The Biological Function And Mechanisms Of Long Noncoding RNA CASC9 In Non-small Cell Lung Cancer

Background and Objectives:Lung cancer ranks as the first cause of cancer related incidence and fatality in the world.The number of lung cancer patients is the largest among men and the second among women(which just less than breast cancer).The five-year survival rate of lung cancer is lower than 15%,which is relatively poor.Recently,researchers have unveiled the fact that there were only 2%of DNA that could code protein while 70%-90%of which were noncoding sequences.These sequences are often transcribed into noncoding RNA actively.LncRNA(long noncoding RNA)is of the biggest part of noncoding RNA,which is longer than 200nt in length.LncRNA,with limited protein coding ability,could exert complex functions during development of diseases through affecting chromatin structure,DNA transcription,mRNA stability,mRNA transcriptional processing and translation etc.Since the categories and functions of IncRNA varied,increasing number of IncRNA molecules have been discovered to be related to biological behavior of cancer.LncRNA CASC9(cancer susceptibility 9)was firstly reported correlated with esophageal squamous carcinoma in 2015.After that,studies that proved CASC9 to serve as an oncogene in hepatoma carcinoma,gastric cancer and breast cancer began to arise.However,the researches related with lung cancer are limited.Researchers found that CASC9 overexpressed in PC9G cell line,which is resistant to EGFR-TKI.Knockdown of CASC9 recovered the sensitivity of PC9G to the drugs.Nevertheless,there exists no well accepted theory of CASC9’s biological function and mechanisms in lung cancer,which need deeper exploration on it.In Chapter one,differentially expressed genes(DEGs)screening was conducted using bioinformatic analysis tools.Then,we correlated CASC9 with other DEGs to perform co-expression genes and functional enrichment analysis,for the purpose of finding possible mechanisms of CASC9.In Chapter two,the expression level of CASC9 was detected in 60 tissue samples of NSCLC(non-small cell lung cancer)patients.The relationship between CASC9’s level and clinicopathological characteristics was studied as well.In Chapter three,in vitro experiments were performed with CASC9 knockdown cell lines in A549,H1299 and SK-MES-1 cells,to research the effects of CASC9 on the biological behavior of NSCLC.In Chapter four,techniques including Western Blot,qPCR,CCK-8 and colony formation were used to learn the possible mechanisms of CASC9.In Chapter five,xenografts in nude mice model was applied to affirm the function of CASC9 in NSCLC.This study provides some evidences for further investigation of CASC9 in NSCLCMethods:(1)DEGs screening and the analysis of correlation of CASC9 with DEGs in NSCLC:Sample data of lung squamous carcinoma(LUSC)and lung adenocarcinoma(LUAD)was extracted from TCGA database.The difference of expression level between tumor samples and control samples was analyzed using R software and online bioinformatic analysis tools.Scatter diagrams were drawn as well METASCAPE was applied to perform function and pathway enrichment analysis.(2)Collection of NSCLC tissue samples and related information:Sixty patients(twenty-three LUSC,thirty-seven LUAD)who were admitted to our hospital from March 1st,2018 to February 15th,2019 and were in accordance with inclusion criteria were collected.Tumor tissues and adjacent normal lung tissues(farther than 5cm from the edge of tumor)were resected and reserved in RNAlater at-80℃.(3)cell culture:Beas-2B and SK-MES-1 cells were cultured with 10%FBS DMEM medium,while A549,SPC-A1,H1299 and GLC cells were cultured with 10%FBS 1640 medium in incubator at temperature of 37℃ and CO2 concentration of 5%.(4)total RNA extraction and qRT-PCR detection:Trizol reagent was used to extract total RNA.The ration of OD260/280 should be within 1.8-2.1.Agarose gel electrophoresis was applied to test the integrity of RNA sample.The relative expression of CASC9 was detected using SYBR reagent and calculated with the 2△-△△CT computational method.