Effect Of Inhibiting SRPK1 Expression On The Proliferation And Apoptosis Of Leukemia K562 Cells

ObjectiveLeukemia accounts for approximately a third of childhood's malignant tumor. In the developed metropolitan region, leukemia is the most common disease of childhood malignant tumor and is the foremost cause of death in children. Therefore it is of great scientific and social significance to research the prevention and cure of childhood leukemia. Chemotherapy is the predomiant method for leukemia, but most chemotherapy drugs are hard to avoid poor selectivity, easy to induce big adverse reactions such as myelosuppression, severe infection and so on. Therefore, there is a developing interest in targeting therapy as a specific treatment against tumor cells.RNA interference (RNAi), which highly conserved during evolution, is a phenomenon of high-efficiency and specific homologous RNA degradation induced by double-stranded RNA (dsRNA). The expression of specific genes can be eliminated or closed by RNAi, so this technology has been widely used in exploration of gene function and gene therapy field of hereditary desease, infectious disease and malignant tumor.Recent years, serine-arginine protein kinases (SRPKs) were found as an important part of serine-threonine protein kinase family, which played an important role in specific phosphorylation of serine. SRPKs aslo showed a vital role in mRNA (Messenger RNA) of alternative splicing, somatic cell and sperm chromatin redistribution, cell cycle, p53 regulation and in the process of cell metabolism signal. SRPK1 is one of SRPKs, which studied most deeply. As a SR splicing factor phosphorylation protein, SRPK1 plays an important role in accumulating function signal and regulating the other members of SRPKs family. Current research has suggested that SRPKs could be potential to be the target of tumor therapy, because of high expression in some tumor cells. After interfering with SRPK1 gene, the proliferation of tumor cells was found to be reduced, apoptosis potential increased, the sensitivity of chemotherapy drugs improved.This study would intend to observe the change of proliferation and apoptosis of leukemia K562 cells as target after SRPK1 gene silencing by using RNAi technology. First, we synthesize SRPKl-siRNA and transfect it into K562 cells. We detected the influence of K562 cells by MTT assay, cell cycle assay and cell apoptosis assay. Second, we investigated the roles of caspase-3, PARP and Bcl-2/Bax proteins in human K562 cells apoptosis using western blot analysis. We provide new experimental basis for targeting therapy for leukemia.Methods1.Effect of inhibiting SRPK1 expression on the proliferation of leukemia K562 cells(1) K562 cells cultureThe cultured cells collected into centrifuge tube,1000 rpm centrifugal for 5 min, added in the RPMI1640 (Roswell Park Memorial Institute 1640) complete medium. Cultures were incubated at the condition of 37? in a fully humidified atmosphere containing 5% CO2.(2) RNA interference assaySRPK1-siRNA was synthesis by company. All transactions were carried out using C10511-1 riboFectTM CP Transfection Kit (333T) according to the manufacturer's instruction. The same volume specific siRNA and control RNA were dissolved in RPMI 1640 medium at 100?L, and then a certain volume C10511-1 riboFectTM was dissolved in 100?L RPMI 1640 medium for 5 minutes at room temperature. After 5 min the dilution of RNA and C10511-1 riboFectTM were blended incubation for 20 minutes at room temperature. The 200?L compound was joined in the cell culture plate containing serum free medium, gently shacked thoroughly incorporated. The cells were harvested after 4-6h, replaced with refresh medium. siRNA transfection efficiency was analyzed using flow cytometer (FCM).(3) Real-time fluorescent quantitative PCRTotal RNA was extracted from the sample using Trizol reagent and was reversed transcribed into cDNA using RevertAidTM First Strand cDNA Synthesi kit according to the manufacture's protocol. The samples of RNA detect on the spectrophotometer determination of 260 nm,280 nm absorbance values after diluted 100 times. RNA concentration calculation, formula (mu g/ml)=A260x 40 x diluted multiples (mu g/ml). A260/A280 is 1.9 to 2.0. The expression of SRPK1 was detected through RT-PCR (real-time polymerase chain reaction) analysis in control and transfection groups.(4) Cell proliferation was tested by MTTThe cells were seeded in 96-well plates at density of 1×105/ml. Each plate was transfection according to the introduction above. Cultures were added MTT continuing for culturing. After centrifugal supernatant, each plate was joined with DMSO. The cells proliferation activity were measured at 490nm OD value, and calculated.