ECEL1 could be a gene involved in the cell proliferation and apoptosis in hepatocellular carcinoma
Authors: Nie Li , Zou Ke Bing , Wang YuanYuan , Zuo Yang , Chen Qing , Long Jian Wu , Yang Xuefeng , Liu LongFei , Peng Liu 
Published: September 2023

Abstract
Background: The expression and role of the endothelin converting Enzyme like1 (ECEL1) gene in hepatocellular carcinoma（HCC）was unclear. The purpose of this study was to understand the expression of ECEL1 in HCC tissues and cells and its effects on the proliferation and apoptosis of HCC cells, so as to provide a new therapeutic target for the prevention and treatment of HCC.
Methods: (1) The expression of ECEL1 between HCC and normal liver tissues was analyzed by TCGA database, and the analysis results of TCGA database were verified by immunohistochemical staining. The correlation between the expression of ECEL1 and the survival time of HCC patients was analyzed by Kaplan Meier plotter database. (2) On the basis of confirming that HCC cell lines could express ECEL 1 gene, ECEL 1 expression in HCC cells were silenced by lentivirus-mediated RNAi technology, and observed the functional changes in the direction of proliferation of HCC cells by clone formation, Celigo count, MTT, etc. At the same time, the functional changes of apoptosis direction of HCC cells were observed, such as apoptosis, Caspase-3/7 activity, after silencing ECEL1. The HCC cells that RNAi effectively silenced ECEL1 were injected subcutaneously into nude mice to observe the ability of ECEL1 to regulate the proliferation of liver cancer in vivo. (3) The expression profile microarray was used to detect the differential expression of the downstream genes after ECEL1 silencing in HCC cells, and quantitative real-time PCR（qRT-PCR）and Western Blot were applied to verify some genes related to HCC proliferation and apoptosis to explore the possible mechanism of ECEL1 regulating the proliferation and apoptosis of hepatocellular carcinoma.
Results: (1) The original data of 374 RNAseq samples (Cancer/Normal) in TCGA database were filtered, standardized, BCV (biological coefficient of variation) quality controlled and statistical analyzed. It showed that the expression level of ECEL1 in HCC tissues were higher than that in normal liver tissue (p<0.05). The results of immunohistochemical staining showed that there were significant differences in the expression of ECEL1 gene between liver cancer tissues and normal liver tissues, which were consistent with the analysis results of TCGA database. (2) The expression of ECEL1 gene in various HCC cell lines were detected by qRT-PCR, and the results using GAPDH as an internal reference showed that ECEL1 gene was expressed in various HCC cells. (3) Clone formation, Celigo and MTT assay showed that the proliferation of BEL-7404 and Huh-7 cells were significantly inhibited after ECEL1 silencing (p<0.05).(4) Flow cytometry showed that the apoptosis rate of BEL-7404 and Huh-7 cells increased significantly after ECEL1 silencing (P<0.01). After ECEL1 silencing, BEL-7404 cells in S phase decreased significantly, while those in G2/M phase increased significantly (P<0.01). (5)Caspase-Glo®3/7 Assay kit showed that the activity of Caspase-3/7 in BEL7404 and Huh-7 cells were significantly increased after silencing the ECEL1 gene (P<0.01). (6) The results of animal tumorigenesis experiment showed that the tumor volume of shECEL1 group was smaller than that of shCtrl group, the tumor weight was lighter, the total fluorescence expression in the tumor area and the average fluorescence expression per cm2in the region were weaker. (7) The expression profile chip detection showed that 371 genes were up-regulated and 377 genes were down regulated in the shECEL1 group. Bioinformatics analysis, qRT-PCR and Western Blot verification showed that the expression of DUSP1, THBS1 and PTEN was up-regulated, while the expression of EGR1 was down regulated.
Conclusions: The ECEL1 was highly expressed in liver cancer tissues and ECEL1 gene was significantly related to survival time of patients with liver cancer;The silencing ECEL1 expression in liver cancer cells could induce cell apoptosis, inhibit cell proliferation and the growth of subcutaneous transplanted tumors; The expression of DUSP1, THBS1, PTEN may participate in the inhibition of liver cancer cell proliferation. These results indicate that ECEL 1 gene is a gene related to the proliferation and apoptosis of hepatoma cells