Experiment Study Of Mic2/CD99 Gene Transforms The H/RS Cells Of L428 Cell Line Into Lymphoma Cell

IntroductionRecently, considerable progresses have been made in the study of the etiology,morphology, and the molecular mechanisms of their growth and regulation of H/RScells. Many researches have showed that majority of H/RS cells are originated from"defect B lymphocytes", and the expression of LMP-1, lost expression of CD99protein and lasting activation of NF-κB are the three key elements of H/RS cellsformation. A recent ground breaking discovery has drawn the public attentions to thefact that the originations of H/RS cells are closely related to the lost expression ofCD99 protein. Some recent researches have reported that the antisense CD99transfected to Human B lymphocytes BJAB and IM9 to knockdown the expression ofCD99 can result in classic characteristics of H/RS cells similar to the ones in thelymph nodes of a HL patient, showing "mirror image cell", which over express CD30and CD15 protein. When CD99 gene was transfected into prior mentioned H/RS-likecells, these cells will return to the original phenotype of the B lymphoma. Thisindicates that the origin of H/RS cells, the morphogenesis and change ofimmunophenotype of H/RS cells is related to the lost expression of CD99 gene. Someresearches showed that down-regulation the expression of CD99 can result in thetransformation of H/RS-like cells. Human CD99 (mic2) is a type of cellular transmembrane protein with 32kDa. Itscoding gene is located at the site of Xp22.32-pter and Yp11-pter in the PAR zones ofthe X and Y chromosome short chains. Genomic sequencing analysis indicated thatPAR is the potential oncogene for HL, participating in the differentiation ofhemocytes. Our previous study showed that using RNAi technology to silence themCD99L2 gene from A20 cells, which result in the transformation of H/RS-like cells,and confirm its immunophenotype characteristic and biological features. So howabout up-regulate CD99 expression in human HL cell line to make these cells returnto the original phenotype of the B lymphoma?We detected the expression of mic2/CD99 gene was negetive in H/RS cells ofhuman HL cell line L428, and constructed the eukaryotic expressing vectorpcDNA3.1(+)-CD99. The L428-CD99 cell clone expressing mic2/CD99 gene wasgain by stable transfection and clone selected. We observed the morphology and theimmunophenotype of H/RS cells of L428-CD99, and constructed the L428-CD99 cellmodel. It's the basic of further to studying the etiology and the molecular mechanismsof H/RS cells of HL.Objectives1. To make clear CD99 mRNA and protein level expression of the H/RS cells of theHuman HL cell line and the cHL tissue sample.2. Construct the mic2/CD99 gene eukaryotic expressing vector.3. Constrsct the L428-CD99 cell model and confirm its character.Method1. Design oligo-nucletide probe, and detect CD99 expression in H/RS cells of HLcell line and 15 samples of cHL tissue by molecular hybridization in situ andimmunohistochemistry.2. Design CD99 gene specific PCR primer, and acquire CD99 cDNA from T lymphoma cell line Jurkat total RNA by RT-PCR, clone it into T vector, get positiveclone, make enzyme cleaving and DNA sequencing confirmation. Design PCR primerwith enzyme cleavage locus, get CD99 gene expression frame by PCR, then combineto the vector pcDNA3.1 (+). The sequence of the PCR product was confirmed byenzyme cleaving and DNA sequencing analysis.3. Stable transfer and selected by G418, up-regulation of CD99 gene in L428, andget the monoclone by using soft agar clone formation protocol and 96 well platelimiting dilution assay, proliferate this clone and named the result cell modelL428-CD99.4. Examine the integrated status of L428-CD99 clone and pcDNA3.1 (+)-CD99vector by using molecular biology technique. Observe the morphologicalcharacteristics of L428-CD99 cells under light microscope. Evaluate transformationrate of H/RS cells of L428 (diameter=25μm) transformed to L428-CD99 by netcounting method. Detect the CD99,CD30 protein expression in L428-CD99 clone andL428 by immunohistochemistry. The expression of CD30 and CD15 of L428-CD99,L428,BJAB cell line was tested with flow cytometer.Result1. CD99 expression in H/RS cells of HL cell lines and cHL tissue sample:CD99 expression was negative in H/RS cells of HL cell line and cHL tissuesample (14/15,93.3%) at mRNA and protein level.2. Construction of CD99 eukaryotic expression vector:Full length cDNA of CD99 was acquired by RT-PCR and gained correctsequencing confirmation. The sequence was totally consistent with the data ofGenBank (NM-002414) by DNA sequencing analysis. The sequence of therecombination plasmid pcDNA3.1 (+)-CD99 was totally consistent with the data ofGenBank through DNA sequencing analysis. Eukaryotic expression vector pcDNA3.1(+)-CD99 was successfully constructed.3. Construst the L428-CD99 cell model and confirm its chatacter:Transfer the L428 cells by liposome and the positive cell clones were selected andobtained by G418 after 10 days. Obvious morphological changes, including volumeand nucleus decrease were observed after 6-7 days. We named this cell clone asL428-CD99. 558 bp fragments were detected in L428-CD99 group by electrophoresis,which served as an evidence of successfully transfection. Cell counting resultsshowed that after 48 h of inoculation in 96 well plate, rate of H/RS-like cells (over 25μm in diameter) in control group L428 was (10.17±0.0194)%, and itwas(3.33±0.0103)% in L428-CD99 group. H/RS-like cells in L428 cells after 96 hinoculation were (11.50±0.0339)%, H/RS-like cells in L428-CD99 were(3.67±0.0126)%. H/RS-like cells in L428 group were significantly more than inL428-CD99 group (P＜0.01). Result of immunohistochemistry showed that CD30protein expression was positive in H/RS cells of L428, and was negative in H/RScells of L428-CD99 cells and CD99 protein expression was positive in H/RS cells ofL428 and was negative in L428-CD99. The expression of CD30 and CD15 of celllines were tested with flow cytometer showed that the expression of CD30 ofL428-CD99 and L428 was respectively 3.17% and 55.24%. The expression of CD30of BJAB cell line was 4.64%. And the expression of CD15 of L428-CD99 and L428was respectively 4.15% and 12.01%, and the expression of CD15 of BJAB was3.44%.Conclusion1. CD99 expression in H/RS cells of Human HL cell line L428 and cHL tissuesamples were both negative at mRNA level and protein level, which may served asone of its phenotype characteristics. 2. Up-regulation CD99 gene expression in human HL cell line L428 will result inthe changes of the morphology, immunophenotype and biological features of H/RScells, which return to the characteristics of B lymphocytes.Original ideas of the study1. Confirmed CD99 expression at mRNA level and protein level was negative orlow in H/RS cells of human HL cell line L428 and cHL tissue samples, indicatedthat CD99 expression was negetive or low may served as one of its phenotypecharacteristics.2. Construct the L428-CD99 cell modle. Observed that mic2/CD99 gene maytransform H/RS cells of human HL cell line L428 into B lymphoma-like cells.