CD44v6 may influence ovarian cancer cell invasion and migration by regulating the NF-κB pathway

Abstract

Ovarian cancer (OC) has the worst prognosis among all malignancy types in females worldwide according to epidemiological studies in 2017. Although radiotherapy, chemotherapy and surgical treatment are the most common treatment methods, their curative effects are not satisfactory. The present study aimed to examine the role of cluster of differentiation 44 variant 6 (CD44v6) in the molecular mechanism of the proliferation and tumorigenicity of OC cells, and provide a novel target for the clinical treatment of OC. A total of 46 clinical samples were collected, including 24 malignant ovarian tumor tissue samples and 22 benign ovarian tissue samples. Expression of CD44v6 and nuclear factor-κB (NF-κB) in these samples was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. The A2780 OC cell line was used to establish a normal control group, a negative control group and a CD44v6-small interfering (si)RNA transfection group. The expression of CD44v6 and NF-κB mRNA was detected in each group by RT-qPCR. The proliferation, invasion and migration abilities of the cells were then assessed by Transwell and colony formation assays. Additionally, immunofluorescence was used to detect nuclear NF-κB expression. CD44v6 and NF-κB mRNA expression levels were significantly increased in malignant ovarian tumor tissues, compared with normal ovarian tissues (P<0.01), and immunohistochemistry demonstrated similar results. In the CD44v6-siRNA group, NF-κB mRNA expression was significantly reduced, compared with the control and negative control (both P<0.01) groups. Transwell and colony formation assays demonstrated that the migration, invasion and colony formation abilities of OC cells in the CD44v6-siRNA group were significantly reduced, compared with the control and negative control (both P<0.01) groups. Immunofluorescence results demonstrated that the expression of NF-κB in the cytoplasm and nucleus of the CD44v6-siRNA group was also markedly reduced, compared with the other two groups. In conclusion, CD44v6 may participate in the proliferation of OC cells through activation of the NF-κB pathway and these observations may provide a novel therapeutic target for the clinical treatment of OC.

Keywords: cluster of differentiation 44 variant 6; invasion; migration; nuclear factor-κB; ovarian cancer.

Figure 1.

CD44v6 and NF-κB expression in ovarian cancer tissues and an ovarian cancer cell line. (A) Immunohistochemical analysis of CD44v6 and NF-κB in benign ovarian tissue and malignant ovarian tumor tissue (×400 magnification). Brown represents positive expression of CD44v6 and NF-κB. CD44v6 was predominantly located on the cell membrane and NF-κB was primarily located in the nucleus of cancer tissue. (B) CD44v6 and NF-κB mRNA expression levels in benign ovarian tissue and malignant ovarian tumor tissue. **P<0.01 vs. benign ovarian tissue. (C) Relative mRNA expression levels of CD44v6 and NF-κB in different groups, including the control, empty vector and CD44v6-siRNA groups. No significant difference was identified between the control and empty vector groups. A significant difference was revealed between the control and CD44v6-siRNA groups. Data are presented as the mean ± standard deviation. **P<0.01 vs. control group. NF-κB, nuclear factor-κB; siRNA, small interfering RNA; CD44v6, cluster of differentiation variant 6.

Figure 2.

CD44v6-siRNA impairs the proliferation, migration and invasion of ovarian cancer cells. (A) Representative figures of invading cells in different groups (magnification, ×200). (B) Quantification of the invasion ability of the control group (134.39±9.69), empty vector group (175.34±29.64) and CD44v6-siRNA group (43.25±3.02) was assessed by 0.1% crystal violet staining. (C) Representative figures of migratory cells in different groups (magnification, ×200). (D) Quantification of the migration ability of the control group (421.67±48.95), empty vector group (403.25±20.75) and CD44v6-siRNA group (154.45±21.75) was assessed by 0.1% crystal violet staining. The Transwell migration assays demonstrated that the number of migratory and invading cells per field in the CD44v6 siRNA group significantly decreased compared with the control group (P<0.01). (E) Representative figures of colony formation ability. (F) Quantification of the colony formation ability of the control group, empty vector group and CD44v6-siRNA group. A total of 300 cells were seeded per well. The colony formation rate=number of colonies/number of cells seeded ×100%. The data are presented as the mean ± standard deviation. **P<0.01 vs. control group. siRNA, small interfering RNA; CD44v6, cluster of differentiation variant 6.

Figure 3.

Immunofluorescence of the control, empty vector and CD44v6-siRNA groups. Blue represents the nucleus, green represents expression of CD44v6 and red represents expression of NF-κB. siRNA, small interfering RNA; NF-κB, nuclear factor-κB; CD44v6, cluster of differentiation variant 6.