| [Background and Objective]:Lung cancer is the most common malignancy and is a leading cause of mortality worldwide due to cancer.Non-small cell lung cancer accounts for 80%of all lung cancer cases.Xuanwei City in Yunnan Province is an area with the highest mortality rate due to lung cancer in China.In Laibin Town of Xuanwei city,the morbidity rate of female lung cancer is 20 times higher than that of national average.Indoor air pollution caused by burning of bituminous coal is the main cause of NSCLC in the population of Xuanwei region.Nano-silica particles,a by-product due to incomplete combustion of coal is the main component of indoor pollution in this area.The special microstructure and forming conditions of nano-silica has been the topic of research by scholars at home and abroad.In an experimental cell toxicity study,it suggests that nano silica particles could enhance the expression of miR-501-5p,and the expression of miR-501-5p was significantly increased in lung adenocarcinoma tissues in Xuanwei area.Likewise,the nano silicon dioxide particles were found in lung adenocarcinoma tissues in patients from Xuanwei area.It revealed the relationship between nano silicon dioxide,miR-501-5p and lung adenocarcinoma in Xuanwei area.It is not clear whether miR-501-5p plays a role in the development of lung cancer or the regulatory mechanism of miR-501-5p that affects on the biological function of lung adenocarcinoma cells.Aim of this study is to establish specific microRNA expression profile of lung adenocarcinoma of Xuanwei area by microRNA chips,to detect the expression of miR-501-5p in lung adenocarcinoma tissues and serum,and to analyze the correlation between the expression of miR-501-5p and the clinical factors.The effects of miR-501-5p on cell proliferation,migration,invasion and apoptosis resistance were observed in vitro.The target genes and signaling pathways of miR-501-5p were studied preliminarily.It is significant to investigate the function and mechanism of miR-501-5p in NSCLC.Section ?:Specific MicroRNA Profiles and their predicted Target Gene and Signaling Pathways in Lung Tissues of Patients with Lung Adenocarcinoma in Xuanwei Region[Objective]:To screen of differential expression of microRNAs?microRNAs?profiles and predict related target genes and signaling pathways of these microRNAs in lung adenocarcinoma tissues from Xuanwei region.[Methods]:Lung cancer tissues and normal lung tissues were collected from 24 cases of lung adenocarcinoma from Xuanwei regions who underwent surgery and 10 cases from non-Xuanwei regions were selected for screening of microRNA expression profiles.The results of microarray were verified by QPCR.By Ago and KEGG pathway analysis,the related target genes and signaling pathways were predicted by bioinformatics.[Results]:1.Compared with the normal lung tissues,153 differential expressed microRNAs of lung adenocarcinoma in Xuanwei region were identified,64 were up-regulated and 89 were down-regulated.83 differential expression of microRNAs were identified in lung adenocarcinoma in non-Xuanwei area,two were up-regulated and 81 were down-regulated.2.The chip verification:Compared with normal lung tissues,the results of the microRNAs expression level in lung cancer tissue were?values of chip and qPCR?:hsa-miR-21-3p?2.564,15.134?,hsa-miR-182-5p?2.426,16.625?,hsa-miR-210-3p?2.742,17.625?,hsa-miR-30a-5p?0.288,0.307?,hsa-miR-126-5p?0.259,0.398?,both methods showed consistent results,supported the chip results.3.Specific microRNA expression profile of lung adenocarcinoma in Xuanwei area showed 34 differential expression of microRNAs.Twenty-three upregulated microRNAs were hsa-miR-504-5p,hsa-miR-501-5p,hsa-miR-147a,hsa-miR-372-3p,hsa-miR-187-3p,hsa-miR-325,hsa-miR-369-3p,hsa-miR-33b-5p,hsa-miR-182-5p,hsa-miR-210-3p,hsa-miR-595,hsa-miR-9-5p,hsa-miR-224-5p,hsa-miR-558,hsa-miR-495-3p,hsa-miR-485-5p,hsa-miR-134-5p,hsa-miR-519d-3p,hsa-miR-96-5p,hsa-miR-375,hsa-miR-645,hsa-miR-938,and hsa-miR-13 5b-5p.The 11 down regulated microRNAs were hsa-miR-598-3p,hsa-miR-138-5p,hsa-miR-99a-5p,hsa-miR-34b-3p,hsa-miR-30d-5p,hsa-miR-140-3p,hsa-miR-34c-3p,hsa-miR-497-5p,hsa-miR585-3p,hsa-miR-30c-5p and hsa-miR-363-3p.The biological function of the predicted target gene on lung cancer may be related with PI3K/Alt,Wnt and MAPK signaling pathway.[Conclusion]:These specific microRNAs may play an important role in carcinogenesis and progression of lung adenocarcinoma in Xuanwei region,which may relate to the regulation of the predicted target genes and PI3K/Alt,WNT and MAPK signaling pathway.Section ?:Exploration of expression and significance of miR-501-5p in lung adenocarcinoma cancer from Xuanwei area[Objective]:To study the expression of miR-501-5p in adenocarcinoma tissues and serum of patients from Xuanwei area and its correlation with clinical characteristics.