3.1. Involvement of ZBTB5 in the mechanism of PTX resistance in cervical squamous cell cancer
3.1.1. Use of cervical squamous carcinoma cell line (SiHa cells) as an in vitro model of ZBTB5 affecting PTX resistance
Using the CRISPR/Cas9 gene-editing tool, six sgRNAs were designed against the ZBTB5 gene, and plasmids were constructed, packaged with lentivirus, and infected the human cervical cancer cell line to obtain SiHa cell lines with knockdown of ZBTB5. The ploidy data of each group of cells were analyzed by treating each of the six SiHa cell lines with 4 ng/mL PTX with drug solvent for 120 h (see Table 1). The optimal ZBTB5 knockdown target was selected, and knockdown of ZBTB5 significantly reduced the PTX resistance of SiHa cervical squamous carcinoma cells.
Table 1 Assessment of optimal ZBTB5 knockdown targets using SiHa cell lines for PTX resistance
|
Target
|
Number of cell doubling (120 h)
|
Sensitization folds
|
Notes
|
sgRNA-NC
|
7.93
|
1.00
|
|
sgRNA-NC-PTX
|
2.24
|
ZBTB5-sgRNA(05084)
|
7.49
|
1.37
|
|
ZBTB5-sgRNA(05084)-PTX
|
1.54
|
ZBTB5-sgRNA(05085)
|
7.52
|
1.57
|
|
ZBTB5-sgRNA(05085)-PTX
|
1.35
|
ZBTB5-sgRNA(05086)
|
7.5
|
1.16
|
|
ZBTB5-sgRNA(05086)-PTX
|
1.82
|
ZBTB5-sgRNA(05087)
|
7.04
|
1.64
|
Final target selection
|
ZBTB5-sgRNA(05087)-PTX
|
1.21
|
ZBTB5-sgRNA(05088)
|
6.62
|
1.34
|
|
ZBTB5-sgRNA(05088)-PTX
|
1.4
|
ZBTB5-sgRNA(05089)
|
7.68
|
1.05
|
|
ZBTB5-sgRNA(05089)-PTX
|
2.07
|
3.1.2. Immunofluorescence shows that overexpression of ZBTB5 enhances PTX resistance in cervical squamous cell cancer by functioning in the nucleus
When cultured in 4 ng/mL PTX for 14 days, normal SiHa cells had a rounded morphology and smaller pseudopods than SiHa cells with ZBTB5 overexpression, indicating that overexpression of ZBTB5 increased cell adhesion and invasion, thereby enhancing the resistance of cervical squamous SiHa cells to PTX and leading to PTX resistance. ZBTB5 was labeled with red light, and cytoplasmic red fluorescence intensity after overexpression of ZBTB5 decreased and accumulated near the punctate nucleus, indicating that the relative amount of ZBTB5 in the cytoplasm decreased, aggregated towards the nucleus, and increased PTX resistance by functioning in the nucleus (see Figure 1).
3.2. Involvement of ZBTB5 in cervical cancer cell function
3.2.1. Effect on the cell clone-forming ability
To investigate the effect of ZBTB5 knockdown on the cloning of SiHa cervical squamous carcinoma cells, the effect of ZBTB5 overexpression on the clone-forming ability of cervical squamous carcinoma cells was observed using a plate clone formation assay, and the number of cell clones was counted after the above cells were cultured for 9 days, where the study cells were incubated with PTX on the fourth day. The results showed that in SiHa cells, the number of clones was 245 ± 11 in the sgCtrl group, 108 ± 8 in the sgCtrl PTX group, 227 ± 19 in the sgZBTB5 group, and 55 ± 7 in the sgZBTB5 PTX group. The difference between the sgCtrl PTX / sgCtrl ratio and the sgZBTB5 PTX / sgZBTB5 ratio was statistically significant with weaker cell cloning ability (p < 0.05) (see Figure 2). ZBTB5 knockdown inhibited cell cloning and increased sensitivity to PTX.
3.2.2. Effect on cell proliferation
To investigate the effect of sgZBTB5 on the proliferation of SiHa cervical squamous carcinoma cells, two groups were incubated with PTX for 6 days and treated with MTT for 4 h. The absorbance of light at a wavelength of 490 nm over time for each group was compared in an enzyme-labeling instrument. Here, OD490 reflected the number of viable cells. As shown in Figure 3-1 and Figure 3-2, the cells in the sgZBTB5+ group were more sensitive to the drug compared to the sgCtrl+ group (p < 0.05).
