SRF Potentiates Colon Cancer Metastasis and Progression in a microRNA-214/PTK6-Dependent Manner

Abstract

Objective: Serum response factor (SRF), a sequence-specific transcription factor, is closely related to metastasis of gastric cancer, a digestive tract cancer. Herein, we probed the effect of SRF on metastasis and progression of colon cancer (CC), another digestive tract disorder, and the detailed mechanism.

Methods: Microarray analysis was conducted on tumor and adjacent tissues to filter differentially expressed miRNA, followed by RT-qPCR validation in CC cell lines. The transcription factor and the target gene of microRNA-214 (miR-214) were predicted, and their binding relationships were tested by luciferase reporter assays and ChIP assays. Subsequently, SRF and protein tyrosine kinase 6 (PTK6) expression in CC patients and cells was evaluated by RT-qPCR, while JAK2 and STAT3 expression in cells by Western blot analysis. To further explore functions of miR-214, PTK6 and SRF on CC, CC cells were delivered with si-PTK6, miR-214 mimic and/or SRF overexpression.

Results: miR-214 expressed poorly in CC tissues and cell lines, which related to advanced TNM staging and survival. miR-214 mimic inhibited proliferation, migration, invasion, xenograft tumor growth and metastasis of CC cells. SRF, overexpressed in CC samples and cells, suppressed the transcription of miR-214. Meanwhile, SRF upregulation counteracted the inhibitory role of miR-214 mimic in CC cell growth. miR-214 negatively regulated PTK6 expression to impair the JAK2/STAT3 pathway activation, thereby halting CC cell proliferation, migration, invasion, xenograft tumor growth and metastasis.

Conclusion: Altogether, miR-214 may perform as a tumor suppressor in CC, and the SRF/miR-214/PTK6/JAK2/STAT3 axis could be applied as a biomarker and potential therapeutic target.

Keywords: Colon cancer; PTK6; SRF; The JAK2/STAT3 pathway; microRNA-214.

Figure 1

The analysis of differentially expressed miRNAs in CC tissues. (A) differentially expressed miRNAs in tumor and adjacent tissues screened out by microarray analysis; (B) miR-214 expression in CC tumor and adjacent tissues evaluated by RT-qPCR (*p < 0.05 according to the two-way ANOVA); (C) the correlation analysis between miR-214 expression and TNM stage of patients with CC; (D) survival analysis of CC patients; (E) miR-214 expression in CC cell lines assessed RT-qPCR analysis (*p < 0.05 according to the one-way ANOVA); (F) miR-214 mimic was transfected into LOVO and SW620 cells (*p < 0.05 according to the two-way ANOVA).

Figure 2

Increased miR-214 is associated with decreased CC cell proliferative, migratory, invasive, tumorigenic and metastatic capacities. (A), CC cell proliferation evaluated by EdU staining; (B), CC cell migration evaluated by Transwell assays; (C), CC cell invasion evaluated by Transwell assays; (D), representative tumor images and tumor volume from mice injected with CC cells overexpressing miR-214; (E), KI67 positive rate of tumors detected by immunohistochemistry; (F), changes of pulmonary nodules detected by HE staining. *p < 0.05 according to the two-way ANOVA. Data represent averages of three independent experiments.

Figure 3

The binding relationship between miR-214 and SRF is identified. (A) the transcription factor that regulate miR-214 and the binding promoter sequences predicted by TransmiR and ALGGEN; (B) the targeting relationship between SRF and miR-214 verified by a dual-luciferase reporting assay; (C) the effect of SRF-NC and SRF-OE on miR-214 promoter validated by ChIP; (D) SRF expression in CC and adjacent tissues evaluated by RT-qPCR detection; (E) SRF expression in CC cell lines evaluated by RT-qPCR detection; (F) SRF expression in CC cell lines overexpressing miR-214 evaluated by RT-qPCR detection; (G) SRF expression in CC cell lines overexpressing miR-214 evaluated by RT-qPCR detection. *p < 0.05 according to one-way (panel (E) or two-way ANOVA (panel BD, F and G). Data represent averages of three independent experiments.

