CASC2c as an unfavorable prognosis factor interacts with miR-101 to mediate astrocytoma tumorigenesis

Abstract

miR-101 has been suggested as a tumor suppressor, but the promoter methylation and loss of heterozygosity didn't contribute to its low expression in astrocytoma. We investigated the role of a new long non-coding RNA CASC2c binding with miR-101. High CASC2c was positively correlated with astrocytoma progression, and an unfavorable prognosis factor for patients. Knockdown CASC2c inhibited proliferation and tumorgenesis. Overexpression of CASC2c promotes the malignant characteristic of astrocytoma cells.CASC2c directly bound miR-101 and mediated pre-miR-101 processing into mature miR-101, and functions as a competitor of miR-101 target genes such as CPEB1. Patients who possessed both low CASC2c and high miR-101 had a longer survival than those of low CASC2c alone or high miR-101 alone. In summary, CASC2c plays the onco-RNA role in the tumorgenesis of astrocytoma by acting as a decoy miR-101 sponge. Combination of low expression of CASC2c and high expression of miR-101 has an important referential significance to evaluate the prognosis of patients.

Figure 1

CASC2c is correlated with tumorigenicity and progression in astrocytoma. (a) Left, the expression of CASC2c in normal brain tissues and astrocytoma was determined by real-time qPCR. Data presented as mean±S.E.M. of three independent experiments; *P<0.05. Right, the level of CASC2c in different status of pathology classification was measured by real-time qPCR. Data presented as mean±S.E.M. of three independent experiments; ***P<0.001. (b) CASC2c expression level was evaluated using real-time qPCR in nc/si-CASC2c-transfected U251 and U87 cells. Data presented as mean±S.E.M. of three independent experiments; *P<0.05, **P<0.01, ***P<0.001. (c) CCK8 assay was performed to determine the viability of U251 and U87 cells that transfected nc/si-CASC2c-2.Data shown are the mean ±S.E.M. of three independent experiments; *P<0.05, **P<0.01. (d) EDU assay was applied to assess cell proliferation of U251 and U87 cells that transfected nc/si-CASC2c-2. Data shown are the mean±S.E.M. of three independent experiments; red scale bars, 200 μm; **P<0.01. (e) Wound healing assay measured cell migration of U251 and U87 cells that transfected nc/si-CASC2c-2. Data shown are the mean±S.E.M. of three independent experiments; red scale bars, 200 μm; **P<0.01. (f) Transwell assay and matrigel-coated transwell assay were performed in U251 and U87 cells that transfected si-CASC2c-2. Data shown are the mean±S.E.M. of three independent experiments; red scale bars, 50 μm; *P<0.05, **P<0.01. (g) H&E staining of shCAS2c-induced tumors in the coronal section of rats. **P<0.01. (h) The expression of CASC2c and miR-101 in intracranial-transplanted tumors were detected by in situ hybridization, the expression of Ki-67 in intracranial-transplanted tumors were detected by immunohistochemical staining. Scale bars, 200 μm

Figure 2

CASC2c and miR-101 existed in RISC complex simultaneously and formed a reciprocal repression loop between miR-101 and CASC2c. (a) Alignment of potential CASC2c base pairing with miR-101 was identified by using DIANA LAB (http://diana.cslab.ece.ntua.gr/microT/). (b) Relative fluorescence activity in U251 cells co-transfected with pMIR-REPORT-WT/mutant CASC2c and miR-101 or the negative control. Data presented as mean±S.E.M. of three independent experiments; *P<0.05. (c) Expression of CASC2c was assayed by real-time qPCR in miR-101 mimics/miR-101 inhibitor-transfected U251 and U87 cells. Data presented as mean±S.E.M. of three independent experiments; *P<0.05, **P<0.01. (d and e) Association of CASC2c and miR-101 with Ago2 in U251 cells. CASC2c and miR-101 expression levels were detected using real-time qPCR. Data presented as mean±S.E.M. of three independent experiments; **P<0.01, ***P<0.001. (f) Relative fluorescence activity in U251 cells co-transfected with the pMIR-miR-101 wild-type reporter plasmid (or the corresponding pMIR-miR-101mutant reporter) and shCASC2c/pcDNA3.1-CASC2c plasmid or the negative control. Data presented as mean±S.E.M. of three independent experiments; **P<0.01. (g) The expression of pri-miR-101, pre-miR-101-1 and mature miR-101 was measured by real-time qPCR in NC- or shCASC2c-transfected U251 cells. Data presented as mean±S.E.M. of three independent experiments; *P<0.05. (h) The expression of miR-101 was analyzed through real-time qPCR in pFRT plasmid, pFRT-Dicer or pFRT-Dicer-transfected U251 cells. Data presented as mean±S.E.M. of three independent experiments; *P<0.05, **P<0.01. (i) The expression of Dicer was measured by western blot in pFRT plasmid, pFRT-Dicer or pFRT-Dicer-transfected U251 cells. (j) The expression of Dicer and TRBP was evaluated using real-time qPCR in control/si-CASC2c-transfected U251 cells. Data presented as mean±S.E.M. of three independent experiments; *P<0.05. (k) The expression of Dicer and TRBP was evaluated using western blot in control/si-CASC2c-transfected U251 cells

