As the most up-regulated circRNA, hsa_circ_0000423 (termed as circPPP1R12A) was back-spliced of exons 24/25 of PPP1R12A gene located at 12q21
July 17, 2021
As the most up-regulated circRNA, hsa_circ_0000423 (termed as circPPP1R12A) was back-spliced of exons 24/25 of PPP1R12A gene located at 12q21.2 (Fig. considered as statistically significant. Results Expression profiles and screening of circRNAs in CC tissues and cells Firstly, circRNA microarray was employed to characterize the expression profiles of circRNAs in paired CC tissues and adjacent non-tumor tissues from 10 patients. A total of 126 circRNAs (P?0.05 and fold change >?1.5) were differentially expressed between the CC tissues and paired adjacent non-tumor tissues. Among the Pyrazinamide 126 differentially expressed circRNAs, 110 circRNAs were up-regulated, while 16 ones were down-regulated in CC tissues compared with the adjacent non-tumor tissues (Fig.?1 a). Additional?file?1: Table S1 lists the detailed information about these dysregulated circRNAs. These circRNAs were mostly located at exonic regions Rabbit Polyclonal to Cytochrome P450 7B1 (Fig. ?(Fig.11 b). As the most up-regulated circRNA, hsa_circ_0000423 (termed as circPPP1R12A) was back-spliced of exons 24/25 of PPP1R12A gene located at 12q21.2 (Fig. ?(Fig.11 c). Next, we re-examined the expression of circPPP1R12A in CC and paired non-tumor tissue samples from 20 patients by quantitative real-time PCR to confirm its elevated expression (Fig. ?(Fig.11 d). We further found that the circPPP1R12A expression was consistently and significantly increased in CC tissues compared with the matched controls, while the expression of PPP1R12A (linear transcript of PPP1R12A gene) was comparable in CC tissues and matched controls (Additional?file?2: Physique S1a). Moreover, the expression of circPPP1R12A was significantly up-regulated in a series of cultured CC cell lines (HT-29, HCT-116, SW480, SW620, LoVo, SW48, DLD-1, Caco2 and HCT-15) compared with a normal human colon mucosal epithelial cell line NCM460 cells. The highest expression of circPPP1R12A was found in HCT-116 cells, followed by LoVo cells (Fig. ?(Fig.11 e). Therefore, our subsequent experiments focused on the role of circPPP1R12A in CC progression. Open in a separate window Fig. 1 CircRNA expression profile in CC and characterization of circPPP1R12A. a Heatmap of the differentially expressed circRNAs in 10 pairs of human CC tissues and matched non-tumor tissues. b Classification of dysregulated circRNAs. c CircPPP1R12A was back-spliced by exons 24 and 25 of PPP1R12A gene located at 12q21.2. d The expression level of circPPP1R12A in CC and matched non-tumor tissue samples from 20 patients was analyzed by real-time PCR. e The expression level of circPPP1R12A in a series of cultured CC cell lines (HT-29, HCT-116, SW480, SW620, LoVo, SW48, DLD-1, Caco2 and HCT-15) was analyzed by real-time PCR. ***P?0.001 Characterization of the existence and subcellular distribution of circPPP1R12A in CC cells and tissues In the present study, we designed two sets of primers to characterize circPPP1R12A. One pair (divergent primers) was used to amplify the circular transcripts, while the other pair (convergent Pyrazinamide primers) was used to detect the linear transcripts. The results suggested that this circular form could be amplified using the convergent primers from both cDNA and gDNA, while it was only amplified from cDNA by divergent primers (Fig.?2 a). To further confirm the presence of circPPP1R12A, the RNase R degradation assay was used to evaluate the resistance of circPPP1R12A to RNase R treatment. Physique?2 b shows that the linear transcripts of PPP1R12A were degraded by RNase R treatment, while such treatment failed to degrade the circular transcripts of circPPP1R12A. Nuclear mass separation assay (Fig. ?(Fig.22 c) and FISH analysis (Fig. ?(Fig.22 d) reveled that over 93% of circPPP1R12A appeared in the cytoplasm of HCT-116 and LoVo cells. We also detected the expression of circPPP1R12A in CC tissues by ISH using TMA consisting of 100 pairs of CC and adjacent non-tumor tissues (Fig. ?(Fig.2e).2e). Table?1 lists the detailed clinical parameters of these patients. Among the clinicopathological variables, pathological stage and circPPP1R12A ISH score were identified as risk factors for predicting overall survival based on univariate analysis, while multivariate analysis with Cox regression model further confirmed that pathological stage III and circPPP1R12A ISH score 3C4 were the impartial poor prognostic factors (Table?2). KaplanCMeier survival curves showed that patients with higher expression of circPPP1R12A had a shorter overall survival [HR?=?1.886; 95% confidence interval (CI), 1.129C3.1529; P?=?0.0154; Fig. ?Fig.22 f]. Open in a separate window Fig. 2 Characterization the presence and subcellular distribution of circPPP1R12A in CC cells and tissues. a The Pyrazinamide divergent primers detected circPPP1R12A in cDNA but not.