1. The expression of TMEM16A in HSCC was higher than that in normal tissues.
We selected 57 patients with HSCC which were diagnosed as primary disease, without systemic disease and treatment. The results of IHC staining confirmed that the expression of TMEM16A in HSCC was higher than that in normal tissues (Fig. 1A,1B and 1E). The expression of TMEM16A in lymph gland was higher in HSCC patients with lymphatic metastasis than that in those without lymphatic metastasis (Fig. 1B and 1C).
In addition, we also carried out Western blot and found that the expression of TMEM16A was consistent with that of IHC (Fig. 1D and 1F).
2. Overexpression and down-regulation of TMEM16A affect cell growth, migration and invasion.
According to our previous detection results of TMEM16A in HSCC, we consider its role in the progression of HSCC. We used western blot to detect the expression of TMEM16A in laryngeal carcinoma cell lines AMC-HN-9, TU212, pharynx carcinoma cell line FaDu and 5-8F. We used the human nasopharyngeal epithelial cell line (NP69) as the normal control. The final results showed that the expression of FaDu was the highest, followed by TU212, and the expression of NP69 was the lowest. Therefore, we used FaDu cell line to carry out related functional experiments in vitro. We constructed FaDu cells with TMEM16A silencing and TMEM16A overexpression (OE) by transfecting TMEM16A shRNA lentivirus and TMEM16A OE lentivirus, respectively. The successful construction of the cell line was verified by Western blot (Fig. 2A).
Then, we used colony formation to analyze cell proliferation. Wound healing migration test, transport cell migration test and invasion test were used to evaluate the migration and invasion ability of FaDu cells in the control group. Clone formation analysis showed that the clone formation rate of FaDu cells TMEM16A OE was significantly higher than that of control cells (181.7 ± 11.02 vs. 110.7 ± 3.48, P < 0.01, Fig. 2B). The clone formation rate of shTMEM16A cells was significantly lower than that of untreated cells (113 ± 9.574 vs. 265 ± 6.608, P < 0.01, Fig. 3B). The migration experiment of wound healing showed that compared with the control group, the cells overexpressed by TMEM16A showed stronger migration ability and faster narrowed healing boundary (Fig. 2C, P < 0.01). This phenomenon was significantly reversed by shTMEM16A(Fig. 2C). Both the number of migrated cells in the Transwell experiment and the number of invasive cells were determined by counting the cells on the lower side of the filter. The results showed that the numbers of migration (93 ± 9.849 vs 263 ± 9.574, P < 0.01, Fig. 2D) and invasion (87 ± 13.3 vs 258.8 ± 15.72, P < 0.01, Fig. 2D) of FaDu cells overexpressed by TMEM16A were significantly higher than those in the control group. Compared with shTMEM16A cells, the number of migrating cells (271 ± 9.983 vs. 69.5 ± 12.74, P < 0.01, Fig. 3D) and invasive cells (275 ± 18.79 vs. 69 ± 13.3, P < 0.01, Fig. 3D) in shTMEM16A group decreased significantly. To sum up, the in vitro experimental results show that the high expression of TMEM16A can promote the proliferation of FaDu cells and enhance the ability of migration and invasion of FaDu cells. Silencing TMEM16A inhibits the proliferation, migration and invasion of FaDu cells.
3. Down-regulation of TMEM16A inhibits tumorigenicity in mice.
In order to verify the role of TMEM16A in tumorigenesis and growth in vivo, FaDu cells transfected with shTMEM16A lentivirus were subcutaneously injected into nude mice. At the same time, the cells transfected with control lentivirus were injected as control experiments. Mice were randomly divided into two groups and injected with shTMEM16A and control lentivirus respectively. The tumor tissue began to form on the 6th day after injection. On the 14th day after injection, the tumor in the control group showed irregular swelling, borderline and keratinization, while the tumor in the shTMEM16A group grew slowly (Fig. 3A). The tumor growth curve of the control group showed that the tumor grew rapidly, and there was a significant difference in tumor growth between the experimental group and the control group on the 14th day of injection (Fig. 3B, P < 0.01). The measurement of tumor weight showed that the tumor weight of mice injected with shTMEM16A cells was significantly lower than that of control mice (Fig. 3C, P < 0.01). The tumor tissues were collected and further analyzed by Western blot(Fig. 3D)and IHC(Fig. 3E). The results showed that the expression of TMEM16A in shTMEM16A group was significantly down-regulated. It is suggested that silencing TMEM16A can reduce the tumorigenicity of FaDu cells in vivo.
4. Down-regulation of TMEM16A induces autophagy to cause cell death in FaDu cell.
Autophagy is a widespread cellular pathway which is associated with tumor progression. When we silenced TMEM16A, we found that FaDu cells did not show typical apoptotic morphological changes. In order to determine whether silent TMEM16A regulates autophagy, we detected autophagy markers p62, LC3-I and LC3-II to observe the occurrence of autophagy by western blot. The results showed that compared with the untreated cells, the expression of p62 was significantly decreased in the cells silenced by TMEM16A, while the expression of LC3-II/LC3-I was significantly decreased and the expression of p62 was increased in the cells with OE of TMEM16A (Fig. 4A). At the same time, autophagy vacuoles increased in the cells silenced by TMEM16A under electron microscope (Fig. 4B). In order to detect the distribution of LC3, we performed immunofluorescence staining and found that the staining spots of LC3 increased significantly in the cells with silenced TMEM16A, while decreased in the untreated cells (Fig. 4C). The accumulation of LC3-II suggests that the formation of upstream autophagosomes is increased or the downstream autophagy-lysosome fusion is impaired. In order to clearly verify the specific effect of knocking down TMEM16A on autophagy, we chose chloroquine, an autophagy inhibitor that can inhibit the fusion of autophagosomes and lysosomes. After the addition of chloroquine to TMEM16A silenced cells, the number of LC3 staining spots in FaDu cells was higher than that in TMEM16A silenced cells (Fig. 4C). This experimental result are consistent with those of western blot (Fig. 4A). These results provide evidence for the autophagy of FaDu cells induced by silenced TMEM16A.
Then, through western blot experiments, we found that the expression of apoptosis marker caspase-3 was significantly decreased (Fig. 4D), which means TMEM16A silence could not induce apoptosis. These indicated that cell death induced by silencing TMEM16A was through the activation of autophagy but not through apoptosis.
5. TMEM16A down-regulates autophagy of hypopharyngeal carcinoma FaDu cells through affecting mTOR pathway
In our experiments, we found that the expression of mTOR decreased when TMEM16A was silenced and increased when overexpressing TMEM16A. Then we added rapamycin to inhibit mTOR in FaDu cells with TMEM16A overexpression and found that the expression of mTOR decreased significantly, while the expression of LC3-II increased (Fig. 5A and.5B). Finally, we re-tested the colony formation test, wound healing test, migration and invasion test of the TMEM16A overexpressed FaDu cells added with rapamycin. The results showed that compared with the control group, the ability of colony formation was decreased when adding rapamycin in TMEM16A overexpressed FaDu cells (figure.5E), the ability of wound healing slowed down (Fig. 5F), and the ability of migration and invasion were significantly inhibited (Fig. 5G). Therefore, it is suggested by in vitro experiments that silent TMEM16A inhibits the invasion and metastasis of FaDu cells through autophagy induced by mTOR.