KRT15 promotes proliferation, migration and invasion of cervical cancer cells

2.1 Bioinformatics analysis

The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) projects’ tumor and normal sample RNA sequencing expression data are available in the GEPIA database (gepia.cancer-pku.cn). Using GEPIA, the expression of KRT15 in cervical cancer tissues was evaluated.

2.2 Cell culture

The human cervical cancer cells HeLa, SiHa, CaSki and C33A, as well as the immortalized keratinocyte cell line HaCaT, were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultivated at 37 °C in a humidified incubator with 5% CO₂ in high-glucose media Dulbecco’s modified eagle medium (DMEM, 2458509, Gibco, Grand Island, NY, USA) with 10% Fetal Bovine Serum (FBS, FB12999102, Gibco, Grand Island, NY, USA) and 1% penicillin-Streptomycin (C0222, Beyotime, Shanghai, China).

2.3 Cell transfection

KRT15-specific siRNA (si-KRT15, 5′-AGGAGTACAAGATGCTGCTTGACAT-3′) and a negative control (si-NC, 5′-AGGATACGATACGGTTCGTAGACAT-3′) were used to knockdown KRT15. Si-KRT15 and si-NC were transfected into cervical cancer cells (HeLa and SiHa) using the Lipofectamine 3000 kit (L3000015, Invitrogen, Carlsbad, CA, USA). KRT15 expression was measured 48 h after knockdown to assess the impact.

2.4 Cell viability assay (CCK-8)

3 × 10³ HeLa and SiHa cells were seeded and cultured for 24 h. Cell viability was calculated using CCK-8 (10 µL/well; C0037, Beyotime, Shanghai, China) by reading optical density using a microplate reader. Six replicate samples were included in each group.

2.5 Colony formation assay

In separate 6-well plates, transfected HeLa and SiHa cells (5 × 10³) were seeded. A 4% paraformaldehyde fixation and 0.1% amethyst staining (548-62-9, Sigma-Aldrich, Shanghai, China) were performed after cell clump formation to determine their colonial number. In colony formation experiments, if cells form colonies, this usually indicates significant proliferative capacity and viability.

2.6 Transwell assay

Invasion experiment: the upper chamber of Matrigel-coated Transwell plates (Corning Company) was injected with transfected HeLa and SiHa cells (4 × 10⁴) suspended in FBS-free medium. 10% FBS-containing medium was added to the lower chamber culture media. An inverted light microscope (Leica DMi1, Leica Microsystems, Wetzlar, HE, Germany) was used to count the invasive cells after they had been cultivated for 24 h and stained with crystal violet (Y268091, Beyotime, Shanghai, China) for 20 minutes at room temperature.

Migration experiment: the experimental method is similar to the invasion experiment, except that the Transwell plate is not coated with Matrigel [11].

2.7 Western blotting

Radioimmunoprecipitation assay (RIPA) buffer (P0013B, Beyotime, Shanghai, China) was used to lyse cervical cancer cells. A Bicinchoninic acid assay (BCA) protein assay kit (P0009, Beyotime, Shanghai, China) was used to measure the total protein concentration. Using 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 20 micrograms of proteins and transferred to a polyvinylidene fluoride (PVDF) membrane. Primary antibodies were incubated overnight at 4 °C after the membrane was blocked for 90 mins at 37 °C using 5% Bovine Serum Albumin (BSA, GC305010, Savis Biotechnology Co., Ltd., Wuhan, China), including KRT15 (ab52816; Abcam, Cambridge, UK), β-catenin (ab32572; Abcam, Cambridge, UK), cyclin D1 (ab239794; Abcam, Cambridge, UK), Wnt3a (ab219412; Abcam, Cambridge, UK), matrix metalloproteinase-2 (MMP-2, ab92536; Abcam, Cambridge, UK), MMP-9 (ab76003; Abcam, Cambridge, UK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ab8245; Abcam, Cambridge, UK). Incubation was conducted for 1.5 h at 37 °C with diluted horseradish peroxidase (HRP)-labeled secondary antibody (ab6721; Abcam, Cambridge, UK). The membrane was detected using an enhanced chemiluminescence (ECL) kit (G2161, Wuhan Saiwei Biotechnology Co., Ltd., Wuhan, China), and grayscale values were analyzed using Image J (1.0, NIH).

