Suppressive Effect of Constructed shRNAs against Apollon Induces Apoptosis and Growth Inhibition in the HeLa Cell Line

Abstract

Background: Cervical cancer is the second most common female cancer worldwide. Inhibitors of apoptosis proteins (IAPs) block apoptosis; therefore, therapeutic strategies targeting IAPs have attracted the interest of researchers in recent years. Apollon, a member of IAPs, inhibits apoptosis and cell death. RNA interference is a pathway in which small interfering RNA (siRNA) or shRNA (short hairpin RNA) inactivates the expression of target genes. The purpose of this study was to determine the effect of constructed shRNAs on apoptosis and growth inhibition through the suppression of apollon mRNA in HeLa cell line.

Methods: Three shRNAs with binding ability to three different target sites of the first region of apollon gene were designed and cloned in pRNAin-H1.2/Neo vector. shRNA plasmids were then transfected in HeLa cells using electroporation. Down-regulation effects of apollon and the viability of HeLa cells were analyzed by RT-PCR, lactate dehydrogenase assay, and MTT assay, respectively. Also, the induction and morphological markers of apoptosis were evaluated by caspase assay and immunocytochemistry method.

Results: The expression of shRNA in HeLa cells caused a significant decrease in the level of apollon mRNA1. In addition, shRNA1 effectively increased the mRNA level of Smac (as the antagonist of apollon), reduced the viability of HeLa cells and exhibited immunocytochemical apoptotic markers in this cell line.

Conclusion: Apollon gene silencing can induce apoptosis and growth impairment in HeLa cells. In this regard, apollon can be considered a candidate therapeutic target in HeLa cells as a positive human papillomavirus cancer cell line.

Keywords: Coronary artery disease; Genetic association study; Iran; Single nucleotide polymorphisms.

Fig. 1

Relative RNA expression of apollon gene in HeLa cells transfected with different shRNAs compared to mock control cells (A). Comparison of apollon mRNA expression in HeLa cells at 48, 72, and 96 h after shRNA1 transfection is shown in the Figure (B). The mRNA expression of apollon was normalized with β-actin. An average expression value (E value) indicating gene regulation was calculated using REST software. Also, 95% confidence intervals were used for expression ratios

Fig. 2

Up-regulation of Smac after apollon knockdown shown 48 h after the transfection of the HeLa cells with shRNA1 plasmid. The mRNA expression of Smac was normalized with β-actin. An average expression value (E value) indicating gene regulation was calculated using REST software, and 95% confidence intervals were used for expression ratios

Fig. 3

Effect of apollon down-regulation on viability of the HeLa cells. Cell viability was measured using MTT and LDH assays. (A) and (B) show LDH and MTT assays, respectively. Each bar represents the mean value±standard deviation (SD) of triplicate. P<0.05 was compared to the control cell group

Fig. 4

Effect of apollon gene silencing on the induction of apoptosis. Immunocytochemical staining using anti-apollon in HeLa cells. DAPI staining was employed as a nuclear counter stain. Arrows show apoptosis induction in cells