| Cold-inducible RNA binding protein(also known as CIRP,CIRBP or A18 hnRNP), a kind of RNA binding protein, belongs to heterogeneous nuclear ribonucleoprotein(hnRNP) family, which is highly conservative and widespread expressed in the spine animal tissues and organs. The previous study have showed that CIRP protect cells when it over-express with a variety of stress conditions. It participate in and control the physiological and pathological process of cell growth, reproductive growth, neuromodulation and cancer. Also, it was reported that CIRP over-expression in mouse fibroblast cells could inhibit cell proliferation with prolong cells in G1 phase. But recent studies have shown that CIRP play a promote role in mouse embryonic fibroblast cells and mice spermatogonium. So CIRP may had different effect on the proliferation which in different types of cells. CIRP were rarely reported association with mammalian kidney but clawed frog former kidney formation process. Therefore, this experiment is to study the explore of CIRP in the impact of cell proliferation and its molecular mechanism to BHK-21, providing new clues for further research of CIRP effects on kidney development and related diseases. Achievements acquired were as follows: 1 Cirp is a positive regulator for BHK-21 cell proliferationWe builted CIRP over expression BHK-21 cells(Cirp+BHK-21)and CIRP knockdown BHK-21 cells(Cirp-BHK-21)to studying the role of CIRP on the proliferation of BHK-21. Green fluorescent expression protein BHK-21 cells(GFP-BHK-21) were the in control cells. Then,we detected living cells number of Cirp+BHK-21 and Cirp-BHK-21 at 0、24 h、48 h、72 h、96 h, and draw the growth curve and calculated the specific growth rate. It showed that in the same conditions, living cells number of Cirp+BHK-21 in 24-72 h was significantly higher than in control cells(P﹤0.05), in 72-96 h reached extremely significant difference(P﹤0.01), between 24-96 h average specific growth rate were 0.023±0.015 h-1 and 0.020±0.012 h-1. The Cirp-BHK-21 number between 0-48 h of living cells were not obvious difference in control cells, but between 48 h–96 h were significantly lower than in control cells(P﹤0.05), between 48-96 h average specific growth rate were 0.010±0.005 h-1 and 0.008±0.003 h-1. Thus it can be seen that CIRP overexpression can promote BHK-21 cell proliferation, and CIRP knockdown can inhibit BHK-21 cells proliferation, which proved that the CIRP do positive regulating effect on BHK-21 cells proliferation. This results suggest CIRP played an important role on mammalian renal cell development. 2 BHK-21 with CIRP over expression changed its cell morphology after continuous cell cultureIn the test process, Cirp+BHK 21 cells, after frozen storage, recovery and continuous subculture to the tenth generation, were surprised that the cell morphology changed from the original spindle gradually to the irregular circle, we named Cirp + BHK- 21 △. Giant nucleation and nucleation, many cells full shape on the whole were observed in Cirp + BHK 21△ nucleus with HE staining. Then, observed by scanning electron microscopy(SEM), the surface of the Cirp + BHK–21△ was smooth, with more bubble around, there were many developed pseudopodia. Almost every cell in the pseudopodia out from edge of cell membrane to cell free area, some pseudopodia which is one of the important structure of tumor cells, were closely connected with neighboring cells or mosaic. Since CIRP itself is a proto-oncogene, has high expression in a wide variety of tumor cells, So, further validation of the CIRP express whether led to BHK- 21 cell cancerization is worth studying. The results show that the CIRP can change the configuration of BHK- 21 cells, and the CIRP of cytoskeleton adjustment has not been reported, it is necessary to further study the molecular mechanism. 3 Over expression of CIRP promote cell proliferation by speeding up BHK- 21 cell cycleIn order to further verify the CIRP and the influence of cell proliferation to BHK-21, flow cytometry for CIRP+BHK-21△, GFP-BHK-21, CIRP-BHK-21 cell cycle were tested. Take these three cells grew well, made after the cell suspension, ethanol fixed, Triton X- 100 processing, propyl iodide organism(PI) staining of DNA, using flow cytometry instrument to detect the cell cycle. Results showed that under the same conditions, the Cirp+BHK-21△, GFP-BHK-21, Cirp-BHK-21 in phase G0-1 the DNA content of proportion(that is, cells number) in turn was 52.4%, 55.3%, 66.7%, in the DNA content of S phase proportion, in turn, was 26.1%, 24.7%, 19.7%, G2- M phase of DNA content in 20.8%, 12.7%, 8.5%, namely the Cirp + BHK-21△ cells in G0-1 stage is reduced, and the S phase and G2- M phase of the increase in the number of cells, Cell cycle progression from G1 phase to S phase, the shift to strengthen instructions CIRP can promote BHK- 21 cells to enter S phase in advance, so as to promote the BHK-21 cell proliferation, but the specific molecular mechanism remains to be further validation. 4 Screening of candidate target protein that interact with CIRP in BHK- 21 cellsIn order to study the molecular mechanism of BHK- 21 cell growth and development with CIRP, immune co-precipitation(Co-IP) combined with mass spectrometry(MS) were used to carry on the preliminary screening of the candidate target protein that CIRP + BHK 21 and CIRP + BHK- 21△ with CIRP interactions. In the Cirp+BHK-21, a total of 24 protein was settling down.According to discharge the Mascot software combined with CIRP protein ability score the top five were: G3bp1, Caprin1, Pabpc1, Alb, G3bp2. The analysis results G3bp1, G3bp2 and Caprin1 have proved closely associated with cell growth and development, so we speculated CIRP possibly through interaction with these proteins to regulate the proliferation of BHK-21 cells.At Cirp + BHK 21△ group, a total of 55 protein were settling down, in which the score in the top five of the protein is respectively: Tpm1, Tpm3, Tpm2, Gapdh, Myh10. The analysis concluded that these five proteins were associated with the regulation of the cytoskeleton, we speculated CIRP possibly through interaction with these proteins to regulate the BHK-21 cell morphology.This results provided a new clue for the further study that CIRP do molecular mechanism of cell growth and development in BHK – 21. |
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