A Preliminary Analysis Of Proliferative Inhibition On Human Lung Cancer Line A549and Subline A549/DDP By Deuterium Depleted Water And Relevant MiRNA Assay

Objective: To detect anti-tumor effect and miRNA expression profiling by DeuteriumDepleted Water in lung cancer A549and A549/DDP cells, and preliminary studypossibility anti-tumor mechanism of Deuterium Depleted Water.Methods: The growth inhibition of lung cancer A549and A549/DDP cells wasexamined by MTT assay and plate colony formation assay; Migration and invasionof lung cancer A549cells were measured by transwell and scratch test; The phasedistribution of cell cycle was analyzed by flow cytometry （FCM）; The activitiesof lung cancer cells’ ATPase were measured by Ultramicro ATPase Kit; The proteinsexpression of NQO1, ATP, MRP1, PTEN, P-PTEN, Non-p-PTEN, VEGF, PDK1,P-PDK1and PCNA was determined by Western Blot. The expression of ATP,VEGF, MMP9, PCNA, NQO1and Nrf2were evaluated using immunofluorescence.Using the Affymetrix microRNA chip to detect the miRNA expression profile of lungcancer cells cultured with DDW; Bioinformatics predict target gene and their function;QPCR verify miRNA differentially expression profile in lung cancer cells; RT-PCRdetect mRNA expression in lung cancer cells transfected miRNA inhibitor and mimics;Western Blot detect NQO1, MRP1, MDR1and PCNA expression of lung cancerA549cells transfected miRNA inhibitor and mimics; Scratch test detect migration oflung cancer A549cells transfected miRNA inhibitor and mimics.Results:As the deuterium concentrations in DDW were reduced, lung cancer A549andA549/DDP cells proliferation, plated colony formation, migration and invasion wassignificantly suppressed （P <0.05）, whereas normal preosteoblast MC3T3-E1cells andnormal human airway epithelial cells （16HBE） were not inhibitted in the same midiacompared with control group. Cell cycle analysis revealed that DDW caused cell cyclearrest in the G1/S transition, reduced the number of cells in the S phase and significantly increased the population of cells in the G1phase in lung cancer A549cells（P <0.05）. Ultramicro ATPase Kit detection results revealed that DDW couldsignificantly inhibit activities of Na⁺K⁺-ATPase （P <0.01）, Ca⁺⁺Mg⁺⁺-ATPase （P <0.01） and T-ATPase （P <0.001） in A549cells. Western blot suggested deuteriumdepleted water down-regulated the protein expression of MRP1, VEGF, ATP, PDK1,P-PDK1and PCNA, meanwhile significantly increased the protein expression of PTEN,P-PTEN, Non-p-PTEN and NQO1. The immunofluorescence result showed deuteriumdepleted water down-regulated the protein expression of ATP, VEGF, MMP9andPCNA, increased the protein expression of NQO1and Nrf2.182miRNA of lung cancercell line A549showed more than twice expression profiling difference （96highexpression and86low expression） cultured with deuterium depleted water （50ppm）compared with normal water （150ppm） by Affymetrix microRNA chip. The expression ofmiR-1291, miR-143, miR-15b and let-7b of lung cancer A549cells among the miRNAwith expression profiling difference cultured with deuterium depleted waterrespectively increased by16.89times,2.29times,6.69times and13.88times validatedby QPCR. QPCR result showed the expression of miR-1291and miR-143of lung cancerA549/DDP cells cultured with DDW respectively increased by3.43times and3.05times, whereas the change expression of miR-15b and let-7b was not obvious. Thebioinformatics prediction results showed that tumor proliferation, apoptosis, metastasisand multidrug resistance were related to the target genes regulated by miR-1291,miR-143, miR-15b and let-7b. MiRNA transfection results showed that TRAF4,MMP24, MDR1, MRP1and BCL2may be the target gene regulated by miR-1291,ABCC4, BCL2and Caspase3may be the target gene regulated by miR-143. Westernblot suggested miR-1291down-regulated the protein expression of MRP1, MDR1andPCNA, increased the protein expression of NQO1. Scratches experimental resultssuggested that miR-143could inhibit invasion and metastasis of lung cancer A549cells.Conclusion:1. Deuterium depleted water showed selective targeted biologicalinhibition effects on lung cancer A549and A549/DDP cell proliferation.2. Significantly increased expression of NQO1, Nrf2, P-PTEN, Non-p-PTEN and PTEN protein, reduced expression of MRP1, VEGF, ATP, PDK1, P-PDK1, PCNAprotein and standing in G1, S phase arrest of lung cancer cells induced by deuteriumdepleted water may contribute to one of molecular mechanisms of the deuteriumdepleted water inhibiting the proliferation and metastasis of lung cancer cell lines.3. Significantly up-regulation of the expression of a large number of tumor suppressormiRNAs, down-regulation of a large number of proto-oncogene miRNAs expressioninduced by deuterium depleted water may be another molecular mechanism of thedeuterium depleted water inhibiting the proliferation and metastasis of lung cancer celllines.4. TRAF4, MMP24, MDR1, MRP1and BCL2may be the target gene regulated bymiR-1291, ABCC4, BCL2and Caspase3may be the target gene regulated by miR-143.