Down-regulation of miR-550a-3-5p correlated with HPV-positive OSCC metastasis
A total of 70 clinical specimens of OSCC were detected for HPV status using PCR analysis of extracted DNA (Supplementary Fig. 1A). HPV was detected in 25/70 (35.7%) patient samples, with HPV16 ranking the most common type in our study which was detected in 23/70 (32.9%) cases. Specimens were grouped into HPV-positive OSCC (n=25) and HPV-negative OSCC (n=45). To identity the differentially expressed miRNAs between HPV-positive and HPV-negative OSCC, an Agilent human miRNA microarray was performed. As shown in Fig. 1A and Supplementary Table 1, 8 miRNAs were significantly up-regulated in HPV-positive OSCC samples, whereas the levels of 11 miRNAs were down-regulated with respect to HPV-negative OSCC samples (Fold change ≥2, P<0.05). By in silico and literature analysis, we selected miR-550a-3-5p, miR-451a, and miR-210-3p to validate their expression differences. qRT-PCR showed that miR-550a-3-5p was down-regulated 10.3-fold in HPV-positive OSCC (n=25) compared to HPV-negative OSCC tissue samples (n=45, P<0.001) and 24.6-fold compared to normal (HPV-negative non-cancerous) tissue samples (n=20, P<0.001), miR-451a was down-regulated 2.6-fold in HPV-positive OSCC compared to HPV-negative OSCC tissue samples (P=0.002) and 4.2-fold compared to normal tissue samples (P<0.001), and miR-210-3p was up-regulated 1.5-fold in HPV-positive OSCC compared to HPV-negative OSCC tissue samples (P<0.001) and 2.1-fold compared to normal tissue samples (P<0.001) (Fig. 1B), which were all consistent with the microarray results. Ranked as the most differentially-expressed miRNAs, we therefore firstly concentrated on miR-550a-3-5p for further studies.
We verified miR-550a-3-5p expression levels and localization by utilizing miRNA fluorescence in situ hybridization (FISH). HPV infection was shown by immunohistochemistry staining of p16. The red fluorescent distribution indicated that miR-550a-3-5p was mainly localized in the cytoplasm of both OSCC and normal cells, and had a highest expression in normal mucosa while lowest expression in HPV-positive OSCC specimens (Fig. 1C). Additionally, miR-550 was also reported to be down-regulated in p16-positive oropharyngeal squamous cell carcinoma (OPSCC) compared to p16-negative OPC samples from other miRNA microarray data (Supplementary Fig. 1B) (20). Then, the qRT-PCR results of miR-550a-3-5p and their associations with corresponding clinicopathological features in HPV-positive and HPV-negtive OSCC patients were summarized. After differentiating miR-550a-3-5p expressions into lower and higher part according to the cut-off point of their median, we found that in HPV-positive OSCC patients, lower miR-550a-3-5p expression was associated with higher tumor size (P=0.025) and the presence of nodal metastasis (P=0.009), but had no correlations with age, gender, drink, smoke, differentiation, clinical stage and recurrence (Table 1). However, miR-550a-3-5p expression was associated with none of these features in HPV-negative OSCC patients. Using the Kaplan-Meier method and log-rank test, we found that HPV-positive OSCC had better survival (HR 0.39; P>0.05) than HPV-negative OSCC. Patients with higher miR-550a-3-5p expression, no matter in HPV-positive OSCC (HR 0.48; P>0.05) or HPV-negative OSCC (HR 0.46; P>0.05), exhibited better survival than those having lower miR-550a-3-5p expression (Fig. 1D). However, there were no statistical significances between groups, which the reason, we speculated, may be due to the small number of clinical specimens. Thus, miR-550a-3-5p levels were frequently down-regulated in HPV-positive OSCC, particularly in patients with higher tumor size, nodal metastasis, which indicated a potential role for miR-550a-3-5p in targeting HPV-positive OSCC progression.