3.2.3. ZBTB5 can negatively regulate the apoptosis ability of the cervical squamous carcinoma cell line SiHa under PTX (4 ng/mL) treatment conditions
Compared with the control group, the apoptosis ability of SiHa cells with ZBTB5 knockdown was significantly enhanced after PTX treatment, which reduced resistance to PTX and reduced PTX resistance in SiHa cells (as shown in Figure 4).
3.3. IPA bioinformatics analysis: The ZBTB5 gene affects downstream gene expression by regulating BCL6 (see Figure 5)
Through genome-wide IPA analysis, ZBTB5 may indirectly affect the expression of downstream genes through the BCL6 gene, which overexpresses to promote tumor proliferation or inhibit apoptosis, and the ZBTB5 gene is consistent with its function.
3.4. ZBTB5 knockdown can downregulate BCL6 gene expression levels (see Figure 6)
The qPCR and WB results suggested that ZBTB5 knockdown downregulated BCL6 gene expression (mRNA) and that ZBTB5 was correlated with BCL6.
3.5. ChIP screening and validation of downstream genes with possible ZBTB5 action
3.5.1. ChIP detection of downstream genes with possible ZBTB5 action
A ChIP assay was performed after overexpression of ZBTB5, where the ZBTB5 protein was screened, gene fragments 0~3 kb away from the transcription start site (TSS) were selected for gene sequencing, and the distribution of reads (sequenced fragments) 0~3 kb close to the TSS was determined. The peak refers to a gene fragment near the ZBTB5 protein promoter (395 kb away from the TSS). GO functional enrichment analysis was performed on peak-related genes, and these were classified according to their functions. Results of the GO functional enrichment analysis are shown in Figure 7-1, and KEGG pathway enrichment analysis was performed on peak-related genes in Figure 7-2, which showed that the BCL6 gene was in the signaling pathway of these genes. The above analyses revealed that the BCL6 gene is a downstream gene of ZBTB5 gene action, and the ZBTB5 gene affects cell proliferation and apoptosis by regulating the signaling pathway of the BCL6 gene, thus, affecting resistance to PTX-based chemotherapy in patients with cervical cancer, which is consistent with the results of IPA bioinformatics analysis (see Table 2).
Table 2 BCL gene sequences that may be regulated downstream of ZBTB5
|
Seq
|
Start
|
End
|
Width
|
Annotation
|
Distance to TSS
|
Gene
|
chr3
|
187843762
|
187843935
|
174
|
Distal Intergenic
|
−98035
|
BCL6 transcription repressor
|
chr3
|
187765104
|
187765282
|
179
|
Distal Intergenic
|
−19377
|
BCL6 transcription repressor
|
chr3
|
187729721
|
187729865
|
145
|
3' UTR
|
−4098
|
BCL6 transcription repressor
|
chr3
|
187725021
|
187725228
|
208
|
Promoter
(≤ 1 kb)
|
395
|
BCL6 transcription repressor
|
3.5.2. Co-IP screening combined with mass spectrometry of target gene interaction complexes
Co-IP was performed in the OE and NC groups, and further in-gel enzymatic digestion and shotgun-mass spectrometry protein identification was performed. Overall, 1,371 proteins were identified in the normal group and 1,336 in the overexpression group, and a total of 322 differential proteins were identified. Bioinformatics analysis was performed on the differential proteins to analyze 247 proteins with possible relevance and their mechanism of action, including their possible involvement in cell proliferation, metastasis, apoptosis, and invasion, as shown in Figure 8.
3.5.3. WB validation of proteins identified by Co-IP screening
From the proteins identified by the above screening, 13 were selected for validation after bioinformatics analysis: U2AF2, RBM5, ILK, ENAH, JUP, RELA/P65, SQSTM1, YY1, STIM1, Integrin alpha V, EED, SUGT1, and NFKB1, among which U2AF2 was successfully detected in the input. The target bands were detected in NC and OE and, compared with NC, U2AF2 expression was significantly increased in the OE group, suggesting that the 3× Flag-ZBTB5 protein pulled down the U2AF2 protein and there may be an interaction between the two proteins (see Figure 9).