| Objective: The incidence and mortality of lung cancer rank the first among malignant tumors all over the world.Among all lung cancers,non-small cell lung cancer accounts for about 85%,and lung adenocarcinoma is the main pathological type of non-small cell lung cancer.Although the diagnosis and treatment of lung cancer have made some progress in recent years,the prognosis of lung cancer is still poor due to its strong invasiveness and easy metastasis.Therefore,it is of great significance to explore the relationship between functional genes and biological characteristics of lung adenocarcinoma,to further understand the mechanism of its proliferation and metastasis,and to find more effective anticancer targets for designing reasonable therapeutic drugs and controlling the occurrence and development of lung adenocarcinoma.The genesis and development of tumor is a multi-stage,multi-factor and multi-step process regulated by complex molecular network.After the hypothesis of competitive endogenous RNA was proposed,a new understanding of the regulation mechanism of lnc RNA and mi RNA was obtained.In recent years,more and more attention has been paid to the important role of Lnc RNA FBXL19-AS1 in tumor genesis and development,but its molecular mechanism in the development and progression of lung adenocarcinoma remains unclear.mi R-203a-3p is a tumor-suppressing mi RNA whose anti-lung cancer effect has been well established.This study will combine in vivo and in vitro experiments to explore whether Lnc RNA FBXL19-AS1 can regulate mi R-203a-3p,thereby affecting the expression of its downstream target genes and promoting the proliferation and metastasis of lung adenocarcinoma,so as to lay the foundation for elucidating the Lnc RNA FBXL19-AS1/mi R-203a-3p signaling pathway and the pathogenesis of lung adenocarcinoma,thus providing a potential therapeutic target for lung adenocarcinoma.Methods: 1.Clinical experiments.We used q RT-PCR to detect the expression of Lnc RNA FBXL19-AS1 and mi R-203a-3p in lung adenocarcinoma tissues and corresponding adjacent tissues.2.Cell experiments.We first carried out the basic work of cell culture,cell line screening and the construction of stable transfected cell lines.Then,CCK-8 assay,flow cytometry,Transwell assay,cell scratch assay,and Western blot were used to evaluate the effect of Lnc RNA FBXL19-AS1 on proliferation,cycle,invasion,migration,and expression of downstream tumorigenic factors of lung adenocarcinoma cells.After that,we used double luciferase assay to verify the binding of Lnc RNA FBXL19-AS1 with mi R-203a-3p,and we also constructed some new stable transfected cell lines.Finally,CCK-8 assay,Transwell assay,cell scratch assay and Western blot were used again to verify whether Lnc RNA FBXL19-AS1 had an effect on the proliferation,invasion,migration and expression of downstream tumorigenic factors of lung adenocarcinoma cells through mi R-203a-3p.3.Animal experiments.We first evaluated the effect of Lnc RNA FBXL19-AS1 on in vivo growth of lung adenocarcinoma through tumor formation in nude mice.Then,we used q RT-PCR to detect the expression of Lnc RNA FBXL19-AS1 and mi R-203a-3p in tumor tissues.Finally,we evaluated the effect of Lnc RNA FBXL19-AS1 on in vivo proliferation of lung adenocarcinoma by immunohistochemistry.4.Statistical methods.According to the nature of the collected clinical information and the data from the experiment results,the statistical method adopted in this study includes: "Chi-square test","Paired t test","Unpaired t test","Ordinary one-way ANOVA","Bonferroni’s multiple comparisons test","Two-way ANOVA",and "Tukey’s multiple comparisons test",etc.Results: 1.Clinical experiments.We used qRT-PCR to detect the expression level of RNA in lung adenocarcinoma tissues and corresponding adjacent tissues.The results showed that the expression level of Lnc RNA FBXL19-AS1 in lung adenocarcinoma tissues was significantly increased(P<0.01),while the expression level of mi R-203a-3p was significantly decreased(P<0.01)comparing with corresponding adjacent tissues.By combining the expression of Lnc RNA FBXL19-AS1 and mi R-203a-3p with various clinicopathological factors,we found that the higher the expression of Lnc RNA FBXL19-AS1,the more likely the lung cancer is to become larger(P<0.01),the more likely the lymph node is to metastasize(P<0.01),and the later the tumor staging(P<0.01).The lower the expression of mi R-203a-3p,the more likely the lung cancer is to become larger(P<0.01),the more likely the lymph node is to metastasize(P<0.01),and the later the tumor staging(P<0.01).2.Cell experiments.First,we selected NCI-H1975(1.00)and SPC(0.69),whose expression of Lnc RNA FBXL19-AS1 were relatively higher in cell