in liver cancer cells in vitro

Abstract

Background: Liver cancer is a type of malignant tumor with high morbidity and mortality in People's Republic of China. Its occurrence and development involve the variation and expression changes of multiple genes, and the pathogenesis and related regulatory networks are complex.

Purpose: In the present research, we investigate the involvement of NEAT1_2 and SFPQ in cisplatin resistance in liver cancer. The effects of LncRNA NEAT1 and SFPQ expression on the chemotherapeutic resistance of liver cancer cells were analyzed.

Methods: The expression level of NEAT1_2 and SFPQ mRNA in tissue specimens or cell lines were examined by RT-qPCR and western blotting. CCK-8 assay was performed to evaluate cell viability. Cell proliferation was performed using the EdU cell proliferation assay.

Results: Our data showed that increase NEAT1_2 and SFPQ expressions in liver cancer specimens were associated with the development of cisplatin resistance; high SFPQ expression level impaired patients' survival from liver cancer. Gain-and loss-of function assay using NEAT1_2 knock-in and knock-out cells constructed using CRISPER/Cas9 system revealed that NEAT1_2 is essential for liver cancer cell survival and mediates cisplatin resistance in liver cancer cells at least partially through SFPQ. Artificial change in NEAT1_2 expression level didn't significantly influence SFPQ transcription or translation level.

Conclusion: Our data revealed NEAT1_2-SFPQ axis as a novel cisplatin resistance mechanism in liver cancer cells in vitro.

Keywords: NEAT1; NEAT1_1; NEAT1_2; SFPQ; cisplatin resistance; liver cancer.

Figure 1

NEAT1_2 or SFPQ expression level change correlates with liver cancer development and cisplatin resistance. Notes: (A and B) Comparison of transcription levels of NEAT1_2 and SFPQ in different tissue specimens; data presented as fold change comparing to naïve-adjacent group after normalization to GAPDH; one-way ANOVA was employed for statistical significance tests between naïve and cisplatin resistance groups. (C) Correlation of NEAT1_2 and SFPQ transcription levels among all obtained tissue specimens. (D) Kaplan–Meier plot of survival of liver cancer patients with different SFPQ transcription levels. Naïve refers to patients diagnosed on the first visit; cisplatin-resistance refers to patients with cisplatin-resistant primary liver cancer. *P<0.05; **P<0.01. Abbreviations: Ca, cancerous (liver cancer tissue specimens); Ad, adjacent (adjacent non-cancerous tissue specimens); NS, normal saline.

Figure 2

NEAT1_2 facilitates liver cancer cell activity in vitro. Notes: (A) NEAT1_2 expression levels in six different liver cancer cell lines, revealed by RT-qPCR. (B) Cell viability of QGY-7703 or HUH-7 cells with different NEAT1 gene modifications. (C and D) Representation and statistics of cell proliferation in different cell groups. (E and F) Representation and statistics of cell apoptosis in different cell groups. (G and H) Representation and statistics of colony formation in different cell groups. **P<0.01; ***P<0.001. Abbreviations: Ctl, knock-out or knock-in control (wild type); KO, NEAT1 knock-out; KI, NEAT1 knock-in.

Figure 3

NEAT1_2 or SFPQ expression is essential for cisplatin resistance in liver cancer cells in vitro. Notes: (A and B) Representation and statistics of cell apoptosis in QGY-7703 or HUH-7 cells with different NEAT1 gene modifications with the presence of 0.25 µg/mL cisplatin in culture media. (C and D) Representation and statistics of colony formation in different cell groups with same cisplatin treatment as in (A or B). *P<0.05. Abbreviations: Ctl, knock-out or knock-in control (wild type); KO, NEAT1 knock-out; KI, NEAT1 knock-in; NC, non-specific control; siSFPQ, SFPQ knock-down by shRNA targeting.

Figure 4

Artificial NEAT1 gene modification did not affect SFPQ transcription or translation. Notes: (A and B) Representation and statistics of SFPQ protein expression in QGY-7703 or HUH-7 cells with different NEAT1 gene modifications. (C) Statistics of SFPQ mRNA expression in different cell groups. Abbreviations: WT, unmodified cells; KO, NEAT1 knock-out; KI, NEAT1 knock-in.