lncRNA small nucleolar RNA host gene 12 promotes renal cell carcinoma progression by modulating the miR‑200c‑5p/collagen type XI α1 chain pathway

Abstract

Renal cell carcinoma (RCC) is a primary malignant kidney cancer subtype. It has been suggested that long non‑coding RNAs (lncRNAs) serve important roles in the progression of kidney cancer. In fact, the lncRNA small nucleolar RNA host gene 12 (SNHG12) was discovered to be overexpressed in various types of cancer. However, to the best of our knowledge, the role of SNHG12 in RCC remains unclear. The present study aimed to investigate the function of SNHG12 and its underlying molecular mechanism of action in RCC. In patient samples and datasets from The Cancer Genome Atlas. Reverse transcription‑quantitative PCR, demonstrated that SNHG12 expression levels were upregulated in RCC tumor tissues, but not in normal kidney tissues. SNHG12 upregulation was also observed in RCC cell lines. Kaplan‑Meier survival analysis indicated a poor prognosis for those patients with RCC who had upregulated SNHG12 expression levels. Following lentivirus transduction, SNHG12 was successfully knocked down (validated by western blot analysis) and cell migration and invasion assays were performed. SNHG12 knockdown markedly inhibited cell viability and invasion, while increasing apoptosis in both A498 and 786O cell lines. The results of the luciferase reporter assay suggested that SNHG12 exerted its role by sponging microRNA (miR)‑200c‑5p, which led to the upregulation of its target gene, collagen type XI α1 chain (COL11A1). This was further validated, as miR‑200c‑5p inhibition reduced the effects of SNHG12 downregulation on cell viability and apoptosis, without affecting SNHG12 expression levels. Furthermore, the findings indicated that SNHG12 may partially exert its role through COL11A1, which was also upregulated in RCC. In conclusion, the results of the present study suggested that the SNHG12/miR‑200c‑5p/COL11A1 axis may be crucial for RCC progression, which provided an insight into potential therapeutic strategies for RCC treatment.

Figure 1.

Relative expression levels of SNHG12 are upregulated in patients with RCC and in RCC cell lines. (A) SNHG12 expression levels in patients with RCC and normal patient samples from TCGA database. (B) SNHG12 expression levels in clinical RCC samples compared with adjacent non-tumor tissues were analyzed using RT-qPCR. Data are expressed in the form of taking logarithm of the counts number to the base 2. (C) SNHG12 expression levels in RCC cell lines and the HK-2 normal kidney cell line were analyzed using RT-qPCR. (D) Kaplan-Meier curve of the survival of patients with RCC from TCGA database according to low and high SNHG12 expression levels. 0, alive; 1, deceased. SNHG12, small nucleolar RNA host gene 12; RCC, renal cell carcinoma; TCGA, The Cancer Genome Atlas; RT-qPCR, reverse transcription-quantitative PCR; Cum, cumulative. *P<0.05, **P<0.01, ***P<0.001.

Figure 2.

Functional role of SNHG12 in RCC cell lines. (A) Transfection efficiency of si-SNHG12 in A498 and 786O cell lines was analyzed using reverse transcription-quantitative PCR. (B) Cell viability was analyzed using an MTT assay following SNHG12 knockdown. (C) Cell invasive ability was detected using a Transwell assay following SNHG12 knockdown. Magnification, ×100; scale bar, 100 µm. (D) Flow cytometric analysis of apoptosis was performed following SNHG12 knockdown. Untransfected RCC cells were used as the ‘blank’ control. *P<0.05. SNHG12, small nucleolar RNA host gene 12; RCC, renal cell carcinoma; si, small interfering RNA; NC, negative control; 7-AAD, 7-Aminoactinomycin D.

Figure 3.

SNHG12 binds to miR-200c-5p and downregulates its expression levels. (A) Inverse correlation was identified between the expression levels of SNHG12 and miR-200c-5p in patient samples. (B) Transfection efficiency of miR-200c-5p mimics in A498 and 786O cell lines was analyzed using reverse transcription-quantitative PCR. (C) Upregulation of miR-200c-5p expression levels in RCC cell lines had no effect on the expression levels of SNHG12. However, si-SNHG12 knockdown significantly upregulated miR-200c-5p expression levels. (D) Luciferase reporter assay demonstrated that the overexpression of miR-200c-5p reduced the intensity of the relative luciferase activity in 293T cells transfected with the SNHG12-WT vector. Untransfected RCC cells were used as the ‘blank’ control. *P<0.05. SNHG12, small nucleolar RNA host gene 12; RCC, renal cell carcinoma; miR, microRNA; NC, negative control; lncRNA, long non-coding RNA; WT, wild-type; MUT, mutant.

Figure 4.

SNHG12 exerts its role by regulating miR-200c-5p. (A) Transfection efficiency of miR-200c-5p inhibitor in A498 and 786O cell lines was determined using reverse transcription-quantitative PCR. (B) MTT assay was used to determine cell viability in RCC cells transfected with si-SNHG12 + miR-200c-5p inhibitor compared with cells transfected with si-SNHG12 alone. (C) Flow cytometric analysis of apoptosis following si-SNHG12 and miR-200c-5p inhibitor co-transfection compared with cells transfected with si-SNHG12 alone. Untransfected RCC cells were used as the ‘blank’ control. *P<0.05. SNHG12, small nucleolar RNA host gene 12; RCC, renal cell carcinoma; miR, microRNA; NC, negative control; si, small interfering RNA; 7-AAD, 7-Aminoactinomycin D.

Figure 5.

COL11A1 is a direct target of miR-200c-5p. (A) Relative COL11A1 expression levels in RCC tissues from The Cancer Genome Atlas database. (P=0.0248) (B) Relative COL11A1 expression levels following the transfection with the miR-200c-5p mimics in RCC cell lines was detected using reverse transcription-quantitative PCR. (C) Western blotting was used to determine the protein expression levels of COL11A1 in the blank, miR-mimics-NC and miR-200c-5p mimics group. (D) Luciferase reporter assay demonstrated a strong binding between the 3′-UTR of COL11A1 and miR-200c-5p. Untransfected RCC cells were used as the ‘blank’ control. *P<0.05. SNHG12, small nucleolar RNA host gene 12; COL11A1, collagen type XI α1 chain; RCC, renal cell carcinoma; miR, microRNA; NC, negative control; WT, wild-type; MUT, mutant; UTR, untranslated region. The binding sites of COL11A1 and miR-200c-5p were discovered using miRanda database.

Figure 6.

COL11A1 participates in the progression of RCC through the SNHG12/miR-200c-5p/COL11A1 axis. (A) COL11A1 expression levels were discovered to be regulated by SNHG12 in a miR-200c-5p-dependent manner. COL11A1 expression levels were analyzed using RT-qPCR. (B) Expression levels of SNHG12 and COL11A1 were analyzed using RT-qPCR following the transfection with pcDNA3.1-SNHG12 or si-COL11A1, respectively. (C) miR-200c-5p inhibitor rescued COL11A1 expression levels downregulated by si-SNHG12. (D) Flow cytometric analysis of apoptosis was performed in cells co-transfected with pcDNA3.1-SNHG12 and si-COL11A1. (E) Quantification of the apoptosis results were shown as bar plots. (F) Cell viability was determined using an MTT assay following the transfection with pcDNA3.1-SNHG12 + si-COL11A1. *P<0.05. SNHG12, small nucleolar RNA host gene 12; COL11A1, collagen type XI α1 chain; miR, microRNA; NC, negative control; si, small interfering RNA; RT-qPCR, reverse transcription-quantitative PCR; 7-AAD, 7-Aminoactinomycin D.