lncRNA HCP5 acts as a ceRNA to regulate EZH2 by sponging miR‑138‑5p in cutaneous squamous cell carcinoma

Abstract

Long non‑coding RNAs (lncRNAs) and microRNAs (miRNAs) are essential for the progression of tumors, including cutaneous squamous cell carcinoma (CSCC). The present study aimed to examine the competing endogenous RNA (ceRNA) network in CSCC. Differentially expressed genes in CSCC were analyzed using the GSE66359 microarray data set, and the upstream miRNAs and lncRNAs were predicted using online database analysis (TargetScan 7.1, mirDIP 4.1, miRSearch V3.0, miRDB and RNA22 2.0) and were verified in clinical tissues. RNA pull‑down and dual luciferase reporter gene assays were used to verify the targeting relationships among lncRNA human histocompatibility leukocyte antigen complex P5 (HCP5), miR‑138‑5p and enhancer of zeste homolog 2 (EZH2). Cell lines with a high and low HCP5 expression were screened, and a pcDNA‑3.1‑HCP5 overexpression vector, small interfering RNA against HCP5, miR‑138‑5p mimics and miR‑138‑5p inhibitors were transfected into the CSCC cells. Cell viability, invasion, migration, apoptotic rate and autophagy were evaluated. The effects of HCP5 on autophagy and apoptosis of CSCC cells were verified in vivo using Ki67 and TUNEL staining. EZH2 was demonstrated to be upregulated in CSCC cells. miR‑138‑5p target sequences were identified in HCP5 and EZH2. HCP5 was revealed to function as a putative ceRNA of miR‑138‑5p to positively regulate EZH2, and EZH2 was shown to regulate autophagy and apoptosis of CSCC cells through the STAT3/VEGFR2 pathway. HCP5 overexpression decreased miR‑138‑5p levels, increased EZH2 levels and promoted cell malignant behaviors and autophagy but decreased the apoptosis rate. These trends were opposite when HCP5 was silenced. In conclusion, HCP5 may competitively bind to miR‑138‑5p to regulate EZH2 in CSCC cells, promoting autophagy and reducing apoptosis through the STAT3/VEGFR2 pathway. This study may provide a new perspective for understanding the molecular mechanism and treatment of CSCC.

Keywords: STAT3/VEGFR2 pathway; autophagy; cutaneous squamous cell carcinoma; enhancer of zeste homolog 2; long non‑coding RNA human histocompatibility leukocyte antigen complex P5; microRNA‑138‑5p.

Figure 1

LncRNA HCP5 may regulate EZH2 expression through miR-138-5p. (A) Differential expression analysis of the volcano plot of the GSE66359 microarray. Red indicates highly expressed genes and green indicates genes with low expression levels. (B) EZH2 was found to be significantly increased in CSCC tissue compared with normal tissue in the GSE66359 data set. (C) mRNA and (D) protein expression levels of EZH2 in tissue and normal adjacent tissue samples from 60 patients with CSCC were upregulated. (E) Four online databases were used to analyze the intersected data to predict putative miRNAs upstream of EZH2; five strong candidates were identified: miR-26a-5p, miR-26b-5p, miR-1297, miR-4465 and miR-138-5p. (F) RT-qPCR was used to detect the expression levels of the five miRNAs; miR-138-5p level was the lowest in CSCC compared with normal tissues. N=60; ^***P<0.001 vs. normal tissue. (G) Eight lncRNAs were predicted to regulate miR-138-5p using RNA22 database, and these were intersected with the upregulated genes expressed in GSE66359 microarray (H) The expression of these eight lncRNAs was detected using RT-qPCR, and HCP5 showed the largest upregulation in CSCC compared with normal tissues. N=60; ^*P<0.05, ^***P<0.001 vs. normal tissue. Data in panel B were analyzed using the Wilcoxon signed-rank test, and data in panels C, D, F and H were analyzed using paired Student's t-test. CSCC, cutaneous squamous cell carcinoma; EZH2, enhancer of zeste homolog 2; FC, fold change; HCP5, human histocompatibility leukocyte antigen complex P5; lncRNA, long non-coding RNA; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR.

