Role of PRDM1{221}-isoform (with a disrupted PR domain) as a negative regulator of the tumor suppressor PRDM1α in NK-cell neoplasms

NK-cell malignancies (NKLL) consist of two separate entities: Extranodal NK-cell lymphoma, nasal type (ENKL) and aggressive NK-cell leukemia (ANKL). ENKL is the second most common group of extranodal lymphomas in Hong Kong. Deletions in the 6q21 region in ENKL have been consistently reported in the literature and differential expression data indicated that the transcription factor PRDM1 (PR domain containing 1, with ZNF domain) located at 6q21-q22.1 is a candidate TSG in NK-cell neoplasms. PRDM1 exists as 2 isoforms generated from the same gene by alternative transcription promoter. PRDM1- differs from PRDM1-βin that it lacks the amino-terminal 101 amino acids with a disrupted PR domain. As the PRDM1- is functionally impaired, with a loss of repressive function on multiple target genes while maintaining normal DNA-binding activity, we hypothesize that the -isoform, which is overexpressed in NKLLs, may act as a negative regulator of the tumor suppressive α-isoform in NKLLs. In this study, we investigated the possible role of PRDM1- as a negative regulator of tumor suppressor PRDM1-α in NK-cell lymphoma by using a gene silencing technique. Short hairpin-RNA (sh-RNA) construct with sequence targeting to PRDM1- purchasing from biotechnology company was used to knockdown of the gene expression. Series of functional assays were then performed to evaluate the effect of the PRDM1- knockdown in two NK cell line, YT and NKYS, which xpress endogenous PRDM1-. Comparison was made between the 1) shRNA targeting to nt65-nt94 of PRDM1- sequence, sh-PRDM1 -pGFP-V-RS (shV2), and 2) scrambled-pGFPV-RS (scrambled shRNA), negative control with a non-effective shRNA cassette in pGFP-VRS plasmid. Western blot analysis was performed to examine the efficiency of shRNA in knockdown the expression of PRDM1- in 293T cells (normal human embryonic kidney cells). The protein expression level for ectopic PRMD1- was reduced in cells expressing shV2 when compared with the negative control. NKYS cell line expressed with shV2 showed a significant reduction in the number of colonies. Percentage of dead cells was found higher in these cells. The proliferation rate of shV2 expressing cells started to retarded significantly on the third day of measurement in the MTS proliferation assay. The cell also underwent G1 cell cycle arrest and had lower proliferation rate, as indicated by cell cycle analysis. For YT cell line expressed with shV2, significant reduction in both colony number and size in methylcellulose colony formation assay was observed. Base on the results obtained from the two NK-cell lines, we suggest that the shV2 inhibit the tumor cell growth. The knockdown of the PRDM1- lead to an increase level of PRDM1- α. PRDM1- α is a tumor suppressor gene with suppressive function by preventing damaged cells from proliferation or inhibiting the clonogenecity of the tumor cells. An imbalance expression of PRDM1- and PRDM1-αplay an important role in tumor growth and formation, and PRDM1 could possibly be the new tumor suppressor gene in NK-cells lymphoma.published_or_final_versionPathologyMasterMaster of Medical Science