Figure 3

CASC2c competes to combine miR-101 by repelling CPEB1. (a or b) The expression of miR-101 was analyzed through real-time qPCR (a) and western blot (b) in si-CASC2c, miRNA-101 inhibitor or miRNA-101 inhibitor-transfected U251 cells. Data presented as mean±S.E.M. of three independent experiments; *P<0.05, **P<0.01. (c) Upper, nucleotide resolution of miRNA-binding sites in CASC2c and CPEB1. Below, relative fluorescence activity in U251 cells co-transfected with pMIR-REPORT-WT/mutant 3′-UTR CPEB1 and miR-101 inhibitor or negative control, pMIR-REPORT-WT/mutant 3′-UTR CPEB1, si-CASC2c and miR-101. Data presented as mean±S.E.M. of three independent experiments; *P<0.05, **P<0.01. (d) RNA fluorescence in situ hybridization showing the localization of CASC2c, the nucleus is counterstained with DAPI. Scale bar, 29μm. (e) Relative fluorescence activity in U251 cells co-transfected with RLuc-CPEB1-wt or RLuc-CPEB1-mut and miR-101 ncor miR-101 mimics. Data presented as mean ±S.E.M. of three independent experiments; *P<0.05, **P<0.01. (f) Relative fluorescence activity in U251 cells co-transfected with RLuc-CPEB1-wt or RLuc-CPEB1-mut and shCASC2c or shCASC2c-△3 plasmid. Data presented as mean±S.E.M. of three independent experiments; *P<0.05, **P<0.01. (g) The expression of CPEB1 was analyzed through western blot in U251 cells transfected with different combinations of shCASC2c and miR-101 mimics. (+) corresponds to 1.5 mg shCASC2c and to 40 ng of miR-101 mimics, whereas (++) corresponds to 200 ng of miR-101 mimics. Data presented as mean±S.E.M. of three independent experiments; *P<0.05. (h) The expression of CASC2c and CPEB1 was detected by real-time qPCR and western blot in U251 cells after adding variant amount of CASC2c into miR-101-CPEB1 reaction mixture. Data presented as mean±S.E.M. of three independent experiments. (i) The expression of CASC2c and CPEB1 was detected by real-time qPCR and western blot in U251 cells after adding variant amount of the fragment of CASC2c that containing miR-101 target sequences into miR-101-CPEB1 reaction mixture. Data presented as mean±S.E.M. of three independent experiments

Figure 4

Association between miR-101 and CASC2c expression in astrocytoma clinical samples. (a and b) The expression level of CASC2c and miR-101 was detected by in situ hybridization. Black scale bars, 200 μm; red scale bars, 20 μm. (c) ISH score breakdown for a panel of normal tissues and astrocytoma. Each patient sample was scored from triplicate representative tumor cores, and the average CASC2c ISH score was recorded as low (score⩽8) or high (score>8). (d) The correlation between CASC2c expression and miR-101 levels were analyzed using Spearman's rank test. (e) Kaplan–Meier analysis for overall survival in 80 astrocytomas in high- and low-risk groups based on CASC2c expression levels. (f) Kaplan–Meier analysis for overall survival in 80 astrocytomas in high- and low-risk groups based on CASC2c and miR-101 expression levels