Identification of differentially expressed lncRNAs in ccRCC
The expression of lncRNAs was detected in a total of 54 pairs of ccRCC tissues and matched para-carcinoma tissues in the TCGA database. With fold change ≥ 4 and p<0.01 as the criteria, it was found that a total of 20 lncRNAs were upregulated and 329 lncRNAs were downregulated in ccRCC tissues (Figure 1A and 1B). The 5 most significantly up-regulated lncRNAs in ccRCC tissues (ENSG00000224490, ENSG00000225174, ENSG00000237471, ENSG00000255774, ENSG00000260877) and the 5 most significantly downregulated lncRNAs (ENSG00000248517, ENSG00000228521, ENSG00000260580, ENSG00000257191, ENSG00000258402) were selected and verified via qRT-PCR in 20 pairs of ccRCC tissues and para-carcinoma tissues. The results showed that the differential expression of ENSG00000255774 (LINC02747) was the most obvious. Compared with that in para-carcinoma tissues, LINC02747 was highly expressed in cancer tissues (Figure 1C). Then, the expression of LINC02747 was detected via qRT-PCR in normal kidney cells (RPTEC/TERT1) and RCC cells (786-O, ACCN, Caki-1, Caki-2). The results revealed that the expression of LINC02747 in RCC cells was significantly higher than that in normal kidney cells. Therefore, LINC02747 was significantly highly expressed in RCC cells and tissues.
The prognostic value of LINC02747
The expression of lncRNAs in TCGA and related clinicopathological characteristics of 447 ccRCC patients were analyzed. The results showed that the expression of LINC02747 in ccRCC patients with tumors > 7 was significantly higher than that in patients with tumors ≤ 7 (Figure 2A). The expression of LINC02747 was significantly higher in patients with Fuhrman grade III-IV ccRCC than Fuhrman grade I-II ccRCC (Figure 2B). The expression of LINC02747 was significantly higher in patients with TNM stage III-IV ccRCC than TNM stage I-II ccRCC (Figure 2C). With the median value as the cut-off value, the overall survival (OS) of patients with high LINC02747 expression was significantly less than that of patients with low LINC02747 expression (p<0.001) (Figure 2D).
Effect of LINC02747 on the proliferation ability of RCC cells
To study the effect of LINC02747 on the proliferation of RCC cells, the expression of LINC02747 in 786-O and Caki-1 RCC cells was upregulated or downregulated by transfection of an overexpression plasmid or siRNA. The expression of LINC02747 after transfection is shown in Figure 3A and 3B. The CCK-8 assay was performed to detect the effect of LINC02747 expression on the proliferation of 786-O cells. It was found that the upregulation of LINC02747 significantly promoted the proliferation of RCC cells (Figure 3C), while the downregulation of LINC02747 inhibited the proliferation of RCC cells (Figure 3D). The same results were obtained in Caki-1 cells (Figure 3E-3F).
Knocking down LINC02747 inhibited the proliferation of RCC cells in nude mice
To further explore the effect of LINC02747 on tumor growth in nude mice, 786-O cells with stably downregulated LINC02747 (LINC02747 siRNA) and control cells (Control) were injected into the subcutaneous tissues of nude mice for 30 days. It was found that the growth rate of tumors in the LINC02747 siRNA group was significantly lower than that in the Control group (Figure 4A and 4B). The results showed that knocking down LINC02747 significantly inhibited the growth of RCC cells in nude mice, confirming the effect of LINC02747 on the growth of RCC cells in vivo.
