| Research Background:Lung cancer is one of the most harmful malignant tumors in the world today.Non-small cell lung cancer(NSCLC)accounts for 75%to 80%of them,and the poor prognosis is still a prominent problem,although the research on NSCLC has made more progress.Therefore,it is of great clinical significance to look for valuable methods for early diagnosis and markers for prognosisto assessment for NSCLC.With the wide-ranging advancement of lung cancer screening,early diagnosis of the lung cancer,in which the lung adenocarcinoma in NSCLC accounts for the vast majority has become a reality.There are two subtypes of lung adenocarcinoma,called the minimally invasive pulmonary adenocarcinoma(MIA)and the invasive pulmonary adenocarcinoma(IAC)respectively;both of them require surgery treatment,but surgical procedures and the five-year survival rate are significantly different;at the same time,their medical imagings are presented as small ground-glass nodules and hard for biopsy,so,the diagnosis often relies on high resolution high-resolution computed tomography(HRCT).In addition,recent studies have found that long noncoding RNAs(lncRNAs)and microRNAs(miRNAs)are involved in biological processes including apoptosis and cell growth cycle,also play key roles in the occurrence and development of human diseases.Whether they have influence on the corresponding biological processes in NSCLC with high incidence rate has become a hot spot now.Research Aim:This study retrospectively analyzed HRCT characteristics of lung adenocarcinoma patients and explored the regulatory roles of 1ncRNAs and miRNAs in lung NSCLC cell lines A549 and H1975.So as to identify MIA and IAC as well as explore the pathological mechanisms of NSCLC and the functions,interrelationships of 1ncRNA-HOTTIP,miR-206 and Epidermal growth factor receptor(EGFR)in A549 and H1975 as well,and thereby providing a new insight for the treatment of NSCLC.Research Methods:Chapter One:The subsolid nodules(SNs)of 251 patients with lung adenocarcinoma confirmed from September 2008 to June 2012 were reviewed retrospectively at the Department of Radiology.When screening research materials,the inclusion criteria were:(1)SNs? 3 cm,characterised as either MGGN or PGGN;(2)surgically resected and confirmed by postoperative histopathological diagnosis to be MIAs(n = 98)or IACs(n = 162);(3)management procedures implemented in strict accordance with the 2013 guidelines of the Fleischner Society.Routine CT examinations were performed initially with a collimation of 128 x 1.25 mm and a field of view(FOV)of 500 mm.When nodules were found,target HRCT was performed with the following parameters:64 × 0.625 mm collimation,0.64 pitch,1 mm section thickness and 1 mm interval,1-3 second scan time,1024×1024 matrix;180 mm FOV,120 kV,and 300 mA.All images were displayed at a lung window width of 1500 HU,window level of-500 HU,mediastinal window width of 350 HU,and window level of 35 HU using the picture archiving and communication system.Two chest radiologists(with 23 and 18 years of experience in chest CT,respectively)blindly and independently evaluated all CT data.In cases of discrepancy,a third radiologist was consulted to reach a consensus.The following CT characteristics were examined:patterns,shapes,proportion of the solid component,borders,margins,air-bronchograms,microvascular signs,vacuole sign and pleural indentation.Quantitative variables included:(1)diameter of the nodule(largest diameter on the axial section);(2)diameter of the solid component(largest diameter of solid component on the axial section);and(3)CT radiodensity values(PGGN,GGO component of MGGN,and solid component of MGGN),in the largest region of interest that excluded pulmonary vessels.All measurements were performed by a single radiologist,repeated three times,and averaged.The histopathological diagnosis and categorisation of MIA and IAC were performed according to the new pulmonary adenocarcinoma classification,2015 edition.GGNs were resected by video-assisted thoracoscopic surgery.Specimens were fixed in 10%formalin and embedded in paraffin.Representative haematoxylin and eosin stained sections were reviewed.In case of equivocal histopathological classification