microRNA-302c-3p inhibits renal cell carcinoma cell proliferation by targeting Grb2-associated binding 2 (Gab2)

Abstract

The expression and biological function of Grb2-associated binding 2 (Gab2) in renal cell carcinoma (RCC) cells was tested here. We showed that Gab2 expression was significantly elevated in human RCC tissues and RCC cells. It was correlated with over-activation of Akt and downregulation of microRNA-302c-3p ("miR-302c-3p"), a putative Gab2-targeting microRNA. Knockdown of Gab2 inhibited Akt activation and 786-O RCC cell proliferation. Reversely, forced over-expression of Gab2 led to Akt hyper-activation to facilitate 786-O cell proliferation. Exogenous expression of miR-302c caused Gab2 downregulation, Akt inhibition and 786-O cell proliferation inhibition. On the other hand, miR-302c-3p depletion by expressing its anti-sense ("antagomiR-302c") led to Gab2 upregulation, Akt activation and increased 786-O cell proliferation. Significantly, miR-302c-3p failed to affect the proliferation of 786-O cells with shRNA-depleted Gab2. Together, we suggest that miR-302c-3p depletion in human RCC cells leads to Gab2 over-expression, Akt hyper-activation and cell proliferation.

Keywords: Akt and cell proliferation; Grb2-associated binding 2 (Gab2); microRNA-302c-3p; renal cell carcinoma (RCC).

Figure 1. Gab2 over-expression correlates with miR-302c-3p downregulation and Akt hyper-activation in RCC tissues and RCC cells

(A) miR-302c-3p putatively targets the 3′ untranslated regions (UTR) of Gab2 (at position 125-131). A total of eleven (11) human RCC tumor tissues (“T”) and their paired surrounding normal renal tissues (“N”) were subjected to quantitative real-time PCR (“qRT-PCR”) assay to test expression of Gab2 mRNA (B) and miR-302c-3p (C); Listen protein expression was tested by Western blot assay, data were quantified (D). Expressions of Gab2 mRNA (E), miR-302c-3p (F) and listed proteins (G) in established human RCC cell lines (786-O and A498), primary human RCC cells [RCC (P1) and RCC (P2)] and HK-2 tubular epithelial cells were tested. Gab2 and p-Akt expressions were quantified (vs. Tubulin, G). *p < 0.05 vs. “N” group (B, C and D) or HK-2 cells (E and F).

Figure 2. Gab2 shRNA knockdown inhibits Akt activation and 786-O RCC cell proliferation

Expressions of Gab2 mRNA (A) and listed proteins (B) in 786-O RCC cells with indicated Gab2 shRNA (“shGab2-a” or “shGab2-b”) or scramble shRNA (“shSCR”) were shown; Cells were also subjected to the listed proliferation assays (C–F). At the beginning of these assays, exact same amount of viable cells with different background were plated (C–F). Gab2 and p-Akt expressions were quantified (vs. Tubulin, B). *p < 0.05 vs. “shSCR” group. Experiments in this figure were repeated five times, and similar results were obtained.

Figure 3. siRNA knockdown of Gab2 inhibits primary RCC cell proliferation

Expressions of Gab2 mRNA (A) and listed proteins (B) in the primary RCC cells (P2), transfected with Gab2 siRNA (“si-Gab2”) or control non-sense siRNA (“si-C”), were shown. Cells were also subjected to MTT assay (C) and BrdU ELISA assay (D) to test cell proliferation. For the proliferation assays, exact same amount of viable cells with “si-C” or “si-Gab2” were initially plated (C–D). Gab2 and p-Akt expressions were quantified (vs. Tubulin, B). *p < 0.05 vs. “si-C” group. Experiments in this figure were repeated five times, and similar results were obtained.

Figure 4. Gab2 over-expression facilitates 786-O cell proliferation

Expressions of Gab2 mRNA (A) and indicated proteins (B) in the stable 786-O RCC cells, with wt-Gab1 (two lines, “-a”/“-b”) or empty vector (“Vec”, pSuper-puro-GFP-Flag), were shown. Above cells were also subjected to the listed proliferation assays (C–E). For the proliferation assays, exact same amount of viable cells with different background were plated (C-E). Gab2 and p-Akt expressions were quantified (vs. Tubulin, B). *p < 0.05 vs. “Vec” group. Experiments in this figure were repeated three times, and similar results were obtained.

Figure 5. Exogenous expression of miR-302c silences Gab2 and inhibits 786-O cell proliferation

Stable 786-O cells, expressing miR-302c, miR-control (“miR-C”) or the empty vector (“Vec”, pSuper-puro-GFP-Flag), were subjected to qRT-PCR assay of miR-302c-3p (A) and Gab2 mRNA (B); Expression of listed proteins was tested, and results were quantified (C); MTT assay (D) and BrdU ELISA (E) were also applied to test cell proliferation. Stable 786-O cells, with indicated Gab2 shRNA (“shGab2-a” or “shGab2-b”), were transfected with miR-302c or miR-control (“miR-C”), miR-302c-3p expression (F and G) and cell proliferation (MTT assay, H and I) were tested. For the proliferation assays, exact same amount of viable cells with different background were initially plated (D, E, G and I). Gab2 and p-Akt expressions were quantified (vs. Tubulin, C). *p < 0.05 vs. “Vec”/“miR-C” group. Experiments in this figure were repeated three times, and similar results were obtained.

Figure 6. AntagomiR-302c depletes miR-302c, causing Gab2 upregulation, Akt activation and 786-O cell proliferation

Stable 786-O cells, expressing antagomiR-302c or antagomiR-control (“antagomiR-C”), were subjected to qRT-PCR assay of miR-302c-3p (A) and Gab2 mRNA (B); Expression of listed proteins was shown (C); MTT assay (D) and BrdU ELISA (E) were employed to test cell proliferation. For the proliferation assays, exact same amount of viable cells with different background were initially plated (D–E). Gab2 and p-Akt expressions were quantified (vs. Tubulin, C). *p < 0.05 vs. “antagomiR-C” group. Experiments in this figure were repeated four times, and similar results were obtained.