Curcumin played an anti-cancer role in colorectal cancer via mediating circ_KIAA1199-related regulatory axis

Curcumin has been found to have anti-cancer role in many cancer, including colorectal cancer (CRC). However, it is not clear whether curcumin can be involved in CRC progression by mediating the circRNA regulatory axis. Curcumin inhibited the proliferation, migration, invasion, and promoted the apoptosis of CRC cells. Circ_KIAA1199 was an upregulated circRNA in CRC, and its overexpression could reverse the negative regulation of curcumin on CRC progression. MiR-433-3p was found to be sponged by circ_KIAA1199, and its mimic abolished the regulation of circ_KIAA1199 overexpression on the progression of curcumin-treated CRC cells. ADAM10 was a target of miR-433-3p and its expression was regulated by circ_KIAA1199 and curcumin. The suppressive effect of miR-433-3p inhibitor on the progression of curcumin-treated CRC cells could be reversed by ADAM10 silencing. Also, curcumin reduced CRC tumor growth by regulating the circ_KIAA1199/miR-433-3p/ADAM10 axis in vivo. Our data showed that curcumin could restrain CRC progression, which was achieved through the circ_KIAA1199/miR-433-3p/ADAM10 pathway.

Keywords:
colorectal cancer; curcumin; circ_KIAA1199; miR-433-3p; ADAM10

Authorship

Xue-Feng SUN

Department of Gastroenterology, Chongqing University Three Gorges Hospital, Chongqing, China.Chongqing University Three Gorges HospitalChinaChongqing, ChinaDepartment of Gastroenterology, Chongqing University Three Gorges Hospital, Chongqing, China.

Liang LI

Yan-Bing LIU ^**Corresponding author: fhycp89@163.com

https://orcid.org/0000-0001-9849-7948

*Corresponding author: fhycp89@163.com

Conflicts of interest

The authors have no conflict of interest to declare.

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Figures

Figures (8)

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Figure 1
Curcumin inhibited CRC cell progression. HCT116 and SW480 cells were treated with different concentrations (5, 10, and 20 μM) of curcumin. Non-treated cells were used as control. (A) The number of colonies was measured using colony formation assay. (B-C) The cell viability was detected by MTT assay. (D-E) The numbers of migrated and invaded cells were determined using transwell assay. (F) Flow cytometry was used to test the apoptotic rate of cells. *P < 0.05.

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Figure 2
The expression of circ_KIAA1199 in CRC tissues and cells. (A) Circ_KIAA1199 expression in CRC tumor tissues (Tumor) and adjacent normal tissues (Normal) was examined using qRT-PCR. (B) The expression of circ_KIAA1199 in FHC cells and CRC cells (HCT116 and SW480) was detected by qRT-PCR. (C) QRT-PCR was used to detect circ_KIAA1199 expression in HCT16 and SW480 cells treated with different concentrations (5, 10, and 20 μM) of curcumin. (D-E) Subcellular localization assay was performed to assess the localization of circ_KIAA1199 in the nucleus and cytoplasm of CRC cells. (F-G) ActD assay was used to evaluate the stability of circ_KIAA1199 in HCT16 and SW480 cells. *P < 0.05.

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Figure 3
Curcumin regulated circ_KIAA1199 to mediate CRC progression. (A-B) After HCT16 and SW480 cells were transfected with circ_KIAA1199 overexpression vector or vector, the expression of circ_KIAA1199 and linear KIAA1199 was measured by qRT-PCR. (C-J) HCT116 and SW480 cells were transfected with or without circ_KIAA1199 overexpression vector or vector, and then treated with curcumin. Non-transfected and non-treated cells were used as control. (C) Colony formation assay was performed to detect the number of colonies. (D-E) MTT assay was used to measure the cell viability. (F-G) The numbers of migrated and invaded cells were analyzed by transwell assay. (H) The apoptotic rate of cells was examined using flow cytometry. (I-J) The protein levels of Bcl-2 and Bax were determined using WB analysis. *P < 0.05.