[Method]:The expression of miR-501-5p was detected in lung adenocarcinoma tissues of 24 patients from Xuanwei area and in serum of 42 patients with lung adenocarcinoma and 17 patients with benign tumor by qPCR technique.The analysis was done by chi square test between the expression of miR-501-5p in Xuanwei lung adenocarcinoma specimens with clinical characteristics?age,gender,TNM staging of lung cancer and serum CEA?.Multiple regression analysis of correlation between miR-501-5p expression and clinical features were done.[Results]:1.The result of microRNA chip and qPCR indicated high expression of miR-501-5p in tumor tissue of lung adenocarcinoma patients from Xuanwei area.The expression of miR-501-5p by qPCR in tumor tissues and normal lung tissues were 3.937±4.507 and 1.000±1.25680,respectively,?t =3.076,p<0.01?.The miR-501-5p expression in microRNA chip in tumor tissues and normal lung tissues was 1.439±2.004 and 0.454±0.323,respectively,?t--2.449,p<0.05?.2.The expression of miR-501-5p in serum of lung adenocarcinoma patients from Xuanwei region,lung adenocarcinoma patients from other area and benign tumor patients was?mean±SD?3.6031±2.32556,2.4200±1.94891 and 2.3023±1.89972,respectively,there was no statistical difference?p>0.05?.There was no difference in expression in three groups.3.Chi-square test suggested that expression of miR-501-5p was significantly correlated with patient's age,TNM stage,and CEA level in serum,but no correlation with patient's gender.The expression of miR-501-5p was lower in patients<65 than those>65?0.95329±1.37376 and 3.28610±3.03752??F=7.168,p<0.05?.The expression of miR-501-5p in stage ?-? patients and ?-? patients were 0.50563±0.24672 and 4.24027±2.39244?F=36.627,p<0.01?,respectively.The expression of miR-501-5p in patients with lymph node positive and negative were 4.21712±2.43735 and 0.5133±0.2405?F=51.337,p<0.01?.The expression of miR-501-5p in patients with less than 5ng and>5ng CEA in serum were 0.64223±0.74506 and 3.37500±2.78409,respectively?F=30.045,p<0.01?.Multiple regression analysis showed that miR-501-5p expression was positively correlated with age of patient,TNM stage,and CEA value?p<0.05?.[Conclusion]:The expression of miR-501-5p in lung adenocarcinoma tissue of patients from Xuanwei area is up-regulated.The expression level of miR-501-5p is significantly correlated with age,TNM stage,lymph node metastasis and CEA level in lung adenocarcinoma patients from Xuanwei area.It suggests that miR-501-5p may act as an oncogene in progression of lung adenocarcinoma in Xuanwei area and the possible biomarker of lung adenocarcinoma in Xuanwei area.Section ?:The effects of miR-501-5p on non-small cell lung cancer cell lines A549 and XWLC-05[Objective]:To study in vitro effect of miR-501-5p on the proliferation,migration,invasion and anti-apoptosis capacity of human non-small cell lung cancer cell lines A549 and XWLC-05.[Methods]:The expression level of miR-501-5p in human non-small cell lung cancer cell lines was detected using real-time reverse transcriptase polymerase chain reaction.Moreover,miR-501-5p expression in A549 and XWLC-05 cells was silenced by tansfecting LV3?H1/GFP&Puro?-hsa-miR-501-5p sponge inhibitor.The transfection efficiency was tested by qPCR and fluorescence microscope.Moreover,MTT assay,transwell migration and invasion assay were also performed to investigate the effect of down-regulated miR-501-5p on its ability to proliferate,migrate and invade using stable transfected A549 and XWLC-05 cells.Apoptosis and cell cycle was detected in A549 and XWLC-05 cells 48h post transfection?transfected LV2?U6/Puro?-has-miR-501-5p sponge inhibitor?by Flow cytometric detection.[Results]:1.Compared with the expression level of miR-501-5p in BEAS-2B cells,the expression level of miR-501-5p in lung cancer cell lines A549 and XWLC-05 was2.827±1.799 and 2.309±0.364,respectively.And it was more than 2 folds higher compared with that of BEAS-2B cells?P<0.05?.2.The expression of miR-501-5p in inhibitor group and NC group of A549 cells 24h post transfection was 0.163±0.044,1.000 ± 0.181,respectively?t=-7.758,p<0.05?.In XWLC-05 cells,the expression of miR-501-5p in inhibitor group and NC group 24h post transfection was 0.212 ± 0.094,1.000 ± 0.273,respectively,?t=-4.734,p<0.05?.Fluorescence microscopy showed that the transfection efficiency was more than 50%.The transfection of lentiviral vector had significant effect.3.In vitro effects of miR-501-5p down-regulation on the biological function of A549?XWLC-05 cells.? Down-regulation of miR-501-5p inhibited the proliferation of A549 cells 24h after transfection?p<0.05?.In the same way,Down-regulation of miR-501-5p inhibited the proliferation of XWLC-05 cells 72h after transfection?p<0.01?.? The knockdown of miR-501-5p led to a significantly