Circular RNA circEGFR regulates tumor progression via the miR-106a-5p/DDX5 axis in colorectal cancer

Recently, an increasing number of studies have reported that dysregulation of circular RNA (circRNA) expression plays critical roles in the progression of several cancers, including colorectal cancer (CRC). However, the detailed molecular mechanisms of circRNAs involvement in CRC remain largely unknown. Here, we confirmed that the level of circEGFR was significantly increased in CRC tissues compared to matched adjacent non-tumor tissues, and a high level of circEGFR was correlated with poor clinicopathological characteristics and poor prognosis in patients with CRC. Moreover, increased circEGFR expression promoted CRC cell proliferation, migration, and invasion in vitro. Mechanistically, circEGFR acted as a ceRNA for miR-106a-5p to relieve the repressive effect of miR-106a-5p on DDX5 mRNA. Moreover, circEGFR enhanced DDX5 expression, thereby upregulating p-AKT levels. Together, these findings showed that circEGFR promoted CRC cell proliferation, migration, and invasion through the miR-106a-5p/DDX5/AKT axis, and may serve as a promising diagnostic marker and therapeutic target for CRC patients.

circEGFR; Colorectal cancer; Progression; DDX5; miR-106a-5p

Authorship

Ping Fu

Department of General Surgery, Jiangxi Provincial People's Hospital Affiliated to Nanchang University, Nanchang, Jiangxi, ChinaJiangxi Provincial People's Hospital Affiliated to Nanchang UniversityChinaNanchang, Jiangxi, ChinaDepartment of General Surgery, Jiangxi Provincial People's Hospital Affiliated to Nanchang University, Nanchang, Jiangxi, China

https://orcid.org/0000-0003-1792-8924

Liangqing Lin

https://orcid.org/0000-0003-1676-8201

Hui Zhou

https://orcid.org/0000-0002-2206-8183

Sijun Zhao

https://orcid.org/0000-0002-0841-6065

Zhigang Jie Correspondence: Zhigang Jie: <jiezg123@126.com>

Department of General Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, ChinaThe First Affiliated Hospital of Nanchang UniversityChinaNanchang, Jiangxi, ChinaDepartment of General Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China

https://orcid.org/0000-0002-6369-7306

Correspondence: Zhigang Jie: <jiezg123@126.com>

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (6)

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Figure 1
circEGFR is overexpressed in colorectal cancer (CRC) tissues and is correlated with poor prognosis. A, circEGFR expression in 90 paired CRC tissues and their adjacent normal tissues. B, circEGFR expression is upregulated in larger tumors. Data are reported as means±SD. ****P<0.0001 (unpaired t-test). C and D, The higher circEGFR expression is correlated with poor prognosis in CRC patients (overall survival: P=0.0033; recurrence rate: P=0.0048, Kaplan-Meier).

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Figure 2
Increased circEGFR expression promoted proliferation, migration, and invasion of colorectal cancer (CRC) cells in vitro. A, The expression level of circEGFR in CRC cell lines. B, The circEGFR expression level in HCT-116 and LOVO cells after transfection with circEGFR overexpression and shRNA lentivirus. C and D, The cell viability of HCT-116-circEGFR and LOVO-circEGFR shRNA was assessed by CCK-8 assay. E, Transwell assays were performed to assess the invasion ability of HCT-116-circEGFR and LOVO-circEGFR shRNA cells. Scale bar: 100 μm. F, Colony formation assays were performed to assess the proliferative ability of CRC cells. Data are reported as means±SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (t-test and ANOVA).

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Figure 3
circEGFR functions as a ceRNA to regulate DDX5 expression in colorectal cancer (CRC). A and B, The potential miRNA and its target gene sequences were explored by starBase 3.0 software. C and D, The relative luciferase activity was detected in CRC cells co-transfected with wild-type (wt) or mutated-type (mu) circEGFR or DDX5 3′-UTR and miR-106a-5p mimic. E, DDX5 expression was significantly increased after overexpression of circEGFR. F, DDX5 expression was significantly reduced after the knockdown of circEGFR. Data are reported as means±SD. **P<0.01; ***P<0.001 (t-test). NS: not significant; NC: negative control.

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Figure 4
circEGFR regulates the expression of the DDX5 and the DDX5-mediated signaling pathway in colorectal cancer (CRC). A and B, circEGFR overexpression upregulated the level of p-AKT while circEGFR knockdown reduced the p-AKT expression; actin served as internal control. Data are reported as means±SD. C and D, A positive correlation was observed between circEGFR and DDX5 while a negative correlation was observed between circEGFR and miR-106a-5p.