(5) Cell cycle testCells were collected after transfection,1000 rpm centrifugal for 10 min.70% ethanol was used to fixing cells for 30 min. Cells were digestive with RNA enzymes at room temperature for 30 min. PI was used to deal with cells for 20 min. The samples were analyzed by flow cytometer (FCM) at 488nm excitation wavelength.2. Effect of inhibiting SRPK1 expression on the apoptosis of leukemia K562 cells(1) Apoptosis detection assayCells apoptosis were measured by fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit. The cells were collected,1000rpm centrifuge for 5 minutes, with precooling PBS wash again. The cells were centrifuged and suspended with 200?L of buffer. Each pipe added with 4?L Annexin-V/FITC and 8?l 20?g/mL propidium iodide (PI) for 15 min at room temperature. The sample was analyzed by FCM.(2) Western blot analysisAfter total protein extraction, according to the western experimental technology of operation steps to detect apoptosis pathway of protein caspase3, cleaved caspase3, PARP, cleaved PARP and Bcl-2/Bax expression level.3.Statistical analysisStatistics were analysised using IBM SPSS Statistics 20.0 software. Double side (p<0.05) was considered the differences were statistically significant. To conform the normal distribution of continuous variables in the form of "mean+standard". Continuous variables were used t test and variance analysis.Results1. Effect of inhibiting SRPK1 expression on the proliferation of leukemia K562 cells(1) siRNA transfection efficiency by FCMIn order to investigate whether siRNA can inhibition K562 cells in vitro, we designed and synthesized scrambled of siRNA with GFP (Green Fluorescent Protein). The result indicated the inhibition rate of 50nM siRNA was 78.14±2.59%, while 100nM was 93.37±3.36%. It was shown that the MFI (Mean Fluorescent Intensity) of 50nM siRNA was 96.43±3.62 and of 100nM siRNA was 125.15±2.73. The result indicated siRNA had been transfected into the cells and 100nM siRNA had higher transfection than 50nM.(2) SRPK1 expression in K562 cells by RT-PCRThe expression of SRPK1 was detected through RT-PCR. The results showed that SRPK1 mRNA levels were lower in transfected cells groups than in reagent group and scrambled siRNA groups. SRPK1-siRNA at a concentration of 100nM siRNA by synthesized s2 was better than 50nM siRNA by s3 in silencing the SRPK1 gene.(3) Cell proliferation assay by MTTThe results revealed the inhibition rate in transfected groups obliviously higher than SRPK1 non-transfected groups. No statistical difference was found between reagent group and scrambled siRNA groups. Compared with SRPK1 non-transfection groups, the maximum inhibition rate of transfection groups was s2 and s3 sequence at 100nM siRNA cultured for 48h (49.62±4.33%,44.88±3.87%). The concentration of 100nM siRNA was superior to 50nM according to our result. The increased inhibition rate occurred at 48h, slightly reduced at 72h. The results indicated s2 sequence is better than s3 to inhibit cells proliferation.(4) Cell cycle assayAccording to previous results by real-time PCR and MTT, we chose regent group as control 1, scrambled siRNA group as control 2, synthesized sequence s2 for 100nM siRNA as RNAi 1 and s3 100nM siRNA as RNAi 2. The rate of cells in S phase were lower in RNAi groups than in control groups, while in G1 phase were higher in RNAi groups than in control groups.2. Effect of inhibiting SRPK1 expression on the apoptosis of leukemia K562 cells(1) Apoptosis detection assayAccording to the first part results by real-time PCR and MTT, we chose regent group as control 1, scrambled siRNA group as control 2, synthesized sequence s2 for 100nM siRNA as RNAi 1 and s3 100nM siRNA as RNAi 2. Compared with control groups, SRPK1 silencing groups(s2 and s3) increased apoptotic cells at 29.95±3.27% and 31.4±2.85%, while early apoptotic cells also increased at 15.13±4.02% and 15.93±3.52%. No statistical difference of apoptotic cells was found between control groups (2.09±1.98% vs. 3.14±2.16%). The results implicate that silencing SRPK1 induced K562 cells apoptosis.(2) Western blot analysisThe experiment was divided into four groups as described before. The results revealed that the cleavage and activation of both caspase3 and PARP was significantly higher in RNAi groups than in control groups, whereas expression of PARP and caspase 3 in four groups has no significant difference. The expression of RNAi 2 group has significant higher than RNAi 1 group. The expression of Bcl-2/Bax was significantly decreased in RNAi groups than control groups, while no statistical difference was found between control groups and RNAi groups.Conclusions1.Silencing SRPK1 gene expression by siRNA can inhibit K562 cells proliferation and induce apoptosis. We synthesized small interference RNA sequence s2 and s3, can continue the next experiment to exam cell apoptosis and the expression of apoptosis related proteins.2. After siRNA interference SRPK1 expression, the apoptosis of the cells was initiated. Apoptosis related proteins test founded that the effect of cell apoptosis protein caspase3, PARP has been started, the antiapoptotic proteins Bcl-2/Bax obviously decrease.