(5)siRNA transient transfection was implemented with Lipofectamine 3000 and OTPI serum decreased medium.(6)cell proliferation experiments:After transfection,cells were seeded in 96 well plates.CCK-8 reagent was used to react with alive cells and the OD value was detected when cultured after 24h,48h,72h and 96h.Colony formation assay:cells were seeded in 6 well plates.Fourteen days later,cells were fixed and dyed with crystal violet.(8)cell cycle detection:After propidium staining,red fluorescence at 488nm of cells was detected.(9)cell apoptosis assay:Annexin V-FITC staining method was used to detect apoptosis cells.(10)Western Blot:SDS-PAGE method was used to perform Semi-quantitative detection of MMP9,CD44,p53,p21,CDK2,c-Myc and GAPDH.(11)rescue assay:p21 expression was inhibited at the same time of CASC9 knockdown.The viability of A549 cell was observed at the circumstance mentioned above.(12)xenograft transplantation assay in nude mice was carried out with A549 stably transfected with shRNA-CASC9.The weight of mice,volume of tumor and weight of tumor were recorded.Thirty-five days later,the mice were sacrificed to take the tumor tissues preserved for detection of related indexes.Chapter 1 The Bioinformatic Analysis of the Relationship Between CASC9 and DEGs in NSCLCResults:(1)There were 864 DEGs in LUSC(612 up-regulated and 252 down-regulated)and 782 DEGs in LUAD(488 up-regulated and 294 down-regulated).Among the two different pathological type,143 DEGs were the same(same 113 up-regulated and same 30 down-regulated).(2)Compared to normal lung tissues,CASC9 expression increased 2.36 folds in LUSC and 1.66 folds in LUAD.(3)The analysis results from GEPIA showed that CASC9 expression obviously increased in stage Ⅳ patients when compared to stage I to Ⅲ patients(p=0.0257).(4)There were 412 DEGs co-expressed with CASC9 in LUSC(323 genes positively correlated,89 negatively correlated)and 204 DEGs co-expressed with CASC9 in LUAD(147 genes positively correlated,57 negatively correlated).(5)The results of enrichment analysis showed that nicotine addiction pathway(hsa05033)and negative regulation of cell differentiation TERM(GO:0045596)may be involved in the effect of CASC9 in LUSC of NSCLC.In LUAD,the DNA Damage/Telomere Stress Induced Senescence pathway(R-HSA-2559586),the negative regulation of gene silencing TERM(GO:0060969)and the cell-matrix adhesion TERM(GO:0007160)may be related to the function of CASC9 in NSCLC.Conclusions:(1)There were vast number of DEGs in NSCLC.Differences and common points exist between LUSC and LUAD.(2)CASC9 overexpressed significantly both in LUSC and LUAD.The expression level of CASC9 was correlated with TNM stage of Ⅳ,which reminded that CASC9 may be involved in metastasis and prognosis of NSCLC patients.(3)The mechanisms of functions of CASC9 may be different among LUSC and LUAD.The mechanisms of CASC9 in LUAD was highly correlated with cell senescence pathway.Chapter 2 The Expression and Clinical Significance of CASC9 in NSCLC Results:(1)Aberrant high expression of CASC9 in NSCLC tissues:CASC9 was significantly higher in NSCLC tumor tissues than normal lung tissue(tumor vs,normal:△Ct:14.01±0.44 vs.10.38±0.37;p<0.001).The same tendency was observed in LUAD(tumor vs.normal:△CT:12.31±0.62 vs.10.43±0.66;p=0.034)and LUSC(tumor vs.normal:△CT:16.18±0.47 vs.10.33±0.31;p<0.001),respectively.(2)ROC curve analysis indicated a good diagnostic value of CASC9 to distinguish tumor tissues from normal tissues in NSCLC.NSCLC:AUC(95%CI):0.734(0.669-0.799);LUAD:AUC(95%CI):0.612(0.515-0.710);LUSC:AUC(95%CI):0.929(0.886-0.973)。(3)In sixty tissue samples(twenty-three LUSC,thirty-seven LUAD),CASC9 expression was correlated with clinicopathological characteristics.Chi-square test:the aberrant higher expression of CASC9 was significantly related to LUSC type(p=0.009),stage Ⅱ to Ⅵ(p=0.017),and non-prevalence areas(p=0.007).