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Figure 5
DDX5 knockdown affected the circEGFR-induced colorectal cancer (CRC) progression. A, qRT-PCR was performed to verify the knockdown efficacy of DDX5. B, Western blot was performed to validate the knockdown efficacy of DDX5, and actin served as the internal control. C and D, The proliferative abilities of HCT-116 and LOVO cells were determined using CCK-8 assays, and downregulated DDX5 suppressed the proliferation of HCT-116 and LOVO cells. E and F, The invasion abilities of HCT-116 and LOVO cells was assessed by transwell assays, and downregulated DDX5 suppressed the invasion of HCT-116 and LOVO cells (scale bar 100 μm). Data are reported as means±SD. *P<0.05, **P<0.01, ***P<0.001 (t-test). NC: negative control.

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Figure 6
Working model and mechanism diagram of colorectal cancer cell proliferation and invasion.

Figure 1 circEGFR is overexpressed in colorectal cancer (CRC) tissues and is correlated with poor prognosis. A, circEGFR expression in 90 paired CRC tissues and their adjacent normal tissues. B, circEGFR expression is upregulated in larger tumors. Data are reported as means±SD. ****P<0.0001 (unpaired t-test). C and D, The higher circEGFR expression is correlated with poor prognosis in CRC patients (overall survival: P=0.0033; recurrence rate: P=0.0048, Kaplan-Meier).

Figure 2 Increased circEGFR expression promoted proliferation, migration, and invasion of colorectal cancer (CRC) cells in vitro. A, The expression level of circEGFR in CRC cell lines. B, The circEGFR expression level in HCT-116 and LOVO cells after transfection with circEGFR overexpression and shRNA lentivirus. C and D, The cell viability of HCT-116-circEGFR and LOVO-circEGFR shRNA was assessed by CCK-8 assay. E, Transwell assays were performed to assess the invasion ability of HCT-116-circEGFR and LOVO-circEGFR shRNA cells. Scale bar: 100 μm. F, Colony formation assays were performed to assess the proliferative ability of CRC cells. Data are reported as means±SD. P<0.05; P<0.01; P<0.001; ****P<0.0001 (t-test and ANOVA).

Figure 3 circEGFR functions as a ceRNA to regulate DDX5 expression in colorectal cancer (CRC). A and B, The potential miRNA and its target gene sequences were explored by starBase 3.0 software. C and D, The relative luciferase activity was detected in CRC cells co-transfected with wild-type (wt) or mutated-type (mu) circEGFR or DDX5 3′-UTR and miR-106a-5p mimic. E, DDX5 expression was significantly increased after overexpression of circEGFR. F, DDX5 expression was significantly reduced after the knockdown of circEGFR. Data are reported as means±SD. P<0.01; *P<0.001 (t-test). NS: not significant; NC: negative control.

Figure 4 circEGFR regulates the expression of the DDX5 and the DDX5-mediated signaling pathway in colorectal cancer (CRC). A and B, circEGFR overexpression upregulated the level of p-AKT while circEGFR knockdown reduced the p-AKT expression; actin served as internal control. Data are reported as means±SD. C and D, A positive correlation was observed between circEGFR and DDX5 while a negative correlation was observed between circEGFR and miR-106a-5p.

Figure 5 DDX5 knockdown affected the circEGFR-induced colorectal cancer (CRC) progression. A, qRT-PCR was performed to verify the knockdown efficacy of DDX5. B, Western blot was performed to validate the knockdown efficacy of DDX5, and actin served as the internal control. C and D, The proliferative abilities of HCT-116 and LOVO cells were determined using CCK-8 assays, and downregulated DDX5 suppressed the proliferation of HCT-116 and LOVO cells. E and F, The invasion abilities of HCT-116 and LOVO cells was assessed by transwell assays, and downregulated DDX5 suppressed the invasion of HCT-116 and LOVO cells (scale bar 100 μm). Data are reported as means±SD. P<0.05, P<0.01, P<0.001 (t-test). NC: negative control.

Figure 6 Working model and mechanism diagram of colorectal cancer cell proliferation and invasion.

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Circular RNA circEGFR regulates tumor progression via the miR-106a-5p/DDX5 axis in colorectal cancer

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[1] Correspondence: Zhigang Jie: <jiezg123@126.com>