The Study On The Radiosensitizing Effect And Mechanism Of Down-regulation Ubiquitin Level In Human Lung Cancer Cells

Objective To study the expression level of ubiquitin in NSCLC and theproliferation and radiosensitivity effect of down-regulation the expression of ubquitingene on human lung cancer A549and H1299cells and discussion it’s probablemechanism. Further investigate the effect of down-regulation Ubquitin gene onradiosensitivity of nude mice transplanted with H1299cells.Methods: Perform immunohistochemical to investigate the expression ofubiquitin in NSCLC. In vitro study (1) Knock down the expression of Ubquitin gene(Ub-KD) in H1299and A549cells by shRNA-UBB and shRNA-UBC. The silenceeffect was verified n by RT-PCR and Western blotting;(2) Study the proliferation andradiosensitivity effect of Ub-KD plasmid shRNA in lung cancer H1299and A549cellsby MTT assay and plate clone formation assay;(3) Cell apoptosis was detected by flowcytometry assay and DNA fragements; Cell cycle distribution was assessed by flowcytometry and PI staining;(4) We performed Western blotting to detect the changeexpression of apoptosis, cell cycle and DNA damage related proteins；(5) Performedimmunofluorescence staining to detect the forming of γH2AX foci after X-rayirradiation. In vivi study: The experiment was based on40nude mice with subcutaneoustransplantation tumor of human lung cancer H1299cells, then the nude mouse weredivided randomly into eight groups as designed:(1) The tumor size were measuredevery three days, calculate tumor inhibitory rate and growth inhibition rate;(2) Thechanges of proliferation rate of transplantation tumor distribution effected by Ub-KDwas detected by immunohistochemistry technique.Results The expression level of ubiquitin is significant increased in NSCLC. Invitro study:(1)The result of RT-PCR showed that transfection with plasmid shRNA-UBBand shRNA-UBC inhibit ubiquitin expression significantly, and had a silence effect about76%;(2)Ub-KD inhibit the proliferation ability of H1299cells，but this effect wasattenuated in A549cells (67.3%VS.32.8%,24h,32.9%VS.16.5%，48h);(3)Ub-KD increase the radiosensitivity of H1299cells （SER=1.44）but not of A549cells（SER=1.05）;(4)Combine with IR, Ub-KD advance the cell apoptosis from8.79%(IR)to30.44%(shRNA-B4/C2+IR);(5)Compare with IR alone, Ub-KD suppressed theclearance ability of radiation induced γH2AX foci in H1299cells;(6)Ub-KD aloneincrease the G1phase arrest, but did not affect the IR induced cell cycle distribution;(7)Western blotting showed that Ub-KD increased the radiosensitivity of H1299cells via decreasingphospho-AKT and NF-κB expression. In vivi study showed that: compared with the IR alonegroup ((51.55±10.85)%), the inhibition effection of shRNA-UBB+IR group, shRNA-UBC+IRgroup, shRNA-UBB/UBC+IR group was：(70.26±5.66)%,(74.61±7.59)%,(79.31±6.48)%.Conclusion The expression level of ubiquitin is increased in NSCLC. Ub-KD bytranfection shRNA-UBB and shRNA-UBC plasmid in lung cancer cells effectively, witha silence effect of76%; Ub-KD inhibit the proliferation ability effect of H1299cells，butthis effect was attenuated in A549cells; Ub-KD increase the radiosensitivity of H1299cells but not of A549cells; Ub-KD advance the radiation induced cell apoptosis andsuppressed the clearance ability of radiation induced γH2AX foci in H1299cells;Ub-KD alone increased the G1phase arrest, but did not affect the IR induced cell cycledistribution; Our results showed that Ub-KD increased the radiosensitivity of H1299cells via decreasing phospho-AKT and NF-κB expression; Ub-KD increased theradiosensitivity of nude mice transplanted with H1299cells. Ub-KD increase theradiosensitivityo of lung tumour cells by advancing the radiation induced cell apoptosisand suppressed the DNA damage repair response to IR.