3.1 Knockdown of HPV16 E6, E7 decreased ICAT but increased miR-23b-3p expression
The constant expression of High-risk HPV16 E6, E7 are considered to be the crucial cause of cervical cancer, and our previous study showed that ICAT was highly expressed in cervical cancer. Herein, we hypothesized the up-regulated expression of ICAT was associated with HPV16 E6, E7. qRT-PCR analysis showed the mRNA levels of E6 and E7 were suppressed in cells treated with the HPV16 E6, E7 small interfering RNA (siRNA) (Figure 1A). Compared with control, the mRNA and protein levels of ICAT were significantly down-regulated in SiHa cells where HPV16 E6, E7 were down-regulated. Similar results were also found in CasKi cells (Figure 1B and 1C). Taken together, the results indicated that HPV16 E6, E7 could regulated the expression of ICAT.
Previous studies has documented that High-risk HPV16 E6, E7 can cause alterations in the expression of microRNAs, and another study confirmed that miR-23b shows significantly reduced expression in cervical cancer18. To determine the possible interaction between HPV16 E6, E7 and miR-23b-3p, the expression level of miR-23b-3p in SiHa and CasKi cells was validated by qRT-PCR after transfected with HPV16 E6, E7 siRNA. As expected, knockdown of HPV16 E6, E7 increased miR-23b-3p expression in the two HPV16 positive cervical cancer cell lines, SiHa and CaSki (Figure 1D).
3.2 miR-23b-3p repressed ICAT expression in SiHa and CasKi cells
As the expression of miR-23b-3p was inversely proportionate to that of ICAT after HPV16 E6, E7 were knocked down in cervical cancer cells, further research was needed to determine whether miR-23b-3p can affect the expression of ICAT in SiHa and CasKi cells. To verify the hypothesis, miR-23b-3p mimic, miR-23b-3p inhibitor or a negative control were transfected into the SiHa and CasKi cells (Figure 2A). qRT-PCR and western blot analysis revealed that the expression levels of ICAT mRNA and protein in the SiHa and CasKi cells transfected with miR-23b-3p mimic were lower than those transfected with the control group. Consistently, results demonstrated that the mRNA and protein expression of ICAT in SiHa and CasKi cells transfected with miR-23b-3p inhibitor was significantly upregulated compared with the negative control (Figure 2B and 2C). Accordingly, these findings indicate that miR-23b-3p can negatively regulate the expression of ICAT in HPV16 positive cervical cancer cells.
3.3 miR-23b-3p targeted the 3’-UTR of ICAT mRNA
To further investigate whether there is a direct interaction between miR-23b-3p and ICAT gene, bioinformatic analysis was performed with TargetScanHuman 7.2 and miRBase database. Results revealed that the 3`-UTR of ICAT contained a potential binding site for miR-23b-3p (Figure 3A). Whereafter, a dual luciferase reporter assay was carried out to verify the prediction. The results of luciferase reporter assay demonstrated that the luciferase activity remarkably decreased compared to the mutant control when HEK 239 cells were co-transfected with miR-23b-3p mimic. In contrast, the activity increased compared to the negative control after HEK 239 cells were co-transfected with miR-23b-3p inhibitor (Figure 3B and 3C). Accordingly, these results demonstrate that ICAT is a novel target gene of miR-23b-3p.
3.4 miR-23b-3p suppresses proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of cervical carcinoma cell
To confirm whether miR-23b-3p affects HPV16 positive cervical cancer cell proliferation, CCK-8 and Western blot assays were performed. The results of CCK-8 tests revealed that the proliferation of cells transfected with miR-23b-3p mimics was significantly lower than that of the vector groups at 48h, 72h, and 96h, while the opposite results were observed in cells transfected with miR-23b-3p inhibitor (Figure 4A). Furthermore, we detected the protein levels of PCNA and Cyclin D1 by western blot. The results showed that the expressions of PCNA and Cyclin D1 were downregulated after cells were treated with miR-23b-3p mimic. However, the treatment with miR-23b-3p inhibitor caused increased expression of PCNA and cyclin D1(Figure 4B).