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Figure 4
Circ_KIAA1199 targeted miR-433-3p. (A) The binding sites and corresponding mutate sites of circ_KIAA1199 and miR-433-3p were shown. (B-C) The interaction between circ_KIAA1199 and miR-433-3p was confirmed by dual-luciferase reporter assay. (D) The expression of miR-433-3p was measured by qRT-PCR in HCT116 and SW480 cells transfected with vector or circ_KIAA1199 overexpression vector. (E) QRT-PCR was used to detect miR-433-3p expression in HCT16 and SW480 cells treated with 20 μM curcumin. (F) QRT-PCR was employed to examine the expression of miR-433-3p in FHC cells and CRC cells (HCT116 and SW480). (G) MiR-433-3p expression in CRC tumor tissues (Tumor) and adjacent normal tissues (Normal) were measured by qRT-PCR. (H) The correlation between circ_KIAA1199 and miR-433-3p was analyzed using Pearson correlation analysis. *P < 0.05.

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Figure 5
Curcumin regulated CRC progression by regulating circ_KIAA1199/miR-433-3p. (A) HCT16 and SW480 cells were transfected with vector, circ_KIAA1199, circ_KIAA1199 + miR-NC or circ_KIAA1199 + miR-433-3p. The expression of miR-433-3p was detected by qRT-PCR. (B-I) After transfection, HCT116 and SW480 cells were treated with curcumin. Non-transfected and non-treated cells were used as control. (B) The number of colonies was examined using colony formation assay. (C-D) MTT assay was employed to determine the cell viability. (E-F) Transwell assay was performed to test the numbers of migrated and invaded cells. (G) The apoptotic rate of cells was determined by flow cytometry. (H-I) WB analysis was utilized for examining the protein levels of Bcl-2 and Bax. *P < 0.05.

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Figure 6
MiR-433-3p targeted ADAM10 in CRC. (A) The binding sites and corresponding mutate sites of miR-433-3p and ADAM10 3’UTR were shown. (B-C) Dual-luciferase reporter assay was utilized for assessing the interaction between miR-433-3p and ADAM10. (D) The transfection efficiencies of miR-433-3p mimic and inhibitor were confirmed by detecting miR-433-3p expression using qRT-PCR. (E-F) The mRNA and protein expression levels of ADAM10 were measured by qRT-PCR and WB analysis in HCT116 and SW480 cells transfected with miR-NC, miR-433-3p, anti-miR-NC or anti-miR-433-3p. (G-H) The mRNA and protein expression of ADAM10 in CRC tumor tissues (Tumor) and adjacent normal tissues (Normal) were determined using qRT-PCR and WB analysis. (I-J) Pearson correlation analysis was utilized for evaluating the correlation between ADAM10 mRNA expression and miR-433-3p or circ_KIAA1199 expression in CRC tumor tissues. (K-L) QRT-PCR and WB analysis were employed to examine the mRNA and protein expression of ADAM10 in FHC cells and CRC cells (HCT116 and SW480). (M-N) QRT-PCR and WB analysis were used to detect the mRNA and protein expression of ADAM10 in HCT16 and SW480 cells treated with 20 μM curcumin. (O-P) HCT116 and SW480 cells were transfected with vector, circ_KIAA1199, circ_KIAA1199 + miR-NC or circ_KIAA1199 + miR-433-3p. The mRNA and protein expression levels of ADAM10 were examined by qRT-PCR and WB analysis. *P < 0.05.

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Figure 7
Curcumin inhibited CRC progression via the miR-433-3p/ADAM10 axis. (A-B) HCT16 and SW480 cells were transfected with anti-miR-NC, anti-miR-433-3p, anti-miR-433-3p + si-NC or anti-miR-433-3p + si-ADAM10. The mRNA and protein expression of ADAM10 was detected by qRT-PCR and WB analysis. (C-J) After transfection, HCT116 and SW480 cells were treated with curcumin. Non-transfected and non-treated cells were used as control. (C) Colony formation assay was employed to measure the number of colonies. (D-E) Cell viability was tested using MTT assay. (F-G) Transwell assay was utilized to assess the numbers of migrated and invaded cells. (H) Flow cytometry was used to detect the apoptotic rate of cells. (I-J) The protein levels of Bcl-2 and Bax were tested using WB analysis. *P < 0.05.

