Effects And Mechanisms Of Up-regulating SPATA5L1 Gene On The Radiosensitivity Of KB Cells

Objective:earlier biochip screening analysis and Western blot assay found that the expression of SPATA5L1 was increased in the KB cells after treated with ionizing radiation and/or moderate paclitaxel.The mRNA level of SPATA5L1 was 6.78 times after 2Gy ionizing radiation.The fuction of this gene in treatment of cancers has not been reported yet.Our works was to construct a stable KB cell line with SPATA5L1 over-expression plasmid and investigate the effects of SPATA5L1 on radiosensitivity as well as the possible molecular mechanism.These findings will provide experimental evidence for SPATA5L1 as a new target for cancer therapy.Method: A recombinant mammalian plasmid pEX-1 carrying a full-length of SPATA5L1 cDNA was transfected into human oral cancer KB cell line with lipofectamine transfection assay.Alterations of SPATA5L1 mRNA and protein level were monitored by real-time PCR and Western blot assay.CCK-8 assay and cell clone formation assay were used to assess the growth and proliferation of KB cells after raidiation.And the cell cycle distribution and apoptosis was detected by flow cytometric analysis.The cell mitochondrial membrane potential of KB cells were observed by immunofluorescence technology with confocal microscopy.The Cyclin B2 related to cell cycle distribution and the LC3 B and Beclin1 related to autophagy were detected by Western blot.Result: The recombinant plasmid pEX-1-SPATA5L1 was successfully transfected into KB cells.The G418-resistant clone were obtained.The mRNA and protein levels of SPATA5L1 in the pEX-1-SPATA5L1 transfected cells were significantly higher than those in the controls.The growth and proliferation of three cell groups were obviously inhibited by ionizing radiation.Compared with the control and blank groups,the KB cells overexpressed with SPATA5L1 were more sensitive,and the survival fraction of those decreased significantly after irradiated with X-rays.Additionally,the G2/M arrest and apoptosis of the KB cells overexpressed with SPATA5L1 were increased signigicantly,along with the decreasing of mitochondrial membrane potential.The cycle regulatory protein Cyclin B2 was low expressed,and the proteins related to autophagy such as Beclin1 and LC3 B ?were overexpressed,the ratio of LC3 B ?/? was increased.Conclusion: The protein SPATA5L1 was over-expressed stably and efficiently in transfected KB cells.The over-expressed SPATA5L1 protein increased the KB cellular radiosensitivity.The mechanisms may relate to the G2/M phase arrest induced by SPATA5L1 mediated via down-regulating cell cycle protein Cyclin B2 and the dysfunction of mitochondrial membrane potential.Moreover,over-expressed SPATA5L1 protein also participated in the progress of autophagy mediated via up-regulating Beclin1 and LC3 B ?,which induced cell death.SPATA5L1 may be the radiation-sensitizing gene used for radiotherapy of cancer,however,the molecular mechanism of the radiation-sensitizing need to be further investigated.