Downregulation of Sec23A Protein by miRNA-375 in Prostate Carcinoma

Abstract

Prostate carcinoma (CaP) is a leading cause of cancer-related death in men. We have previously determined the microRNA (miRNA) profile of primary CaP in comparison with nontumor prostate tissue. miRNAs are small, noncoding RNAs that inhibit protein synthesis on a posttranscriptional level by binding to the 3 0 -untranslated region (3 0 -UTR) of their target genes. In primary CaP tissue, we have previously found by miRNA sequencing that miR-375 and miR-200c were upregulated 9.1-and 4.5-fold, respectively. A computational analysis predicted the 3 0 -UTR of the SEC23A gene as a potential target for both miR-375 and miR-200c. Here, we show that the 3 0 -UTR of SEC23A mRNA is indeed a target for miR-375 and miR-200c and that both miRNAs downregulate Sec23A protein expression when ectopically expressed in human 293T cells. In primary samples of CaP, we found a direct correlation between reduction of SEC23A mRNA and overexpression of miR-375 but not of miR-200c. The reduced levels of Sec23A protein were inversely correlated to the increased amount of miR-375 in the LNCaP and DU145 CaP cell lines when compared with normal prostate fibroblasts.

Comparative microRNA Profiling of Prostate Carcinomas with Increasing Tumor Stage by Deep Sequencing

“…3A), which are identical for miR-200b and miR-200c. We could previously show that miR-200c functionally interacts with one of these binding sites (9). In the present analysis, we found an upregulation of miR-200b, which shares the seed sequence with miR-200c.…”

“…To obtain the pSG5-miR200b expression construct, the nucleotides 1102390-1102757 of chromosome 1 (NC_000001.10) were amplified by PCR with specific primers and inserted into the pSG5 expression vector. The pSG5-miR-375 expression vector was described previously (9). The dual luciferase reporter plasmid pMIR-RNL, the pMIR-RNL-SEC23A reporter vector, and the appropriately mutated pMIR-RNL-SEC23A reporter constructs were described elsewhere (9).…”

“…The pSG5-miR-375 expression vector was described previously (9). The dual luciferase reporter plasmid pMIR-RNL, the pMIR-RNL-SEC23A reporter vector, and the appropriately mutated pMIR-RNL-SEC23A reporter constructs were described elsewhere (9). The nucleotides 39-905 of the PHLPP1 3 0 UTR (accession number: NM_194449.3) and the nucleotides 1-968 of the PHLPP2 3 0 UTR (accession number: NM_015020.2) were amplified via PCR using specific primers from testis cDNA and inserted via the SpeI, SacI, or NaeI restriction sites in pMIR-RL.…”

“…Furthermore, the SEC23A mRNA is a target for the induced miR-200c and miR-375 with concomitant protein downregulation of SEC23A protein in prostate carcinoma. Accordingly, the overexpression of Sec23A resulted in reduced growth of prostate cancer cell lines (9). In addition, the downderegulated miRNA miR-24 targets the ZNF217 mRNA; the subsequently induced ZNF217 protein is a known oncogene in other tumors and probably also plays a role in prostate carcinoma (10 and references therein).…”

Circulating microRNAs: macro-utility as markers of prostate cancer?

“…For example, miR-375 is a serum/plasma marker of PCa and is also consistently upregulated in tumors (Szczyrba et al 2010, Brase et al 2011, Selth et al 2011, Szczyrba et al 2011, Bryant et al 2012. By interrogating data from the Prostate Cancer OncoGenome Project (Taylor et al 2010), we found that the intratumoral expression of miR-375 at the time of RP can predict biochemical relapse, with high expression being associated with an unfavorable outcome (hazard ratioZ3.49; Selth et al 2011).…”