Lentivirus-mediated knockdown of eukaryotic translation initiation factor 3 subunit D inhibits proliferation of HCT116 colon cancer cells.

Xiaojun Yu,Bo’An Zheng,Rui Chai

doi:10.1042/bsr20140078

Abstract

Dysregulation of protein synthesis is emerging as a major contributory factor in cancer development. eIF3D (eukaryotic translation initiation factor 3 subunit D) is one member of the eIF3 (eukaryotic translation initiation factor 3) family, which is essential for initiation of protein synthesis in eukaryotic cells. Acquaintance with eIF3D is little since it has been identified as a dispensable subunit of eIF3 complex. Recently, eIF3D was found to embed somatic mutations in human colorectal cancers, indicating its importance for tumour progression. To further probe into its action in colon cancer, we utilized lentivirus-mediated RNA interference to knock down eIF3D expression in one colon cancer cell line HCT116. Knockdown of eIF3D in HCT116 cells significantly inhibited cell proliferation and colony formation in vitro. Flow cytometry analysis indicated that depletion of eIF3D led to cell-cycle arrest in the G2/M phase, and induced an excess accumulation of HCT116 cells in the sub-G1 phase representing apoptotic cells. Signalling pathways responsible for cell growth and apoptosis have also been found altered after eIF3D silencing, such as AMPKα (AMP-activated protein kinase alpha), Bad, PRAS40 [proline-rich Akt (PKB) substrate of 40 kDa], SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase), GSK3β and PARP [poly(ADP-ribose) polymerase]. Taken together, these findings suggest that eIF3D might play an important role in colon cancer progression.

Highlights

Colon cancer, known as colorectal cancer, rectal cancer or bowel cancer, is a cancer from uncontrolled cell growth in the colon or rectum or in the appendix
Lentivirus-mediated RNAi effectively decreases eukaryotic translation initiation factor 3 subunit D (eIF3D) expression in HCT116 cells eIF3D, which is of our interest, has been reported to mutate at two points in human colorectal cancer identified by mutation spectrum [17]
Efficiency of lentivirus infection was reflected from green fluorescence of green fluorescent protein (GFP). (B) eIF3D mRNA levels were measured by RT–qPCR in three groups of HCT116 cells after 5 days infection. β-actin was used as an internal gene. (C) eIF3D protein levels were detected by western blot analysis in three groups of HCT116 cells after 5 days infection

Summary

Introduction

Known as colorectal cancer, rectal cancer or bowel cancer, is a cancer from uncontrolled cell growth in the colon or rectum or in the appendix. Most colon cancers originate from small, non-cancerous (benign) tumours called adenomatous polyps that form on the inner walls of the large intestine. Some of these polyps may grow into malignant colon cancers over time if they are not removed during colonoscopy. As of 2008 it is the second most common cause of cancer in women and the third most common in men [6] with it being the fourth most common cause of cancer death after lung, stomach and liver cancer.

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