Asparaginase and isoaspartyl peptidase 1 RNA interference suppresses the growth of nasopharyngeal carcinoma cells

Abstract

Nasopharyngeal carcinoma (NPC) is one of the common malignant tumors, and its pathogenesis has not been fully clarified. This study aims to explore the impact of RNA interference on the growth and invasion of NPC cells. Asparaginase and isoaspartyl peptidase 1 (ASRGL1)-short hairpin(sh) RNA expressing lentivirus was used to investigate the effect of ASRGL1 knockdown on NPC cells (C666-1 and SUN-1). The target shASRGL1 gene was determined by mRNA and protein expression in nasopharyngeal carcinoma cells; nasopharyngeal carcinoma cell proliferation viability, migration, invasion, apoptosis, ATP levels, and oxidative stress were examined. The results found that ASRGL1 was found to be highly expressed in NPC tissues and cell lines. shASRGL1 exhibited a high gene expression knockdown efficiency, downregulated the ASRGL1 protein expression in the nasopharyngeal carcinoma cells, suppressed proliferation viability of transfected nasopharyngeal carcinoma cells, inhibited their migration and invasion and ATP levels, promoted nasopharyngeal carcinoma cell apoptosis, ROS, and ferroptosis. shASRGL1 plays a role in protecting against NPC cell growth and invasion.

Keywords: Asparaginase and isoaspartyl peptidase 1; Nasopharyngeal carcinoma cell; RNA interference.

Fig. 1

ASRGL1 was highly expressed in Nasopharyngeal carcinoma tissues and cell lines. A The mRNA expression level of ASRGL1 in NPC patients was detected by RT-qPCR. Relative mRNA levels were compared in different groups, two-tailed, unpaired t-test, ***p < 0.001. B The protein expression level of ASRGL1 in NPC patients was detected by WB. Representative bands for the 5 cases are shown in the figure. Relative protein levels were compared in different groups, two-tailed, unpaired t-test, *p < 0.05. C The mRNA expression level of ASRGL1 in NPC cell lines was detected by RT-qPCR. Relative mRNA levels were compared in different groups. One-way ANOVA, **p < 0.01, ***p < 0.001. D The protein expression level of ASRGL1 in NPC cell lines was detected by WB. Relative protein levels were compared in different groups. One-way ANOVA, ***p < 0.001

Fig. 2

The shASRGL1 suppressed C666-1 and SUN-1 cell proliferation viability and division. A ASRGL1 RNA interference genes were tested. The mRNA expression level of ASRGL1 was measured by RT-qPCR. Relative mRNA levels were compared in different groups, two-tailed, unpaired t-test, ***p < 0.001. B The protein expression level of ASRGL1 was measured by WB. Relative protein levels were compared in different groups, two-tailed, unpaired t-test, ***p < 0.001. C Cell proliferation was measured using a CCK-8 assay. The cell viability of the control group and the shASRGL1 group was compared by measuring the detection absorbance at 450 nm at different time points. Two-way ANOVA, ***p < 0.001, D The percentage of cell population in different phases of the cell cycle was quantified and compared between the control group and the shASRGL1 group. Two-tailed, unpaired t-test, *p < 0.05, *p < 0.01, ***p < 0.001

Fig. 3

shASRGL1 inhibited C666-1 and SUN-1 cell migration and invasion. A Cell migration was assessed using the scratch test. The migration rate was compared between the shCtrl and shASRGL1 groups after 8 h of cell culture, two-tailed, unpaired t-test. The results showed a significant reduction in migration rate in the shASRGL1 group compared to the shCtrl group (***p < 0.001). B Cell invasion was performed using a transwell assay. Comparison of the migratory cells between shCtrl and shASRGL1 group. Two-tailed, unpaired t-test, *** p < 0.001

Fig. 4

shASRGL1 promoted nasopharyngeal carcinoma cell apoptosis, ROS, and ferroptosis. A Apoptosis was assigned with Annexin V-FITC and PI double staining. Histograms of the shCtrl and shASRGL1 groups are shown. The percentage of early, late, and total apoptosis was quantified and compared between the shCtrl and shASRGL1 groups. Two-tailed, unpaired t-test, **p < 0.01, ***p < 0.001. B The protein expression level of Cleaved caspase3 and Cleaved caspase9 in NPC cell lines was detected by WB. Relative protein levels were compared between the shCtrl and shASRGL1 groups. Two-tailed, unpaired t-test, ***p < 0.001. C The level of ATP was quantified and compared between the shCtrl and shASRGL1 groups. Two-tailed, unpaired t-test, ***p < 0.001. D The protein expressions of ACSL4, FTH1 and GPX4 in cells were detected by WB. E ROS level was assigned with IF. Histograms of the shCtrl and shASRGL1 groups are shown