title: RNA sequence analyses of r-Moj-DM treated cells: TXNIP is required to induce apoptosis of SK-Mel-28.
abstract: RNA sequencing of untreated and r-Moj-DM treated SK-Mel-28 cells was performed after 6 h, to begin unraveling the apoptotic pathway induced by r-Moj-DM. Bioinformatic analyses of RNA sequencing data yielded 40 genes that were differentially expressed. Nine genes were upregulated and 31 were downregulated. qRT-PCR was used to validate differential expression of 13 genes with known survival or apoptotic-inducing activities. Expression of BNiP3, IGFBP3, PTPSF, Prune 2, TGF-ß, and TXNIP were compared from cells treated with r-Moj-DN (a strong apoptotic inducer) or r-Moj-DA (a non-apoptotic inducer) for 1 h, 2 h, 4 h, and 6 h after treatment. Our results demonstrate that significant differences in expression are only detected after 4 h of treatment. In addition, expression of TXNIP (an apoptotic inducer) remains elevated at 4 h and 6 h only in r-Moj-DN treated cells. Based on the consistency of elevated TXNIP expression, we further studied TXNIP as a novel target of disintegrin activation. Confocal microscopy of anti-TXNIP stained SK-Mel-28 cells suggests nuclear localization of TXNIP after r-Moj-DM treatment. A stable TXNIP knockdown SK-Mel-28 cell line was produced to test TXNIP' role in the apoptotic induction by r-Moj-DM. High cell viability (74.3% ±9.1) was obtained after r-Moj-DM treatment of TXNIP knocked down SK-Mel-28 cells, compared to 34% ±0.187 for untransduced cells. These results suggest that TXNIP is required early in the apoptotic-inducing pathway resulting from r-Moj-DM binding to the αv integrin subunit.
authors: Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
authors: Terri D
authors: TD
authors: McBride
authors: Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
authors: U
authors: U
authors: Andrew
authors: Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
authors: Nicko
authors: N
authors: Ly
authors: Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA. Electronic address: Julio.Soto@sjsu.edu.
authors: Julio G
authors: JG
authors: Soto
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D019483:Disintegrins / Q000633:toxicity*; D055785:Gene Knockdown Techniques; D006801:Humans; D017423:Sequence Analysis, RNA
chemical_list: D002352:Carrier Proteins; D019483:Disintegrins; C486715:TXNIP protein, human; C554078:mojastin protein, Crotalus scutulatus
keywords: Cell viability; RNA sequencing; Recombinant RGD disintegrin; TXNIP knockdowns; TXNIP protein localization