lncRNA MIR4435-2HG promoted clear cell renal cell carcinoma malignant progression via miR-513a-5p/KLF6 axis

Abstract

Long non-coding RNAs (lncRNAs) take various biological effects in clear cell renal cell carcinoma (ccRCC) mostly through sponging with microRNAs (miRNAs). lncRNA MIR4435-2HG is found to promote tumour progression in gastric cancer, glioblastoma and hepatocellular carcinoma. However, the role of lncRNA MIR4435-2HG in ccRCC progression remains unknown. The purpose of this research was to investigate the potential molecular mechanism of lncRNA MIR4435-2HG regarding the regulation of ccRCC initiation and progression. In this study, we found the up-regulation of MIR4435-2HG in ccRCC tissues and cell lines. Functionally, overexpression of MIR4435-2HG promoted the proliferation as well as the metastasis in ccRCC cell lines, whereas knockdown of MIR4435-2HG inhibited the above changes. Then, bioinformatic analysis and luciferase reporter assays confirmed the negative regulation effect of MIR4435-2HG on miR-513a-5p. And further investigations showed that KLF6, which collected from the intersection of databases, was the potential conjugated mRNAs of miR-513a-5p. Finally, the rescue experiments revealed the relation among MIR4435-2HG and KLF6, which showed that KLF6 could reverse the promoting effect of MIR4435-2HG on ccRCC in vitro and in vivo. Therefore, our findings provided insight into the mechanisms of MIR4435-2HG in ccRCC and revealed an alternative target for the clinical diagnosis and treatment of ccRCC.

Keywords: KLF6; MIR4435‐2HG; ccRCC; invasion; long non‐coding RNA; proliferation.

Figure 1

Identification of MIR4435‐2HG as an lncRNA up‐regulated in ccRCC. A‐B: GEPIA results of up‐expression of MIR4435‐2HG in ccRCC tissues. C: The subcellular localization of MIR4435‐2HG predicted on lncATLAS website. D: The subcellular localization of MIR4435‐2HG detected by FISH assay (400×). E: qRT‐PCR analysis of the expression levels of MIR4435‐2HG in 40 paired ccRCC tissues and the adjacent normal tissues. F: MIR4435‐2HG expression levels in ccRCC cell lines and human normal renal tubular epithelial cell line HK‐2 were detected by qRT‐PCR analysis. G‐H: The relative expression of MIR4435‐2HG determined by RT‐qPCR analysis following the treatment of knocking down MIR4435‐2HG in 769‐P cells (sh‐MIR4435‐2HG) or over‐expressing MIR4435‐2HG in ACHN cells (oe‐MIR4435‐2HG). All of the data were analysed from three independent experiments. * P < 0.05; ** P < 0.01;**** P < 0.001 vs control group

Figure 2

Long non‐coding RNA MIR4435‐2HG increased proliferation and invasion abilities of ccRCC cells. A: Cell proliferation was examined by CCK‐8 assays in sh‐MIR4435‐2HG group or oe‐MIR4435‐2HG group at the indicated time‐points. The shRNA control 769‐P cells or oe‐control ACHN cells were as control. B: Cell proliferation was determined by colony formation assay of impacts of MIR4435‐2HG in 769‐P and ACHN cells. C: Representative images revealing the invasion capacities of impacts of MIR4435‐2HG in 769‐P and ACHN cells. All of the data were analysed from three independent experiments. * P < 0.05; ** P < 0.01

Figure 3

MIR4435‐2HG interacted with miR‐513a‐5p and repressed its expression in ccRCC. A: miRNA profiling analysis result of TCGA database showing abnormal downstream expressed target miRNAs of MIR4435‐2HG in paracancerous or ccRCC tumour tissues. miR‐513a‐5p is one of the most low‐expressed miRNAs in ccRCC tumour tissues. B: qRT‐PCR assay confirmed the relative expression of miR‐513a‐5p in 40 paired ccRCC cancer tissues compared with corresponding paracancerous normal tissues. C: Correlation between MIR4435‐2HG and miR‐513a‐5p expression in 40 ccRCC tumour tissues. D: miR‐513a‐5p expression level in ccRCC cell lines (769‐P and ACHN cells) and human normal renal tubular epithelial cell line HK‐2 was detected by qRT‐PCR assay. E: Schematic illustration of the predicted binding sites between MIR4435‐2HG and miR‐513a‐5p, and mutation of potential miR‐513a‐5p‐binding sequence in MIR4435‐2HG. Relative luciferase activities of wild‐type (WT) and mutated (MUT) MIR4435‐2HG reporter plasmid in human embryonic kidney (HEK) 293T cells co‐transfected with miR‐513a‐5p mimic. F: Co‐localization between MIR4435‐2HG and miR‐513a‐5p was observed by FISH. Nuclei were stained with DAPI. All of the data were analysed from three independent experiments. ** P < 0.01; *** P < 0.001; **** P < 0.001

