The Diagnostic And Therapeutic Value Of Hsa_circ₀016866 In Colorectal Cancer

BackgroundColorectal cancer（CRC）is one of the most common malignancies,which ranks the third incidence and the second mortality worldwide.The risk factors of CRC include heredity,eating habits,obesity and smoking.At present,the clinical treatment of CRC is a combined treatment mode including surgery,radiotherapy and chemotherapy.Despite the advances in surgical techniques,chemotherapy and radiotherapy in the past few decades,the prognosis for CRC remains poor.According to statistics,a total of 880,000 people died of CRC in 2018.Tumor recurrence and metastasis are the leading causes of death in CRC patients.Therefore,it is crucial to explore a new way for diagnose and treatmentThe development of CRC is a multi-step and multi-pathway biological process with gene mutation,and the dysregulation of cell proliferation,differentiation and apoptosis.Circular RNA（circRNA）is a circular non-coding RNA that widely expressed in various cells.Unlike general linear RNA molecules,circRNA do not have a 5’ and 3’ polar structure,but a closed covalent structure.Recent studies have found that circRNA is involved in regulating tumor-related genes and protein expression,and is closely related to the development of CRC.However,the details of circRNA in the development of CRC is still obscure.circRNA has the characteristics of structural stability,high expression level,and tissue-specific and stage-specific expression,making it a promising tumor marker for the diagnosis of CRC.The study of circRNA can not only understand the pathogenesis of CRC,but also have important significance in the diagnosis and treatment of CRC.PurposeGene chip analysis of CRC and adjacent tissues was performed to screen differentially expressed circRNAs.The selected circRNA was validated in CRC specimens,and their diagnostic and therapeutic roles in CRC were further explored.Methods1.circRNA expression profiling of CRC tissuesSix pairs of CRC tissues and their adjacent normal mucosa tissues were collected,and the expression profile of circRNA was detected by gene chip sequencing,which confirmed abnormally expressed circRNAs in CRC.Hsa_circ₀016866,which has obviously differential expression,was selected as the target molecule for further study.2.Expression level of hsa circ₀016866 in CRC cell linesThe expression of hsa_circ₀016866 was detected by quantitative real-time polymerase chain reaction（qRT-PCR）in three CRC cell lines DLD1,SW480 and HCT8,and one human intestinal epithelial cell line HCO.3.Expression level of hsa_circ₀016866 in CRC tissues and serumThe expression level of hsa_circ₀016866 was detected by qRT-PCR in CRC tissues and adjacent normal mucosa tissues.The expression level of hsa_circ₀016866 was detected in the serum of CRC patients and the healthy people.We also compared the expression level of hsa_circ₀016866 in the same patients between preoperative serum and postoperative serum.4.Functional study of hsa_circ₀016866 with CRC cellsCRC cell lines with high hsa_circ₀016866 expression was selected by qRT-PCR for cell function experiments.Specifically designed small interfering RNA（siRNA）was transfected into CRC cell lines to knockdown the expression level of hsa_circ₀016866.The cell counting kit-8（CCK-8）assay and cell clone formation assay were used to detect the proliferative capacity of CRC cells transfected with si-circRNA.The transwell assay was used to detect the capacity of migration and invasion of cells with si-circRNA.Flow cytometry was used to detected the cell cycle and apoptosis of cells with si-circRNA.5.The value of hsa_circ₀016866 expression level in CRC as a biomarkerWe analyzed the correlation between the expression level of Hsa_circ₀016866 and clinicopathological parameters in patients with CRC.The expression level of hsa_circ₀016866 and the prognosis of patients with CRC were analyzed by survival analysis.Finally,the value of hsa_circ₀016866 as a biomarker for the diagnosis of CRC was evaluated by receiver operating characteristic curve（ROC）.Results1.circRNA expression profiles in CRC tissuesAnalysis of 6 pairs of CRC tissues and adjacent normal mucosa showed that a total of 67 circRNAs were differentially expressed in CRC tissues,among which 16 were upregulated and 51 were downregulated.Hsa_circ₀016866 was selected for further study.2.Hsa_circ₀016866 was upregulated in CRC cellsThe expression of hsa_circ₀016866 in CRC cells SW480 and HCT8 was significantly higher than that in human intestinal epithelial cells.3.Hsa_circ₀016866 was upregulated in CRC tissues and serumThe expression level of circ0016866 in CRC tissues was significantly higher than that in adjacent normal mucosa.Hsa_circ₀016866 was significantly upregulated in serum of CRC patients.The expression level of Hsa_circ₀016866 in post-operative serum was significantly lower than that in pre-operative serum.4.Hsa_circ₀016866 promotes CRC cells proliferation,invasion and migrationThe three cell lines were transfected with siRNA,respectively.It was found that si-hsa_circ₀016866-1 could significantly knockdown the expression level the hsa_circ₀016866 in SW480 and DLD1 cells.Therefore,si-hsa_circ₀016866-1,SW480 and DLD1 cells were used for further cell function assays.CCK-8 cell proliferation experiment and cell clone formation experiment showed that hsa_circ₀016866 expression level was positively correlated with colorectal cancer cell proliferation ability.Trans well assay showed that knockdown of hsa_circ₀016866 could inhibit the migration and invasion of CRC cells.Cell cycle and apoptosis assay showed that knockdown of hsa_circ₀016866 could induce CRC cells G0/G1 phase arrest.However,knockdown of hsa_circ₀016866 had no effect on apoptosis.5.Hsa_circ₀016866 could be used as a biomarker for CRCStatistical analysis showed that the expression level of hsa_circ₀016866 was related to distant metastasis,TNM stage and perineural invasion.Univariate and multivariate analysis showed that the level of expression of hsa_circ₀016866 was an independent prognostic factor for CRC.The ROC curve showed that the area under the curve（Area Under Curve,AUC）of hsa_circ₀016866 was 0.675,which has promising value in the diagnosis of CRC.ConclusionHsa_circ₀016866 could be used as a biomarker for CRC.The expression of hsa_circ₀016866 was upregulated in CRC tissues,serum and cells,and was correlated with poor prognosis.Knockdown of hsa_circ₀016866 could significantly inhibit the proliferation,invasion and migration ability of CRC cells,suggesting that hsa_circ₀016866 promoted CRC cells proliferation,invasion and migration.This study provided the basis for the diagnosis and treatment of CRC.