Long non-coding RNA OIP5-AS1 promotes the progression of esophageal cancer by regulating miR-30a/VOPP1 expression

Abstract

Long non-coding RNAs (lncRNAs) serve an important role in the development of esophageal cancer (EC), which is the eighth most common type of cancer worldwide. lncRNA opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) is associated with human malignancy. However, the biological roles of OIP5-AS1 in the development of EC remain unclear. In the present study, transfection was conducted, and reverse transcription-quantitative PCR and western blot analysis were used for the detection of mRNA and protein expression, respectively. Furthermore, dual-luciferase reporter and RNA immunoprecipitation assays were used to study the interaction between miRNA and lncRNA or genes. The results revealed that OIP5-AS1 expression in EC tissues and cultured EC cells was upregulated, microRNA-30a (miR-30a) expression was downregulated. OIP5-AS1-knockdown suppressed the proliferation, migration and invasion of EC9706 and EC109 cells. miR-30a was confirmed to interact with OIP5-AS1, and miR-30a-mimics transfection ameliorated the effects of OIP5-AS1 in EC cells. Vesicular overexpressed in cancer prosurvival protein 1 (VOPP1) was verified as the direct target of miR-30a. VOPP1 expression was positively correlated with OIP5-AS1 expression in EC cells. Overexpression of VOPP1 ameliorated the negative effects of OIP5-AS1-knockdown on EC9706 and EC109 cells. In conclusion, OIP5-AS1 promoted the proliferation, migration and invasion of EC cells by increasing VOPP1 expression by sponging miR-30a.

Keywords: esophageal cancer; invasion; microRNA-30a; migration; opa-interacting protein 5 antisense transcript 1; vesicular overexpressed in cancer prosurvival protein 1.

Figure 1.

OIP5-AS1/miR-30a/VOPP1 expression in EC tissues and cells. Relative expression levels of (A) OIP5-AS1, (B) VOPP1 and (C) miR-30a in EC tissues (n=32) and adjacent normal tissues (n=32) were detected by RT-qPCR. Pearson's correlation analysis between OIP5-AS1 and (D) VOPP1 or (E) miR-30a expression in EC tissues was conducted. (F) OIP5-AS1 expression in human EC cell lines (EC109, EC9706 and TE-1) and human immortalized normal esophageal epithelial cells (Het-1A) cells was determined by RT-qPCR. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. EC, esophageal cancer; RT-qPCR, reverse transcription-quantitative PCR; OIP5-AS1, opa-interacting protein 5 antisense transcript 1; miR-30a, microRNA-30a; VOPP1, vesicular overexpressed in cancer prosurvival protein 1.

Figure 2.

Knockdown of OIP5-AS1 expression decreases the proliferation, migration and invasion of EC9706 and EC109 cells. (A) Efficiency of sh-OIP5-AS1-knockdown was determined by reverse transcription-quantitative PCR. MTT assays were employed to detect cell viability in (B) EC9706 and (C) EC109 cells. Transwell assays were used to determine the (D and E) migration and (F and G) invasion in sh-OIP5-AS1-transfected EC9706 and EC109 cells; magnification, ×400. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. OD, optical density; OIP5-AS1, opa-interacting protein 5 antisense transcript 1; sh, short hairpin RNA; NC, negative control.

Figure 3.

miR-30a is a target of OIP5-AS1. (A) Potential interaction between OIP5-AS1 and miR-30a was predicted by Starbase2.0 software. Relative luciferase activity was detected in (B) EC9706 and (C) EC109 cells co-transfected with both WT/MUT-OIP5-AS1 and miR-30a mimics/miR-NC. Firefly luciferase reporter activity was normalized to Renilla luciferase activity. Interaction between OIP5-AS1 and miR-30a was confirmed by RNA immunoprecipitation assays in (D) EC9706 and (E) EC109 cells. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. **P<0.01. OIP5-AS1, opa-interacting protein 5 antisense transcript 1; miR-30a, microRNA-30a; NC, negative control; WT, wild-type; MUT, mutant; Ago2, argonaute 2.

Figure 4.

miR-30a overexpression ameliorates the migration and invasiveness of esophageal cancer cells. (A) miR-30a expression in sh-OIP5-AS1-transfected EC9706 and EC109 cells. (B) OIP5-AS1 expression in miR-30a mimics-transfected EC9706 and EC109 cells. (C) miR-30a expression in miR-30a mimics-transfected EC9706 and EC109 cells was detected by reverse transcription-quantitative PCR. Transwell assays were used to determine the (D and E) migration and (F and G) invasion in miR-30a mimics-transfected EC9706 and EC109 cells; magnification, ×400. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. OIP5-AS1, opa-interacting protein 5 antisense transcript 1; sh, short hairpin RNA; NC, negative control; miR-30a, microRNA-30a.

Figure 5.

VOPP1 is a direct target of miR-30a. (A) Potential interaction between miR-30a and VOPP1 was predicted by TargetScan7.2 software. Relative luciferase activity was detected in (B) EC9706 and (C) EC109 cells co-transfected with both WT/MUT-VOPP1 and miR-30a mimics/miR-NC. (D) VOPP1 mRNA expression was detected in miR-30a/miR-NC-transfected EC9706 and EC109 cells. (E) VOPP1 protein expression was detected in miR-30a/miR-NC-transfected EC9706 and EC109 cells. (F) The fold-changes of VOPP1 protein expression were indicated. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. **P<0.01. miR-30a, microRNA-30a; NC, negative control; WT, wild-type; MUT, mutant; VOPP1, vesicular overexpressed in cancer prosurvival protein 1.

Figure 6.

Overexpression of VOPP1 in EC cells. (A) VOPP1 mRNA expression was detected in EC cells. (B and C) VOPP1 protein expression was determined in EC cells. (D) VOPP1 mRNA expression was detected in NC, sh-OIP5-AS1-, sh-OIP5-AS1+pcDNA3.1- and sh-OIP5-AS1+pcDNA3.1-VOPP1-transfected EC cells. (E and F) VOPP1 protein expression was determined. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. EC, esophageal cancer; OIP5-AS1, opa-interacting protein 5 antisense transcript 1; sh, short hairpin RNA; NC, negative control; VOPP1, vesicular overexpressed in cancer prosurvival protein 1.

Figure 7.

VOPP1 overexpression improves the negative effects of OIP5-AS1-knockdown on EC cells. Proliferative activity was detected by MTT in sh-OIP5-AS1- and sh-OIP5-AS1+pcDNA3.1-VOPP1-transfected (A) EC9706 and (B) EC109 cells. (C and D) Migration (magnification, ×200) and (E and F) invasion (magnification, ×400) were determined in sh-OIP5-AS1- and sh-OIP5-AS1+pcDNA3.1-VOPP1-transfected EC cells. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. OD, optical density; EC, esophageal cancer; OIP5-AS1, opa-interacting protein 5 antisense transcript 1; sh, short hairpin RNA; NC, negative control; VOPP1, vesicular overexpressed in cancer prosurvival protein 1.