lines NCI-H1975,SPC,NCI-H1299 and A549,for subsequent experiments.After transfection of FBXL19-AS1 sh RNA and NC sh RNA into cell lines,we used q RT-PCR to detect the expression levels of RNA in each group,and found that the expression of Lnc RNA FBXL19-AS1 were decreased(P<0.01 and P<0.01)and the expression of mi R-203a-3p were increased(P<0.01 and P<0.01)in both NCI-H1975 and SPC.The results of CCK-8 assay showed that interfering with the expression of Lnc RNA FBXL19-AS1 reduced the proliferation rate of NCI-H1975 and SPC.The results of flow cytometry showed that interfering with the expression of Lnc RNA FBXL19-AS1 caused more NCI-H1975 and SPC to be in G0/G1 phase(P<0.01 and P<0.05).The results of Transwell assay showed that interfering with the expression of Lnc RNA FBXL19-AS1 reduced the invasiveness of NCI-H1975 and SPC(P<0.01 and P<0.01).The results of cell scratch assay showed that interfering with the expression of Lnc RNA FBXL19-AS1 reduced migration of NCI-H1975 and SPC(P<0.05 and P<0.01).The results of Western blot showed that interfering with the expression of Lnc RNA FBXL19-AS1down-regulated the expression of survivin(P<0.01 and P<0.01),DLX5(P<0.01 and P<0.01),E2F1(P<0.01 and P<0.01)and ZEB2(P<0.01 and P<0.01)in NCI-H1975 and SPC.The results of double luciferase assay showed that mi R-203a-3p was the target sequence of wild-type Lnc RNA FBXL19-AS1,rather than the mutant Lnc RNA FBXL19-AS1.Then,we constructed new stable transfected cell lines: FBXL19-AS1 sh RNA+NC inhibitor and FBXL19-AS1 sh RNA+mi R-203a-3p inhibitor.The results of CCK-8 assay showed that down-regulating the expression of mi R-203a-3p reversed the inhibitory effect of Lnc RNA FBXL19-AS1 on proliferation in NCI-H1975 and SPC when at its low expression(P<0.01 and P<0.05).The results of Transwell assay showed that down-regulating the expression of mi R-203a-3p reversed the inhibitory effect of Lnc RNA FBXL19-AS1 on invasion in NCI-H1975 and SPC when at its low expression(P<0.01 and P<0.05).The results of cell scratch assay showed that down-regulating the expression of mi R-203a-3p reversed the inhibitory effect of Lnc RNA FBXL19-AS1 on migration in NCI-H1975 and SPC when at its low expression(P<0.01 and P<0.05).The results of Western blot showed that down-regulating the expression of mi R-203a-3p reversed the inhibitory effect of Lnc RNA FBXL19-AS1 on the expression of survivin(P<0.01 and P<0.01),DLX5(P<0.05 and P<0.01),E2F1(P<0.01 and P<0.01)and ZEB2(P<0.01 and P<0.01)in NCI-H1975 and SPC when at its low expression.3.Animal experiments.The results of tumor formation in nude mice showed that interfering with the expression of Lnc RNA FBXL19-AS1 in NCI-H1975 and SPC reduced their growth rate in vivo.q RT-PCR was used to detect the expression of RNA in tumor tissues.The results showed that interfering with the expression of Lnc RNA FBXL19-AS1 in NCI-H1975 and SPC down-regulated their expression of Lnc RNA FBXL19-AS1 in vivo(P<0.01 and P<0.01),while up-regulated their expression of mi R-203a-3p in vivo(P<0.01 and P<0.01).The results of immunohistochemical showed that interfering with the expression of Lnc RNA FBXL19-AS1 in NCI-H1975 and SPC reduced their proliferation rate in vivo(P<0.01 and P<0.01).Conclusion: In this study,we confirmed that Lnc RNA FBXL19-AS1 had an effect on the proliferation,invasion,migration,and the expression of its downstream tumorigenic factors through mi R-203a-3p in lung adenocarcinoma,using the experimental methods of q RT-PCR,CCK-8 assay,flow cytometry,Transwell assay,cell scratch assay,Western blot,double luciferase assay,tumor formation in nude mice,and immunohistochemistry,at the three levels of clinical experiments,cell experiments,and animal experiments,in both in vivo and in vitro environment.In this study,for the first time,we presented a novel ce RNA mechanism,namely,Lnc RNA FBXL19-AS1 can regulate the expression of related genes and further affect the proliferation and metastasis of lung adenocarcinoma by regulating mi R-203a-3p.The findings will open up new ideas for designing novel treatment strategies and improving the prognosis of lung adenocarcinoma.However,this study also has its shortcomings waiting for further improvement.Although we have found some cancer-related genes through experiments,the precise function and mechanism of action of each gene at the cellular level still needs to be further explored.At the same time,other downstream target genes that may affect the proliferation and metastasis of lung adenocarcinoma in the Lnc RNA FBXL19-AS1/mi R-203a-3p pathway should also be included.In the future research,the research focus should be on the deeper analysis of molecular mechanism. |
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