| Objective:Human hypopharyngeal squamous cell carcinoma (HSCC) as a most common malignant tumor of head and neck cancers continues to show low response to treatment and poor outcome. Serine/threonine kinase 33 (STK33) may be a valuable molecular target for the therapy, but little is known about its biological function and molecular mechanisms in cancer. We designed the present study to determine whether STK33 gene can affect the biological traits, such as apoptosis and proliferation, cell cycle, metastasis and invasion, as well as epithelial mesenchymal transition (EMT) in Fadu cells, a cell line of hypopharygeal cancinoma, and its relationship with extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling activation.Materials and methods:The protein expression of STK33 in 30 matched human hypopharyngeal carcinoma and normal tissues were detected by immunohistochemistry (IHC) analysis. After the construction of lentiviral vectors of STK33-RNA interference (STK33-RNAi) and mock, the lentiviral vectors were transfected into Fadu cells and the transfected effect was examined. Meanwhile, The STK33 protein expression was assessed by Western blot. Next, the effects of STK33-RNAi and PD98059 on the apoptosis and proliferation in Fadu cells were investigated by MTT, Hoechst. 33342 & PI double staining, flow cytometry, colony formation experiments. Real-time PCR and western blot were performed to evaluate the relationship between STK33 and ERK1/2, and the effect of STK33-RNAi and 5 μM PD98059 on the apoptosis- and proliferation-related genes as Caspase-3, Bcl-2 and Ki-67 in Fadu cells. On the other hand, the effects of STK33-RNAi and 5 μM PD98059 on the migration and invasion in Fadu cells were examined using the Transwell assay. The mRNA and protein expressions of EMT-related genes as E-cadherin and Vimentin, as well the metastasis-related gene as Nm-23 were investigated in Fadu cells treated with STK33-RNAi and 5μM PD98059.Results:IHC score of STK33 expression was significantly decreased in normal (4.17±3.38) compared with CIS (11.63 ±3.56, P<0.05) or IC (13.97±3.47, P<0.05), otherwise, there was statistical difference between CIS and IC (P<0.05). For the further research, the lentiviral vectors of STK33-RNAi and mock were constructed successfully and transfected into Fadu cells effectively. The STK33 protein expression was obviously decreased in Fadu cells treated with STK33-RNAi compared with mock. Furthermore, STK33-RNAi and 5 μM PD98059 significantly decreased the abilities of cell viability and colony formation, induced apoptosis in Fadu cells differently and synergeticly compared with mock. As for the effect of STK33-RNAi and 5μM PD98059 on cell cycle in Fadu cells, STK33-RNAi blocked the G2/M phase and 5 uM PD98059 arrested G0/G1 phase in Fadu cells. Real-time PCR and western blot analyses showed STK33 expression was markedly reduced in Fadu cells treated with 5 μM PD98059 comparing with mock, but there was not significant difference of ERK1/2 expression between STK33-RNAi and mock. Moreover, STK33-RNAi and 5 μM PD98059 obviously inhibited Bcl-2 expression and elevated Ki-67 expression differently and synergeticly in Fadu cells, while, STK33-RNAi significantly enhanced Caspase-3 expression compared with mock. On the other hand, IHC staining showed that the intensity of STK33 expression was much stronger in non-keratinizing type than keratinizing type of HSCC, with a statistically significant difference in IHC score between the two different types (P< 0.05). In addition, STK33 IHC scores were markedly increased in HSCC specimens in size>2 cm, compared with those≤ 2 cm (P< 0.05). Also, IHC score of STK33 was significantly higher in patients at advanced HSCC stages (stageⅡ, Ⅲ and Ⅳ) than that in stage Ⅰ (P< 0.05). Similarly, IHC score was significantly increased with metastasis to the lymph nodes compared with non-metastasis (P< 0.05). Both STK33-RNAi and 5 μM PD98059 greatly decreased the ability of migration and invasion of Fadu cells differently and synergeticly compared with mock. The mRNA and protein expressions of Nm-23 and E-cadherin was significantly increased in Fadu cells treated with STK33-RNAi or 5 μM PD98059 differently and synergeticly, as well Vimentin expression was obviously decreased in Fadu cells treated with STK33-RNAi or 5μM PD98059.Conclusions:Our study firstly demonstrates STK33 protein really exists in human hypopharyngeal carcinoma and indicates STK33 may play an important role in carcinogenesis and progresssion. The lentiviarl vectors of STK33-RNAi and mock was constructed and transfected into Fadu cells successfully. STK33-RNAi may decrease the cell viability of Fadu cells obviously via the elevation of Caspase-3 expression and the suppression of Bcl-2 expression. STK33-RNAi reducing the expression of Ki-67 may block the G2/M phase and down regulate the ability of colony formation in Fadu cells. As STK33 may be a downstream of ERK1/2, STK33 may promote the proliferation, enhance the ability of colony formation, affect cell cycle and suppress apoptosis in Fadu cells via ERK1/2 signal pathway. On the other hand, high STK33 expression was related with poor differentiation, metastasis and advanced clinical stages of HSCC. STK33 may induce EMT by inhibiting E-cadherin expression synergeticly via participation of ERK1/2 signal pathway and enhancing Vimentin expression, meanwhile decrease Nm-23 expression via ERK1/2 signal pathway in Fadu cells. Thus, STK33 may increase the migratory and invasion of Fadu cells to promote the progression of human hypopharyngeal carcinoma. All the results provide a basement for the potential molecular therapy of STK33 in human hypopharyngeal carcinoma. |
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