Roles Of RPS6KA2 In RNA Polymerase ? And ?-directed Gene Transcription And Regulatory Mechanisms

RPS6KA2 gene is a member of the RSK(ribosomal S6 kinase)gene family encoding serine and threonine kinases.RPS6KA2,is a kinase containing two different kinase catalytic domains,participates in a variety of physiological and biochemical activities,including cell growth and cell differentiation,through phosphorylation different substrates.The results from the previous studies in our laboratory have shown that filamin A(FLNA)can inhibit expression of the genes transcribed by RNA polymerase I(Pol I)and RNA polymerase ?(Pol ?).Recent analysis with FLNA shRNA-expressing cell line by mRNA-seq showed that RPS6KA2 mRNA expression in SaOS2 cells was significantly increased by knocked-down of FLNA.This result was confirmed by analyzing different cell lines with the silence of FLNA expression.Previous studies have revealed that Ras and ERK signaling are involved in the regulation of Pol I and Pol ? gene transcription,and RPS6KA2 is one of the effectors in the signaling pathway.These information suggests that RPS6KA2 may be involved in the regulation of gene transcription directed by Pol ? and Pol ? or is involved in the regulation of Pol? and ? gene transcription mediated by FLNA.To investigate whether RPS6KA2 participates in Pol ? and Pol ?-directed gene transcription,we prepared RPS6KA2 shRNA –expressing lentiviral vector and used it to established RPS6KA2 shRNA-expressing stable cell line using this vector.RT-qPCR and Western blot were used to detect Pol ? and Pol ? gene expression in the cell lines stably expressing RPS6KA2 shRNA,and cell count and MTT assays were used to detect cell proliferation rate.The results showed that knockdown of RPS6KA2 significantly decreased expression of Pol ? and Pol ?-transcribed genes and cell proliferation,suggesting that RPS6KA2 may have a positive effect on the expression of Pol ? and Pol ?-transcribed genes and cell proliferation.To confirm this finding further,the stable cell lines with RPS6KA2 overexpression were established,and Pol ? and ? genetranscription was analyzed.It was observed that overexpression of RPS6KA2 significantly increased expression for a subset of the genes transcribed by Pol ? and ?,and enhanced cell proliferation.The above results confirm that RPS6KA2 can positively regulate Pol ? and ?-directed gene transcription and cell proliferation.To explore the mechanism by which RPS6KA2 regulates gene transcription directed by Pol ? and Pol?,the RPS6KA2 sh RNA-expressing stable cell line was used to analyze both transcription factors and signaling factors regulating Pol ? and ? gene transcription using RT-qPCR and Western blot The result showed that expression of Rho A and C-MYC was significantly reduced in the RPS6KA2 shRNA stable cell line,suggesting that RPS6KA2 possibly regulates Pol ? and ? gene transcription by controlling expression of Rho A and C-MYC.In summary,RPS6KA2 can positively regulate Pol?and?-gene transcription and cell proliferation in which the regulatory role of RPS6KA2 in these processes,is achieved by regulating expression of Rho A and C-MYC.The findings from this project broaden our understanding to the regulatory role of RPS6KA2 and the molecular mechanism of Pol ? and ? gene transcription,and provide a novel insight into the mechanism of cancer cell proliferation.