miR-550a-3-5p inhibited tumor growth and progression in nude mice models without altering the in vitro migration, invasion and EMT in HPV-positive OSCC cells
To better investigate the biological behavior of miR-550a-3-5p in HPV-related OSCC, we transfected miR-550a-3-5p mimic and inhibitor into OSCC cell lines which express different endogenous miR-550a-3-5p levels. The same as expressional differences in tissue specimens, miR-550a-3-5p levels were highest in normal oral keratinocytes (NOK), then dysplasia oral keratinocytes (DOK), HPV-negative OSCC cell lines, and lowest in HPV-positive OSCC cell lines (Supplementary Fig. 1C). Up-regulation and down-regulation of miR-550a-3-5p in HPV-positive cell lines UPCI:SCC090 and UM-SCC-47, HPV-negative cell lines Cal-27 and SCC25 were confirmed by qRT-PCR (Supplementary Fig. 1D). miR-550a-3-5p overexpression decreased cell proliferation rate in HPV-positive (-25.01%, UPCI:SCC090; -40.08%, UM-SCC-47) and HPV-negative cells (-36.69%, Cal-27; -13.13%, SCC25), while its inhibition increased cell proliferation rate in HPV-positive (15.56%, UPCI:SCC090; 54.49%, UM-SCC-47) and HPV-negative cells (36.03%, Cal-27; 31.97%, SCC25) (Fig. 2A, Supplementary Fig. 2A). Using Flow cytometry assay, miR-550a-3-5p overexpression led to significantly increased apoptosis in HPV-positive (62.39%, UPCI:SCC090; 81.82%, UM-SCC-47) and HPV-negative cells (95.21%, Cal-27; 76.27%, SCC25), and its inhibition decreased cell apoptosis (-45.55%, UPCI:SCC090; -62.63%, UM-SCC-47; -55.43%, Cal-27; -45.9%, SCC25) (Fig. 2B, Supplementary Fig. 2B). Furthermore, we performed Wound healing and Transwell invasion assays to detect the abilities of migration and invasion in miR-550a-3-5p-transfected HPV-positive and HPV-negative OSCC cells. The results showed that miR-550a-3-5p overexpression decreased the migrated area (14.64%, Cal-27; 11.04%, SCC25) and invasive ability (68.29%, Cal-27; 58.88%, SCC25) in HPV-negative cells, while had no evident impacts on HPV-positive cells. In contrast, miR-550a-3-5p inhibition increased the migrated area (22.68%, Cal-27; 10.34%, SCC25) and invasive ability (58.60%, Cal-27; 106.33%, SCC25) in HPV-negative cells, but still did not affect those in HPV-positive cells (Fig. 2C, D). As EMT leads a crucial role in the process of epithelial cancer progression (24), we examined both the epithelial and mesenchymal markers in miR-550a-3-5p-overexpressed HPV-positive and negative OSCC cells by qRT-PCR. As can be seen in Figure 2E, miR-550a-3-5p overexpression resulted in a significant increase in E-cadherin and decrease in Vimentin in HPV-negative cells, while had no effects on EMT of HPV-positive cells.
After in vitro studies, we stably transfected HPV-positive OSCC cells UPCI:SCC090 and HPV-negative OSCC cells Cal-27 with a miR-550a-3-5p lentiviral vector, which was confirmed by qRT-PCR (Supplementary Fig. 1E), and injected them into nude mice subcutaneously to establish the xenograft model. miR-550a-3-5p overexpression, both in HPV-positive and HPV-negative OSCC cells, led to a decrease in tumor volume and tumor weight with respect to the vector control group (Fig. 3A-C). HE staining and fluorescence in situ hybridization of miR-550a-3-5p were carried out to verify the xenografts (Supplementary Fig. 1F). Then, immunohistochemistry staining was used to analyze the expression of proliferative indicator Ki-67, EMT markers E-cadherin and Vimentin, and angiogenesis-related markers CD31 and CD34. Interestingly, Ki-67 and Vimentin were reduced but E-cadherin was increased, along with inhibited angiogenesis (CD31 and CD34) in both miR-550a-3-5p-overexpressed HPV-positive and HPV-negative OSCC cells-derived xenografts (Fig. 3D).
