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Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells
Published on: May 9, 2020
[Small interfering RNA inhibits cell proliferation in gastric cancer cell lines highly expressing RegIα]
Xiao-feng Lu1, Liang-jing Wang, Wun-yan Zhou
1Laboratory of Digestive Disease, Clinical Institution of Sir Run Run Shaw Hospital, Zhejiang University and Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou 310006, China.
Objective:
To investigate the effect of small interfering RNA on cell proliferation and apoptosis in gastric cancer cell lines with high expression of RegIα.
Methods:
Total RNA was isolated from six gastric cancer cell lines,and the expression of RegI α mRNA was detected by RT-PCR. RegI α RNAi expression vector was constructed and stably transfected into MKN45 and AGS cells with high RegI α expression, empty-vector was used as control. RegI α mRNA and protein expression was measured by RT-PCR and Western blot respectively in stable transfected cell lines. Cell proliferation and apoptosis were detected with MTT assay and flow cytometry.
Result:
RT-PCR results indicated that RegI α mRNA expression was significantly inhibited by RNAi in both cell lines compared with empty-vector. Western blot results showed that RegIα protein was down-regulated to (44 ± 4)% and (25 ± 4)% respectively in MKN45 and AGS cells compared to empty-vector. MTT results showed that cell growth was significantly inhibited in MKN45 and AGS cells. The apoptosis rate in MKN45 and AGS cells was remarkable increased compared to that of empty-vector (12.96 ± 0.50)% compared with (3.99 ± 0.30)% and (11.59 ± 1.10)% compared with (4.22 ± 0.40)% (P < 0.05).
Conclusion:
Small interfering RNA of RegI α gene can efficiently down-regulate RegI α expression in MKN45 and AGS cell lines, and further inhibit cell growth and induce cell apoptosis.
Insights
Small interfering RNA (siRNA) targeting the RegIα gene effectively reduces RegIα expression in gastric cancer cells. This inhibition significantly suppresses cell proliferation and promotes apoptosis, offering a potential therapeutic strategy.
Area of Science:
- Molecular Biology
- Cancer Research
- RNA Interference
Context:
- Gastric cancer is a significant global health concern with limited effective targeted therapies.
- Regenerative protein alpha (RegIα) is implicated in the proliferation of various cancers, including gastric cancer.
- Understanding the role of RegIα in gastric cancer pathogenesis is crucial for developing novel treatment strategies.
Purpose:
- To investigate the therapeutic potential of small interfering RNA (siRNA) targeting RegIα in gastric cancer cell lines.
- To evaluate the impact of RegIα knockdown on cell proliferation and apoptosis in MKN45 and AGS gastric cancer cells.
Summary:
- RegIα mRNA and protein expression were significantly inhibited in MKN45 and AGS cells following transfection with a RegIα RNAi expression vector.
- MTT assays demonstrated a marked inhibition of cell proliferation in RegIα-silenced gastric cancer cells.
- Flow cytometry analysis revealed a significant increase in apoptosis rates in MKN45 and AGS cells with reduced RegIα expression compared to controls.
Impact:
- These findings highlight the efficacy of RegIα-targeted siRNA in suppressing gastric cancer cell growth and inducing apoptosis.
- The study provides a strong basis for exploring RegIα as a therapeutic target for gastric cancer treatment.
- siRNA-mediated gene silencing represents a promising approach for future gastric cancer therapies.
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