LAUSR

Introduction Clear cell renal cell carcinoma (ccRCC) is the leading cause of cancer related deaths among urological malignancies. ccRCC arises through the acquisition of genetic and epigenetic alterations, of which histone modifications are emerging filed of interest. Histone methylation has been implemented in renal cancer but its clinical value and underlying pathology is still unexplored. Hence, the goal of present study was to elucidate the expression profile of histone 3 lysine 4 (H3K4) modifiers in ccRCC and to evaluate their role in tumour biology. Material and methods The expression profile of 20 H3K4 modifiers, including 13 methylases (HMTs) and 7 demethylases (HDMs), was estimated in 50 cases of ccRCC and adjacent normal tissues using RT-PCR. For functional analysis, siRNAs mediated gene silencing was used in A498 and ACHN cell lines followed by evaluation of tumour characteristics by FACS and MTT assay. The study was approved by Institute Ethics Committee (ref. no- NK/1597/Ph.D/10916). Results and discussions The data showed the differential expression of histone modifiers in ccRCC. Among 13 HMTs, 4 genes viz, MLL1 (p=0.012), MLL2 (p=0.024), SMYD2 (p=0.016) and NSD2 (p=0.004) was significantly up regulated in ccRCC in comparison to normal tissue. Similarly, out of 7 HDMs, the mRNA levels of 4 genes, KDM5A (p=0.001), KDM5B (p=0.047), LSD2 (p=0.002) and FBXL10 (p=0.000) was significantly augmented in ccRCC. Interestingly, when HMTs and HDMs were compared with one another, it was found that higher percentage of histone demethylases was significantly over-expressed with cumulative expression of 1.31 times elevated compared to methylases. This over-expression of HDMs might be engaged in the reduction of H3K4me code in ccRCC. Further, two demethylases LSD2 and KDM5A were selected for functional characterisation. Cell viability of A498 and ACHN cells was reduced after 48 hours of inhibition of LSD2 and KDM5A genes. This decreased in cell viability was due to induction of early apoptosis as revealed by annexin-V/PI staining using FACS. Further, cell cycle analysis showed the arrest of A498 and ACHN cells at S and sub-G1 phase after LSD2 and KDM5A knockdown. Taken together, these data suggest that LSD2 and KDM5A inhibition impaired cell proliferation with the induction of apoptosis and corresponding cell cycle arrest. Conclusion Our findings provide the novel insights behind the pathology involved in H3K4 methylation code alteration in ccRCC and further provide the drugable targets with therapeutic potential.