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Figure 8
Curcumin reduced CRC tumor growth through regulating the circ_KIAA1199/miR-433-3p/ADAM10 axis in vivo. Tumor volume (A) and tumor weight (B) were detected in each group. (C-E) The expression levels of circ_KIAA1199, miR-433-3p and ADAM10 in the tumor tissues of each group were measured by qRT-PCR. (F) The ADAM10 protein level in the tumor tissues of each group was determined by WB analysis. (G) IHC staining was used to detect the Ki67 and Cleaved caspase3 positive cells to assess the proliferation and apoptosis of tumor cells. *P < 0.05.

Figure 1 Curcumin inhibited CRC cell progression. HCT116 and SW480 cells were treated with different concentrations (5, 10, and 20 μM) of curcumin. Non-treated cells were used as control. (A) The number of colonies was measured using colony formation assay. (B-C) The cell viability was detected by MTT assay. (D-E) The numbers of migrated and invaded cells were determined using transwell assay. (F) Flow cytometry was used to test the apoptotic rate of cells. *P < 0.05.

Figure 2 The expression of circ_KIAA1199 in CRC tissues and cells. (A) Circ_KIAA1199 expression in CRC tumor tissues (Tumor) and adjacent normal tissues (Normal) was examined using qRT-PCR. (B) The expression of circ_KIAA1199 in FHC cells and CRC cells (HCT116 and SW480) was detected by qRT-PCR. (C) QRT-PCR was used to detect circ_KIAA1199 expression in HCT16 and SW480 cells treated with different concentrations (5, 10, and 20 μM) of curcumin. (D-E) Subcellular localization assay was performed to assess the localization of circ_KIAA1199 in the nucleus and cytoplasm of CRC cells. (F-G) ActD assay was used to evaluate the stability of circ_KIAA1199 in HCT16 and SW480 cells. *P < 0.05.

Figure 3 Curcumin regulated circ_KIAA1199 to mediate CRC progression. (A-B) After HCT16 and SW480 cells were transfected with circ_KIAA1199 overexpression vector or vector, the expression of circ_KIAA1199 and linear KIAA1199 was measured by qRT-PCR. (C-J) HCT116 and SW480 cells were transfected with or without circ_KIAA1199 overexpression vector or vector, and then treated with curcumin. Non-transfected and non-treated cells were used as control. (C) Colony formation assay was performed to detect the number of colonies. (D-E) MTT assay was used to measure the cell viability. (F-G) The numbers of migrated and invaded cells were analyzed by transwell assay. (H) The apoptotic rate of cells was examined using flow cytometry. (I-J) The protein levels of Bcl-2 and Bax were determined using WB analysis. *P < 0.05.

Figure 4 Circ_KIAA1199 targeted miR-433-3p. (A) The binding sites and corresponding mutate sites of circ_KIAA1199 and miR-433-3p were shown. (B-C) The interaction between circ_KIAA1199 and miR-433-3p was confirmed by dual-luciferase reporter assay. (D) The expression of miR-433-3p was measured by qRT-PCR in HCT116 and SW480 cells transfected with vector or circ_KIAA1199 overexpression vector. (E) QRT-PCR was used to detect miR-433-3p expression in HCT16 and SW480 cells treated with 20 μM curcumin. (F) QRT-PCR was employed to examine the expression of miR-433-3p in FHC cells and CRC cells (HCT116 and SW480). (G) MiR-433-3p expression in CRC tumor tissues (Tumor) and adjacent normal tissues (Normal) were measured by qRT-PCR. (H) The correlation between circ_KIAA1199 and miR-433-3p was analyzed using Pearson correlation analysis. *P < 0.05.

