Experimental Study On The Radiation Sensitivity And Mechanism Of LKB1 In Lung Adenocarcinoma

BackgroundAt present in the world, lung cancer has been the highest incidence of malignant tumors, morbidity and mortality rate is increasing year by year, but its 5 year survival rate has been hovering around 15%-20%, the curative effect needs to be improved urgently. Radiotherapy, as one of the main treatment methods of lung cancer, plays an important role in the comprehensive treatment of lung cancer. How to improve the radiation sensitivity of lung cancer has been a hot issue in the field of cancer research. Liver kinase B1 (LKB1) belongs to calcium/calmodulin dependent protein kinase like family, many studies suggest thatLKB1participateoccurrence, development andmetastasisof lung cancerthrough multiple signaling pathways which correlate with p53 and p21 signals not seldom, and which are radiosensitivity related agentsas all known. Therefore, we hypothesize that LKB1 could enhance lung cancer cell radiosensitivity, and have some preliminary explorations through construction of fusion expression plasmid pEGFP-LKB1 which was transfected to LKB1 mutations A549 and H460 cell line. The experimentindicated that LKB1 overexpressionenhanced the radiosensitivity of A549 and H460 cells. On the basis, in this studyLKB1 small interfere fusion plasmid was constructed and screened, human lung adenocarcinoma cell line H1299 and 95-D which LKB1 expressnormally were transfected by the fusion plasmidagainst human LKB1, the biological behaviors and radiation sensitivity, and possible mechanism were observedin vitro. In addition, the subcutaneous tumor H460 cell model in nude mice was constructed to carry out experiments on LKB1 overexpressionenhanced lung adenocarcinomaradiosensitivity in vivo.After definitive radiotherapy, up to one third of patients will have local recurrence and distant metastasis. Radioresistant cancer cells likely play a crucial role in the recurrence and metastasis of lung cancer after radiotherapy. Several studies suggested that radioresistant cancer cells have enhancedinvasion and metastasis potential. It is thought that radioresistance and metastasis could be potentially linked by epithelial-mesenchymal transition (EMT). In our previous work, A549 radiation resistant cell lines were constructed which LKB1 deficient, and transfected by LKB1 overexpression plasmid,the results showed that upregulation of LKB1 inhibited EMT and weakened the invasion and migration.On the basis of previous experiments,we established the H1299 radioresistant cell line and downregulated LKB1 expression, to investigate the potential relationship among radioresistance. EMT. and enhanced metastatic potential and the underlying mechanism involving liver kinase B1 (LKB1)-Salt-inducible kinase 1 (SIK1)and Zinc-finger E-box-binding homeobox factor (ZEB1)signaling.This study has a certain practical significance and application prospects for improving theirradiation sensitivity of lung cancer cells, and obtains a theoretical and experimental foundation for improving the curative effect of lung cancer. This study was divided into the following two parts. PART I:Study on the effect of LKB1 on radiation sensitivity of lung adenocarcinoma, and PART ?:Study on LKB1 regulation of epithelial mesenchymal transition in lung adenocarcinoma radiation resistant cells.PART I:Study on the effect of LKB1 on radiation sensitivity of lung adenocarcinomaObjective:To observe the effect of LKB1 interference on the biological behaviors andradiosensitivity lung adenocarcinoma H1299 and 95-D cell lines which LKB1 expressnormallym vitro.And to investigate the radiosensitivity by LKB1 ovrrexpression in nude mice transplanted tumor model with H460 cell which has LKB1 deletion in vivo.Methods:The pshLKB1 small interfering plasmid with enhanced green fluorescent protein was constructed and identified, then was transfected to HI299 and 95-D cells, fluorescence microscope was used to observe the expression of green fluorescent protein. The radiosensitivity was evaluated by Cell Counting Kit-8 assayandclonogenic cell survival assay, cell cycle distribution analysis was observed by flow cytometer.The ability ofmigration was evaluated bywound healing assays. The LKB1, y-H2AX, p-Chk2, p53 and p21 protein expressionchange were assessed by Western blotting. The nude miceswith lung cancer H460 subcutaneous transplantation tumor were randomly divided into empty plasmid control (pEGFP-Ctrl) group, overexpression LKB1 (pEGFP-LKB1) group, irradiationcombined empty plasmid (IR+pEGFP-Ctrl) group, and irradiation combined overexpression LKB1 (IR+pEGFP-LKB1) group, the tumor growth and radiosensitivty were observed, HE staining, immunohistochemical