2.8 Statistical analysis

Data plotting and statistical analysis were performed using GraphPad Prism 8 (Harvey Motulsky, San Diego, CA, USA). Group comparisons were analyzed using Student’s t-test. Data are presented as the mean ± Standard Deviation (SD) from three independent experiments. Statistical significance was defined as p < 0.05. Each experiment was conducted three times independently.

3.1 KRT15 was highly expressed in cervical cancer

Using GEPIA website analysis, we found higher KRT15 expression in tumor tissues of cervical cancer patients than in healthy subjects (Fig. 1A). Compared with immortalized keratinocytes (HaCaT cells), HeLa, SiHa (p < 0.001), CaSki and C33A cells (p < 0.01) showed increased KRT15 expression (Fig. 1B). In summary, cervical carcinoma showed elevated expression of KRT15. Since HeLa and SiHa cells expressed KRT15 at the highest levels, they were chosen for additional functional investigations.

Fig. 1.KRT15 expression in cervical cancer. (A) GEPIA database analysis of KRT15 expression in healthy subjects and cervical cancer patients. (B) Western blot analysis of KRT15 expression in the Human immortalized keratinocyte cell line HaCaT and human cervical cancer cell lines HeLa, SiHa, CaSki and C33A. Values are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. n = 3. TPM: Transcripts Per Million; CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma; KRT15: Keratin 15; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

3.2 Knockdown of KRT15 inhibited the proliferation of cervical cancer cells

After transfecting cells with si-KRT15, KRT15 expression decreased, indicating protein knockdown was effective (p < 0.01) (Fig. 2A). After transfection, si-KRT15 decreased the ability of cells to form colonies and maintain cell viability (p < 0.01) (Fig. 2B,C). Consequently, KRT15 plays an active role in cervical cancer cell proliferation.

Fig. 2.The role of KRT15 in cervical cancer cell proliferation. (A) KRT15 expression in transfected and untransfected cells. (B) CCK-8 kit detects cell viability. n = 6. (C) Crystal violet staining detects cell colony formation ability. Values are presented as mean ± SD. **p < 0.01. n = 3. KRT15: Keratin 15; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; si-NC: siRNA Control.

3.3 Knockdown of KRT15 inhibited cervical cancer cell migration and invasion

This study investigated KRT15’s role in cell invasion and migration. Transwell experiments showed that HeLa and SiHa cells’ capacity for invasion and migration was significantly reduced following KRT15 knockdown (p < 0.01) (Fig. 3A). KRT15 knockdown could decrease the expression of MMP-2 and MMP-9 proteins (p < 0.01) (Fig. 3B). Consequently, KRT15 actively contributed to cervical cancer cell invasion and migration. Enhanced migration and invasion capabilities may be indicators of tumor cells’ metastatic potential.

Fig. 3.The role of KRT15 in the cervical cancer cell migration and invasion. (A) Cell migration and invasion numbers. (B) Western blot analysis of MMP-2 and MMP-9 expression in cells. Values are presented as mean ± SD. **p < 0.01. n = 3. si-NC: siRNA Control; KRT15: Keratin 15; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MMP: matrix metalloproteinase.

3.4 Knockdown of KRT15 downregulated Wnt/β-catenin signaling pathway

KRT15 uses β-catenin to control cell invasion and migration in colorectal cancer [7]. Therefore, it is necessary to determine whether KRT15 regulates β-catenin. Western blotting was used to measure the amounts of β-catenin, Wnt3a and Cyclin D1 proteins. KRT15 knockdown suppressed β-catenin, Wnt3a and Cyclin D1 expression in HeLa and SiHa (p < 0.01) (Fig. 4). In cervical cancer cells, KRT15 controlled the Wnt/β-catenin signaling pathway.

Fig. 4.Effect of KRT15 on Wnt/β-catenin pathway in cervical cancer cells. Western blot analysis of β-catenin, Wnt3a, Cyclin D1 expression in cells. Values are presented as mean ± SD. **p < 0.01. n = 3. si-NC: siRNA Control; KRT15: Keratin 15; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.