MicroRNA-421 Inhibits Apoptosis by Downregulating Caspase-3 in Human Colorectal Cancer

Abstract

Purpose: Dysregulated microRNAs (miRNAs/miRs) have been reported to play significant roles in pathogenesis of colorectal cancer (CRC). Previous studies have demonstrated that miR-421 regulates apoptosis in some cancers. Caspase-3 plays a key role in apoptosis, but the relationship between miR-421 and caspase-3 in CRC has not been determined. In this study, we investigated the role of miR-421 in CRC and the relationship between miR-421 and caspase-3.

Methods: Expression of miR-421 and caspase-3 were detected in human paired CRC cancer tissues and corresponding paracancerous tissues. In situ detection of tissue, apoptosis was performed via the TUNEL assay. HCT116 and SW480 cell lines were subjected to several in vitro experiments to explore the relationship between miRNA421 and caspase-3 during apoptosis using miR421 mimics/antagomir and luciferase reporter assay. Apoptosis was measured by determining the levels and activity of caspase-3 as well as DNA fragmentation. Luciferase reporter assay was performed to determine the potential interaction of miR-421 with caspase-3.

Results: The results showed that the expression of miR-421 in cancer tissues was higher than that in corresponding paracancerous tissues. Inhibition of miR-421 induced apoptosis, as shown by the upregulation of caspase-3 activity and expression as well as DNA fragmentation, which were attenuated by miR-421 mimic. We further showed that miR-421 targeted and inhibited CASP3 expression by targeting sites located in the 3'-untranslated region (3'-UTR) of CASP3 mRNA.

Conclusion: This study demonstrated an anti-apoptotic role of miR-421 in CRC and identified caspase-3 gene as a direct target of miR-421. These findings provide a potential treatment strategy using miR-421 as a therapeutic target for CRC.

Keywords: apoptosis; caspase-3; colorectal cancer; microRNA-421.

Figure 1

MiRNA-421 and caspase-3 may involved in inducing apoptosis of colorectal cancer. (A) MiR-421 expression in paired colorectal cancer and adjacent tissues (n=33, p=0.042). (B) Detection in situ of colorectal cancer tissue cell apoptosis by TUNEL assay. Magnification = ×100. (C) Bar graph of apoptosis index. (p=0.007) (D) Representative immunohistochemical data of caspase-3 expression in paired CRC cancer and the corresponding adjacent tissues. Magnification = 100×. *P<0.05 (p=0.042), **P < 0.01 (p=0.007).

Figure 2

MicroRNA-421 expression in HCT116 cells and SW480 cells. Total RNA was extracted from HCT116 and SW480 cells using TRIzol reagent. Expression of miR-421 was determined using the miRNA plate assay kit, which was normalized to U6 snRNA as the internal control. (A) and (B) miR-421 expression in HCT116 cells. (p=0.027; p=0.034). (C) and (D) miR-421 expression in SW480 cells. A1, B1: HCT116 cells; A2, B2: HCT116 cells with control miRNA; A3: HCT116 cells with miR-421 mimics; B3: HCT116 cells with miR-421 antagomir; C1, D1: SW480 cells; C2, D2: SW480 cells with control miRNA; C3: SW480 cells with miR-421 mimics; D3: SW480 cells with miR-421 antagomir. *P < 0.05 vs control miRNA. (C: p=0.029; D: p=0.044).

Figure 3

The effect of miR-421 on apoptosis of colorectal cancer (CRC) cells. (A) and (B) Caspase-3 activity in HCT116 cells. (C) and (D) Caspase-3 activity in SW480 cells. (E) and (F) Caspase-3 protein level in HCT116 cells. (G) and (H) Caspase-3 protein level in SW480 cells. (I) and (J) DNA fragments in HCT116 cells. (K) and (L) DNA fragments in SW480 cells. A1, B1, E1, F1, I1, J1: HCT116 cells; A2, B2, E2, F2, I2, J2: HCT116 cells with control miRNA; A3, E3, I3: HCT116 cells with miR-421 mimics; B3, F3, J3: HCT116 cells with miR-421 antagomir; C1, D1, G1, H1, K1, L1:SW480 cells; C2, D2, G2, K2, L2: SW480 cells with control miRNA; C3, G3, K3: SW480 cells with miR-421 mimics; D3, H3, L3: SW480 cells with miR-421 antagomir. *P < 0.05 vs control miRNA. (A: p=0.025; B: p=0.021; C: p=0.022; D: p=0.035; E: p=0.016; F: p=0.019; G: p=0.013; H: p=0.030; I: p=0.028; J: p=0.029; K: p=0.026; L: p=0.031).

Figure 4

MicroRNA-421 targeted and inhibited caspase-3 expression. A dual-luciferase activity assay was performed. (A) A segment of caspase-3 3ʹ-UTR was inserted downstream of the luciferase-coding sequence. Sequence alignment of miR-421 and the 3ʹ-UTR of caspase-3 showed complementarity with the 5ʹ end of miR-421. (B) Sequence alignment of miR-421 and the mutated 3ʹ-UTR of caspase-3 of showed no complementarity with the 5ʹend of miR-421. (C) HCT116 cells were co-transfected with the plasmid containing the wild-type 3ʹ-UTR of caspase-3 (wt-Luc-Caspase-3) or the mutated 3ʹ-UTR of caspase-3 (mu-Luc-Caspase-3) and miR-421 mimic or a scrambled oligonucleotide as a control. (D) HCT116 cells were transfected with either wt-Luc-Caspase-3 or mu-Luc-Caspase-3, and then incubated with the miR-421 antagomir. (E) The protein levels of caspase-3 in miR-421 or miR-421 antagomir-transfected HCT116 cells. *P < 0.05 vs control miRNA. ^#P < 0.05 vs control miRNA and miRNA mimic (p=0.027).