Figure 4

KLF6 was a direct target of miR‐513a‐5p in ccRCC progression. A: A schematic diagram used to search the target mRNAs of miR‐513a‐5p in four databases. B: qRT‐PCR assay confirmed the relative expression of five candidate target mRNAs of miR‐513a‐5p in 40 paired ccRCC cancer tissues and corresponding paracancerous normal tissues. C: Correlation analysis between KLF6 expression and miR‐513a‐5p expression in 40 ccRCC tumour tissues. D: Relative expression of KLF6 in ccRCC cell lines (769‐P and ACHN cells) and HK‐2 cells. E: Relative expression of KLF6 in 769‐P cells transfected with pcDNA3.1‐KLF6 and ACHN cells transfected with si‐KLF6. F: The relative expression of miR‐513a‐5p determined by qRT‐PCR analysis following the treatment of overexpressing miR‐513a‐5p in 769‐P cells (miR‐513a‐5p mimics) or knocking down miR‐513a‐5p in ACHN cells (miR‐513a‐5p inhibitor). G: Relative expression of KLF6 in 769‐P cells transfected with miR‐513a‐5p mimics and ACHN cells transfected with miR‐513a‐5p inhibitor. H: Schematic illustration of the predicted binding sites between miR‐513a‐5p and KLF6, and mutation of potential miR‐513a‐5p–binding sequence in KLF6. Relative luciferase activities of wild‐type (WT) and mutated (MUT) KLF6 reporter plasmid in human embryonic kidney (HEK) 293T cells co‐transfected with miR‐513a‐5p mimic. All of the data were analysed from three independent experiments. * P < 0.05; ** P < 0.01

Figure 5

miR‐513a‐5p inhibited proliferation and invasion abilities of ccRCC cells. A: Cell proliferation was examined by CCK‐8 assays in miR‐513a‐5p mimics+pcDNA3.1‐KLF6 group or miR‐513a‐5p inhibitor+si‐KLF6 group at the indicated time‐points. The NC cells were as control. B: Cell proliferation was determined by colony formation assay of impacts of miR‐513a‐5p and KLF6 in 769‐P and ACHN cells. C: Representative images revealing the invasion capacities of impacts of miR‐513a‐5p and KLF6 in 769‐P and ACHN cells. D: Western blotting results of KLF6 in different transfected groups of 769‐P and ACHN cells. E: Tumour volume of the xenograft in each group. The tumour sections from different transfected groups of xenograft mouse models were subjected to immunohistochemistry staining using antibodies against KLF6 (400×). All of the data were analysed from three independent experiments. * P < 0.05; ** P < 0.01

Figure 6

MIR4435‐2HG enhanced the proliferation and invasion abilities of ccRCC cells by regulating KLF6. A: qRT‐PCR was conducted to verify the relative expression of KLF6 in 769‐P transfected with NC, sh‐MIR4435‐2HG, sh‐MIR4435‐2HG+pcDNA3.1‐KLF6 and ACHN cells transfected with NC, oe‐MIR4435‐2HG and oe‐MIR4435‐2HG+si‐KLF6. B: The expression of KLF6 was analysed by Western blotting with the indicated antibodies and samples from the 769‐P and ACHN cells in different transfected groups. C: CCK‐8 assay of 769‐P and ACHN cells in different transfected groups. D: Cell proliferation was determined by colony formation assay of different transfected groups in 769‐P and ACHN cells. E: Representative images revealing the invasion capacities of impacts of different transfected groups in 769‐P and ACHN cells. All of the data were analysed from three independent experiments. * P < 0.05; ** P < 0.01

Figure 7

Decreasing MIR4435‐2HG contributed to suppress tumorigenesis and tumour progression by down‐regulating KLF6 in vivo. A: Representative images of the xenograft tumours in subcutaneous xenograft mouse model injected with 769‐P cells transfected with NC, sh‐MIR4435‐2HG and sh‐MIR4435‐2HG+pcDNA3.1‐KLF6. B: Tumour volume and weight of the xenograft in each group. C: The tumour sections from different transfected groups of xenograft mouse models were subjected to H&E staining and immunohistochemistry staining using antibodies against KLF6 (400×). All of the data were analysed from three independent experiments. * P < 0.05; ** P < 0.01