under light microscopy,immunohistochemistry was performed for clarification.All histological preparations and analyses were performed by two senior pathologists.Disagreements were resolved in consensus,which was reached after discussion and/or consultation with a third pathologist.Quantitative variables were assessed by two-sample Wilcoxon's rank-sum tests,and qualitative variables by Pearson's chi-square test and Fisher's exact test,as appropriate.The variables that exhibited statistically significant differences were included in multivariate logistic regression analysis.Receiver operating characteristic(ROC)analyses were conducted for variables that exhibited statistically significant differences in multivariate logistic regression analysis.The cut-off values were defined as those for which sensitivity plus specificity were maximal.p<0.05 was considered statistically significant.All statistical analyses were performed using the SPSS 19.0 software.Chapter two:In this experiment,human lung NSCLC cell lines A549 and H1975 were used as research materials.Cells were transferred to RPMI 1640 medium(Roswell Park Memorial Institute 1640)containing 10%fetal bovine serum(FBS)and antibacterial-antifungal mixture solution,when the cell traits were stable;conventional continuous culture was carried out by means of a carbon dioxide cell incubator(37?,containing 95%air and 5%CO2),and the cells grown under the above conditions were used as the "control group".We detected the relative expression of HOXA transcript at the distal tip(HOTTIP)in normal tissues and tumor tissues by quantitative real-time polymerase chain reaction(qRT-PCR).To further explore whether knockdown HOTTIP had an effect on NSCLC cells A549 and H197,we constructed si-HOTTIP and its negative control(si-NC)to inhibit the expression of HOTTIP,and HOTOIP low expression mutants were formed by transfecting them into A549 and H1975 cells through lipofectamine 3000 reagent.The cells were referred to as "si-HOTTIP group" and"si-NC group",respectively.After the transfection was completed,the relative expression of HOTTIP at the RNA level in the cells of different treatment groups were measured again by qRT-PCR to determine the transfection efficiency.The viability of cells in the "control group","si-HOTTIP group" and "si-NC group" were evaluated by cell counting kit-8(CCK-8),respectively.The apoptosis rate of cells were measured by fluorescein Isothiocynate(FITC)coupled annexin V(Annexin V)and Propidium iodide(PI)double staining(Annexin V-FITC/PI)combined with flow cytometry.The protein levels of Bax,cleaved-Caspase-3 and cleaved-Caspase-9 in each group as well as the expression of key proteins in the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB/AKT)signaling pathway and adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)signaling pathway,including total PI3K(t-PI3K),phosphorylated PI3K(p-PI3K),total AKT(t-AKT),phosphorylated AKT(p-AKT),phosphorylated AMPK(p-AMPK)and total AMPK(t-AMPK)were detected and analyzed by western blot.The ratio of p-PI3K to t-PI3K(p/t-PI3K)and the ratio of p-AMPK to t-AMPK(p/t-AMPK)were quantitatively analyzed by Image LabTM software.The transwell cell culture chamber(polycarbonate membrane with 8 micron pore size at the bottom)and 24-well Millicell hanging cell culture chamber(PET membrane with 8 ?wM pore size)were used to test cells migration and invasion in vitro.All experiments were repeated at least three times and statistical analysis was performed using SPSS 19.0 software.All results were expressed as mean± standard deviation(SD),and the P values were calculated by one-way analysis of variance(ANOVA).Chapter three:To further explore the effect of knockdown HOTTIP on the pathological mechanism of NSCLC,also to reveal the relationship between HOTTIP and miR-206,the qRT-PCR was used to detect the relative expression levels of miR-206 in A549 and H1975 cells in the "control group","si-HOTTIP" treatment group and "si-NC"treatment group respectively,in this study.In addition,the miR-206 in(miR-206 inhibitor)which could inhibit miR-206 expression,and its corresponding negative control NC in(NC inhibitor)were constructed and then transfected into A549 and H1975 