The Effects Of Mechanical Stretch On Gene Expression And Alternative Splicing In Osteoblasts

Many tissues and organs in the human body are subjected to stress in differenttypes and sizes from the physiological environment, and the bone is an importantstructural and functional tissue in the human body, the research that whether and howdoes the stress influence the growth and development of bone tissues will promote thedevelopment of bone tissue engineering. The expression and alternative splicing ofgenes can be changed by stress stimulation. This paper studied the effect of stressstimulation on alternative splicing in osteoblasts, as well as the possible alternativesplicing regulatory mechanisms.The mouse MC3T3-E1cells and human HaCaT cells are cultured and thenobserved with inverted microscope, MC3T3-E1cells were fibroblast-like cell type andHaCaT cells were epithelioid cell type.MC3T3-E1cells were digested and then inoculated onto the silicone membrane,then a stretch stimulation about15%strain and30times/min frequency was loaded toit respectively for3and6hours, using the stress stretching device that designedindependently. Then the cells were collected to studying the effect of tensile stimulationon the expression of VEGF, CD44and cyclinD1. The RT-PCR results indicated thatVEGF was mainly express the splice variants of VEGF188, VEGF164and VEGF120,CD44gene was mainly express the splice variants of CD44S and CD44E, and cyclinD1gene was mainly express the splice variants of cyclinD1a. The changes of expressionwere measured by q-PCR and western blot. The results revealed that after stretch, theexpression of VEGF and its splice variants VEGF164and VEGF120were significantlyup-regulated, but the expression of cyclinD1and its splice variant cyclinD1a weresignificantly descented, while the expression of CD44and its splice variants CD44S andCD44E were increased un-obviously.The eukaryotic expression plasmids of splicing factors ASF/SF2, SC35andhnRNPA1were constructed, named as pcDNA3.1(+)-EGFP-ASF/SF2, pcDNA3.1(+)-EGFP-SC35and pcDNA3.1(+)-EGFP-hnRNPA1. The plasmids were transfectedinto HaCaT cells to mimic the increase of ASF/SF2, SC35and hnRNPA1afterstretching stimulus, and research the effect of splicing factors ASF/SF2, SC35andhnRNPA1on the expression and alternative splicing of VEGF under the tensile stressenvironment. The transfection efficiency attainableness70%that observed under a fluorescence microscope. The protein expressions of ASF/SF2, SC35and hnRNPA1were detected by western blot, and the results proved that ASF/SF2, SC35andhnRNPA1were significantly increased. The expressions of VEGF188, VEGF164andVEGF120were all significantly decreased after over-expressing of ASF/SF2and SC35,but not hnRNPA1. This suggested that the splicing factors ASF/SF2and SC35wereinvolved in the regulation of VEGF alternative splicing under tensile stress environment,while hnRNPA1was not.