Figure 2

Long non-coding RNA HCP5 regulates the malignant behavior, autophagy and apoptosis of CSCC cells. (A) RT-qPCR detection of HCP5 expression in CSCC cell lines. ^*P<0.05, ^***P<0.001 vs. HaCaT. The transfection efficiency of (B) two siRNAs against HCP5 and (C) the HCP5 overexpression vector were verified using RT-qPCR. ^***P<0.001 vs. si-NC or pcDNA3.1-NC. (D) Cell Counting Kit-8 assays, (E) Transwell assays and (F) scratch tests were used to detect the cell viability, migration and invasion, respectively. ^**P<0.01, ^***P<0.001 vs. si-NC or pcDNA3.1-NC. (G) Representative images of mRFP-GFP-LC3 fluorescence tracer assay, in which green and red indicate the observed fluorescence of LC3 under different excitation wavelengths; yellow fluorescence indicates autophagosomes and autolysosomes are represented by red dots. (H) Western blot analysis was used to detect the levels of autophagy-related proteins. ^**P<0.01, ^***P<0.001 vs. si-NC or pcDNA3.1-NC. (I) Flow cytometry was used to determine the apoptotic rates in transfected CSCC cells. ^*P<0.05, ^**P<0.01 si-NC or pcDNA3.1-NC. (J) Western blot analysis was used to detect the levels of apoptosis-related proteins. ^**P<0.01, ^***P<0.001 vs. si-NC or pcDNA3.1-NC. Data in panels A, B and D were analyzed using one-way ANOVA, followed by Tukey's multiple comparisons test, and data in panels C, E, F, H, I and J were analyzed using the Mann-Whitney U-test. Data are representative of three experimental repeats. GFP green fluorescent protein; HCP5, human histocompatibility leukocyte antigen complex P5; LC3, light chain 3; mRFP, monomeric red fluorescent protein; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA.

Figure 3

LncRNA HCP5 competes with EZH2 to bind to miR-138-5p. Binding sites between (A) HCP5 and miR-138-5p, and (B) the miR-138-5p target sites in EZH2 3′UTR and were predicted using the online databases RNA22 and TargetScan. Dual luciferase report gene assay verified the relation between (A) HCP5 and miR-138-5p and (B) EZH2 and miR-138-5p. ^***P<0.001 vs. mimics-NC. (C) miR-138-5p was significantly enriched by HCP5 using RNA pull-down assay. ^***P<0.001 vs. Control. (D) miR-138-3p, (E) EZH2 mRNA and (F) EZH2 protein expression levels in A431 cells following the various transfections were detected using reverse transcription-quantitative PCR or western blotting. ^**P<0.01, ^***P<0.001. Data are representative of three experimental repeats. Data were analyzed by Mann-Whitney U-test. EZH2, enhancer of zeste homolog 2; HCP5, human histocompatibility leukocyte antigen complex P5; inhi, inhibitor; lncRNA, long non-coding RNA; miR, microRNA; MUT, mutant; UTR, untranslated region; WT, wild-type.

