Investigation Of The Roles Of PCBP1 In Skin Squamous-Cell Carcinoma Stem Cell Maintenance

Objective:To examine the expression of poly C binding protein PCBP1 in the stem cell maintenance phase of the skin squamous-cell carcinoma (SCC). We also investigated its roles in the in vitro and in vivo tumorigenesis of the SCC, and further studied the intrinsic molecular mechanisms responsible for the repression of PCBP1 expression in the stem cell maintenance of the SCC.Methods:1. CSCs (Cancer Stem Cells) was enriched from the SCC cell line COLO-16 cells by applying the serum-free sphere growth method, followed by continued amplication, dedifferentiation. Cells were collected for flow cytometric and immunofluorescent confocal microscopy to detect the pluripotency-associated markers.2. The above CSCs were maintained in culture over 3 days, RNA and total protein were respectively exacted from these cells every day. Quantitative PCR (qPCR) and western blot were carried out to examine the mRNA and protein expression of PCBP1 in the time course.3. PCBP1 overexpressing CSCs and the related negative control cells were constructed. Soft agar and tumorigenesis in nude mice experiments were performed to characterize the roles of PCBP1 in SCC progression.4. Targetscan revealed that PCBP1 3’-UTR contained a highly conserved seed region for miR-490-3p. The expression of miR-490-3p was also analyzed during the CSC stem cell maintenance phase by qPCR.5. Polysome-associated mRNA was enriched and used for qPCR to detect the PCBP1 mRNA expression, to confirm the translation regulation of PCBP1 repression in the CSC stem cell maintenance.6. The wide-type and mutant luciferase reporters containing PCBP1 3’-UTR were constructed to study the direct regulation of PCBP1 expression by altered miR-490-3p levels in CSCs.7. Both PCBP1 and miR-490-3p expression were determined in a cohort of 20 SCC samples. The expression correlation of these two molecules was also analyzed.Results:1. Enrichment and amplification of CD34 positive CSC cells from the initial SCC COLO-16 cell line was successful. The pluripotency-associated markers SSEA4, TR1-60 and TR1-81 were all detected in these CSCs.2. During the stem cell maintenance phase, PCBP1 mRNA expression depicted stable, whereaus its protein level revealed a surprising downregulation.3. PCBP1 overexpressing CD34+CD44+ CSCs exhibited significantly reduced tumorigenic potential both in in vitro and in vivo studies.4. PCBP1 was a possible target of miR-490-3p by base pairing to its 3’-UTR region. The relative expression of miR-490-3p was increased steadily over the stem cell maintenance phase.5. Among the polysome-associated mRNA, PCBP1 mRNA was observed to be decreased during the late phase of SCC stem cell maintenance, which demonstrated that the downregulation of PCBP1 protein level might be translational repression after transcription.6. PCBP1 is a bona-fide target of miR-490-3p by applying in vitro luciferase reporter assays. The abnormal altered expression of miR-490-3p could directly impact the PCBP13’-UTR luciferase activity.7. In clinical SCC samples we found that miR-490-3p was upregulated and PCBP1 was downregulated at mRNA level. The negative correlation was found for these two molecules.Conclusion:PCBP1 is downregulated during SCC stem cell maintenance phase. It could dictate the in vitro proliferation and in vivo tumorigenic potentials of the CSC cells, and it was a bona-fide target of miR-490-3p in the SCC stem cells. The expression of PCBP1 and miR-490-3p negatively correlated with each other.