Effects of ALKBH5 on the malignant biological behavior of esophageal squamous cell carcinoma and the related mechanism

doi:10.3760/cma.j.cn371439-20240607-00012

Abstract

Abstract:

Objective To investigate the role and potential mechanism of m⁶A demethylase ALKBH5 in esophageal squamous cell carcinoma （ESCC）. Methods Real time fluorogenic quantitative PCR and Western blotting were used to detect ALKBH5 expression in normal esophageal epithelial cells （Het-1A） and ESCC cell lines （Eca109， KYSE30， KYSE150， KYSE410）. Transient cell lines with overexpression/knockdown of ALKBH5 （siRNA transfection was divided into si-ALKBH5-1 group and si-ALKBH5-2 group） and control cell lines were constructed. The effects of ALKBH5 on ESCC cell proliferation， migration and apoptosis were studied by MTT assay， cell scratch assay and cell apoptosis assay respectively. The differentially expressed gene was screened by the intersection of RNA sequencing （RNA-seq） and methylated RNA immunoprecipitation sequencing （MeRIP-seq） techniques， and the effect of ALKBH5 on the gene expression was detected by RT-qPCR. Results Real time fluorogenic quantitative PCR results showed that， the relative expression levels of ALKBH5 RNA in Het-1A， Eca109， KYSE30， KYSE150 and KYSE410 were 1.03±0.28， 0.46±0.02， 0.23±0.10， 0.04±0.02， 0.05±0.00， respectively， with a statistically significant difference （F=444.60， P<0.001）. Western blotting showed that， the relative expression levels of ALKBH5 protein in Het-1A， Eca109， KYSE30， KYSE150 and KYSE410 were 1.14±0.03， 0.88±0.04， 0.66±0.01， 0.69±0.01， 0.95±0.01， respectively， with a statistically significant difference （F=139.90， P<0.001）. MTT test showed that the absorbance （A） values of KYSE30 control group and ALKBH5 overexpression group were 0.86±0.01 and 1.25±0.01 after 72 hours， respectively， with a statistically significant difference （t=46.93， P<0.001）. The A values of KYSE150 control group and ALKBH5 overexpression group were 1.00±0.03 and 1.43±0.02 after 72 hours， respectively， with a statistically significant difference （t=16.80， P<0.001）. The A values of KYSE30 control group， si-ALKBH5-1 group and si-ALKBH5-2 group were 0.98±0.01， 0.85±0.02 and 0.80±0.09 after 96 hours， respectively， with a statistically significant difference （F=72.97， P<0.001）. The A values of KYSE30 control group were higher than those of si-ALKBH5-1 and si-ALKBH5-2 groups （both P<0.001）. The A values of KYSE410 control group， si-ALKBH5-1 group and si-ALKBH5-2 group were 1.28±0.02， 1.15±0.02 and 1.08±0.05 after 72 hours， respectively， with a statistically significant difference （F=16.97， P=0.003）. The A values in KYSE410 control group were higher than those in si-ALKBH5-1 group and si-ALKBH5-2 group （P=0.020； P=0.003）. The cell scratch test showed that 48 hours after scratch， the migration rates of KYSE30 cells in control group and ALKBH5 overexpression group were （27.39±0.54）% and （48.89±5.12）%， respectively， with a statistically significant difference （t=5.90， P=0.004）. The migration rates of KYSE150 cells in control group and ALKBH5 overexpression group were （39.67±0.43）% and （62.20±0.60）%， respectively， with a statistically significant difference （t=43.15， P<0.001）. The migration rates of KYSE30 cells in control group， si-ALKBH5-1 group and si-ALKBH5-2 group were （25.08±1.86）%，（18.75±1.59）% and （7.67±0.52）%， respectively， with a statistically significant difference （F=74.28， P<0.001）. The migration rates of KYSE30 cells in control group were higher than those of si-ALKBH5-1 group and si-ALKBH5-2 group （P=0.010； P<0.001）. The migration rates of KYSE410 cells in control group and si-ALKBH5-1 group， si-ALKBH5-2 group were （38.70±0.41）%，（28.27±1.01）% and （19.40±0.47）%， respectively， with a statistically significant difference （F=400.20， P<0.001）. The migration rates of KYSE410 cells in control group were higher than those of si-ALKBH5-1 group and si-ALKBH5-2 group （both P<0.001）. Apoptosis test showed that the apoptosis rates of KYSE30 cells in control group and ALKBH5 overexpression group were （9.59±0.88）% and （4.81±0.89）%， respectively， with a statistically significant difference （t=6.23， P=0.006）. The apoptosis rates of KYSE150 cells in control group and ALKBH5 overexpression group were （8.36±0.09）% and （6.42±0.19）%， respectively， with a statistically significant difference （t=12.90， P<0.001）. The apoptosis rates of KYSE30 cells in control group， si-ALKBH5-1 group and si-ALKBH5-2 group were （4.31±0.19）%，（5.72±0.30）% and （8.94±0.71）%， respectively， with a statistically significant difference （F=53.46， P<0.001）. The apoptosis rates in KYSE30 cells in control group were lower than those in si-ALKBH5-1 group and si-ALKBH5-2 group （P=0.049； P<0.001）. The apoptosis rates of KYSE410 control group， si-ALKBH5-1 group and si-ALKBH5-2 group were （4.45±0.36）%，（5.40±0.11）% and （6.64±0.15）%， respectively， with a statistically significant difference （F=43.36， P<0.001）. The apoptosis rates in KYSE410 cells in control group were lower than those in si-ALKBH5-1 group and si-ALKBH5-2 group （P=0.016； P<0.001）. The differentially expressed gene IGF2BP3 was screened by the intersection of RNA-seq and MeRIP-seq techniques， and the RT-qPCR results showed that， the relative expression levels of IGF2BP3 in KYSE30 were 1.01±0.10 and 1.41±0.10 in control group and ALKBH5 overexpression group， respectively， with a statistically significant difference （t=4.06， P=0.015）. The relative expression levels of IGF2BP3 in KYSE150 were 1.00±0.10 and 1.94±0.24 in control group and ALKBH5 overexpression group， respectively， with a statistically significant difference （t=5.08， P=0.007）. The relative expression levels of IGF2BP3 in KYSE410 were 1.01±0.14， 0.67±0.04 and 0.41±0.04 in control group， si-ALKBH5-1 group and si-ALKBH5-2 group， respectively， with a statistically significant difference （F=24.36， P=0.001）. The relative expression levels of IGF2BP3 in KYSE410 control group were higher than those in si-ALKBH5-1 group and si-ALKBH5-2 group （P=0.017； P=0.001）. Conclusions ALKBH5 is underexpressed in ESCC cell lines， but the overexpression of ALKBH5 can promote the proliferation and migration of ESCC cells and inhibit cell apoptosis， which may be related to some negative feedback regulation mechanism. IGF2BP3 may be the downstream target of ALKBH5.

Key words: ALKBH5, Esophageal squamous cell carcinoma, IGF2BP3

Ma Peihan, Zhang Lingmin, Li Qian, Lu Ning, Wen Hua, Zhang Mingxin. Effects of ALKBH5 on the malignant biological behavior of esophageal squamous cell carcinoma and the related mechanism[J]. Journal of International Oncology, 2025, 52(2): 79-88.

Figures/Tables 4

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