LINC02747 regulated the proliferation of RCC cells by inhibiting miR-608
The nuclear-cytoplasmic fractionation assay demonstrated that LINC02747 was mainly located in the cytoplasm (Figure 4A). Therefore, it was speculated that LINC02747 may regulate the function of RCC cells by inhibiting miRNA. Using the RegRNA2.0 database (http://regrna2.mbc.nctu.edu.tw/), the miRNAs (miR-187, miR-330, miR-608, miR-593, miR-661) most likely to bind to LINC02747 were identified. Then, LINC02747 was overexpressed in 786-O and Caki-1 cell lines, and the expression changes of the above miRNAs were detected by qRT-PCR. The results showed that miR-608 expression was significantly downregulated (Figure 5B). Then, LINC02747-WT and LINC02747-MUT were cloned into plasmid psiCHECK-2 (Figure 5C), and the constructs were cotransfected with miR-608 mimic into 786-O and Caki-1 cells for the luciferase reporter assay. The results showed that miR-608 overexpression decreased the luciferase activity of the LINC02747-WT vector (p<0.01) but did not reduce the luciferase activity of the mutant vector or empty vector (Figure 5D). This result showed that LINC02747 can directly bind to miR-608, and the mutated sites are the binding sites between the two molecules. In addition, miR-608 expression is lower in cancer tissues (Figure 6A), and the expression levels of LINC02747 and miR-608 were negatively correlated in 20 ccRCC tissues (Figure 6B).
Effect of miR-608 on the proliferation ability of RCC cells
To study the effect of miR-608 on the proliferation ability of RCC cells, miR-608 mimic and miR-608 inhibitor were transfected into 786-O and Caki-1 cell lines, and then the expression of miR-608 was detected using qRT-PCR. The expression of miR-608 after transfection is shown in Figure 6C and 6D. In the CCK-8 proliferation assay, it was found that upregulation of miR-608 significantly promoted the proliferation of RCC cells (Figure 6E), while downregulation of miR-608 inhibited the proliferation of RCC cells (Figure 6F). The same results were obtained in Caki-1 cells (Figure 6G-6H).
LINC02747 regulated the proliferation of RCC cells by inhibiting miR-608
To explore whether LINC02747 regulates the proliferation of RCC cells by inhibiting miR-608, 786-O and Caki-1 cells were transfected with Control+miR-NC, LINC02747+miR-NC, Control+miR-608 mimic or LINC02747+miR-608 mimic. The expression levels of LINC02747 and miR-608 in the four groups after transfection are shown in Figure 7A and 7B. The CCK-8 proliferation assay showed that in the cell lines with upregulated miR-608, the upregulation of LINC02747 had no significant impact on the proliferation ability of RCC cells (Figure 7C and 7D). These results indicate that LINC02747 regulates the proliferation ability of RCC cells by inhibiting miR-608.
Screening and verification of miR-608 target genes
The 5 most likely target genes of miR-608, namely, BRD4, SSBP3, FOXO6, SOD3 and TFE3, were selected as candidate target genes using TargetScan (http://www.targetscan.org/) and miRDB (http://mirdb.org/). Then, the expression of miR-608 in 786-O and Caki-1 cells was upregulated, and it was found that the mRNA expression of TFE3 changed most significantly, and the difference was statistically significant (p<0.01, Figure 8A). Then, the expression of miR-608 was upregulated in 786-O and Caki-1 cells, and it was found that the expression of TFE3 protein decreased, while the expression of TFE3 increased after downregulating miR-608 expression (Figure 8B). Then, TFE3 wild-type and mutant sequences were cloned into the luciferase reporter plasmid psiCHECK-2 (Figure 8C), and the constructs were cotransfected with miR-608 mimic into 786-O and Caki-1 cells for the luciferase reporter assay. The results showed that overexpression of miR-608 could greatly lower the luciferase activity of the wild-type TFE3 vector (p<0.01) but did not reduce that of the mutant or empty vector (Figure 8D). This result proved that TFE3 is the direct target gene of miR-608, and the mutated sites are the binding sites between the two.
MiR-608 regulated the proliferation of RCC cells by inhibiting TFE3
To verify whether miR-608 can inhibit the proliferation of RCC by inhibiting TFE3, 786-O and Caki-1 cells were transfected with Control+miR-NC, Control+miR-608 mimic, TFE3+miR-NC or TFE3+miR-608 mimics. The mRNA and protein expression levels of TFE3 in the four groups after transfection are shown in Figure 9A and 9B. The CCK-8 assay showed that upregulation of miR-608 expression had no significant impact on the proliferation ability of RCC cells when TFE3 was upregulated (Figure 9C and 9D). These findings show that miR-608 regulates the proliferation of RCC by inhibiting TFE3. In summary, LINC02747 upregulates the expression of TFE3 by adsorbing miR-608, ultimately promoting the proliferation ability of RCC cells.