The Src family of protein kinases (SFKs) plays key roles in regulating fundamental cellular processes, including cell growth, differentiation, cell shape, migration and survival, and specialized cell signals in various malignancies. The pleotropic functions of SFKs in cancer make them promising targets for intervention. Here we sought to investigate the role of miR-205 in inhibition of Src-mediated oncogenic pathways in renal cancer. We report that expression of miR-205 was significantly suppressed in renal cancer cell lines and tumors when compared with normal tissues and a non-malignant cell line, and is correlated inversely with the expression of SFKs. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3’UTR sequences complementary to either Src, Lyn or Yes, which was abolished by mutations in these 3’UTR regions. Over-expression of miR-205 in A498 cells reduced Src, Lyn and Yes expression both at mRNA and protein levels. Proliferation of renal cancer cells was suppressed by miR-205, mediated by the phosphoSrc-regulated ERK1/2 pathway. Cell motility factor- FAK and STAT3 activation was also inhibited by miR-205. Transient as well as stable over-expression of miR-205 in A498 cells resulted in induction of G0/G1 cell cycle arrest and apoptosis as indicated by decreased levels of cyclin D1 and cMyc, suppressed cell proliferation, colony formation, migration, and invasion in renal cancer cells. miR-205 also inhibited tumor cell growth in vivo. This is the first study demonstrating that miRNA-205 inhibits protooncogenic Src family of kinases indicating a therapeutic potential of miR-205 in the treatment of renal cancer.

BackgroundmiR-23b is located on chromosome number 9 and plays different roles in different organs especially with regards to cancer development. However, the functional significance of miR-23b-3p in renal cell carcinoma (RCC) has not been reported.Methods and ResultsWe measured miR-23b-3p levels in 29 pairs of renal cell carcinoma and their normal matched tissues using real-time PCR. The expression level of miR-23b-3p was correlated with the 5 year survival rate of renal cancer patients. In 15 cases (52%), miR-23b-3p expression was found to be high. All patients with moderate to low miR-23b-3p expression survived 5 years, while those with high miR-23b-3p expression, only 50% survived. After knocking down miRNA-23b-3p expression in RCC cell lines, there was an induction of apoptosis and reduced invasive capabilities. MiR-23b-3p was shown to directly target PTEN gene through 3′UTR reporter assays. Inhibition of miR-23b-3p induces PTEN gene expression with a concomitant reduction in PI3-kinase, total Akt and IL-32. Immunohistochemistry showed the lack of PTEN protein expression in cancerous regions of tissue samples where the expression of miR-23b-3p was high. We studied the in vitro effects of the dietary chemo preventive agent genistein on miR-23b-3p expression and found that it inhibited expression of miR-23b-3p in RCC cell lines.ConclusionsThe current study shows that miR-23b-3p is an oncogenic miRNA and inhibits PTEN tumor suppressor gene in RCC. Therefore, inhibition of miR-23b-3p may be a useful therapeutic target for the treatment of renal cell carcinoma.

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.

Background:The purpose of this study was to identify new tumour suppressor microRNAs (miRs) in clear cell renal cell carcinoma (ccRCC), carry out functional analysis of their suppressive role and identify their specific target genes.Methods:To explore suppressor miRs in RCC, miR microarray and real-time PCR were performed using HK-2 and A-498 cells. Cell viability, invasion and wound healing assays were carried out for functional analysis after miR transfection. To determine target genes of miR, we used messenger RNA (mRNA) microarray and target scan algorithms to identify target oncogenes. A 3′UTR luciferase assay was also performed. Protein expression of target genes in ccRCC tissues was confirmed by immunohistochemistry and was compared with miR-584 expression in ccRCC tissues.Results:Expression of miR-584 in RCC (A-498 and 769-P) cells was downregulated compared with HK-2 cells. Transfection of miR-584 dramatically decreased cell motility. The ROCK-1 mRNA was inhibited by miR-584 and predicted to be target gene. The miR-584 decreased 3′UTR luciferase activity of ROCK-1 and ROCK-1 protein expression. Low expression of miR-584 in ccRCC tissues was correlated with high expression of ROCK-1 protein. The knockdown of ROCK-1 by siRNA inhibited cell motility.Conclusion:miR-584 is a new tumour suppressor miR in ccRCC and inhibits cell motility through downregulation of ROCK-1.

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Brassica juncea is a major oilseed crop in tropical and subtropical countries, especially in south-east Asia like India, China, Bangladesh, and Pakistan. The widespread cultivation of genetically similar varieties tends to attract fungal pathogens which cause heavy yield losses in the absence of resistant sources. The conventional disease management techniques are often expensive, have limited efficacy, and cause additional harm to the environment. A substantial approach is to identify and use of resistance sources within the Brassica hosts and other non-hosts to ensure sustainable oilseed crop production. In the present review, we discuss six major fungal pathogens of B. juncea: Sclerotinia stem rot (Sclerotinia sclerotiorum), Alternaria blight (Alternaria brassicae), White rust (Albugo candida), Downy mildew (Hyaloperonospora parasitica), Powdery mildew (Erysiphe cruciferarum), and Blackleg (Leptoshaeria maculans). From discussing studies on pathogen prevalence in B. juncea, the review then focuses on highlighting the resistance sources and quantitative trait loci/gene identified so far from Brassicaceae and non-filial sources against these fungal pathogens. The problems in the identification of resistance sources for B. juncea concerning genome complexity in host subpopulation and pathotypes were addressed. Emphasis has been laid on more elaborate and coordinated research to identify and deploy R genes, robust techniques, and research materials. Examples of fully characterized genes conferring resistance have been discussed that can be transformed into B. juncea using advanced genomics tools. Lastly, effective strategies for B. juncea improvement through introgression of novel R genes, development of pre-breeding resistant lines, characterization of pathotypes, and defense-related secondary metabolites have been provided suggesting the plan for the development of resistant B. juncea.

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