miR-550a-3-5p inhibited M2 macrophages polarization to suppress the migration, invasion, and EMT of HPV-positive OSCC cells
The above contradiction of in vitro and in vivo results in HPV-positive OSCC suggested that miR-550a-3-5p might participate in a cell nonautonomous mechanism to exert its tumor-suppressive effects. One possibility for miRNA to execute this function is to mediate the crosstalk between cancer cells and inflammatory cells, especially TAMs, which exhibit one of the most abundant types in tumor microenvironment (25). Thus, we utilized a model of TAMs to investigate whether there were underlying interactions (Fig. 4A). The human monocyte cell line THP-1 was incubated with PMA for 24 h and differentiated into macrophages, which then turned into TAMs after being cultured with conditioned media (CM) from HPV-positive OSCC cell lines (UPCI:SCC090 or UM-SCC-47). Morphologically, TAMs were stretched and elongated compared to PMA-induced THP-1 cells (Fig. 4B). Consistent with the observation above, M1 and M2 markers were both increased in OSCC cells-primed macrophages in contrast with that of PMA-induced THP-1 cells using qRT-PCR. However, macrophages treated with CM from miR-550a-3-5p mimic-transfected HPV-positive cells exhibited lower levels of M2 macrophage markers including CD163, IL-10 and Arginase-1 when compared with those treated with CM from mimic NC-transfected HPV-positive cells, while M1 macrophage markers such as CD80, IL-12p40 and TNF-α showed no evident differences (Fig. 4C). These indicated that TAMs of a mixed M1/M2 phenotype were induced by HPV-positive OSCC cells, while miR-550a-3-5p overexpression down-regulated the polarization of M2 macrophages.
To investigate whether miR-550a-3-5p could suppress migration, invasion and EMT of HPV-positive OSCC cells by inhibiting M2 macrophages polarization, Wound healing and Transwell invasion assays were performed in miR-550a-3-5p-overexpressed HPV-positive cells after being co-cultured with corresponding TAMs for 48h. As shown in Figure 4D, miR-550a-3-5p overexpression decreased the migrated area (14.10%, UPCI:SCC090; 23.12%, UM-SCC-47) and invasive ability (68.74%, UPCI:SCC090; 62.39%, UM-SCC-47) in HPV-positive cells. Meanwhile, we verified whether TAMs could suppress the EMT of miR-550a-3-5p-overexpressed HPV-positive cells. qRT-PCR showed that TAMs co-culture significantly increased the epithelial markers such as E-cadherin and β-catenin, and decreased the mesenchymal markers such as N-cadherin and Vimentin in miR-550a-3-5p-overexpressed HPV-positive cells. Up-regulated E-cadherin and down-regulated Vimentin protein levels were also detected in miR-550a-3-5p-overexpressed HPV-positive cells by Western blot (Fig. 4E). Taken together, our findings revealed that miR-550a-3-5p suppressed migration, invasion and EMT of HPV-positive OSCC cells by inhibiting M2 polarization.
miR-550a-3-5p, down-regulated by oncoprotein E6, directly targeted YAP in HPV-positive OSCC cells
We hypothesized that a mechanism between HPV-positive OSCC cells and TAMs exists that explains at least partly the previously depicted tumor-suppressive behaviors of miR-550a-3-5p. HPV virus expresses two main oncogenic proteins, E6 and E7 that target host tumor suppressor proteins to promote tumorigenesis and progression (26). We found that miR-550a-3-5p expression was induced after E6 knockdown, while not influenced after E7 inhibiting in HPV-positive cells (Fig. 5A, Supplementary Fig. 3A). Then we detected miR-550a-3-5p levels after overexpressing HPV16 E6 in HPV-negative OSCC cells using Lv201-HPV16 E6 plasmid. Results showed that miR-550a-3-5p expression was significantly decreased after E6 overexpressing (Fig. 5B). Moreover, TAMs which were treated with CM from E6-silencing HPV-positive cells, exhibited lower levels of CD163, while this effect was mostly abrogated when treated with CM from miR-550a-3-5p inhibitor and E6 siRNA-cotransfected HPV-positive cells (Fig. 5C). These suggested that miR-550a-3-5p was negatively regulated by E6 oncoprotein in HPV-positive OSCC cells.
