Detection of Lb-miR166a expression in RCC cells
qRT-PCR showed that Lb-miR166a was not expressed in RCC cells and L-Con group cells, whereas the expression of Lb-miR166a was significantly upregulated in Lb-miR166a group cells. This shows that the transferred Lb-miR166a could be successfully expressed in tumor cells (Figure 2).
Transcriptome sequencing and differential gene screening analysis
The Hisat2 software was used to compare the reads obtained from the sequencing data with the reference genome for similarity analysis. By quickly comparing each “read” with the reference genome sequence, the position and matching quality of the “read” on the compared genome or other reference sequences are finally obtained, and the gene or transcript can be annotated and quantified. A total of 19,225 gene entries were measured in this study.
There were differences in the gene expression among the different samples. Based on the comparison results, the differential expression of genes in each sample was analyzed using edgeR software, and the adjusted P (Padj) value of differential expression was calculated. The Padj value indicated that the gene expression difference was more significant. In this experiment, genes with |log2 fold change| ≥ 1 and Padj < 0.05 were selected as significant differentially expressed genes. The differences in mRNA expression between the two groups were statistically analyzed. A total of 1,813 differentially expressed mRNAs were screened. Cluster analysis of differentially expressed mRNAs showed that 1,232 mRNAs were significantly upregulated and 581 were significantly downregulated (Figure 3a).
To better understand the biological function of differentially expressed mRNA, we analyzed the differentially expressed mRNA using GO. A total of 1,813 differentially expressed genes were annotated into 1,094 GO entries, of which 1,074 were significantly enriched (P < 0.05), including 605 physiological process entries, 44 cell component entries, and 48 molecular function entries (Figure 3b).
The differentially expressed genes were enriched in 43 KEGG pathways, of which 30 were significantly enriched (P < 0.05). It can be seen that the upregulated genes were enriched in tumor-related cell adhesion molecules and apoptosis; the downregulated genes were significantly enriched in cell proliferation and other pathways (Figure 3c).
Lb-miR166a regulates hsa05200 pathway gene expression
Hsa05200 (a pathway in cancer) has been identified as the most important pathway in tumor pathogenesis. The activation of many genes and signaling pathways plays a key role in tumor cell proliferation and differentiation; apoptosis; and tumor infiltration, growth, recurrence, and metastasis.
Through differential gene annotation, as shown in Figure 4, Lb-miR166a can regulate the expression of multiple genes of hsa05200 pathway in cancer, so as to regulate tumorigenesis and development.
Lb-miR166a inhibits proliferation of Caki-1 cells
Cell proliferation depends mainly on the normal operation of the cell cycle. Mitosis entering the cell cycle is strictly controlled by the promotion or inhibition of growth signals, which are important for cell proliferation and division. To further clarify the effect of Lb-miR166a on cell proliferation-related genes and pathways, we screened out cell proliferation-related differential genes based on gene function annotation and constructed a circular heat map. There were 88 proliferation-related differential genes in the Lb-miR166a group compared with the N-Con group, including 29 upregulated genes and 59 downregulated genes (Figure 5a).
The above differentially expressed proliferation genes were annotated and analyzed by GO and KEGG enrichment analysis, and 6 proliferation-related GO entries and 1 KEGG pathway were enriched (P < 0.05), as shown in Figure 5b,c.
To verify the effect of Lb-miR166a on cell proliferation, we first evaluated the proliferation activity of the three groups using a Cell Counting Kit-8 cell proliferation assay. The results showed that the cell optical density value in the Lb-miR166a group was significantly lower than that in the N-Con and L-Con groups (P < 0.05), whereas there was no significant difference between the N-Con and L-Con groups (P > 0.05; Figure 6a). The results showed that Lb-miR166a significantly inhibited renal cell viability. Simultaneously, we compared the cell cycle distribution of the three groups using flow cytometry (Figure 6b,c). The results showed that the proportion of cells in the G0/G1 phase increased in the Lb-miR166a group, whereas the proportion of cells in the S and G2/M phases decreased, indicating that the cells in the Lb-miR166a group were blocked in the G0/G1 phase. Western blotting showed that the expression levels of ID4, CCND2, CCNB1, CCNA2, CDC20, and CCNE2 in the Lb-miR166a group were downregulated (P < 0.05); this trend was consistent with the results of transcriptome sequencing (Figure 6d,e).