staining and Western blotting were used to detect the expression of LKB1.Results:The pshLKB1 plasmid was successfully constructed by enzyme digestion and sequencing. After transfection of pshLKB1 plasmid,12,24,48 and 72 h, green fluorescent protein expression was observed, green fluorescent protein and LKB1 protein expression reached a peak at 24 h after transfected.Proliferation activity of HI299 and 95-Dcells downregulatedLKB1 enhanced by CCK-8 assay, cells in G1 period and G2/M phase cell ratio decreased significantly, while the proportion of cells in S phase increased through flow cytometer, migration ability upgraded bywound healing assays, the difference were statistically significant (P<0.05, respectively).Colony forming rate of H1299 and 95-D cellsdownregulatedLKB 1 increased, Do and Dq increased,?/?value decreased, and SER was 0.809 and 0.735 respectively by clonogenic cell survival assay.Combined with X-ray irradiation, the survival rates of H1299 and 95-d cells downregulatedLKB1 increased for variousdosesby CCK-8 assayrespectively, the difference was maximum for 8 Gyirradiation dose (P<0.05); cell cycle distributionshowed that after downregulation with LKB1 andirradiation, cellsin G0/G1 period and G2/M phase cell proportionreduced significantly, while the proportion of cells in S phase increased through flow cytometer (P<0.05); Western blotting showedthatLKB1,?-H2AX, p-Chk2,and p21 protein expressiondecreased (P<0.05);H1299 cells were not detected p53 protein expression, reduced expression ofp53 protein for 95-D(P<0.05).Nude mice transplanted tumor experiments showed that pEGFP-LKB1 group, IR+pEGFP-Ctrl group and IR+pEGFP-LKB1 group tumor growth were inhibitedat differently degree. Tumor weight and tumor volume of IR+pEGFP-LKB1 groupwere the lowest among these groups. UpregulationLKBl and irradiationhadsynergistic inhibition forlung cancer (P<0.05). The tumor tissues of pEGFP-LKB1 group and IR+pEGFP-LKB1 groupexpressed LKB1 protein, the other two groups without LKB1 expression by immunohistochemical staining and Western blotting.Conclusions:Interference of LKB1 might regulate the proliferation, cycle distribution and migration of lung adenocarcinoma cells in vitro.LKB1 may have the effect of radiation sensitization,which possibly regulates p53 and p21 signals to affect the radiosensitivity of lung adenocarcinoma cells.PART II:Study on LKB1 regulation of epithelial mesenchymal transition in lung adenocarcinoma radiation resistant cellsObjective:To establish radioresistant human lung cancer H1299 cells and to detect the cells'biological characteristicschanges such as proliferation, apoptosis, invasion and migration.Then, to interfere the expression of LKB1 investigating the potential relationship between radioresistance and enhanced metastatic potential, and the underlying mechanism involving the LKB1-SIK1 signalingin vitro.Methods:The radioresistant cell line H1299R was established by exposure to conventional dose fractionation mode,sublethal dose mode and gradiently increased dose mode,respectively.Microscope was used to monitor the morphological changes.Colony formation assay,CCK-8 cell viability kit and FACScan were used to confirm the capacity of radioresistance.Transwell cell assay was applied to test the invasion and migration abilities.The expression of epithelial cell marker and mesenchymal cell marker were detected by western blotting.Results:Gradiently increased dose mode successfully induced the radioresistance of H1299R.In terms of biomorphology,the morphology of radioresistant cells was transition to mesenchymal cell morphology. The colony forming abilities and survival rates of H1299R were higher than the control groups,respectively (P<0.05), whereas the apoptosis rates were decrease (P<0.05). By used of transwell cell assay, the invasion and migration abilities were increased,respectively(P<0.05). Over-expression of vimentin andZinc-finger E-box-binding homeobox factor 1(ZEB1) were observed in radioresistant cells, while E-cadherin,LKBl andSIKlwere down-regulated,respectively (P<0.05).Knockdown of LKB1 in H1299R cells led to further promotion ofthe invasion and migration abilities, enhancement of the EMT phenotype, downregulation of LKB1 andSIK1, and upregulation ofZEBl,respectively (P<0.05).Conclusions:Radioresistant lung adenocarcinoma cell lines were established successfully,which showed more invasion and migration abilities than non-radioresistant cells which correlated with epithelial-mesenchymal transition potentially.Our work suggests attenuated LKB1-SIK1 signaling contributes to enhanced metastatic potential of radioresistant cancer cells.Targeting the LKB1-SIK1-ZEB1 pathway might reduce the risk of metastasis after radiotherapy.