To determine what downstream signals of miR-550a-3-5p contributed to inhibition of M2 macrophages polarization, we looked at the GO (molecular function) and KEGG (signaling pathway) analysis of miR-550a-3-5p. Results found that miR-550a-3-5p was related to the cytokine, chemokine activity, and Hippo signaling pathway (Supplementary Fig. 3D). As recent studies demonstrated critical functions of Hippo signaling in cancer immunity (27), we firstly detected expressions of its central transcription factors, YAP and TAZ. Overexpression of miR-550a-3-5p significantly inhibited YAP mRNA and protein levels in HPV-positive cells, but had no effects on TAZ expression (Fig. 5D). Then in silico analysis revealed that miR-550a-3-5p had three putative targets in 3’UTR of YAP mRNA, which were further validated by Dual luciferase reporter gene assays. As shown in Figure 5E, Luc-YAP-3’UTR (wild type, WT) cotransfected with miR-550a-3-5p mimics in UPCI:SCC090 and UM-SCC-47 cells showed an obvious decrease of luciferase activity, while Luc-YAP-3’UTR (mutant, MUT) group had a minimal effect on that.
YAP/CCL2 was required for miR-550a-3-5p deficiency-mediated M2 macrophages polarization and M2 macrophages-induced EMT
Our results revealed that YAP was directly regulated by miR-550a-3-5p. Next, we performed qRT-PCR to assess several critical factors which have been reported to be regulated by YAP and affect cancer immunity (28-31). Results found that CCL2 and CXCL5 were significantly inhibited after YAP knockdown in both UPCI:SCC090 and UM-SCC-47 cells, while TLR9 and CSF1 were not changed evidently in both of them. Moreover, only CCL2 expression showed an obvious decrease in miR-550a-3-5p-overexpressed UPCI:SCC090 and UM-SCC-47 cells, which led us to speculate that CCL2 may serve as a downstream factor of miR-550a-3-5p/YAP in HPV-positive OSCC (Fig. 5F, Supplementary Fig. 3B). As YAP regulates gene expression largely through binding to TEAD family transcription factors, we utilized a small molecule called verteporfin (VP) which could inhibit YAP-TEAD association to investigate whether CCL2 was regulated by YAP-TEAD complex (32). Results showed that mRNA and protein levels of CCL2 were significantly decreased upon VP treatment, clarifying the role of YAP-TEAD transcription activity in mediating CCL2 expression (Fig. 5G). To further analyze whether CCL2 was regulated by miR-550a-3-5p/YAP signaling, the YAP-5SA active mutant (33) (serine-to-alanine mutation at all putative LATS kinase phosphorylation sites on YAP including S61A, S109A, S127/128A, S131A, S163/164A, and S381A, thus leading to constitutive YAP activation) was used in the miR-550a-3-5p-overexpressed HPV-positive cells. YAP-5SA transfection obviously rescued miR-550a-3-5p-induced repression of CCL2, which identified the role of miR-550a-3-5p/YAP in regulating CCL2 expression (Fig. 5H). Furthermore, classical target genes of YAP were also detected (34), and the ability of YAP-5SA rescuing miR-550a-3-5p-induced repression of CCNE2 and CDC6 might indicate the reason why miR-550a-3-5p affected proliferation and apoptosis in OSCC cells (Supplementary Fig. 3C).