Figure 4

Increased SRF is associated with elevated CC cell proliferative, migratory, invasive, tumorigenic and metastatic capacities. SRF-OE or SRF-NC was delivered into CC cell lines. (A) CC cell proliferation determined by EdU staining; (B) CC cell migration evaluated by Transwell assays; (C) CC cell invasion evaluated by Transwell assays; (D) representative tumor images and tumor volume from mice injected with CC cells overexpressing SRF; (E) KI67 positive rate of tumors detected by immunohistochemistry; (F) changes of pulmonary nodules detected by HE staining. *p < 0.05 according to the two-way ANOVA. Data represent averages of three independent experiments.

Figure 5

SRF overexpression rescues the repressive role of miR-214 mimic on CC cell viability. CC cell lines were transfected with SRF-OE or SRF-NC in the presence of miR-214 mimic. (A) SRF expression in CC cell lines co-transfected with miR-214 mimic and SRF-OE evaluated by RT-qPCR detection; (B) CC cell proliferation determined by EdU staining; (C) CC cell migration evaluated by Transwell assays; (D) CC cell invasion evaluated by Transwell assays; (E) representative tumor images and tumor volume from mice injected with CC cells overexpressing SRF; (F), KI67 positive rate of tumors detected by immunohistochemistry; (G) changes of pulmonary nodules detected by HE staining. *p < 0.05 according to the two-way ANOVA. Data represent averages of three independent experiments.

Figure 6

The binding relationship between miR-214 and PTK6 is identified. (A) the targeting relationship between PTK6 and miR-214 predicted by bioinformatic analysis and verified by a dual-luciferase reporting assay; (B) no direct targeting relationship between PTK6 and SRF verified by a dual-luciferase reporting assay; (C) PTK6 expression in CC and adjacent tissues evaluated by RT-qPCR detection; (D) PTK6 expression in CC cell lines evaluated by RT-qPCR detection; (E) PTK6 expression in CC cell lines overexpressing miR-214 or SRF evaluated by RT-qPCR detection; (F) PTK6 expression in CC cell lines with PTK6 knockdown evaluated by RT-qPCR detection. *p < 0.05 according to one-way (panel (D) or two-way ANOVA (panel AC, E and F). Data represent averages of three independent experiments.

Figure 7

PTK6 knockdown is associated with reduced CC cell proliferative, migratory, invasive, tumorigenic and metastatic capacities. Si-PTK6 or PTK6-NC was delivered into CC cell lines. (A) CC cell proliferation determined by EdU staining; (B) CC cell migration evaluated by Transwell assays; (C) CC cell invasion evaluated by Transwell assays; (D) representative tumor images and tumor volume from mice injected with CC cells with PTK6 knockdown; (E) KI67 positive rate of tumors detected by immunohistochemistry; (F) changes of pulmonary nodules detected by HE staining. *p < 0.05 according to the two-way ANOVA. Data represent averages of three independent experiments.

Figure 8

The JAK2/STAT3 pathway is regulated by the SRF/miR-214/PTK6 axis. The JAK2 and STAT3 protein expression and phosphorylation in LOVO (A) and SW620 (B) cells transfected with SRF-NC, SRF-OE, miR-214 control, miR-214 mimic, miR-214 mimic + SRF-NC, miR-214 mimic + SRF-OE, PTK6-NC or si-PTK6. *p < 0.05 according to the two-way ANOVA. Data represent averages of three independent experiments.

Figure 9

The model for miR-214 regulation upon CC cell progression. SRF could bind to the miR-214 promoter region, located on chromosome 1 q24.3, and repress its expression. The restoration of miR-214 lowers the expression PTK6, thereby inhibiting the activity of the JAK2/STAT3 pathway, which in turn inhibits CC cell proliferation, migration, invasion, tumorigenesis, and metastasis.