reduced migration of A549 cells and XWLC-05 after.48h.The count of migration in negative control group and inhibitor group of A549 cells was 466±188.4 and 191.1±49.4,respectively,p<0.05.Similarly,in XWLC-05,the count of migration was 477.8±110.2 and 249.3±89.2,respectively,p<0.05.? Knockdown of miR-501-5p led to a significantly decreased invasion of A549 cells and XWLC-05 cells after 48h.The count of invasion in negative control group and inhibitor group of A549 cells was 170.5±23.4 and 126.1±53.2,respectively,p<0.05,Similarly,in XWLC-05,the count of migration was 230.4±20.1 and 185±26.2,respectively,p<0.05.? Down-regulation of miR-501-5p could increase A549/XWLC-05 cell apoptosis.There was a statistically significant increase of apoptosis ratio in A549 cells 48h post-transfection.The apoptosis ratio of negative controls and inhibitor group was 5.810±3.500?%?;16.850±6.547?%?,respectively,the difference was statistically significant?p<0.05?.There was also a statistically significant increase of apoptosis ratio in inhibitor group of XWLC-05 cells 48h post-transfection compared to negative controls.The apoptosis ratio of negative controls and inhibitor group was 7.923±1.374?%?;13.140±4.923?%?,respectively,p<0.05.? There was no statistical difference on cell cycle?G0/G1,S,G2/M?of A549?XWLC-05 cells between down-regulation of miR-501-5p group and negative control group.[Conclusion]:The expression level of miR-501-5p in A549 and XWLC-05 cells was up-regulated.Furthermore,the miR-501-5p could enhance the ability of proliferation,migration,invasion and apoptosis resistance.Thus,miR-501-5p may play an important role in NSCLC progression.Section ?:Studies on the potential target genes and signaling pathway of miR-501-5p in NSCLC progression[Objective]:To investigate the potential target genes and signaling pathway of miR-501-5p in A549 and XWLC-05 cells.[Methods]:The target genes and signaling pathway of miR-501-5p was predicted by bioinformatics analysis.Expression of mRNA of predicted target genes and genes related to the predicted signaling pathway in A549 and XWLC-05 cells were detected by qPCR.The protein expression of genes related to predicted signaling pathway in A549 and XWLC-05 cells were detected by Western blot.[Results]:1.The bioinformatics analysis data showed that the target genes of miR-501-5p included DCC,ERRFI1,UBAP1.Furthermore,the predicted signaling pathway of miR-501-5p was both cancer-related signaling pathway and WNT signaling pathway.2.Compared with the expression in NC group,the down-regulation of miR-501-5p in A549 cells reduced the mRNA expression level of DCC,UBAP1 and ERRFI1.The expression of these genes in inhibitor group and NC group of A549 was that DCC?0.803±0.539,1.000±0.788,p>0.05?,UBAP1?1.157±1.242,1.0000±785,p>0.05?and ERRFI1?0.804±0.387,1.000±0.515,p>0.05?.In XWLC-05 cells,the expression in inhibitor group and NC group was that DCC?0.922±0.702,1.000±0.671,p>0.05?,UBAP1?0.820±0.528,1.000±0.622,p>0.05?and ERRF1?1.117±0.440,1.000±0.549,p>0.05?.The difference had no statistical significant in all paired samples.It showed that the down-regulated miR-501-5p had no effect on the expression of DCC,UBAP1 and ERRFI1.3.Down-regulation of miR-501-5p in A549 cells could remarkably reduce the expression level of WNT signaling pathway key genes ?-catenin,APPL1,LRP6.The expression of genes in inhibitor group and NC group was that?-catenin?0.150±0.075,1.000±0.494,p<0.05?;APPL1?0.109±0.023,1.O00±0.354,p<0.05?;LRP6?0.123±0.040,1.000±0.254,p<0.05?and GSK-3??0.557±0.746,1.000±1.177,p>0.05?.Similarly,in XWLC-05 cells,the expression of genes in inhibitor group and NC group was that?-catenin?0.132±0.050,1.000±350,p<0.05?;APPL1?0.090±0.079,1.000±0.435,p<0.05?;LRP6?0.112±0.022,1.000±0.431,p<0.05?and GSK-3??0.648±0.332,1.000±0.606,p>0.05?.There difference of DCC,APPL1 and LRP6 expression had statistical significant.It showed that the down-regulated expression of miR-501-5p has negative effect on the expression of?-catenin,APPL1 and LRP6.4.Compared with negative control groups,moreover,down-regulated expression of miR-501-5p in A549 and XWLC-05 cells could significantly increase the protein expression level of Caspase-3,and decrease the protein expression level of WNT signaling pathway key genes LRP6,APPL1 and ?-catenin.[Conclusion]:Down-regulation of miR-501-5p has no significant effect on mRNA expression of DCC,ERRFI1 and UBAP1,suggested that they might not be potential target genes of miR-501-5p.Furthermore,down-regulation of miR-501-5p could decrease the mRNA and protein expression level of LRP6,APPL1,?-catenin,and enhance protein expression of caspase-3.It suggested that miR-501-5p could play a critical role in NSCLC progression via activation of WNT/?-catenin signaling pathway. |
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