MiR-155-5p exerts tumor-suppressing functions in Wilms tumor by targeting IGF2 via the PI3K signaling pathway.
MicroRNA-155-5p (miR-155-5p) has been reported to play an oncogenic role in different human malignancies; however, its role in Wilms tumor (WT) remains unclear.
Differentially expressed miRNAs (DE-miRNAs) and mRNAs (DEGs) in WT blood and tissues were identified by using miRNA microarray and RNA-sequencing. Bioinformatics prediction and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to predict the potential functions of DE-miRNAs. DE-miRNAs and DEGs in WT obtained from Gene Expression Omnibus (GEO) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) were identified by using the "edgeR" package. RT-qPCR was used to explore miR-155-5p and IGF2 expression and their clinical significance in WT specimens. A rhabdoid cell line (G401) and Ewing sarcoma cell line (SK-NEP-1) were used. Immunohistochemical staining, western blotting and dual-luciferase reporter assays were performed to study the mechanisms involved. The CCK-8, flow cytometry, wound healing and transwell assays were performed to identify the effects of miR-155-5p and IGF2 knockdown on cell proliferation, apoptosis, migration and invasion, respectively.
MiR-155-5p was downregulated in both blood and tissues from WT patients who did not receive chemotherapy before surgery but was upregulated in tissues from WT patients who had received chemotherapy before surgery. IGF2, PI3K, AKT and mTOR were found to be upregulated in WT tissues. Additionally, miR-155-5p and IGF2 were significantly correlated with TNM stage and lymphatic metastasis in WT patients. Molecular mechanism exploration indicated that IGF2 was downregulated by miR-155-5p via direct binding to its 3' untranslated region in cell lines. Furthermore, IGF2, PI3K, AKT and mTOR expression was inversely correlated with miR-155-5p expression, and PI3K, AKT and mTOR expression was positively correlated with IGF2 expression in cell culture. Functional studies demonstrated that miR-155-5p upregulation and IGF2 knockdown suppressed cell proliferation, migration and invasion and induced cell apoptosis. Moreover, the tumor-suppressing effects of miR-155-5p in cells were abrogated by miR-155-5p inhibitor treatment.
Taken together, these findings suggest that miR-155-5p functions as a tumor suppressor in WT through inactivating the PI3K/AKT/mTOR signaling pathway by directly targeting IGF2. Thus, miR-155-5p might be a novel therapeutic target for WT.
Luo X
，Dong J
，He X
，Shen L
，Long C
，Liu F
，Liu X
，Lin T
，He D
，Wei G
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Effects of miR-202-5p silencing PIK3CA gene expression on proliferation, invasion, and epithelial-mesenchymal transition of cervical cancer SiHa cells through inhibiting PI3K/Akt/mTOR signaling pathway activation.
To explore the mechanism of miR-202-5p targeting the expression of PIK3CA and mediating the activation of PI3K/Akt/mTOR signaling pathway on the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of cervical cancer. The objects of study were 105 cases of cervical cancer and their corresponding normal tissues. qRT-PCR was used to detect the expression of miR-202-5p and PIK3CA in adjacent normal tissue and cervical cancer tissue. Dual luciferase reporter assay was used to verify the targeting relationship between miR-202-5p and PIK3CA gene. Human cervical cancer cell lines HPV-16E6, SiHa, HeLa, and CaSki were purchased for our cell experiments. The expression levels of PIK3CA in the cells were detected by qRT-PCR. The cell line with higher expression levels was selected to complete the follow-up experiment. The cultured cells were transfected and divided into the miR-202-5p mimic NC group, miR-202-5p mimic group, miR-202-5p inhibitor NC group, miR-202-5p inhibitor group, siRNA-PIK3CA NC group, siRNA-PIK3CA group, miR-202-5p inhibitor NC + siRNA-PIK3CA NC group, miR-202-5p inhibitor + siRNA-PIK3CA NC group, and miR-202-5p inhibitor + siRNA-PIK3CA group. QRT-PCR was used to detect the expression of miR-202-5p. Western blot and qRT-PCR were applied to detect the mRNA and protein expression levels of related pathway proteins (PIK3CA, PI3K, PTEN, p-Akt1, and p-mTOR) and epithelial-mesenchymal transition-related factors (N-cadherin, E-cadherin, and vimentin). Cell proliferation was detected by plate colony formation assay. Transwell assay was used to detect the invasion ability of each group. When compared with the adjacent tissues, PIK3CA mRNA expression level was significantly increased and miR-202-5p expression level was significantly decreased in cervical cancer tissues (all P < 0.05). PIK3CA was a target gene of miR-202-5p. The mRNA expression level of PIK3CA in SiHa cervical cancer cells was significantly higher than that in CaSki, HeLa, and HPV-16E6 cells (all P < 0.05), and SiHa cervical cancer cells were selected to complete the follow-up experiments. When compared with the corresponding NC group, the expression of miR-202-5p in miR-202-5p mimic group was increased. In addition, the mRNA and protein expression levels of E-cadherin and PTEN in miR-202-5p mimic and siRNA-PIK3CA groups were increased, and the protein expression of p-Akt1 and p-mTOR was decreased, and also, the mRNA and protein expression levels of PIK3CA, PI3K, N-cadherin, and vimentin were decreased (all P < 0.05); in miR-202-5p inhibitor group, the expression levels of miR-202-5p, E-cadherin, and PTEN decreased, the protein expression of p-Akt1 and p-mTOR increased, and the mRNA and protein expression of PIK3CA, PI3K, N-cadherin, and vimentin increased in miR-202-5p inhibitor group (all P < 0.05); in miR-202-5p inhibitor + siRNA-PIK3CA group, the expression of miR-202-5p decreased (P < 0.05), but the mRNA and protein expression of PIK3CA, PI3K, p-Akt1, p-mTOR, N-cadherin, E-cadherin, and vimentin had no significant changes (all P > 0.05). When compared with the corresponding NC group, the number of cell clones in miR-202-5p mimic group and siRNA-PIK3CA group was decreased, and the invasion ability of miR-202-5p inhibitor group was increased, and the invasion ability was enhanced (all P < 0.05); miR-202-5p inhibitor + siRNA-PIK3CA group showed no significant change in the number of cell clones and the rate of invasion (P > 0.05). In conclusion, the overexpression of miR-202-5p can suppress PIK3CA gene expression and the activation of PI3K/Akt/mTOR signaling pathway to suppress the proliferation, invasion, and EMT of cervical cancer.
Zheng Y
，Xie L
，Xu S
，Yan W
，Zhang H
，Meng Y
，Liu J
，Wei X
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