Silencing of PRR11 suppresses cell proliferation and induces autophagy in NSCLC cells

Abstract

Our previous studies have demonstrated that proline-rich protein 11 (PRR11) is a novel tumor-related gene and implicates in regulating the proliferation in lung cancer. However, its precise role in cell cycle progression remains unclear. Our recent evidences show that PRR11 silencing has an effect on autophagy in non-small-cell lung cancer (NSCLC) cells. Two human NSCLC cell lines, H1299 and A549 were transiently transfected with against PRR11 siRNA. The Cell Counting Kit-8 and plate clone formation assay showed that downregulation of PRR11 inhibited the cell proliferation associated with cell cycle related genes reduced. And our data suggested that PRR11 depletion expression enhanced the autophagosomes formation, followed with downregulation of P62 and upregulation of LC3-II protein. Furthermore, the immunoblotting results indicated that silencing of PRR11 inactivated the Akt/mTOR signaling pathway. Collectively, these results demonstrated PRR11 had an effective role in autophagy in NSCLC cells through Akt/mTOR autophagy signaling pathways. Therefore, it is helpful to clarify the function of PRR11 in tumorigenesis of NSCLC.

Keywords: Akt/mTOR; Autophagy; Cell Counting Kit-8, (CCK8); Cell proliferation; Non-small-cell lung cancer; PRR11; chromosomal instability, (CIN); lysosome-associated membrane protein 1, (Lamp1); non-small-cell lung cancer, (NSCLC); proline-rich protein 11, (PRR11); small cell lung cancer, (SCLC).

Figure 1

Silencing of PRR11 inhibits cell viability in NSCLC cells. (A) siRNA-mediated silencing of PRR11. H1299 and A549 cells were transiently transfected with a negative control siRNA (NC siRNA) or with siRNA against PRR11. Forty-eight hours after transfection, total RNA and whole cell lysates were prepared and subjected to RT-PCR (left) and immunoblotting (right), respectively (B) The effect of PRR11 depletion expression with the cellular proliferation. Cells as siNC and siPRR11 treatment was determined by CCK8 assay at indicated timepoints. *,p < 0.01, **,p < 0.001 (C) Silencing of PRR11 expression suppressed colony formation in lung cancer cells. Cells were cultured for 8 days (D) Depletion of PRR11 expression inhibited lung cancer cells proliferation measured BrdU labeling. Scale bars, 50 μm***,p < 0.0001 (E) H1299 and A549 cells were transiently transfected with a negative control siRNA (NC siRNA) or with siRNA against PRR11. Forty-eight hours after transfection, total RNA and whole cell lysates were prepared, and RT-PCR (above) and immunoblotting (under) was used to determine the expression levels of indicated genes, respectively. GAPDH was used as an internal control (F) Cell apoptosis analysis in H1299 and A549 cells. Cells were transiently transfected with siRNA. Forty-eight hours after transfection, attached and suspension cells were harvested, and then the apoptosis were analyzed by FACS.

Figure 2

Silencing of PRR11 stimulates autophagy in NSCLC cells. (A) Immunoblot analysis expression of LC3, Atg5, Beclin1 and p62 in H1299 and A549 cells. Cells were transiently transfected with a negative control siRNA (NC siRNA) or with siRNA against PRR11. Forty-eight hours after transfection, whole cell lysates were prepared and subjected to immunoblotting as indicated proteins (B) PRR11 depletion expression promotes autophagy. Left, Representative images of the formation of endogenous LC3 puncta in cells treated with PRR11 siNC or siRNA for 48 h. Right, total number of endogenous LC3 puncta per cells. Scale bars, 20 μm (C) Autophagy measured by transmission electron microscopy in cells treated with as in (B). Arrows, autophagosomes/autolysomes. Right, total number of autophagosomes per cell. ***,p < 0.0001. Scale bars, 1 μm.

Figure 3

Depletion of PRR11 expression promotes autophagy flux in NSCLCs. (A) and (B) Cells were transiently transfected with an RFP-GFP-LC3 tandem fluorescent-tagged LC3 (RFP-GFP-LC3). In addition, cells were treated with PRR11 siRNA alone for 48 h or in combination with 10 μmol/L chloroquine (CQ) for 24 h. Scale bars, 10 μm (C) Immunofluorescent staining was performed for LAMP1 and analyzed by microscopy. Scale bars, 20 μm (D) and (E) Left, immunofluorescence analysis of endogenous LC3 puncta in H1299 and A549 cells. Scale bars, 20 μm ***,p < 0.0001. Scale bars, 20 μm.

Figure 4

Autophagy is involved in inhibiting cell proliferation by PRR11 silencing in NSCLC cells through Akt/mTOR signaling pathway. (A) Cells were transiently transfected with a negative control siRNA (NC siRNA) or with siRNA against ATG5. Forty-eight hours after transfection, whole cell lysates were prepared and subjected to immunoblotting in H1299 and A549 cells. Cells were transfected with Atg5 siRNA in combination with PRR11 siRNA. Cellular proliferation was detected by CCK8 assay (B), colony formation (C) and BrdU labeling (D). Scale bars, 50 μm *,p < 0.01,**,p < 0.001, ***,p < 0.0001 (E) Immunoblot analysis of phosphorylation of Akt (S473) and mTOR (S2448) in cells treated with PRR11 siRNA for 72 h. Total Akt and mTOR was used as the internal control, respectively.