cells as the "miR-206 in" treatment group and the "NC in" treatment group.At the same time,in order to further verify whether miR-206 mediated the function of HOTTIP,we co-transfected the si-HOTTIP and si-NC with NC in or miR-206 in into A549 or H1975 cells respectively,called "si-NC + NC in" group,"si-HOTTIP+ NC in" group and "si-HOTTIP + miR-206 in" group.The transfection efficiency was determined by qRT-PCR.As in the previous experiments,we used the CCK-8 method to examine A549 and H1975 cells viability of the "control group",the "si-NC + NC in" treatment group,the "si-HOTTIP+ NC in" treatment group and the "si-HOTTIP + miR-206 in" group,respectively.The apoptosis rate of was measured by Annexin V-FITC/PI double staining and flow cytometry.The protein expression levels of Bax,cleaved-Caspase-3 and cleaved-Caspase-9 in each group were determined by Western blot and the corresponding protein bands were analysed by Image LabTM software.The migration and invasion tests were carried out using transwell cell culture chamber and 24-well Millicell hanging cell culture chamber.In order to verify whether the epidermal growth factor receptor(EGFR)is a target of miR-206,we constructed and transfected the miR-206 mimic,the NC mimic,the wild-type EGFR luciferase reporter plasmid(EGFR-wt),and the mutant-EGFR luciferase reporter plasmid(EGFR-mut)follwed by a dual luciferase assay.The "miR-206 mimic + EGFR-wt" treatment group,the"NC mimic + EGFR-wt" treatment group,the "miR-206 mimic + EGFR-mut"treatment group,and the "NC mimic + EGFR-mut" treatment group were defined,respectively.Research result:Chapter 1:microinvasive lung adenocarcinoma and invasive lung adenocarcinoma with subsolid nodules(diameter? 3 cm)can be distinguished according to HRCT featuresThe experiment results showed that there were significant differences in age between the patients of the two group.Patients with MIA(53.4 ± 12.5)were younger than patients with IAC(60.4± 8.0)(P<0.001).In addition,compared with the IAC group(la,131;Ib,31),the tumour node metastasis(TNM)stage(Ia,98;lb,0)was significantly lower in the MIA group(P<0.001).There were no pleural invasion(P<0.001)and disease recurrence(P = 0.047)in the MIA group compared to the IAC group.86%of IAC patients underwent lobar resection compared with only 30%of MIA patients(P<0.001).Meanwhile,56%of the MIA patients had a wedge resection compared with 14%of IAC patients(P<0.001).There were no differences between the two groups in sex and laterality.There was a significant difference in the frequency of PGGNs between the MIA group and the IAC group(P<0.001).The frequency of PGGNs in the nodules of the MIA group was 4.9%,compared with 60.2%in the IAC group.Compared with patients in the IAC group,patients with MIA showed a higher frequency of regular shape(P<0.001),smaller diameter(P<0.001),smaller solid component(P<0.001),lower CT radiodensity value of the GGO component of MGGNs(P<0.001),lower CT radiodensity value of the solid component of MGGNs(P<0.001),lower frequency of undefined borders(P = 0.02),more regular margins(P<0.001),lower frequency of microvascular sign(P<0.001),higher frequency of air bronchogram in the GGO component(P<0.001),less vacuoles(P<0.001),and lower frequency of pleural indentation(P<0.001).For patients in the MIA group,the volume of the nodule and the diameter of the solid component were significantly reduced(P = 0.029 or 0.031);the ratio of the solid component was significantly lower(P = 0.002),less than 50%;the CT radioactivity value of the sexual components was significantly lower(P = 0.005);the frequency of bronchial aeration and microvascular signs was higher(P = 0.008 or 0.006).Chapter 2:knockdown HOTTIP inhibited NSCLC cell proliferation,migration and invasion,and activity of PI3K/AKT and AMPK signaling pathways but promoted apoptosisThe expression level of HOTTIP was significantly increased in lung cancer tissue cells compared to normal lung tissue cells(P<0.001).In A549 cells,si-NC transfection did not significantly affect the relative expression of HOTTIP at the RNA level compared to control cells;however,compared with the "si-NC" group,si-HOTTIP transfection remarkably decreased the relative expression of HOTTIP at