Circular RNA hsa_circ_0006401 promotes proliferation and metastasis in colorectal carcinoma

Abstract

Dysregulation of circular RNA (circRNA) expression is involved in the progression of cancer. Here, we aimed to study the potential function of hsa_circ_0006401 in colorectal cancer (CRC). CircRNA hsa_circ_0006401 expression levels in CRC and adjacent nontumor tissues were analyzed by real-time quantitative PCR (qRT-PCR) and circRNA in situ hybridization (RNA-ISH). Then, CRC cell proliferation was assessed by cell counting. Wound-healing and transwell assays were utilized to detect the effect of hsa_circ_0006401 on CRC migration. A circRNA-ORF construct was created, and a specific antibody against the splice junction of hsa_circ_0006401 was prepared. Finally, the proteins directly binding to hsa_circ_0006401 peptides were identified by immunoprecipitation combined with mass spectrometry. In our study, we found hsa_circ_0006401 was closely related to CRC metastasis and exhibited upregulated expression in metastatic CRC tissue samples. Proliferation and migration were inhibited in vitro when hsa_circ_0006401 expression was silenced. Downregulation of hsa_circ_0006401 expression decreased CRC proliferation and liver metastasis in vivo. A 198-aa peptide was encoded by sequences of the splice junction absent from col6a3. Hsa_circ_0006401 promoted CRC proliferation and migration by encoding the hsa_circ_0006401 peptide. Hsa_circ_0006401 peptides decreased the mRNA and protein level of the host gene col6a3 by promoting col6a3 mRNA stabilation. In conclusion, our study revealed that circRNAs generated from col6a3 that contain an open-reading frame (ORF) encode a novel 198-aa functional peptide and hsa_circ_0006401 peptides promote stability of the host gene col6a3 mRNA to promote CRC proliferation and metastasis.

Fig. 1. Analysis of the expression level of hsa_circ_0006401 in CRC and corresponding normal tissue specimens.

A Schematic representation of the back splicing of hsa_circ_0006401. B Gene expression level of hsa_circ_0006401 in CRC patients in either the metastasis or control group. C Primers for hsa_circ_0006401. D ROC curve analysis of hsa_circ_0006401 in CRC patients. E Reprehensive images of hsa_circ_0006401 in metastatic and nonmetastatic CRC tissue samples. F Sequencing of PCR products with a splice junction. Error bars indicate SD, *P < 0.05.

Fig. 2. Hsa_circ_0006401 promoted the proliferation and migration of CRC cells in vitro.

A Representative images of the expression and location of hsa_circ_0006401 in SW480 and SW620 cells. B Silencing efficiency of two siRNAs in the CRC cell lines SW480 and SW620 by qRT-PCR. C Cell counts of CRC cells in the control group and hsa_circ_0006401 downregulation group. D Clone forming assay to detect the proliferative ability of CRC cells in the control group and hsa_circ_0006401 downregulation group. E Transwell assay to detect the migratory ability of CRC cells in the control group and hsa_circ_0006401 downregulation group. F Wound-healing assay to detect the migratory ability of CRC cells in the control group and hsa_circ_0006401 downregulation group. G Flow cytometry to assess cell apoptosis in the control group and hsa_circ_0006401 downregulation group. Error bars indicate SD, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. Hsa_circ_0006401 promoted the proliferation and metastasis of CRC in vivo.

A Cells were subcutaneously injected into nude mice. All injected mice formed tumors. Tumor were isolated, and tumor volume was measured. B Box plot of tumor volume in nude mice. n = 6. *p < 0.05. C Tumors and the surrounding tissues from mice in the control and hsa_circ_0006401-silenced groups were fixed and subjected to hematoxylin and eosin staining. D The number of liver metastases and the metastatic burden was determined and graphed. *p < 0.05. E Representative images of the livers of mice from different groups are shown. F Representative images of hematoxylin and eosin staining of the livers of mice from different groups are shown. Error bars indicate SD.

Fig. 4. Hsa_circ_0006401 encoded a novel peptide.

