Study On Biological Functions And Relevant Mechanisms Of THOC1in Lung Cancer

ObjectiveThis experiment was designed to study the effects and the possible mechanisms ofTHOC1on cell proliferation, migration, invasion and radiosensitivity in human lungcancer cells. These findings will provide theoretical foundation for the clinicalapplication of THOC1as a new target gene in the future.Methods1.The recombinant plasmid pcDNA3-THOC1was transfected into the lung cancercell lines, SPC-A-1and NCI-H1975; U6/GFP-shTHOC1was transfected into95D,through Lipofectamine2000transfection assay. The G418resistant clones werecollected. The mRNA and protein expression of THOC1was tested by qPCR andWestern blot assay, respectively.2. MTT assay was used to detect the cell viability; the alteration of cell cycledistribution and apoptosis were assessed by Flow cytometric analysis; Wound healingassay and Transwell experiment assay were performed to study cell migration andinvasion.Western blot assay was used to evaluate the changes of protein expressionrelated to cell cycle, apoptosis, invasion and migration.3. The SPC-A-1, SPC-A-1/Neo and SPC-A-1/THOC1cells were subcutaneouslyinoculated to establish the lung tumor model in BALB/c nude mice, The effect ofTHOC1on tumor growth was examined. The weight of nude mice, organs index andthe main biochemical indexes in serum were observed; the pathological changes of themain organs and the tumors were detected by HE staining, Immunofluorescence assaywas used to study the expression of THOC1, Ki-67and VEGF.4. After irradiation with X-ray, colony formation method was used to evaluated thesurvival fraction, the cell cycle distribution, apoptosis and ROS were assessed by flow cytometric analysis. Comet assay and-H2AX foci was used to detect DNA damagerepair after irradiation. Western blot assay was employed to study the changes of theprotein level related to DNA damage repair and NEJH.Results1. pcDNA3-THOC1was successfully constructed and transfected into lung cancercell lines, SPC-A-1and NCI-H1975. A higher expression of THOC1at both mRNA andprotein levels was observed; U6/GFP-shTHOC1was successfully transfected into95D,a lower expression of THOC1at both mRNA and protein levels was detected.2. Compared with the blank cells and Neo cells, overexpression of THOC1inhibited the vability of SPC-A-1and NCI-H1975, the protein expression of p-Akt,p-GSK-3β and p-JNK decreased; p-c-Raf, p-PDK1and p-P38increased inSPC-A-1/THOC1cells. The protein expression of p-GSK-3β and p-JNK wasdown-regulated; p-Erk1/2, p-PDK1and p-P38was up-regulated in NCI-H1975/THOC1cells. The cell proliferation of95D/shTHOC1increased compared with blank cells andNeo cells; an increased expression of p-GSK-3β and a decreased expression of p-P38were detected in95D/shTHOC1cells. Overexpression of THOC1caused a G2/M phasearrest, an increase of protein expression in CyclinA and CyclinB1.95D/shTHOC1displayed an increase of cells at G0/G1phase, with a decreased protein expression ofCyclinD1and an increased expression of CyclinE1.3. Compared with the blank cells and Neo cells, the apoptosis rate increased inSPC-A-1/THOC1cells and NCI-H1975/THOC1cells, accompanying with an increaseof protein exprssion of Bax and caspase-3.4. Overexpression of THOC1inhibited invasion and migration of SPC-A-1cells,with a decreased protein expression of NF-kB, MMP-2and MMP-9.5. Overexpression of THOC1inhibited the tumor growth vability of SPC-A-1cells,a lower average weight and volume of the THOC1-overexpressed tumors were observed.There was no obvious effets on the weight, viscera index of main organs, bloodbiochemical indexes including AST, ALT, CREA, UREA and ALP. The morphologicalchanges of major organs had not been observed; Immunofluorescence experimentshowed that overexpression of THOC1caused a significant decrease in Ki-67and VEGF expression.6.After X-rays exposure, overexpression of THOC1increased the radiosensitivityin both of the SPC-A-1and NCI-H1975cells, caused a G2/M arrest, with an increasedexpression of cyclinB1and a decrease of cyclinD1. Enhanced expression of THOC1promoted apoptosis rate and the mean fluorescence intensity of ROS, with a reducedexpression of p-Akt, an increased expression of p-P38and p-Erk1/2.7.Compared with blank cells and Neo cells, the "comet" tail length, tail momentand the number of-H2AX foci had no obviouse changes in THOC1-overexpressedcells after irradiation with X-rays. Moreover, there was no significant alterations of aseries of DNA damage repair protein levels including Ku-70, Ku-80, DNA-PKcs,XRCC4, Rad50, Mre11.Conclusion1. SPC-A-1/THOC1cells, NCI-H1975/THOC1cells and95D/shTHOC1cellshave been obtained in lung cancer cell lines.2. Overexpression of THOC1inhibits the growth of lung cancer cells in vitro andin vivo, which is probably related to a change of cell cycle distribution, apoptosisinduction and the regulation of PI3K-Akt and MAPK signaling pathway.3. Obvious suppression of invasion, metastasis is observed in lung cancer cellswith enforced expression of THOC1, accompanying with a decrease of NF-kB, MMP-2and MMP-9.4. Overexpression of THOC1increases cell sensitivity to X-rays, which may berelated with its regulation of cell cycle progression, apoptosis induction and PI3K-Aktand MAPK signaling pathway. The participation of THOC1in the DNA damage repairneeds to furher experiments.