Figure 5 Curcumin regulated CRC progression by regulating circ_KIAA1199/miR-433-3p. (A) HCT16 and SW480 cells were transfected with vector, circ_KIAA1199, circ_KIAA1199 + miR-NC or circ_KIAA1199 + miR-433-3p. The expression of miR-433-3p was detected by qRT-PCR. (B-I) After transfection, HCT116 and SW480 cells were treated with curcumin. Non-transfected and non-treated cells were used as control. (B) The number of colonies was examined using colony formation assay. (C-D) MTT assay was employed to determine the cell viability. (E-F) Transwell assay was performed to test the numbers of migrated and invaded cells. (G) The apoptotic rate of cells was determined by flow cytometry. (H-I) WB analysis was utilized for examining the protein levels of Bcl-2 and Bax. *P < 0.05.

Figure 6 MiR-433-3p targeted ADAM10 in CRC. (A) The binding sites and corresponding mutate sites of miR-433-3p and ADAM10 3’UTR were shown. (B-C) Dual-luciferase reporter assay was utilized for assessing the interaction between miR-433-3p and ADAM10. (D) The transfection efficiencies of miR-433-3p mimic and inhibitor were confirmed by detecting miR-433-3p expression using qRT-PCR. (E-F) The mRNA and protein expression levels of ADAM10 were measured by qRT-PCR and WB analysis in HCT116 and SW480 cells transfected with miR-NC, miR-433-3p, anti-miR-NC or anti-miR-433-3p. (G-H) The mRNA and protein expression of ADAM10 in CRC tumor tissues (Tumor) and adjacent normal tissues (Normal) were determined using qRT-PCR and WB analysis. (I-J) Pearson correlation analysis was utilized for evaluating the correlation between ADAM10 mRNA expression and miR-433-3p or circ_KIAA1199 expression in CRC tumor tissues. (K-L) QRT-PCR and WB analysis were employed to examine the mRNA and protein expression of ADAM10 in FHC cells and CRC cells (HCT116 and SW480). (M-N) QRT-PCR and WB analysis were used to detect the mRNA and protein expression of ADAM10 in HCT16 and SW480 cells treated with 20 μM curcumin. (O-P) HCT116 and SW480 cells were transfected with vector, circ_KIAA1199, circ_KIAA1199 + miR-NC or circ_KIAA1199 + miR-433-3p. The mRNA and protein expression levels of ADAM10 were examined by qRT-PCR and WB analysis. *P < 0.05.

Figure 7 Curcumin inhibited CRC progression via the miR-433-3p/ADAM10 axis. (A-B) HCT16 and SW480 cells were transfected with anti-miR-NC, anti-miR-433-3p, anti-miR-433-3p + si-NC or anti-miR-433-3p + si-ADAM10. The mRNA and protein expression of ADAM10 was detected by qRT-PCR and WB analysis. (C-J) After transfection, HCT116 and SW480 cells were treated with curcumin. Non-transfected and non-treated cells were used as control. (C) Colony formation assay was employed to measure the number of colonies. (D-E) Cell viability was tested using MTT assay. (F-G) Transwell assay was utilized to assess the numbers of migrated and invaded cells. (H) Flow cytometry was used to detect the apoptotic rate of cells. (I-J) The protein levels of Bcl-2 and Bax were tested using WB analysis. *P < 0.05.

Figure 8 Curcumin reduced CRC tumor growth through regulating the circ_KIAA1199/miR-433-3p/ADAM10 axis in vivo. Tumor volume (A) and tumor weight (B) were detected in each group. (C-E) The expression levels of circ_KIAA1199, miR-433-3p and ADAM10 in the tumor tissues of each group were measured by qRT-PCR. (F) The ADAM10 protein level in the tumor tissues of each group was determined by WB analysis. (G) IHC staining was used to detect the Ki67 and Cleaved caspase3 positive cells to assess the proliferation and apoptosis of tumor cells. *P < 0.05.

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Curcumin played an anti-cancer role in colorectal cancer via mediating circ_KIAA1199-related regulatory axis

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[1] *Corresponding author: fhycp89@163.com