SEH1L siliencing induces ferroptosis and suppresses hepatocellular carcinoma progression via ATF3/HMOX1/GPX4 axis

Abstract

SEH1 like nucleoporin (SEH1L) is an important component of nuclear pore complex (NPC), which is crucial in the regulation of cell division. However, the interrelation between SEH1L expression and tumor progression is less studied. In this research, we performed a systematic bioinformatic analysis about SEH1L using TCGA, Timer 2.0, Cbioportal, UCLAN and CellMiner™ databases in pan-cancer. Besides, we further validated the bioinformatic results through in vitro and in vivo experiments in HCC, including transcriptome sequencing, real-time quantitative PCR (RT-qPCR), western blotting (WB), immunohistochemistry (IHC), cell proliferation assays, clone formation, EdU, transwell, flow cytometry and subcutaneous tumor model. Our results suggested that SEH1L was significantly up-regulated and related to poor prognosis in most cancers, and may serve as a potential biomarker. SEH1L could promote HCC progression in vitro and in vivo. Besides, the next generation sequencing suggested that 684 genes was significantly up-regulated and 678 genes was down-regulated after the knock down of SEH1L. SEH1L siliencing could activate ATF3/HMOX1/GPX4 axis, decrease mitochondrial membrane potential and GSH, but increase ROS and MDA, and these effects could be reversed by the knock down of ATF3. This study indicated that SEH1L siliencing could induce ferroptosis and suppresses hepatocellular carcinoma (HCC) progression via ATF3/HMOX1/GPX4 axis.

Keywords: ATF3; Ferroptosis; HCC; HMOX1; SEH1L.

Fig. 1

The mRNA expression level of SEH1L in normal tissue and pan-cancer. (A) The expression of SEH1L in normal tissue. (B) SEH1L was up-regulated in most cancer (C) The ROC curves of SEH1L in COAD, ESCA, KICH, LIHC, LUSC and STAD

Fig. 2

SEH1L may function as a prognostic biomarker in pan-cancer. (A) Overall survival. (B) Progression free interval. (C) Disease free interval. (DD) Disease specific survival

Fig. 3

The promoter methylation characteristic of SEH1L in pan-cancer. (A-I) The promoter methylation level of SEH1L in KIRC, COAD, KIRP, LIHC, LUSC, PAAD, PRAD, READ and UCEC

Fig. 4

The drug sensitivity analysis of SEH1L. (A-D, F-I, K-M, O) A negative correlation was shown between SEH1L expression and drug IC50. (E, G, N, P) A positive correlation was shown between SEH1L expression and drug IC50

Fig. 5

SEH1L was up-regulated in HCC. (A-B) The protein expression of SEH1L in HCC (C: cancer P: paracancerous non-cancer). (C) The mRNA expression of SEH1L in HCC cells. (D-E) The SEH1L mRNA knock down and over-expression efficiency. (F) The protein expression of SEH1L in HCC cells. (G-H) The SEH1L protein knock down and over-expression efficiency. (I) Next generation sequencing showed 684 up-regulated genes and 678 down-regulated genes after SEH1L knock down. (J-K) GO and KEGG analysis of the different expressed genes

Fig. 6

Over-expression of SEH1L promoted HCC progression. (A-C, F-G) SEH1L promoted the proliferation of HCC. (D-E) SEH1L promoted HCC migration. (H-I) Over-expression of SEH1L increased HCC GSH, but decreased MDA. (J-K) Over-expression of SEH1L increased HCC mitochondrial membrane potential. (L-N) Over-expression of SEH1L decreased HCC ROS. (* represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001)

Fig. 7

Knock down of SEH1L suppressed HCC progression. (A-C, F-G) Knock down of SEH1L suppressed the proliferation of HCC. (D-E) Knock down of SEH1L suppressed HCC migration. (H-I) Knock down of SEH1L decreased HCC GSH, but increased MDA. (J-K) Knock down of SEH1L decreased HCC mitochondrial membrane potential. (L-N) Knock down of SEH1L increased HCC ROS. (O-P) Knock down of SEH1L suppressed HCC progression in vivo. (* represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001)

Fig. 8

SEH1L siliencing activated ATF3/HMOX1/GPX4 pathway. (A-B) The mRNA level of ATF3 and HMOX1 were up-regulated after the knock down of SEH1L. (C-D) The protein level of ATF3, HMOX1 were up-regulated and GPX4 was down-regulated after the knock down of SEH1L. (E-F) The ATF3 knock down efficiency. (G-H) Knock down of ATF3 reversed the influence of SEH1L on HMOX1. (* represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001)

Fig. 9

SEH1L siliencing induced HCC ferroptosis via ATF3/HMOX1/GPX4 axis. (A, E) Knock down of ATF3 rescued the influence of SEH1L on HCC proliferation. (B, C, F) ATF3 siliencing rescued the influence of SEH1L on HCC mitochondrial membrane potential. (D, G) ATF3 siliencing rescued the influence of SEH1L on HCC ROS. (* represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001)