SPLUNC1 regulates cell progression and apoptosis through the miR-141-PTEN/p27 pathway, but is hindered by LMP1

Abstract

Little is known about the role of the host defensive protein short palate, lung and nasal epithelium clone 1 (SPLUNC1) in the carcinogenesis of nasopharyngeal carcinoma (NPC). Here we report that SPLUNC1 plays a role at a very early stage of NPC carcinogenesis. SPLUNC1 regulates NPC cell proliferation, differentiation and apoptosis through miR-141, which in turn regulates PTEN and p27 expression. This signaling axis is negatively regulated by the EBV-coded gene LMP1. Therefore we propose that SPLUNC1 suppresses NPC tumor formation and its inhibition by LMP1 provides a route for NPC tumorigenesis.

Figure 1. SPLUNC1 inhibited the course of EBV infection.

(A, B) Human peripheral blood lymphocytes (LCs) were infected with green fluorescence protein (GFP)-tagged EBV. Approximately 70% of LCs showed green fluorescence after infection, but only 28% of LCs treated with the recombinant SPLUNC1 protein showed green fluorescence. (C, D)HNE-2 cells were co-cultured with B95-8 cells, which product EBV, for 1, 2, 3, 5, and 7 days, and B95-8 cells were then removed by complement-activated cellular cytotoxicity test. (C) Expression of EBER, BZLF1 and LMP1 was lower in the presence of enforced SPLUNC1 expression versus the control in HNE-2 cells. (D) SPLUNC1 expression in the HNE-2 cells was increased significantly 1 day after the addition of B95-8 cells to the HNE-2 culture system, and then decreased. After 3 days of co-culture with B95-8, SPLUNC1 expression decreased to its lowest level.

Figure 2. SPLUNC1 protein expression was decreased in highly differentiated nasopharyngeal carcinoma.

(A) SPLUNC1 protein was high expressed in normal human nasopharyngeal epithelium, SPLUNC1 expression was gradually downregulated with progression from (B) mild to (C) severe atypical hyperplasia of nasopharyngeal tissues. (D) No positive staining for SPLUNC1 was observed in the epithelium of nasopharyngeal carcinoma.

Figure 3. SPLUNC1 inhibited miR-141 expression and the effects of miR-141 and SPLUNC1 on PTEN expression in nasopharyngeal carcinoma cells.

(A) SPLUNC1 expression in CNE-1 nasopharyngeal carcinoma cells was higher than that in the HNE-2 and 5–8 F cells, while the miR-141 expression in CNE-1 cells was lower than that in the HNE-2 and 5–8 F cells (P<0.05). (B) When SPLUNC1 was overexpressed in HNE-2 and 5–8 F cells, miR-141 expression was decreased (P<0.05). (C) Expression of miR-141 mimics in NPC cells decreased the expression of PTEN; expression of miR-141 inhibitor increased the expression of PTEN. (D) Enforced expression of SPLUNC1 in NPC cells increased PTEN expression. (E) miR-141 mimics inhibited PTEN expression and increased Akt phosphorylation in the presence of enforced SPLUNC1-expression in NPC 5–8 F cells. (F) Enforced expression of SPLUNC1 decreased Akt expression in NPC cells; and decreased the levels of phosphorylated Akt and MDM2 (G), but had little effect on GSK3β. SPLUNC1 inhibited the baculovirus phosphatase (BVP)-induced phosphorylation of PTEN and Akt(H).

Figure 4. SPLUNC1 induced cellular apoptosis.

(A) SPLUNC1 and the BPI domain-deleted mutant, ΔSPLUNC1, increased the expression of the proapoptotic proteins BAX, BAD caspase-3 and 8, and decreased the antiapoptotic protein Bcl-2 expression. (B) Enforced expression of SPLUNC1 and ΔSPLUNC1 in HNE-2 cells increased cellular apoptosis.

Figure 5. SPLUNC1 promoted cell differentiation and inhibits tumor growth.

(A) Enforced expression of SPLUNC1 increased JNK2 and NFκB protein expression, and inhibited phosphorylation of ERK (p-Tyr-204) and Iκα (Ser-32). ΔSPLUNC1 had no effect on phosphorylation of ERK (p-Tyr-204) and Iκα (Ser-32). (B) SPLUNC1 also increased p27 expression and decreased the expression of CCND1, CCND2, CCND3, CCNE2, and CDK2. (C, D) clone formation in soft agar; tumors formed in the presence of full length SPLUNC1 or ΔSPLUNC1 were smaller versus control. (E, F) SPLUNC1 and ΔSPLUNC1 inhibited tumor formation in nude mice and (G) SPLUNC1 expression in the transplant tumor was validated by immunohistochemical analysis.

Figure 6. The interaction between SPLUNC1 and LMP1.

(A) SPLUNC1 expression was significantly decreased in NP-69 cells transfected with LMP1. (B) miR-141 expression was significantly increased in NP-69 cells transfected with LMP1. (C) PTEN expression was decreased and the Akt pathway was activated by foreign LMP1 expression. (D) Re-expression of SPLUNC1 in LMP1-transfected cells led to inhibition of LMP1 expression and induction of PTEN.