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. 2020 Feb 20:12:1281-1292.
doi: 10.2147/CMAR.S234620. eCollection 2020.

Long Non-Coding RNA Cancer Susceptibility 9 (CASC9) Up-Regulates the Expression of ERBB2 by Inhibiting miR-193a-5p in Colorectal Cancer

Yuansheng Ding  1 , Xiaoyan Li  1 , Yucui Zhang  1 , Jie Zhang  1
Affiliations

Long Non-Coding RNA Cancer Susceptibility 9 (CASC9) Up-Regulates the Expression of ERBB2 by Inhibiting miR-193a-5p in Colorectal Cancer

Yuansheng Ding et al. Cancer Manag Res. .

Retraction in

Abstract

Background: Emerging studies have reported that long non-coding RNAs (lncRNAs) were crucial regulators in the progression of colorectal cancer (CRC). LncRNA susceptibility 9 (CASC9) was involved in several cancers; however, its role in CRC remains unknown.

Methods: RT-PCR was done to probe the expression of CASC9 and miR-193a-5p in CRC samples. CRC cell lines (HCT116 and SW480) were used as cell models. The biological influence of CASC9 on cancer cells was studied using CCK-8 assay, Transwell assay and TUNEL assay in vitro, and subcutaneous xenotransplanted tumor model in vivo. Interaction between CASC9 and miR-193a-5p was investigated by bioinformatics analysis, RT-PCR, and luciferase reporter assay. The expression level of the downstream gene of miR-193a-5p, erb-b2 receptor tyrosine kinase 2 (ERBB2), was tested by Western blot.

Results: CASC9 was significantly up-regulated in CRC samples, while miR-193a-5p was markedly down-regulated. Overexpression of CASC9 promoted viability, migration and invasion of CRC cells, while overexpression of miR-193a-5p had the opposite effect. CASC9 could down-regulate miR-193a-5p via sponging it, and there was a negative relevancy between CASC9 and miR-193a-5p in CRC samples. CASC9 also enhanced the expression levels of ERBB2, while this effect could be reversed by co-transfection with miR-193a-5p.

Conclusion: CASC9, an oncogenic lncRNA, was abnormally up-regulated in CRC tissues, and it could indirectly modulate the expression of ERBB2 via reducing the expression level of miR-193a-5p.

Keywords: CASC9; ERBB2; colorectal cancer; erb-b2 receptor tyrosine kinase 2; long non coding RNA cancer susceptibility 9; microRNA-193a-5p.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
The expression levels of CASC9 and miR-193a-5p in CRC tissues and cell lines. (A, B) qRT-PCR was applied to detect the expression of CASC9 and miR-193a-5p in CRC tissues and adjacent normal tissues. (C) In CRC tissues, the expression of CASC9 and miR-193a-5p was negatively correlated. (D, E) qRT-PCR was performed to detect the expression of CASC9 and miR-193a-5p in CRC cell lines. **, ***Represent P < 0.01 and <0.001, respectively.
Figure 2
Figure 2
The effect of CASC9 on CRC cells. (A) HCT116 cells with high expression of CASC9 and SW480 cells with low expression were successfully constructed. (B, C) CCK-8 assay was used to detect the effect of CASC9 on the proliferation of CRC cells. (D, E) The effect of CASC9 on the migration and invasion of CRC cells was detected by transwell assay. (F)The effect of CASC9 on apoptosis of CRC cells was detected by TUNEL assay. *, **, ***Represent P < 0.05, P< 0.001, <0.001, respectively.
Figure 3
Figure 3
Effect of miR-193a-5p on CRC cells. (A) HCT116 cells with high expression of miR-193a-5p and SW480 cells with miR-193a-5p inhibited were successfully constructed. (B, C) CCK-8 assay was used to detect the effect of miR-193a-5p on the proliferation of CRC cells. (D, E) The effect of miR-193a-5p overexpression or inhibition on the migration and invasion of CRC cells was detected by transwell assay. (F) The effect of miR-193a-5p on apoptosis of CRC cells was detected by TUNEL assay. *, **, ***Represent P < 0.05, P< 0.001, <0.001, respectively.
Figure 4
Figure 4
The interaction between CASC9 and miR-193a-5p. (A) The binding site between CASC9 and miR-193a-5p was predicted by DIANA database. (B, C) The binding between CASC9 and miR-193a-5p was verified by dual-luciferase reporter assay. (C) The effect of over-expression or knockdown of CASC9 on the expression of miR-193a-5p was detected by qRT-PCR. **, ***Represent P < 0.01 and <0.001, respectively.
Figure 5
Figure 5
The function of CASC9/miR-193a-5p axis on the malignant phenotypes of HCT116 cells. (A) CCK-8 assay was used to detect the proliferation of HCT116 cells. (B, C) The migration and invasion of HCT116 cells were evaluated by Transwell assay. (D) The effect of miR-193a-5p on the inhibition of CRC cell apoptosis by CASC9 was detected by TUNEL. In Figure (A), ** represents NC group compared with pcDNA3.1-CASC9 group (P < 0.01). & represents that the pcDNA3.1-CASC9 group compared with the pcDNA3.1-CASC9 + microRNA-193a-5p mimics group (P < 0.05). In Figure (A–D), *, **, ***Represent P < 0.05, P< 0.001, <0.001, respectively.
Figure 6
Figure 6
Interaction between miR-193a-5p and ERBB2. (A) Binding site between miR-193a-5p and 3ʹUTR of ERBB2 was predicted through TargetScan database. (B) The binding of miR-193a-5p to the 3ʹUTR of ERBB2 was validated by dual-luciferase reporter assay. (C, D) The effects of transfection of miR-193a-5p mimics or miR-193a-5p inhibitors on ERBB2 mRNA and its protein expression were detected by qRT-PCR and Western blot, respectively. (E, F) The influence of transfected miR-193a-5p mimics on the promotion of ERBB2 mRNA and protein expression by CASC9 was detected by qRT-PCR and Western blot, respectively. **, ***Represent P < 0.01 and < 0.001, respectively.
Figure 7
Figure 7
The effect of CASC9 on the growth of xenograft tumors in mice. (A, B) The effect of CASC9 overexpression on the size and weight of tumors. (C, D) The expression of CASC9 and miR-193a-5p in xenograft tumors was detected by qRT-PCR. (E, F) The expression of ERBB2 mRNA and its protein in xenograft tumors was detected by qRT-PCR and Western blot. **, ***Represent P < 0.01 and <0.001, respectively.
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