CCL2 is a well-established chemokine important for the recruitment of macrophages (35). Accordingly, we examined whether the role of miR-550a-3-5p in inhibiting migration, invasion and EMT of HPV-positive OSCC cells was dependent on YAP/CCL2-induced M2 macrophages infiltration. In CM from HPV-positive OSCC cells, miR-550a-3-5p overexpression or YAP knockdown significantly abrogated expressions of M2 macrophages marker CD163 on PMA-induced THP-1 cells, whereas YAP-activation using YAP-5SA in miR-550a-3-5p-overexpressed cells or adding recombinant human CCL2 protein to miR-550a-3-5p-overexpressed or YAP-knockdown cells could rescue CD163 expressions (Fig. 5I). Then, after co-culturing with these CM-induced TMAs for 48h, we found that increased mRNA expressions of E-cadherin in both UPCI:SCC090 and UM-SCC-47 cells by miR-550a-3-5p overexpression or YAP knockdown were mostly abolished when co-transfected with YAP-5SA or added CCL2. Completely opposite results were observed in Vimentin expressions, and protein levels of E-cadherin and Vimentin were confirmed by Western blot, suggesting that the effects of miR-550a-3-5p on inhibiting EMT of HPV-positive OSCC cells were dependent on suppression of YAP/CCL2-mediated M2 macrophages polarization (Fig. 5J).
Verteporfin treatment inhibited tumor development in E6-E7 knock-in mice with 4NQO-induced OSCC
VP, previously identified as a transcriptional inhibitor of YAP, was assessed in a 4NQO-induced model of OSCC in Rosa26-E6-E7 constitutive knock-in mice. After 8 weeks of drinking water with 4NQO and 8 weeks with distilled water, VP was injected intraperitoneally at 3-day intervals, and the mice were assessed after 6 doses of VP (Fig. 6A). Results showed that tongue lesions caused by 4NQO treatment were more obvious in vehicle-treated mice than VP-treated mice. A total of 62.5% (5/8) mice showed lesions in both VP and Vehicle group, while the tumor size was (1.70±0.97) mm on average in VP group and (2.59±0.82) mm in Vehicle group, and the average number of lesions per mouse was 1.00 in VP group and (2.20±1.09) in Vehicle group (Fig. 6B, C). These revealed that VP weakened the formation of tongue lesions induced by 4NQO.
Then, HE staining demonstrated that the tumors present in the VP-treated mice were minimally dysplastic as compared to the more advanced in Vehicle-treated mice (Fig. 6D). Results of histopathological analysis of VP and Vehicle group were described in Table 2, which showed that in VP group, 37.5% of the mice exhibited a high risk of oral carcinogenesis, with respect to 62.5% of the mice in Vehicle group, according to a previously reported two-category system (normal/hyperplasia/mild or moderate dysplasia: low risk; severe dysplasia/carcinoma: high risk) (36). In addition, a significant decrease in the expression of CCL2 and CD163 was observed in tumors from VP-treated mice (Fig. 6E). These indicated that VP suppressed the 4NQO-induced tongue carcinogenesis by down-regulating CCL2 production and M2 macrophages in transgenic mice with E6/7 knock-in.
Correlations of miR-550a-3-5p, YAP, CCL2 and M2 macrophages in vivo and in HPV-positive OSCC patients
We investigated the expression of YAP, CCL2 and CD163 in miR-550a-3-5p-overexpressed HPV-positive OSCC cells-derived xenografts of nude mice. Results of immunohistochemistry staining showed that miR-550a-3-5p overexpression was significantly correlated with decreased nuclear-stained of active YAP, cytoplasmic-stained of CCL2 and CD163. Consistently, miR-550a-3-5p overexpression was associated with decreased mRNA levels of YAP, CCL2 and CD163 (Fig. 7A, B). Furthermore, staining of active YAP, CCL2 and CD163 were also carried out in 25 cases of HPV-positive OSCC specimens which we have used in analyzing miR-550a-3-5p expression. As shown in Figure 7C, patient 1 referred to representative case with low miR-550a-3-5p expression, while patient 2 had higher miR-550a-3-5p expression. Similarly, low miR-550a-3-5p expressions tended to be associated with high levels of YAP and CCL2, and more M2 macrophages infiltration in HPV-positive OSCC patients (Figure 7D).