Silencing of ENO1 by shRNA Inhibits the Proliferation of Gastric Cancer Cells
Hui Qiao1, Yu-Feng Wang2,3, Wen-Zhen Yuan4
11 Department of Oncology, The First Hospital of Lanzhou University, Lanzhou, China.
Abstract:
α-Enolase is a significant subunit of enolase and acts as a glycolytic enzyme responsible for catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the anaerobic glycolysis pathway. The research about their role is known little in tumor invasion and metastasis. This research analyzed the effect of α-enolase in proliferation and progression of human gastric cancer. The constructed PLKO.1-ENO1 shRNA vector was transfected into 293 T cells and used to infect gastric cancer cells, MKN45, by using lentivirus method. Negative controls were generated by infection with viruses containing empty vector PLKO.1-scramble-shRNA by the same protocol and using wild-type MKN45 cells as blank control. The silencing effect was confirmed by reverse transcription polymerase chain reaction and Western blotting at messenger RNA and protein levels, respectively. Cell proliferation and chemosensitivity were tested by methyl-thiazolyl-tetrazolium assay. Cell apoptosis was tested by flow cytometry. The cell line α-enolase short hairpin RNA stabling silence α-enolase was successfully constructed. In the α-enolase short hairpin RNA cell lines, messenger RNA and protein expression of α-enolase were significantly lower than those in negative control and blank control groups. The proliferation and clone formation ability were significantly inhibited, cell apoptosis was increased significantly, and the inhibition rate of chemotherapy drugs was increased ( P < .05). Our data provide strong evidence that α-enolase short hairpin RNA interference vector can effectively suppress the proliferation and increase chemosensitivity of MKN45 cells, which may provide a novel gene therapy for gastric cancer.
Insights
Silencing alpha-enolase (ENO1) in gastric cancer cells significantly reduced proliferation and increased sensitivity to chemotherapy. This suggests ENO1 short hairpin RNA (shRNA) may offer novel gene therapy for gastric cancer.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- Alpha-enolase (ENO1) is a key glycolytic enzyme with largely unknown roles in tumor invasion and metastasis.
- Gastric cancer progression involves complex molecular mechanisms that are not fully understood.
Purpose of the Study:
- To investigate the effect of alpha-enolase (ENO1) on the proliferation and progression of human gastric cancer.
- To evaluate the potential of ENO1 gene silencing as a therapeutic strategy for gastric cancer.
Main Methods:
- Constructed a PLKO.1-ENO1 shRNA lentiviral vector to silence ENO1 expression in MKN45 gastric cancer cells.
- Confirmed ENO1 knockdown at both mRNA and protein levels using RT-PCR and Western blotting.
- Assessed cell proliferation, apoptosis, and chemosensitivity using MTT assays and flow cytometry.
Main Results:
- Successful construction of stable ENO1-silenced MKN45 cell lines with significantly reduced ENO1 expression.
- ENO1 knockdown led to significant inhibition of cell proliferation and clone formation.
- Increased apoptosis and enhanced sensitivity to chemotherapy drugs were observed in ENO1-silenced cells.
Conclusions:
- Alpha-enolase (ENO1) plays a crucial role in promoting gastric cancer cell proliferation and chemoresistance.
- ENO1 short hairpin RNA (shRNA) interference effectively suppresses gastric cancer progression.
- ENO1 shRNA represents a promising candidate for novel gene therapy in gastric cancer treatment.
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