Up-regulation of microRNA-21 correlates with lower kidney cancer survival.

BackgroundMicroRNA-21 is up-regulated in a variety of cancers like, breast, colorectal, lung, head and neck etc. However, the regulation of miR-21 in renal cell carcinoma (RCC) has not yet been studied systematically.Methods and resultsWe measured miR-21 levels in 54 pairs of kidney cancers and their normal matched tissues by real-time PCR. The expression level of miR-21 was correlated with 5 year survival and the pathological stage. Functional studies were done after inhibiting miR-21 in RCC cell lines. We studied in vitro and in vivo effects of the chemo preventive agent genistein on miR-21 expression. In 48 cases (90%), miR-21 was increased. All patients with low miR-21 expression survived 5 years, while with high miR-21 expression, only 50% survived. Higher expression of miR-21 is associated with an increase in the stage of renal cancer. Functional studies after inhibiting miRNA-21 in RCC cell lines show cell cycle arrest, induction of apoptosis and reduced invasive and migratory capabilities. Western blot analysis showed an increase in the expression of p21 and p38 MAP kinase genes and a reduction in cyclin E2. Genistein inhibited the expression of miR-21 in A-498 cells and in the tumors formed after injecting genistein treated A-498 cells in nude mice besides inhibiting tumor formation.ConclusionsThe current study shows a clear correlation between miR-21 expression and clinical characteristics of renal cancer. Thus we believe that miR-21 can be used as a tumor marker and its inhibition may prove to be useful in controlling cancers with up-regulated miR-21

Up-Regulation of MicroRNA-21 Correlates with Lower Kidney Cancer Survival

MicroRNA-21 is up-regulated in a variety of cancers like, breast, colorectal, lung, head and neck etc. However, the regulation of miR-21 in renal cell carcinoma (RCC) has not yet been studied systematically.We measured miR-21 levels in 54 pairs of kidney cancers and their normal matched tissues by real-time PCR. The expression level of miR-21 was correlated with 5 year survival and the pathological stage. Functional studies were done after inhibiting miR-21 in RCC cell lines. We studied in vitro and in vivo effects of the chemo preventive agent genistein on miR-21 expression. In 48 cases (90%), miR-21 was increased. All patients with low miR-21 expression survived 5 years, while with high miR-21 expression, only 50% survived. Higher expression of miR-21 is associated with an increase in the stage of renal cancer. Functional studies after inhibiting miRNA-21 in RCC cell lines show cell cycle arrest, induction of apoptosis and reduced invasive and migratory capabilities. Western blot analysis showed an increase in the expression of p21 and p38 MAP kinase genes and a reduction in cyclin E2. Genistein inhibited the expression of miR-21 in A-498 cells and in the tumors formed after injecting genistein treated A-498 cells in nude mice besides inhibiting tumor formation.The current study shows a clear correlation between miR-21 expression and clinical characteristics of renal cancer. Thus we believe that miR-21 can be used as a tumor marker and its inhibition may prove to be useful in controlling cancers with up-regulated miR-21

Correlation of miR-21 expression with 5 year survival of renal cell carcinoma patients.

<p>Correlation of miR-21 expression with 5 year survival of renal cell carcinoma patients.</p

Effect on cell cycle and apoptosis after knocking down miR-21 in A-498 cells.

<p>(a) Effect on cell cycle: Flow cytometry analysis of the cell cycle showed a significant increase in G0/G1 phase (9.1%), with a concomitant reduction in the G2/M phase (6.8%), indicating that the forced reduction of miR-21 expression in A-498 cells could inhibit the cell growth and cause G0/G1 arrest (p value 0.010). (b) Effect on apoptosis: Apoptosis assay showed a marginal increase in the number of total apoptotic cells (5.17%) in the miR-21 anti-miRNA inhibitor transfected cells as compared to the negative control (1.79%) and mock (2.49%) (p value 0.017).</p

<i>In vitro</i> and <i>In vivo</i> effect of genistein on the expression of miR-21.

<p>(a) Effect of genistein (25 µM) on the expression of miR-21 in A-498 cells: miR-21 expression was found to be reduced by ∼30% in genistein treated A-498 cells as compared to untreated cells. (b) <i>In vivo</i> effect of genistein on tumor size in nude mice: Genistein was able to reduce tumor formation significantly (black arrows, right panel).</p

Western analysis after the knock down of miR-21 in A-498 cells.

<p>Expression of p21, p38MAP kinase and cyclin E2 after the inhibition of miR-21 in A-498 cells, as compared to negative control. GAPDH was used as a normalizing control. p21 and p38 MAP kinase were up regulated, whereas cyclin E2 expression was found to be down regulated.</p

Effect on cell invasion and migration on A-498 cells after miR-21 inhibition.

<p>(a) Effect on cell invasion: Invasive properties of A-498 cells were decreased after the inhibition of miR-21 as compared to the negative control, Stained A-498 cells (Magnification-40×); Absorbance at 560 nm (p value 0.026). (b) Effect on cell migration: Migratory properties of A-498 cells were also decreased after the inhibition of miR-21 as compared to the negative control, Stained A-498 cells (Magnification-40×); Absorbance at 560 nm (p value 0.016).</p