A Schematic representation of plasmid construction. B Detection of GFP fluorescence. The indicated constructs were transfected into HeLa cells for 24 h. C Western blot analysis with anti-hsa_circ_0006401 antibodies to evaluate proteins from SW620 cells transfected with different constructs. NC (negative control), p-Circ (hsa_circ_0006401 circRNA level was highly expressed), ORFmut (ATG start codon of p-Circ was mutated to TTG), siRNA (hsa_circ_0006401 circRNA level was silenced by siRNA). GAPDH was used as a loading control. D qRT-PCR analysis of hsa_circ_0006401 cirRNA expression level of different groups. E qRT-PCR analysis of hsa_circ_0006401 cirRNA expression level of different groups of SW480 cells. NC (negative control), col6a3 (col6a3 overexpression), p-Circ (hsa_circ_0006401 circRNA level was highly expressed), ORFmut (ATG start codon of p-Circ was mutated to TTG). F Western blot analysis with anti-hsa_circ_0006401 antibodies (HAPL0559) to evaluate proteins from SW480 cells transfected with different constructs. NC (negative control), col6a3 (col6a3 overexpression), p-Circ (hsa_circ_0006401 circRNA level was highly expressed), ORFmut (ATG start codon of p-Circ was mutated to TTG). G Reprehensive images of IHC analysis with an antibody (HAPM0617) to evaluate proteins from colon cancer tissues and normal colon tissues (left panel). IHC scores were calculated (right panel). Error bars indicate SD, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5. Function of the ORF in CRC.

A Cell counts of SW480 CRC cells transfected with different constructs. B Clone forming assay to detect the proliferative ability of SW480 CRC cells transfected with different constructs. C Transwell assay to detect the migratory ability of SW480 CRC cells transfected with different constructs. D Wound-healing assay to detect the migratory ability of SW480 CRC cells transfected with different constructs. E Flow cytometry assay to assess cell apoptosis in SW480 CRC cells transfected with different constructs. NC (negative control), p-Circ (hsa_circ_0006401 circRNA level was highly expressed), ORFmut (ATG start codon of p-Circ was mutated to TTG). Error bars indicate SD, *p < 0.05, ns represents no significance.

Fig. 6. Hsa_circ_0006401 peptides regulate COL6A3 mRNA expression.

A Gene set analysis of COL6A3 expression in normal tissue and colon cancer using TCGA cancer browser. The number of patients from each subtype is indicated below the box plot. B COL6A3 expression was assessed by Kaplan–Meier survival analysis for 5-year overall survival outcome in 270 colon cancer patients. C The correlation of hsa_circ_0006401 and COL6A3 mRNA in 12 human CRC tissues. *p < 0.05. D COL6A3 mRNA expression level in negative control SW620 cancer cells (NC) and hsa_circ_0006401 silencing SW620 cancer cells (siRNA). **p < 0.01. E COL6A3 protein expression level in SW620 cells transfected with different constructs. NC (negative control), p-Circ (hsa_circ_0006401 circRNA level was highly expressed), ORFmut (ATG start codon of p-Circ was mutated to TTG), siRNA (hsa_circ_0006401 circRNA level was silenced by siRNA). F TGFβ1 mRNA expression level in negative control SW620 cancer cells (NC) and hsa_circ_0006401 silencing SW620 cancer cells (siRNA). *p < 0.05. Error bars indicate SD.

Fig. 7. Hsa_circ_0006401 peptides promote COL6A3 mRNA stabilization.

A HA tag expression in negative control SW480 cancer cells (NC) and hsa_circ_0006401 ORF-198aa-HA tag plasmid transfected SW480 cancer cells (ORF-198aa-HA). B Representative images of the location of hsa_circ_0006401 peptide in SW480 cells. C Gene ontology (GO) analysis for hsa_circ_0006401 peptide combined proteins. BP represents biological processes, CC represents cellular components and MF represents molecular functions. D Decay of col6a3 and GAPDH mRNA was monitored in SW480 cells treated with Actinomycin D with indicated time points. NC (negative control), siRNA (hsa_circ_0006401 circRNA level was silenced by siRNA). Min represents minutes. Error bars indicate SD, and data were fitted by linear regression. *p < 0.05.