the RNA level(P<0.01).The situation in H1975 cells was consistent with A549 cells.These results indicated that we successfully transfected si-HOTTIP and si-NC into A549 and H1975 cells.The cell viability assay showed that in A549 cells,si-NC transfection had no impact on the cell viability when comparing with the "control"group,but,in comparation with the "si-NC" group,the viability of cells in the"si-HOTTIP" group was significantly decreased(P<0.01).In H1975 cells,compared with the "control" group,the "si-NC" group cells had markedly lower viability;the cell viability in the "si-HOTTIP" group was significantly lower than that in the"si-NC" group(P<0.05).These results indicated that knockdown HOTTIP significantly inhibited cell proliferation.The apoptosis assay showed that the apoptosis rate of the "control" group cells was basically in accordance with the "si-NC" group cells.Compared with the "si-NC"group,the apoptosis rate of the A549 and H1975 cells in the "si-HOTTIP" treatment group was significantly improved(both P<0.001).The expression of Bax,cleaved-caspase 3 and cleaved-caspase 9 were significantly promoted compared with the "si-NC" group(all P<0.001);there was no significant difference between the"si-NC" group and the "control group".The migration and invasion test results illustrated that compared with the"control" group,si-NC did not significantly affect the relative mobility of A549 and H1975 cells;compared with the "si-NC" group,knockdown HOTTIP notably decreased the relative mobility of the cells(both P<0.05).For A549 and H1975 cells,there were no significant differences in the invasion between the "control" group and the "si-NC" group;in comparison with the "si-NC" group,the relative invasive rate of cells in the "si-HOTTIP" group was significantly decreased(both P<0.01).Western blot analysis of PI3K/AKT signaling pathway proteins demonstrated that the expression levels of the p-PI3K and the p-AKT,the p/t-PI3K and the p/t-AKT as well in the "si-HOTTIP" group were significantly reduced(both P<0.01)when comparing with that of the "si-NC group A549 cells;there was no apparent difference between the "control"group cells and the "si-NC" group cells.For H1975 cells,the relative expression levels of all proteins and p/t-PI3K or p/t-AKT in the "control"group cells were essentially the same as the cells in the "si-NC" group;compared to the "si-NC" group,the relative expression levels of p-PI3K and p-AKT as well as p/t-PI3K(P<0.01)and p/t-AKT(P<0.05)in the "si-HOTTIP" group were significantly reduced.But there was no significant change in the relative expression levels of t-PI3K and t-AKT in H1975 cells of all treatment groups.For A549 cells,the relative expression levels of each protein and p/t-AMPK in the "control",group cells were basically consistent with the cells in the "si-NC" group.Compared with the'"si-NC" group,the expression level of p-AMPK and p/t-AMPK(P<0.05)in the"si-HOTTIP" group cells were significantly decreased.However,there was no significant difference in the expression level of t-AMPK between the cells in each treatment group.For H1975 cells,the "control" group was in keeping with the "si-NC"group.But compared with the "si-NC" group,knokdown HOTTIP significantly decreased the relative expression of p-AMPK and p/t-AMPK(P<0.01)Chapter 3:knockdown HOTTIP inhibited cell proliferation,migration and invasion but promoted apoptosis by up-regulating expression of miR-206 targeting EGFRqRT-PCR revealed that compared with the "control" group cells,si-NC did not significantly alter the expression of miR-206 at the RNA le,vel in A549 and H1975 cells.However,compared with the "si-NC" treatment group,the relative expression of miR-206 were significantly promoted in the "si-HOTTIP" treated group A549 and H1975 cells(P<0.01 or P<0.001).At the same time,we also found that compared with the "control" group,NC in did not affect the expression of miR-206.In comparison with the "NC in" treatment group,the relative expression levels of miR-206 at the RNA level were significantly decreased in the "miR-206 in" treatment group A549 and H1975 cells(both P<0.01).The CCK-8 test results indicated that the cells viability in the "si-NC + NC in" treatment