1.1 The mRNA level of BMI1 in human cancers from TCGA database
Through TCGA database data analysis, we identified BMI1 as an oncogene in several human cancers, including COAD, COADREAD, BRCA, ESCA, STES, STAD, HNSC, LIHC, and CHOL (Figure. S1, Table S2). We further analyzed the mRNA levels of BMI1 in CC and EC tissues. We compared BMI1 mRNA levels in 304 CC tumor specimens, 3 healthy cervical tissue specimens, 546 EC tumor specimens, and 35 healthy endometrial tissue specimens from TCGA database. The former showed no statistical difference owing to the small number of healthy cervical tissue specimens (Figure. S2A), whereas the mRNA level of BMI1 was significantly higher in normal endometrial tissues than in EC tissues (Figure. S2B). Furthermore, the mRNA level of BMI1 in paired CC and EC samples from TCGA database was also analyzed, with no significant difference in the transcriptional level between normal and tumor tissues (Figure. S2 C, D).
1.2 The correlation between BMI1 and clinical features in CC and EC from TCGA database
The UALCAN platform (http://ualcan.path.uab.edu/index.html) was used to analyze the correlation between BMI1 mRNA level and tumor stage and pathological type in CC and EC patients from the TCGA database (data type: TCGA-UCEC/TCGA-CESC, project: RNA-COUNTS). The transcription level of BMI1 was not significantly correlated with the tumor stage of CC but was related to the pathological type of CC. BMI1 mRNA levels were significantly increased in the endometrioid adenocarcinoma subtypes (Figure. 1A, B). However, in EC, the transcription level of BMI1 was not significantly correlated with tumor stage or the pathological type of EC (Figure. 1C, D).
The UALCAN platform (http://ualcan.path.uab.edu/analysis-prot.html) was used to analyze the correlation between BMI1 protein expression and EC clinical features from the CPTAC database (data type: CPTAC-UCEC, project: BMI1 (NP_005171.4:S251)), and the results showed that BMI1 protein is highly expressed in endometrioid carcinoma and serous carcinoma subtypes compared with normal tissues (Figure. 1E). In addition, higher BMI1 expression was associated with a high tumor grade in patientswith EC (Figure. 1F). However, data on the expression of the BMI1 protein in CC were not found in the CPTAC database.
2.1 Protein level of BMI1 in CC and EC tissues
Since the mRNA level of BMI1 in CC and EC samples from TCGA database exhibits no significant difference, the protein level of BMI1 indicates potential clinical value. We next attempted to detect the expression level of BMI1 protein in CC and normal tissues using IHC analysis. BMI1 protein expression levels were significantly higher in CC tissues than in normal tissues (χ2=25.1, P<0.001) (Figure. 2A, B). A total of 52.5% (21/40) of CC tissues exhibited high expression of BMI1, whereas the other CC tissues (47.5%, 19/40) had negative or low expression levels of BMI1. In contrast, 2.5% (1/40) of normal CC tissues exhibited strong staining for BMI1 and 97.5% (39/40) exhibited negative and low staining.
Similarly, BMI1 protein expression levels were significantly higher in EC tissues than in normal tissues (χ2=11.06, P<0.01) (Figure. 2C, D). A total of 75% (9/12) of EC tissues exhibited high BMI1 expression, whereas the other EC tissues (25%, 3/12) had negative or low expression levels of BMI1. In contrast, 2.5% (1/12) of normal EC tissues exhibited strong staining for BMI1 and 97.5% (11/40) exhibited negative and low staining. These findings indicated that BMI1 protein expression in CC and EC tissues was higher than that in normal CC and EC tissues.
2.2 Clinical value of BMI1 in CC and EC
The possible correlations between BMI1 protein expression and clinicopathological features in CC and EC tissues were evaluated. The protein level of BMI1 in CC tissues was closely related to the degree of pathological tumor differentiation (χ2=10.455, P<0.05). There was no significant correlation with clinicopathological characteristics (age, height, weight, BMI, tumor size, tumor invasion depth, pathological type, TNM stage, FIGO stage, ER expression, and PR expression) (Table 1). In EC tissues, the expression level of BMI1 was not significantly correlated with clinicopathological characteristics (tumor pathological differentiation degree, age, height, weight, BMI, tumor size, tumor invasion depth, pathological type, TNM stage, FIGO stage, ER expression, and PR expression) (Table 2).
