RNAi-mediated TCF-3 gene silencing inhibits proliferation of Eca-109 esophageal cancer cells by inducing apoptosis.

Jie Ma,Lian Li,Rui Li,Fang Wang,Xian-Bin Wang,Shu-Hong Xuan,Zhi-Ping Zhang,Lu Tan,Xin Hua Li

doi:10.1042/bsr20170799

Jie Ma, Lian Li + Show 7 more

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https://doi.org/10.1042/bsr20170799

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Abstract

Esophageal cancer (EC) remains an important health problem in China. In the present study, through the use of siRNA, specific gene knockdown of transcription factor 3 gene (TCF-3) was achieved in vitro and the effect of TCF-3 gene on human EC Eca-109 cell proliferation and apoptosis. Eca-109 cells were treated using negative control (NC) of siRNA against TCF-3 (siTCF-3) and siTCF-3 group. Colony formation assay was used to detect the colony formation ability in Eca-109 cells. MTT assay was used to measure the cell growth and viability, whereas BrDU assay was used to evaluate cell proliferation, and flow cytometry (FCM) to assess cell apoptosis. Reverse-transcription quantitative PCR (RT-qPCR) was applied to measure TCF-3 gene expression. Protein expressions of TCF-3, apoptosis-related proteins, Bcl-2, Bax, and caspase-3 were determined using Western blotting. Transfection of siTCF-3 successfully down-regulated TCF-3 gene expression. In addition, siTCF-3, reduced Eca-109 cell viability and proliferation, in a time-dependent manner, and inhibited progression of cell cycle from G0/G1 to S-stage. When treated with siTCF-3, the Eca-109 cells exhibited increased apoptosis, with up-regulated cleaved caspase and Bax expressions, whereas Bcl-2 expression was down-regulated. The present study shows that TCF-3 gene silencing inhibits Eca-109 cell growth and proliferation, suppresses cell cycle progression, and promotes apoptosis, which might serve as a new objective for EC treatment.

Highlights

Esophageal cancer (EC), mostly occurs as esophageal squamous cell carcinoma, which is the eighth most common type of cancer all over the world with 482300 new cases every year
Forty-eight hours after the liposome was transfected with Eca-109 cells, Reverse-transcription quantitative PCR (RT-qPCR) was used to detect transcription factor 3 gene (TCF-3) mRNA level, which revealed that compared with the control and siRNA/control groups, the mRNA levels of TCF-3 in the siTCF-1, and siTCF-2 groups remarkably decreased, but there was no significant difference in the mRNA level between control and negative control (NC) groups (P>0.01) (Figure 1A)
Compared with the control and NC groups, Eca-109 cells in the siRNA against TCF-3 (siTCF-3) group were irregular with cell membrane crinkled and broken, cells dropped from the bottom of the plate and the cell density decreased due to many dead cells (Figure 2)

Summary

Introduction

Esophageal cancer (EC), mostly occurs as esophageal squamous cell carcinoma, which is the eighth most common type of cancer all over the world with 482300 new cases every year. Traditional therapies such as surgery, chemotherapy, and radiotherapy have been regarded as effective treatments for EC, the serious side effects and long-term survival showed that these therapies had poor prognosis after surgery [4,5] Nowadays, regulatory factors such as miRNAs, genes, and signaling pathways have provoked wide concern in treating cancer as it is safer than traditional therapies due to fewer side effects [6,7,8]. Various types of gene silencing might have a potential in suppressing EC [11,12]

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