The Potential Role of CERS1 in Autophagy Through PI3K/AKT Signaling Pathway in Hypophysoma

Abstract

To explore the role and mechanism of CERS1 in hypophysoma and investigate whether CERS1 overexpression can change the autophagy process of hypophysoma, and then to explore whether CERS1's effect was regulated by the PI3K/AKT signaling pathway. Western blot and RT-PCR were used to analyze the expression or mRNA level of CERS1 at different tissues or cell lines. Afterwards, the occurrence and development of hypophysoma in vivo and in vitro, respectively, was observed by using CERS1 overexpression by lentivirus. Finally, MK-2206 and LY294002 were applied to discuss whether the role of CERS1 was regulated by the PI3K/AKT signaling pathway. Results show that the CERS1 expression and mRNA level in tumor or AtT-20 cells were decreased. CERS1 over-expressed by lentivirus could inhibit hypophysoma development in vivo and in vitro by reducing tumor volume and weight, weakening tumor proliferation and invasion, and enhancing apoptosis. In addition, shCERS1 could reverse the process. The above results indicate that CERS1 is possibly able to enhance autophagy in hypophysoma through the PI3K/AKT signaling pathway.

Keywords: CERS1; PI3K/AKT signaling pathway; autophagy; hypophysoma.

Figure 1.

(Figure 1A) Western blot assay of CERS1 expression in tumor and para-tumor tissue of invasive growth hormone adenoma, non-invasive growth hormone adenoma, invasive nonfunctional adenoma and non-invasive nonfunctional adenoma. (Figure 1B) Quantification of CERS1 in different tissues. (Figure 1C) Quantitative RT-PCR assay for mRNA level of CERS1. (Figure 1D) Western blot assay of CERS1 expression in Astrocytes and AtT-20 cells. (Figure 1E) Quantification of CERS1 in different cells. (Figure 1F) Quantitative RT-PCR assay for mRNA level of CERS1. Protein and mRNA levels were normalized to β-actin. (Tumor or AtT-20 cells vs. Para-tumor or Astrocytes, ^{*, #, &, ^} P < 0.05, n = 6 per group, all data was represented as Mean ± Standard error).

Figure 2.

(Figure 2A) Mouse weight and (Figure 2B) tumor volume in the 42-day period and (Figure 2C) tumor weight at 42 days of Con, oe-CERS1 and control-CERS1 mice. (Figure 2D) Immunofluorescence assay of TUNEL (×400) and (Figure 2E) TUNEL (+) cells assay in Con, oe-CERS1 and control-CERS1 mice. (Figure 2F-G) Survival analysis of Con, oe-CERS1, control-CERS1, sh-CERS1, and control-shCERS1 mice. (oe-CERS1 versus the Con and control-CERS1 group, ^*P or ^**P < 0.05, n = 6 per group).

Figure 3.

(Figure 3A) Proliferation assay in 72 hours, (Figure 3B) cell cycle assay and (Figure3C) invasion assay at 72 hours of Con, oe-CERS1 and control-CERS1 group. (Figure 3D-E) The flow cytometry assay of apoptosis in Con, oe-CERS1 and control-CERS1 group. (Figure 3F) Proliferation assay in 72 hours, (Figure 3G) cell cycle assay and (Figure 3H) invasion assay at 72 hours of Con, sh-CERS1 and control-shCERS1 group. (Figure 3I-J) the flow cytometry assay of apoptosis in Con, sh-CERS1 and control-shCERS1 group. (oe-CERS1 or sh-CERS1 vs. Con and control-CERS1 or control-shCERS1 group, ^*P or ^**P < 0.05, n = 6 per group).

Figure 4.

(Figure 4A) Western blot assay of Beclin-1, LC3-Ⅰ and LC3-Ⅱ expression in vivo. (Figure 4B and C) Quantification of Beclin-1 and LC3-Ⅱ / LC3-Ⅰ. (Figure 4D) Immunofluorescence assay of Beclin-1 (×400) and (Figure 4E) Beclin-1 (+) cells assay in Con, oe-CERS1 and control-CERS1 mice. (Figure4F) Western blot assay of Beclin-1, LC3-Ⅰ and LC3-Ⅱ expression in vitro. (Figure 4G,H) Quantification of Beclin-1 and LC3-Ⅱ / LC3-Ⅰ. (Figure 4I) MDC staining assay and (Figure 4J) fluorescence intensity in Con, oe-CERS1, and control-CERS1 group. (oe-CERS1 vs. Con and control-CERS1 group, ^**P < 0.05, n = 6 per group).

Figure 5.

(Figure 5A) Western blot assay of PI3K-P85, p-PI3K-P85, AKT, p-AKT, CERS1, Beclin-1, LC3-Ⅰ and LC3-Ⅱ expression in Vehicle, LY., MK. and LY.+MK. group. (Figure 5B) Immunofluorescence assay of Beclin-1 (×400) and (Figure 5C) Beclin-1 (+) cells assay in the Vehicle, LY., MK. and LY.+MK. group. (Figure 5D) MDC staining assay and (Figure 5E) fluorescence intensity in Vehicle, LY., MK. and LY.+MK. group. (vs. Vehicle group, ^**P < 0.05; vs. LY. and MK. group, ^##P < 0.05, n = 6 per group).

Figure 6.

The potential mechanisms of autophagy by CERS1 through PI3K/AKT signaling pathway in hypophysoma. PI3K/AKT signaling pathway could increase CERS1 expression, enhance autophagy and autophagosome progress by rough endoplasmic reticulum, induce apoptosis and prolong the life of mice. LY294002 and MK-2206 2HCL reverse the process through reducing p-PI3 K and p-AKT expression, causing CERS1 to decrease and inhibit the autophagy.