CALR Gene Expression In MDS Patients And Influence Of CALR-RNAi Lentiviral Vector On Cell Growth And Apoptosis

Objective: To study the expression of calreticulin(CALR) in patients with myelodysplastic syndrome(MDS), and construct RNAi lentiviral vector targeting CALR gene in SKM-1. Then investigate the effect of CLAR-RNAi on cells apoptosis and proliferation.Methods: Reverse transcription PCR( RT-PCR) was used to detect CALR expression in bone marrow specimens obtained from 34 MDS patients and 8 non-MDS patients; Three kinds of single-stranded primers were designed and synthesized, then annealed to double-stranded oligo sequences and subcloned into linear GV248 lentiviral plasmid digested by enzyme to produce GV-CALR-RNAi lentiviral vector. The lentiviral vector was cotransferred into 293 T cells. After being identified by PCR and sequencing, recombinant lentiviral vector expressing CALR RNAi were obtained. This lentiviral vector was transferred into human SKM-1 cells,and the transfection efficiency was detected by flow cytometry. The interference efficiency of CALR gene was determined by quantitative reverse transcription PCR and Western blotting, and the most effectivetargeted sequence was screened. CCK8 assay and Annexin V/7-AAD staining were conducted to observe the effects of CALR gene silencing on cell growth and apoptosis.Results: CALR gene expression was significantly decreased(P < 0.01)in 31 of 34 patients with MDS compared with controls. Three kinds of CALR-RNAi-expressing lentiviral vector was successfully constructed, and the transfection efficiency was more than 70%. Quantitative reverse transcription PCR and Western blot results showed that CALR-RNAi(3)lentiviral vector was the most effective vector for interfering the expression of CALR gene. Moreover, CALR-RNAi(3) could promote cell growth and inhibit apoptosis in SKM-1 cells(P < 0.05). The percentage of SKM-1 cells in G0/G1 phase was decreased and that in S phase was increased. Western blot showed that downregulation of CALR resulted in the decreased expression level of cleaved-caspase-3.Conclusion: CALR is an important tumor suppressor gene in MDS,and the CALR-RNAi(3) lentivirus vector will be helpful in further studying the functional role of CALR gene in MDS.