miR-155 regulates the proliferation and invasion of clear cell renal cell carcinoma cells by targeting E2F2

Abstract

MicroRNAs (miRNAs) have emerged as critical modulators of carcinogenesis and tumor progression. In the present work, we sought to identify the biological function of miR-155 as well as its underlying mechanism in clear cell renal cell carcinoma (ccRCC). We examined the expression of miR-155 in clear cell RCC (ccRCC) and adjacent normal tissues and then explored the roles of miR-155 both in vitro and in vivo. The results of this analysis indicated that miR-155 activity was significantly upregulated in ccRCC tissues compared with the corresponding normal tissues. miR-155 was associated with ccRCC aggressiveness in both cell lines and clinical specimens, and a specific and inverse correlation between miR-155 and E2F2 expression was found in human ccRCC samples. Overexpression of miR-155 in 786-O cells decreased E2F2 expression while reduction of miR-155 by anti-miR-155 in ACHN cells elevated E2F2 expression. Re-expression of E2F2 in 786-O cells repressed the cell migration/invasion abilities elevated by miR-155, whereas knockdown of E2F2 in ACHN cells restored these cellular functions hampered by the miR-155 inhibitor. Using Western blot and luciferase reporter assays, we determined that E2F2 was a direct target of miR-155. Taken together, the in vitro and in vivo results demonstrate that miR-155 functions as a tumor-promoting miRNA by targeting E2F2 in ccRCC.

Keywords: E2F2; clear cell renal cell carcinoma; miR-155; proliferation and invasion.

Figure 1. Expression of miR-155 in clinical samples and various cell lines and its relationship with E2F2 expression

(A) miR-155 expression levels were significantly upregulated in ccRCC cancer tissues compared with normal tissues. (B–D) Correlations between miR-155 expression level and the clinical stage, T stage, Fuhrman grades of ccRCC respectively. (E) miR-155 expressions in various RCC cell lines compared with that in HKC. (F) Downregulation of E2F2 mRNA levels in ccRCC cancer tissues; here, normal tissues served as controls. (G) Correlations between E2F2 expression level and the clinical stage of ccRCC. (H) Negative correlation of E2F2 mRNA levels and miR-155 levels (n = 54, r² = 0.4121, P < 0.0001). (I) Representative images of E2F2 IHC in ccRCC cancer tissues and their paired normal tissues. (J) Western blot of E2F2 showed alterations in protein levels consistent with variations in mRNA levels in clinical samples. Data represent the mean ± SD. (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 2. Influence of miR-155 on tumor cell proliferation, migration, and invasion

(A) Alteration of the miR-155 expression levels of 786-O cells 48 h after transfection with the miR-155 or NC mimics. (B) Alteration of the miR-155 expression levels of ACHN cells 48 h after transfection with the miR-155 or NC inhibitors. (C) MTS assay showed that transfection of the miR-155 mimic can significantly increase the proliferation velocity of 786-O cells and that the miR-155 inhibitor can attenuate proliferation in ACHN cells. (D) Effect of miR-155 overexpression and suppression on the colony formation of 786-O and ACHN cells, respectively. The number of foci of > 50 cells was counted after 14 d. (E) Representative photographs of transwell assays (magnification, ×100) of 786-O and ACHN cells to determine the oncogenic function of miR-155. (F) Wound healing assay was performed to reveal that the miR-155 mimic increases the cell viability of 786-O cells. In ACHN cells, miR-155 inhibition hampered cell motility. Data represent the mean ± SD. (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3. Effects of E2F2 on in vitro cell proliferation, motility, and migration

(A) Alteration of E2F2 expression levels in 786-O cells 48 h after siRNA treatment compared with siNC. (B) Alteration of E2F2 expression levels in ACHN cells 48 h after transfection with lentiviral E2F2 plasmids compared with the empty vector. (C) MTS assay revealed that transfection of the siRNA of E2F2 significantly accelerated proliferation velocity in 786-O cells while E2F2 overexpression attenuated proliferation in ACHN cells. (D) Effect of E2F2 overexpression or suppression on the colony formation of ACHN and 786-O cells, respectively. The number of foci of > 50 cells was counted after 14 d. (E) Representative photographs of transwell assays (magnification, ×100) of 786-O and ACHN cells identifying E2F2 as a tumor suppressor. (F) E2F2 knockdown in 786-O cells significantly increased the number of viable cells; in ACHN cells, E2F2 overexpression largely decreased cell viability. Data represent the mean ± SD. Each experiment was performed in triplicate. (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 4. miR-155 downregulated E2F2 expression by specifically targeting its 3′UTR

(A) Sequence alignment of the E2F2 3′UTR with wild-type (WT) versus mutant (MUT) potential miR-155 targeting sites. (B) mRNA levels of E2F2 were examined by qRT-PCR in 786-O and ACHN cells with different interferences. (C) Transfection of the miR-155 mimic or miR-155 inhibitor in 786-O and ACHN greatly changed E2F2 protein levels. (D) Immunofluorescence staining results showed the inverse effect of miR-155 on E2F2 in different cell lines. (E) Luciferase reporter assay showed decreased reporter activity after transfection of the wild-type E2F2 3′UTR reporter construct in 293T cells overexpressing miR-155. The E2F2 3′UTR MUT and control constructs showed no effect on reporter activity. Here, the Renilla luciferase construct was used as an internal control. The normalized luciferase activity of the control construct in each experiment was set to 1. Data represent the mean ± SD. (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 5. miR-155 regulated the proliferation, migration, and invason of ccRCC by targeting E2F2

(A) miR-155 and E2F2 mRNA level changes in 786-O cells with the miR-155 mimic (versus the control) and the E2F2 plasmid (versus the control) 48 h after transfection. (B) The mRNA levels of miR-155 and E2F2 in ACHN were analyzed by qRT-PCR after the use of miR-155 inhibitor and siE2F2. (C) E2F2 protein level alterations in 786-O cells with the miR-155 mimic (versus the control) and the E2F2 plasmid (versus the control) 48 h after transfection. After co-transfection of ACHN cells with siRNA duplexes (siE2F2 or the negative control) and miR-155 inhibitors (miR-155 or the negative control), the levels of E2F2 protein and EMT-related proteins were measured by Western blot. (D) E2F2 overexpression counteracted the positive proliferative effects of miR-155 in 786-O cells. (E) E2F2 inhibition hampered the negative proliferative function of miR-155 inhibitor in ACHN cells. (F) Cell migration and invasion in 786-O cells were analyzed by transwell assays. Cells were co-transfected with lentiviral particles (E2F2 or the empty vector) and miR-155 (mimic or the NC mimic). (G) Knockdown of E2F2 may reverse the effect of the miR-155 inhibitor on migration and invasion in ACHN cells. Data represent the mean ± SD. (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 6. Overexpression miR-155 promoted tumor growth in vivo

(A) Mouse tumors were obtained and dissected 4 weeks after subcutaneous injection of the transfected 786-O/miR-155 plasmid or 786-O/EV transfected cells. (B, C) Comparison of tumor weights and volumes between the 786-O/miR-155 and 786-O/EV groups. (D) Protein levels of E2F2 were immunohistochemically evaluated in the experimental and control groups. (E) EMT-related markers, including E-cadherin, ZEB1, and vimentin, were detected by Western blot between the groups. Data represent the mean ± SD. (*P < 0.05; **P < 0.01; ***P < 0.001).