Cell migration and invasion are considered to be key events in tumor progression. Thus, wound healing assay and transwell assay were further performed to ascertain whether miR-23b-3p is involved in HPV16 positive cervical cancer cells migration and invasion. The results revealed that the overexpression of miR-23b-3p significantly impaired the migration and invasion abilities of CasKi and SiHa cells, while the miR-23b-3p inhibitor markedly improved the abilities of migration and invasion in CaSki and SiHa cells (Figure 4C and 4D).
EMT is demonstrated to be a critical step in the metastatic cascade. To future investigate whether miR-23b-3p suppressed cell migration and invasion through inhibition of EMT, we measured the expressions of related markers by western blot. The results showed that the expression of E-cadherin increased in response to the overexpression of miR-23b-3p, accompanied by decreased expression of N-cadherin and vimentin, and vice versa (Figure 4E).
Taken together, these data suggest that miR-23b-3p inhibits HPV16 positive cervical cancer cells proliferation, invasion, migration and EMT.
3.5 ICAT overexpression reversed the antitumor impact of miR-23b-3p
Since ICAT was found to be the direct target of miR-23b-3p, we further verified whether the miR-23b-3p impairs the progression of cervical carcinoma cells via regulating ICAT. Hence, miR-23b-3p mimic + AdRFP and miR-23b-3p mimic + AdICAT were co-transfected into SiHa and CasKi cells. Western blot analysis showed that the expression of ICAT was significantly up-regulated in cells transfected with the AdICAT (Figure 5B). CCK-8 and western blot assays showed that the effect of miR-23b-3p on the proliferation of SiHa and CasKi cells was inhibited by overregulation of ICAT (Figure 5A and 5B). Migration and invasion assays showed that the cellular migratory and invasive abilities were strikingly reserved by co-transfection with miR-23b-3p and ICAT in cells (Figure 5C and 5D). Furthermore, western blot data showed that overexpression of ICAT significantly rescued the effect of miR-23b-3p on the EMT in the SiHa and CasKi cells (Figure 5E). Above results demonstrate that ICAT overexpression partially reverse the inhibitory effects of miR-23b-3p on the HPV16 positive cervical cancer cells.
3.6 HPV16 E6, E7 regulated the progression of cervical carcinoma cell through miR-23b-3p and ICAT
HPV16 E6, E7 are documented to be important oncoproteins in cervical carcinoma. To further verify whether miR-23b-3p and ICAT were involved in the effect of HPV16 E6, E7 on the progression of cervical cancer cells, SiHa and CasKi cells were co-transfected HPV16 E6, E7 siRNA with miR-23b-3p inhibitor and inhibitor NC respectively. CCK-8 and western blot assays revealed that knockdown of miR-23b-3p could reverse the inhibition effects of siRNA HPV16 E6, E7 on proliferation of cervical cancer cells (Figure 6A and 6B). Similar results were also observed in wound healing and transwell assays, that reduced migration and invasion abilities induced by HPV16 E6, E7 knockdown could be rescued by miR-23b-3p inhibitor (Figure 6C and 6D). Besides, co-transfected with miR-23b-3p inhibitor could decrease siRNA HPV16 E6, E7-mediated down-regulation of N-cadherin and Vimentin expressions and could reverse siRNA HPV16 E6, E7-mediated upregulation of E-cadherin expression (Figure 6E). Furthermore, western blot analysis showed that the protein expression of ICAT was significantly increased in the HPV16 E6, E7 + miR-23b-3p inhibitor group compared with that in the control group (Figure 6B), indicating that HPV16 E6, E7 regulated ICAT via miR-23b-3p. These results demonstrate that HPV16 E6, E7 can regulate ICAT via miR-23b-3p and affect the progression of cervical cancer cells.