Expression of ZNF695 Transcript Variants in Childhood B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia is the most commonly diagnosed childhood malignancy worldwide; more than 50% of these cases are diagnosed in Mexico. Although the five-year survival rate is >80%, 30% of patients experience relapse with poor prognosis. Cancer-associated gene expression profiles have been identified in several malignancies, and some transcripts have been used to predict disease prognosis. The human transcriptome is incompletely elucidated; moreover, more than 80% of transcripts can be processed via alternative splicing (AS), which increases transcript and protein diversity. The human transcriptome is divided; coding RNA accounts for 2%, and the remaining 98% is noncoding RNA. Noncoding RNA can undergo AS, promoting the diversity of noncoding transcripts. We designed specific primers to amplify previously reported alternative transcript variants of ZNF695 and showed that six ZNF695 transcript variants are co-expressed in cancer cell lines. The amplicons were sequenced and identified. Additionally, we analyzed the expression of these six transcript variants in bone marrow from B-cell acute lymphoblastic leukemia patients and observed that ZNF695 transcript variants one and three were the predominant variants expressed in leukemia. Moreover, our results showed the co-expression of coding and long noncoding RNA. Finally, we observed that long noncoding RNA ZNF695 expression predicted survival rates.

Identification and Validation of Novel Reference Genes in Acute Lymphoblastic Leukemia for Droplet Digital PCR.

Droplet digital PCR is the most robust method for absolute nucleic acid quantification. However, RNA is a very versatile molecule and its abundance is tissue-dependent. RNA quantification is dependent on a reference control to estimate the abundance. Additionally, in cancer, many cellular processes are deregulated which consequently affects the gene expression profiles. In this work, we performed microarray data mining of different childhood cancers and healthy controls. We selected four genes that showed no gene expression variations (PSMB6, PGGT1B, UBQLN2 and UQCR2) and four classical reference genes (ACTB, GAPDH, RPL4 and RPS18). Gene expression was validated in 40 acute lymphoblastic leukemia samples by means of droplet digital PCR. We observed that PSMB6, PGGT1B, UBQLN2 and UQCR2 were expressed ~100 times less than ACTB, GAPDH, RPL4 and RPS18. However, we observed excellent correlations among the new reference genes (p < 0.0001). We propose that PSMB6, PGGT1B, UBQLN2 and UQCR2 are housekeeping genes with low expression in childhood cancer.