3.1 High expression of BMI1 promotes migration of CC and EC cells
To determine the potential functional role of BMI1 in CC and EC cell lines, we utilized the HeLa, HEC-1-A and SPEC-2 cell lines, and transfected myc-BMI1 and shBMI1 into these cells to increase the expression level of BMI1 or decrease the expression level of BMI1, respectively. The efficiency of shBMI1 was first detected in HEK293T cell, and shBMI1#2 (referred to as shBMI1 in the following text) was chosen for the subsequent BMI1 knockdown experiments because of the best knockdown outcome (Figure.S3). The further BMI1 expression was assessed using RT-PCR and WB, and the results showed that BMI1 mRNA was significantly increased in HeLa, HEC-1-A and SPEC-2cells after transfection with myc-BMI1, and decreased after transfection with shBMI1 (Figure. 3A, Figure. 4A, Figure.S4A); accordingly, the BMI1 protein was consistent with BMI1 mRNA (Figure. 3B, Figure. 4B, Figure.S4B). After the transfection of cells with myc-BMI1 and shBMI1, the effect of BMI1 on cell migration was assessed using a wound-healing closure assay. The results showed that after 24 hours of culture, high expression of BMI1 promoted the migration of HeLa cells, whereas downregulation of BMI1 significantly reduced the migration of HeLa cells (Figure. 3C). However, wound healing assays were not performed in HEC-1-A and SPEC-2 cells because they could not be fully fused in 6-well plate. In addition, the same results were obtained from the transwell migration assay (Figure. 3D). Similar results of the transwell migration of HEC-1-A cells were found, further proving the role of BMI1 in the migration of CC and EC cells (Figure. 4C).
3.2 High expression of BMI1 in vitro promotes the growth and proliferation of CC and EC cells
The results of the CCK-8 assay showed that cell growth and proliferation were increased in myc-BMI1-transfected cells but decreased in shBMI1#2-transfected HeLa, HEC-1-A, and SPEC-2 cells compared with the respective controls (Figure. 3E, Figure. 4D, Figure.S4C). In addition, the results of the HeLa, HEC-1-A, and SPEC-2 cell colony formation assays showed that high expression of BMI1 promoted cell growth and proliferation, and low expression of BMI1 inhibited cell growth and proliferation(Figure. 3F, Figure. 4E). SPEC-2 cell colony formation assays also showed that high BMI1 expression promoted cell growth and proliferation (Figure. S4D).
4. The synergic anti-growth effect of shBMI1 combine with PTX treatment
PTX is a commonly used chemotherapeutic drug that has a certain effect on the development of CC and EC. To determine the potential functional role of BMI1 in PTX resistance, we transfected with empty plasmids and shBMI1 plasmids in HeLa and HEC-1-A cells. Next, we added PTX (5 nmol/L) after transfected plasmids and shBMI1 plasmids 24 hours. Then, the effect of co-treatment of shBMI1 and 5 nmol/L PTX on cell migration was assessed using a transwell assay. The results showed that after 24 hours of culture, compare with respective control, shBMI1 inhibited the migration of HeLa and HEC-1-A cells, and co-treatment of shBMI1 and 5 nmol/L PTX has a more significant decrease in cell migration of HeLa and HEC-1-A cells (Figure. 5A, Figure. 6A). Then, we used a CCK-8 assay to detect the growth and proliferation of HeLa and HEC-1-A cells treated with PTX. The results of the CCK-8 assay showed that cell growth and proliferation were decreased in shBMI1-transfected HeLa and HEC-1-A cells compared with the respective controls. Importantly, the growth and proliferation of shBMI1-transfected HeLa and HEC-1-A cells treated with 5 nmol/L PTX further decreased (Figure. 5B, Figure. 6B). Similarly, in colony formation assay, the number of average colonies counts of shBMI1 knockdown group of HeLa and HEC-1-A cells was apparently lower than respective control, and the co-treatment of shBMI1 and 5 nmol/L PTX has a more significant decrease in cell colony formation (Figure. 5C, Figure. 6C). These results suggest that BMI1 knockdown may be sensitive to the PTX.