Fig 2: miR-7 Regulates the Sp1/KLK10 Axis and Inhibits MTA2-Induced Cell Metastasis(A) The MTA2-silenced stable HeLa cells were transfected with the antagomiR-NC or antagomiR-7 for 48 h followed by determination of the expression of miR-7 by qRT-PCR, as well as the expressions of MTA2, Sp1, and KLK10 by western blotting. (B) The migratory and invasive abilities of indicated cells were measured by migration and invasion assays. (C) The MTA2-silenced stable HeLa cells were co-transfected with MTA2 plasmid and miR-7 mimic for 48 h, followed by determination of the expression of miR-7 by qRT-PCR, the expressions of Sp1 and KLK10 by western blotting, and (D) the migratory and invasive ability were determined by migration and invasion assays. (E) Kaplan-Meier curves for overall survival of human cervical cancer patients. (F) Analysis of the correlation between miR-7 expression and Sp1 expression in human cervical cancer tissues from TCGA (p < 0.01). ∗∗p < 0.01, compared with shLuc cells; #p < 0.05, compared with shMTA2 stable cells transfected with antagomiR-NC or MTA2 plasmid.

Fig 3: Sp1 Is Involved in MTA2-Mediated Transcriptional Regulation of KLK10 and Subsequent Cell Migration and Invasion(A) The protein extracted from shLuc and shMTA2 cells was separated into the nuclear part and total lysate and then subjected to western blotting to analyze the expression of MTA2 and Sp1. β-Actin was used as an internal control. Nuclear proteins were resolved and the levels of Sp1 were analyzed. Lamin B and α-tubulin were used as internal controls for nuclear and cytosolic fractions, respectively. (B) Detected expression of Sp1 and KLK10 in HeLa and SiHa cells after siRNA transfection. (C) MTA2, Sp1, and KLK10 expression levels were analyzed by western blotting in indicated cells. (D) Sp1 and KLK10 expression levels after siRNA transfection were analyzed by immunofluorescence staining. Scale bars, 50 μm. (E) The migratory and invasive abilities of indicated cells were analyzed by migration and invasion assays. (F) Expression status of Sp1 in Oncomine and TCGA databases. (G) The luciferase reporter constructs of the truncated KLK10 promoters were introduced into shLuc and shMTA2 cells. Luciferase activity was measured 48 h post-transfection. (H) Predicted Sp1 binding sites in the promoter region of KLK10. (I) The association between Sp1 and KLK10 in shMTA2 cells was examined by a chromatin immunoprecipitation (ChIP) assay with anti-Sp1 antibody, as well as PCR analysis of the genes encoding the promoter regions of KLK10 (−206/−6 and −692/−493). (J) Ectopic expression of MTA2 in shMTA2 cells and detection of the binding activity of Sp1 to the KLK10 promoter by a ChIP assay. ∗∗p < 0.01, compared with control.

Fig 4: KLK10 Is Critical for MTA2-Mediated Cell Migration/Invasion and Acts as the Prognosis Marker for Cervical Cancer Patients(A) The total protein lysates of shLuc and shMTA2 stably transfected cells were collected for a human protein array assay. (B) Quantification of the protein expression of KLK10 was presented in terms of percentage of shLuc is shown as a histogram. (C–E) The expression levels of MTA2 and KLK10 in mRNA and protein levels of shLuc and shMTA2 cells were examined by a qRT-PCR assay (C), western blotting (D), and ELISA (E), respectively. β-Actin acted as an internal control. ∗∗p < 0.01, compared with shLuc cells. (F and G) The protein and mRNA expression levels of MTA2 and KLK10 of three cervical cancer cell lines were examined by western blotting (F) and qRT-PCR (G). β-Actin acted as the loading control. (H–J) Expression of MTA2 and KLK10 in protein and mRNA levels were examined by a western blot assay (H), ELISA (I), and qRT-PCR (J) with ectopic expression of MTA2. β-Actin acted as the protein loading control. GAPDH was used for normalization of the MTA2 and KLK10 mRNA levels. ∗p < 0.05, ∗∗p < 0.01, compared with shLuc cells transfected with Neo plasmid; #p < 0.05 compared with shMTA2 cells transfected with Neo plasmid. (K and L) The expression levels of MTA2 and KLK10 were detected by western blotting (K) and qRT-PCR assays (L) of shMTA cells transfected with KLK10 siRNA. (M) Effects of migratory and invasive abilities of KLK10 siRNA on shMTA2 cells were measured by a migration assay and invasion assay. ∗∗p < 0.01, compared with shLuc cells transfected with si-Control; #p < 0.05, compared with shMTA2 cells transfected with si-Control. (N) KLK10 expression levels of cervical tumor tissue and normal tissue were analyzed using TCGA database. ∗p < 0.05, compared with normal tissue. (O) Representative IHC staining of KLK10 from tumor grade I to grade III. Scale bars, 100 μm. (P) Expression of KLK10 correlated with tumor grade. ∗p < 0.05, grade III compared with grade I+II. (Q) Kaplan-Meier curves for overall survival of human cervical cancer patients.