Figure 4

Upregulation of miR-138-5p in cutaneous squamous cell carcinoma cells inhibits autophagy and promotes apoptosis. (A) miR-138-5p inhibitor was co-transfected into A431 cells with si-HCP5, and the expression levels of miR-138-5p were detected by reverse transcription-quantitative PCR. ^***P<0.001. (B) Cell Counting Kit-8 assay was used to measure cell viability at each time point. ^***P<0.001. (C) Western blot analysis was used to determine the expression levels of autophagy-related proteins. ^*P<0.05, ^**P<0.01, ^***P<0.001. (D) Flow cytometry was used to determine the apoptotic rates in transfected cells. ^**P<0.01, ^***P<0.001. (E) Western blot analysis was used to determine the expression levels of apoptosis-related proteins. ^**P<0.01, ^***P<0.001. Data are representative of three experimental repeats. Data in panels A and E were analyzed by Mann-Whitney U-test, and data in panels B-D were analyzed using the one-way ANOVA followed by Tukey's multiple comparisons test. EZH2, enhancer of zeste homolog 2; HCP5, human histocompatibility leukocyte antigen complex P5; inhi, inhibitor; LC3, light chain 3; miR, microRNA; NC, negative control; si, small interfering RNA.

Figure 5

Upregulation of EZH2 counteracts the effects of HCP5 silencing in cutaneous squamous cell carcinoma cells. (A) mRNA and (B) protein expression levels of EZH2 were detected using reverse transcription-quantitative PCR and western blot analysis, respectively. ^***P<0.001. (C) Cell Counting Kit-8 assays were used to measure cell viability at each time point. ^***P<0.001. (D) Western blot analysis was used to determine the expression levels of autophagy-related proteins. ^**P<0.01, ^***P<0.001. (E) Flow cytometry was used to determine the apoptotic rates in transfected cells. ^**P<0.01. (F) Western blot analysis was used to determine the expression levels of apoptosis-related proteins. ^*P<0.05, ^**P<0.01, ^***P<0.001. Data in panel C were analyzed using one-way ANOVA, followed by Tukey's multiple comparisons test, and data in panels A, B and D-F were analyzed using the Mann-Whitney U-test. EZH2, enhancer of zeste homolog 2; HCP5, human histocompatibility leukocyte antigen complex P5; inhi, inhibitor; LC3, light chain 3; miR, microRNA; NC, negative control; si, small interfering RNA.

Figure 6

EZH2 regulates autophagy and apoptosis in cutaneous squamous cell carcinoma cells through the STAT3/VEGFR2 pathway. Western blot analysis was used to determine the expression levels of (A) p-STAT3, t-STAT3 and VEGFR2 proteins and (B) autophagy-related proteins. ^*P<0.05, ^***P<0.001 vs. pcDNA3.1-NC; ^###P<0.001 vs. EZH2. (C) Flow cytometry was used to determine the apoptotic rates in transfected cells. ^***P<0.001 vs. pcDNA3.1-NC; ^##P<0.01 vs. EZH2. Data are representative of three experimental repeats. Data were analyzed using one-way ANOVA, followed by Tukey's multiple comparisons test. EZH2, enhancer of zeste homolog 2; LC3, light chain 3; NC, negative control; p-, phosphorylated, t-, total.

Figure 7

HCP5 promotes EZH2 expression to activate the STAT3/VEGFR2 pathway in vivo. Tumor (A) volume and (B) weight after HCP5 silencing in nude mice. ^**P<0.01, ^***P<0.001 vs. si-NC. (C) HCP5 expression levels in the excised tumors were detected using reverse transcription-quantitative PCR. ^***P<0.001 vs. si-NC. (D) Western blot analysis was used to determine the expression levels of the p-STAT3, t-STAT3 and VEGFR2 proteins and autophagy-related proteins. ^***P<0.001 vs. si-NC. (E) Immunohistochemistry was used to detect the Ki67-positive expression rate. ^***P<0.001 vs. si-NC. (F) TUNEL staining was used to detect cell apoptosis in tumors. ^***P<0.001 vs. si-NC. N=5. Data in panel A were analyzed using one-way ANOVA, followed by Tukey's multiple comparisons test, and data in panels B-E were analyzed using Mann-Whitney U-test. EZH2, enhancer of zeste homolog 2; HCP5, human histocompatibility leukocyte antigen complex P5; LC3, light chain 3; NC, negative control; p-, phosphorylated; si, small interfering RNA; t-, total.