Lb-miR166a promotes apoptosis of Caki-1 cells
Apoptosis is a process of spontaneous programmed cell death, which is closely related to the occurrence and development of tumors. To further clarify the effect of Lb-miR166a on apoptosis-related genes and pathways, we screened out apoptosis-related differential genes based on gene function annotation and drew a circular heat map. It can be seen that there were 89 differential genes in the Lb-miR166a group compared with the N-Con group, including 60 upregulated and 29 downregulated apoptosis-related genes (Figure 7a).
The above differentially expressed apoptosis genes were annotated and analyzed by GO and KEGG pathways, and 11 apoptosis-related GO entries and one KEGG pathway were enriched (P < 0.05), as shown in Figure 7b,c.
To verify the effect of Lb-miR166a on RCC cell apoptosis, we compared the apoptosis rates of the three groups using flow cytometry. The results showed that the apoptosis rate of the Lb-miR166a group was significantly higher than that of the N-Con and L-Con groups (P < 0.05); however, there was no significant difference between the N-Con and L-Con groups (P > 0.05) (Figure 8a,b).
We then detected the protein expression of apoptosis-related genes in the three groups of cells using western blotting. The results showed that the Lb-miR166a group inhibited the downregulation of the expression of the apoptosis protein Bcl2 (P < 0.05), while the expression levels of the apoptosis proteins Bad and Bax and the content of the cleaved Caspase-3 protein in the cells were significantly upregulated (P < 0.05); this trend was consistent with the transcriptome sequencing results (Figure 8c,d).
Gene interaction network reveals the effect of Lb-miR166a on cell apoptosis and proliferation
By selecting the key GO and KEGG pathways for cell proliferation and apoptosis, we demonstrated the effect of Lb-miR166a on the gene table of proliferation and apoptosis of RCC cells through the gene protein–protein interaction network. The squares in the figure represent different GO and KEGG pathways and the dots represent genes. Red indicates that the expression of the Lb-miR166a group is upregulated compared with that of the N-Con group, and green indicates that the expression of the Lb-miR166a group is downregulated compared with that of the N-Con group (Figure 9).
Lb-miR166a inhibits cell metastasis and invasion
Migration/invasion, the key initial step in tumor cell metastasis, is of great significance in the occurrence and development of tumors. To further verify the effect of Lb-miR166a on the migration and invasion of RCC cells, we determined the scratch healing rate of the three groups using a cell scratch experiment. The results showed that the scratch healing rate of cells in the Lb-miR166a group decreased significantly compared to that of those in the N-Con and L-Con groups (P < 0.05; Figure 10a,b).
The Transwell invasion experiment showed that compared to those in the N-Con and L-Con groups, the number of cancer cells migrating to the lower compartment in the Lb-miR166a group decreased significantly (P < 0.05). These results showed that Lb-miR166a inhibited the migration and invasion of RCC cells (Figure 10c,d).
Lb-miR166a inhibits tumor proliferation and metastasis in nude mice
The tumor-bearing experiment in nude mice showed that the subcutaneous tumor forming volume in the Lb-miR166a group was significantly smaller than that in the N-Con group (P < 0.05) (Figure 11a,b). The number of liver metastases in the Lb-miR166a group was significantly lower than that in the N-Con group (P < 0.05) (Figure 11c,d). Immunohistochemical detection of subcutaneous tumorigenesis in nude mice showed that the expression of Ki67 was decreased in the subcutaneous tumorigenesis of the Lb-miR166a group mice (Figure 11e,f).