Knockdown of mitofilin inhibits autophagy and facilitates starvation-induced apoptosis in HeLa cells

Abstract

Objectives: Mitofilin contributes to the maintenance of mitochondrial structure and functions. This study was undertaken to determine the mechanisms underlying its regulation of apoptosis.

Materials and methods: Mitofilin was knockdowned by specific short hairpin RNA (shRNA) and the stable HeLa cell clone was selected. The autophagy activity were assessed with LC3-II conversion and puncta formation by western blot and fluorescence imaging in starved and normal cultured HeLa cells. Autophagy flux was measured in the presence of NH₄Cl. Wortmannin was used to inhibit autophagy. Cell viability and apoptosis were detected with cell counting kit-8 (CCK-8) and fluorescence-activated cell sorting (FACS) assay, respectively.

Results: Mitofilin expression was down-regulated in starved HeLa cells. In established mitofilin stable knockdown cell lines, LC3-II conversion and puncta formation were detected, which are both hallmarks of autophagy, under both basal and starvation conditions. Mitofilin down-regulation decreased LC3-II conversion and puncta formation, which indicates that loss of mitofilin function inhibits both basal and starvation-induced autophagy activity. CCK-8 and FACS analysis confirmed mitofilin involvement in the regulation of cell survival since mitofilin down-regulation facilitated starvation-induced apoptosis in HeLa cells.

Conclusion: Taken together, mitofilin is a potent regulator of autophagy and it may modulate cell survival through regulation of autophagy.

Keywords: Apoptosis; Autophagy; HeLa cell; Mitofilin; Starvation.

Figure 1

Mitofilin is down-regulated in starved HeLa cells. HeLa cells were cultured in Hank’s balanced salt solution for 2 hr. The cell lysate and total RNA were extracted and subjected to western blotting (A) and real-time PCR assay (B). β-actin was detected as a loading control in immunoblot. The mRNA level of mitofilin was normalized to the level of β-actin. Images are representative of three independent experiments. Data are expressed as mean±SD. * P<0.05

Figure 2

Knockdown of mitofilin inhibits basal autophagy activity. HeLa cells were transfected with mitofilin specific short hairpin RNA (shRNA) and selected with G418. Mitofilin expression level in the established stable cell line was detected at mRNA (A) and protein (B) expression levels. β-actin was used as internal control. (C) The LC3-II conversion was detected with western blotting. For the measurement of autophagy flux, cells were pretreated with 20 mM NH₄Cl. (D) The LC3-II puncta distribution was assessed with immunofluorescence (400X), and (E) the LC3 puncta per cell were counted. Pretreatment of NH₄Cl was used to evaluate the autophagy flux. Images are representative of three independent experiments. Data are expressed as mean±SD. * P<0.05

Figure 3

Mitofilin down-regulation inhibits starvation-induced autophagy. Mitofilin knockdown cells and vector cells were starved for 2 hr, and the LC3 conversion and puncta formation were detected with immunoblot (A) and immunofluorescence (B, 400×). NH₄Cl was used for the assessment of autophagy flux. (C) The LC3 puncta were counted. β-actin was used as internal control. Images are representative of three independent experiments. Data are expressed as mean±SD. * P<0.05