Abstract: Objective: To construct the recombinant expression vector of mitochondria-targeted KillerRed (KR), and to explore the producing regularity of reactive oxygen species (ROS) induced by visible light exposure, and its enhancement in the proliferation inhibition of radiation on the HeLa cells. Methods: Gene recombination technique was used to build the recombinant vectors plxsp-flag-Sarm1-KR and plxsp-flag-Sarm1-mCherry targeting mitochondria. The cervical cancer HeLa cells were divided into control, plxsp-flag, plxsp-flag-Sarm1-KR, 4 Gy, plxsp-flag + 4 Gy and plxsp-flag-Sarm1-KR + 4 Gy groups. After the vectors were transfected into the cells for 24 h, the expression levels of mCherry and mitochondrial track COXⅣ proteins were determined by fluorescence microscope;DCFH-DA probe was used to detect the mean fluorescence intensity(MFI) to indicate the ROS producing level. After 4 Gy X-ray irradiation,the cell proliferation activity was measured by CCK8 kits. Results: After the vectors were sequenced and identified, the sequence was consistent with that in GenBank, and the results showed the fusion expression vector plxsp-flag-Sarm1-KR(mCherry) was successfully constructed. After the plasmids were transfected into the HeLa cells, under fluorescence microscope, mCherry protein and COXⅣ protein had the same expression location. From ROS producing level, in control and plxsp-flag groups, after the cells were exposed to visble light for 10, 30 and 60 min, the MFI had no obvious changes after the probe was incorporated for 10, 30 and 60 min; but in plxsp-flag-Sarm1-KR group, the MFIs were increased with the time prolongation; compared with control group,the cells were exposed to visble light for 10 and 30 min, and the MFIs were significantly increased after the probe was incorporated for 60 min (P<0.05); when the cells were exposed to visble light for 60 min, the MFIs were significantly decreased after the probe was incorporated for 30 and 60 min (P<0.05). Compared with control group, the A(450) in plxsp-flag group had no obvious change(P>0.05); the proliferation activities were significantly decreased at 10 and 24 h in plxsp-flag-Sarm1-KR group (P<0.05); the proliferation activities were significantly decreased at 10 h in 4 Gy and plxsp-flag + 4 Gy groups (P<0.01); the proliferation activities were significantly decreased at 10, 24 and 48 h in plxsp-flag-Sarm1-KR + 4 Gy group (P<0.05 or P<0.01). The proliferation activity in plxsp-flag-Sarm1-KR + 4 Gy group was lower than those in plxsp-flag-Sarm1-KR and 4 Gy groups (P<0.05). Conclusion:The mitochondria-targeted fusion expression vectors are successfully constructed,and the KR proteins can induce the ROS producing and enhance the inhibitory effect of proliferation of ionizing radiation on the HeLa cells.

Key words: KillerRed, mitochondria, radiation, cell proliferation, reactive oxygen species