group did not change notably,compared with the "control" group;moerover the viability of A549 and H1975 cells(both P<0.01)in the "si-HOTTIP +NC in" treatment group were significantly lower than that in the "si-NC + NC in" treatment group;compared with the "si-HOTTIP +NC in" treatment group,co-transfection of si-HOTTIP and miR-206 in resulted in a significant elevation in the viability of both A549 and H1975 cells(both P<0.05).Similarly,the apoptosis rate of A549 and H1975 cells in the "si-NC + NC in"treatment group was consistent with the "control" group;compared with the "si-NC+NC in" treatment group,the apoptotic rates of A549 and H1975 were significantly elevated after the si-HOTTIP and NC in co-transfection(both P<0.001);in comparison with the "si-HOTTIP + NC in" treatment group,the apoptosis rates of A549 and H1975 cells in the "si-HOTTIP + miR-206 in" treatment group were significantly decreased(both P<0.05).Western blot suggested that the relative expression levels of Bax,cleaved-Caspase-3 and cleaved-Caspase-9 in A549 and H1975 cells did not occur obvious change in the "si-NC + NC in" treatment group,compared with the "control group";in contrast with the "si-NC + NC in"treatment group,the expression levels of the apoptosis-related proteins in the"si-HOTTIP + NC in" treatment group A549 cells were significantly elevated(all P<0.001);besides,the expression of proteins in H1975 cells were also markedly improved(all P<0.001).Compared with the "si-HOTTIP+ NC in" treatment group,the relative expression of apoptotic proteins were significantly attenuated(all P<0.01)in the "si-HOTTIP + miR-206 in" treatment group A549 cells.In H1975 cells,the relative expression of each apoptotic protein was significantly reduced(P<0.05).The cell migration and invasion assays showed that there was no significant change in the relative mobility rate and invasive rate of A549 and H1975 cells in the "si-NC + NC in" treatment group,compared with the "control" group;and the relative mobility rate and invasive rate of the cells in "si-HOTTIP + NC in" treatment group were significantly lower(all P<0.01)than that in the "si-NC + NC in" treatment group.The relative mobility rate and invasive rate of A549 and H1975 cells in the"si-HOTTIP + miR-206 in" treatment group were significantly higher than those in the "si-HOTTIP + NC in" treatment group(all P<0.05).The EGFR expression were detected by the western blot.For the A549 and H1975 cells,there was no significant difference between the "control" group and the"si-NC + NC in" treatment group.However,compared with the "si-NC + NC in"treatment group,knockdown HOTTIP significantly inhibited the expression of EGFR protein(both P<0.01);compared with the "si-HOTTIP + NC in" treatment group,the relative expression level of EGFR was significantly increased when knockdown HOTTIP but silence miR-206 at the same time in the "si-HOTTIP + miR-206 in"treatment group.(both P<0.05).The dual luciferase activity assay suggested that co-transfection of NC mimic and EGFR-wt did not make sense.In contrast,the luciferase activity were significantly reduced after co-transfection of miR-206 mimic and EGFR-wt(P<0.05).What's more,compared with the control group,co-transfection of the NC mimic and the EGFR-mut or co-transfection of the miR-206 mimic and the EGFR-mut did not significantly change the luciferase activity in cells.Conclusions:HRCT could be used for the preoperative differential diagnosis for MIA and IAC patients who have subsolid nodules with a diameter of?3 cm.Knockdown HOTTIP inhibited the proliferation,the migration and the invasion but promoted the apoptosis of NSCLC cells by upregulating miR-206,whereas EGFR was the target of miR-206.Significance:The present study systematically compared the image characteristics of MIAs and IACs appearing as SNs of ?3 cm in diameter by the HRCT,which provided a basis to help selecting accurate treatment plans for MIA and IAC patients.What's more,the mechanisms of action of HOTTIP,miR-206 and EGFR in the NSCLC cell lines A549 and H1975 were revealed at the molecular level,and this provided a new target for the intervention or treatment of lung cancer. |
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