(4)The expression of CASC9 was higher in LUSC when compared with LUAD(LUSC vs.LUAD:10.47±7.10 vs.3.67±4.95;P<0.001).Conclusions:(1)The overexpression of CASC9 in tumor tissues and the results derived from ROC curve analysis imply that it may serve as a potential diagnostic indicator in NSCLC.(2)The fact that higher expression of CASC9 correlated with pathology type LUSC,later TNM stages and non-prevalence patient areas supplied its possible effect on the development and prognosis of NSCLC patients.(3)It was the first time for the discover of relationship between CASC9 and patients’source areas,which reminded that it may play different roles in different populations.(4)The expression level was higher in LUSC than LUAD,which indicated that the mechanisms of CASC9 may differed among different pathological type of NSCLC.Chapter 3 The Study of Biological Functions of CASC9 in NSCLCResults:(1)CASC9 overexpressed significantly in A549,SPC-A1,H1299,GLC and SK-MES-1 cells compared to normal lung epithelial cell Beas-2B.(2)The proliferation,migration and invasion of A549,H1299 and SK-MES-1 cells was decreased when CASC9 was knocked down.CCK-8 assay:Knockdown of CASC9 inhibited proliferation of NSCLC cells and the effect could be observed most obviously in A549 at the second to the fourth day(p<0.05;p<0.01;p<0.01),in H1299 at the fourth day(p<0.05)and in SK-MES-1 at the second to the fourth day(p<0.001;p<0.001;p<0.01).Colony formation:Knockdown of CASC9 significantly inhibited colony formation of A549(p<0.01)and H1299(p<0.05)cells.Transwell assay:Knockdown of CASC9 significantly decreased migration(p<0.001;p<0.001;p<0.01)and invasion(p<0.001;p<0.01;p<0.01)of A549,H1299 and SK-MES-1 cell.(3)Knockdown of CASC9 caused NSCLC cell cycle arrest at G1/G0 period:The ratio of G1/G0 cells is listed respectively:A549-NC vs.A549-SH1:63.17%±6.12%vs.74.92±5.41%,p<0.01;H1299-NC vs.H1299-SH1:70.88%±2.24%vs.75.78±1.88%,p<0.01;SK-MES-1-NC vs.SK-MES-1-SH1:57.32%±0.18%vs.63.79±1.02%,p<0.01.(4)Knockdown of CASC9 did not affect NSCLC cell apoptosis significantly.Conclusions:CASC9 overexpressed significantly in multiple NSCLC cell lines.Knockdown of CASC9 inhibited the malignant phenotypes in NSCLC and make the cell cycle arrested at G0/G1 period.The results implied that CASC9 played a role as oncogene in NSCLC.Chapter 4 The Study of Biological Functions of CASC9 in NSCLC Results:(1)p53/p21/CDK2 axis is the key points of cell senescence pathway R-HSA-2559586.Decreasing CASC9 expression would increase p53 and p21,while subscequently inhibit CDK2 expression.(2)The rescue experiments showed that inhibition of p21 will recover CDK2 expression which had been attenuated by CASC9 knockdown.(3)CDK2 is necessary for the effect of cell proliferation promotion induced by c-Myc.In our study,CASC9 knockdown would reduce the expressions of c-Myc and CDK2.(4)CD44 is directly targeted by MMP9.In our study,CASC9 knockdown could attenuate NMP9 and CD44 expressions.Conclusions:(1)The mechanism of CASC9 may be involved in p53/p21/CDK2 axis and the effect on cell senescence pathway R-HSA-2559586 in NSCLC.(2)The oncogenic function of CASC9 could be related with increasing the relative molecules’ expression of c-Myc/CDK2 axis.(3)The effect of CASC9 on inducing cell migration and invasion in NSCLC may be correlated with up-regulation of relative molecules’ expression of MMP9/CD44 axis.Chapter 5 The In Vivo Study of CASC9 Effects on the Viability in NSCLCResults:(1)No significance of mice weight was observed among three groups.However,seventeen days after cell injection,the tumor enlargement was faster in A549 and A549-NC group,while much slower in A549-SH1 group.(2)The mice were sacrificed on the 35th day.Data including tumor weight was recorded and analyzed statistically.The average tumor weight of CASC9 knockdown group was obviously lighter than control groups:A549-NC vs.A549-SH1:0.259±0.059 vs.0.083±0.013,p<0.01;A549 vs.A549-SH1:0.267±0.040 vs.0.083±0.0135,p<0.001.Conclusions:Knockdown of CASC9 could inhibit the tumor formation ability in vivo,which indicated the oncogenic role of CASC9 in NSCLC.