Long noncoding RNA HCP5 suppresses skin cutaneous melanoma development by regulating RARRES3 gene expression via sponging miR-12

Abstract

Objective: This research aimed to investigate the role and mechanism of long noncoding RNA (lncRNA) HCP5 in skin cutaneous melanoma (SKCM).

Materials and methods: Survival analysis was performed using The Cancer Genome Atlas (TCGA)-SKCM data and SKCM patients' clinical data. Primary SKCM cells were derived from patients' pathologic tissue specimens. HCP5 overexpression was achieved by lentiviral transduction. Malignancy of SKCM cells was evaluated in vitro by cell proliferation, colony formation, apoptosis and transwell invasion assays. RARRES3 knockdown was achieved by siRNA transfection. DIANA microT-CDS algorithm was used to predict miRNAs that might interact with HCP5 and 3' untranslated region of RARRES3 mRNA. microRNA target luciferase reporter assay and AGO2-RNA immunoprecipitation were used to verify the interaction between HCP5, 3' UTR of RARRES3 mRNA and miR-1286.

Results: HCP5 level was decreased in SKCM tissue specimens compared to noncancerous counterparts. Low expression of HCP5 was associated with SKCM patients' poor overall survival and disease progression. HCP5 overexpression significantly reduced the malignancy of primary SKCM cells in vitro. RARRES3 was found as a HCP5-co-expressing gene in SKCM cells. HCP5 overexpression significantly increased RARRES3 expression in SKCM cells. RARRES3 knockdown partially abolished the anti-SKCM effect of HCP5 overexpression. MiR-1286 was found interacting with both HCP5 and 3' UTR of RARRES3 mRNA.

Conclusion: HCP5 is a cancer-suppressive lncRNA in SKCM. HCP5 overexpression decreased SKCM cell malignancy in vitro by upregulating RARRES3, possibly via sponging miR-1286.

Keywords: HCP5; RARRES3; long noncoding RNA; miR-1286; skin cutaneous melanoma.

Figure 1

Clinical significance of HCP5 expression in SKCM. (A) Survival analysis of HCP5 using TCGA-SKCM data. (B) HCP5 levels in 54 pairs of SKCM tissue specimens and normal skin tissue specimens were compared by qRT-PCR. Each dot represents one tissue specimen. (C) Survival analysis of HCP5 using the 54 SKCM patients’ clinical data. *p<0.05. Abbreviation: TCGA-SKCM, The Cancer Genome Atlas-skin cutaneous melanoma.

Figure 2

HCP5 overexpression inhibited primary SKCM cell proliferation, survival, clonogenicity and invasiveness in vitro. SKCM cells were transduced with empty (EV) or HCP5 (overexpressing; OE) lentiviral vectors for 24 hrs before assay. (A) HCP5 levels in SKCM cells with or without HCP5 overexpression were compared by qRT-PCR. (B) SKCM cell proliferation was assessed by cell proliferation assay. (C and D) Clonogenicity of SKCM cells was assessed by colony formation assay. (E and F) Survival of SKCM cells was assessed by apoptosis assay. Cell apoptosis was induced by serum deprivation for 8 hrs. G and H, invasiveness of SKCM cells was assessed by transwell invasion assays. Bar indicates 100 μM. *p<0.05. Abbreviations: SKCM, skin cutaneous melanoma; NC, normal control.

Figure 3

RARRES3 mRNA expression correlated with that of HCP5 in SKCM. (A) RARRES3 mRNA levels in 54 pairs of SKCM tissue specimens and normal skin tissue specimens were compared by qRT-PCR. (B) Correlation analysis of RARRES3 mRNA and HCP5 expression levels. Each dot represents one tissue specimen. *p<0.05. Abbreviation: SKCM, skin cutaneous melanoma.

Figure 4

HCP5 overexpression reduced SKCM cell malignancy through RARRES3. After transducing with HCP5 overexpressing lentiviral vector (HCP5 OE), SKCM cells were transfected with siRNA for RARRES3 knockdown (si-RARRES3) for 24 hrs. (A and B) Upregulation of RARRES3 mRNA and protein level by HCP5 overexpression was abolished by RARRES3 knockdown. (C–I) SKCM cell malignancy was evaluated by cell proliferation, colony formation, survival and transwell invasion assays, as described in Figure 2. In panel 4C, red asterisks labeled the comparison to NC group, while the green asterisks labeled the comparison to HCP5 OE group. Bar indicates 100 μM. *p<0.05. Abbreviations: SKCM, skin cutaneous melanoma; NC, normal control.

Figure 5

HCP5-regulated RARRES3 expression in SKCM cells by sponging miR-1286. (A) HCP5 overexpression increased the expression of Gaussia luciferase (Gluc) governed by the 3ʹ UTR of RARRES3 mRNA. (B) HCP5 overexpression reduced the association of RARRES3 mRNA with AGO2 protein. (C) Putative HCP5 and RARRES3 mRNA interacting miRNAs, predicted by the DIANA microT-CDS algorithm with prediction score >0.6. (D) HCP5 overexpression increased the association of miR-1286 as well as other miRNAs with AGO2 protein in SKCM cells. (E) Putative miR-1286-binding site on the wild type 3ʹ UTR of RARRES3 (WT), which was mutated (MUT). (F) miR-1286 mimic transfection reduced the-expression of Gluc governed by wild-type 3ʹ UTR of RARRES3. (G and H) RARRES3 mRNA and protein level in SKCM was reduced by miR-1286 mimic transfection. *p<0.05. Abbreviations: SKCM, skin cutaneous melanoma; NC, normal control.