| The treatment of lung cancer has already entered the era of targeted therapy.However,despite the successful clinical application of a large number of targeted drugs,such as epidermal growth factor receptor tyrosine kinase inhibitors,anaplastic lymphoma kinase inhibitors and immune checkpoint inhibitors,the overall five-year survival rate of lung cancer is still as low as 15.9%.This indicates that we still lack sufficient understanding of the pathogenesis of lung cancer,as well as the lack of diagnostic markers and therapeutic targets for early lung cancer.As we all know,genes usually do not act alone,and there are often very complex regulatory networks behind them.In recent years,more and more studies have shown that non-coding RNA plays an important regulatory role in the initiation of gene expression and other processes.Non-coding RNA is mainly divided into two subtypes: non-coding small RNA(microRNA)with less than 200 nt transcripts and long non-coding RNA(lncRNA)with transcripts between 200 nt and 100 KB.Previously,the function of lncRNA has been largely ignored as the "noise" of transcription without any biological function.In recent years,a large number of studies have shown that lncRNA in X chromosome silence,genomic imprinting and chromatin modification and transcription activation of transcription,interference,intranuclear transport and other important regulatory process,and control of various cellular functions,including cell proliferation,growth,migration,maintenance,transformation of epithelial mesenchymal stem cells and apoptosis,etc.These results make lncRNA as a new tumor marker and therapeutic target become a new research hotspot,and show a good clinical application prospect.Objective: We used GEO database gene chip and TCGA database analysis to indicate that CCNE1 is highly expressed in lung adenocarcinoma,which is closely related to poor prognosis.Through bioinformatics prediction,it was found that CCNE1 might be the target gene of miR-195,and further prediction found that LINC00355,a long non-coding RNA,could bind to miR-195 and had the same binding site as miR-195/CCNE1.Meanwhile,TCGA analysis showed that LINC00355 and CCNE1 were highly expressed in lung adenocarcinoma,while miR-195 was poorly expressed.Correlation analysissuggested that miR-195 expression level was negatively correlated with LINC00355 and CCNE1.Based on the above data,we hypothesized that LINC00355 may promote the proliferation of lung adenocarcinoma cells by down-regulating miR-195 and then up-regulating the expression of CCNE1.Currently,there is no research report on the specific biological function of LINC00355.Some database analysis on LINC00355 suggests that LINC00355 is highly expressed in colorectal adenocarcinoma and bladder cancer.Therefore,this study detected the expression of LINC00355 in lung adenocarcinoma tissues and cells and its relationship with clinicopathological factors and prognosis,and observed the biological function of LINC00355 through in vivo and in vitro experiments.Through the construction and transfection of mutated/wild-type LINC00355 and CCNE1 plasmid of mir-195 complementary sequence respectively,the targeting relationship and regulatory effect were verified to clarify the possible mechanism of LINC00355 in lung adenocarcinoma.Methods: 1.Lung adenocarcinoma gene chip was downloaded from pubmed GEO database to screen mRNA-CCNE1 with significant expression difference in lung adenocarcinoma and adjacent tissues,and the expression of CCNE1 and its relationship with prognosis were verified by using TCGA database.2.Bioinformatics predicted that CCNE1 may be the target gene of miR-195,while miR-195 may be the target gene of long non-coding RNA LINC00355.3.The expressions of LINC00355 and miR-195 in lung adenocarcinoma were analyzed with the TCGA database,and the correlation was analyzed.4.Real-time PCR was used to detect the expression of LINC00355 in 103 cases of lung adenocarcinoma and adjacent tissues,and the correlation between the clinical pathological data of the patients and the expression level of LINC00355 in lung cancer tissues was statistically analyzed.5.EDU assay,cell clonal formation assay,and cell cycle assay were used to detect the changes of proliferation activity of lung adenocarcinoma cells before and after LINC00355 down-regulated.6.Flow cytometry was used to detect the early apoptosis of lung adenocarcinoma cells before and after LINC00355 down-regulated.7.Western Blot assay was used to detect the changes of proliferation,cycle and apoptosis-related protein expression in lung adenocarcinoma cells before and after LINC00355 down-regulated.8 LncAtlas predicted and detected thesublocalization of LINC00355 in lung adenocarcinoma cells by FISH experiment.9.Bioinformatics predicted the binding sites of miR-195 with CCNE1 and LINC00355,and constructed the sites containing miR-195 binding sites respectively LINC00355 and CCNE1 fragment vectors.Double luciferase reporter gene assay verified the targeting relationship between miR-195 and CCNE1 and LINC00355.10.RNA pull-down and RIP experiments were performed to further verify the binding of LINC00355 to miR-195 and the presence of the same RISC complex.11.Cells were cultured and divided into Blank group,NC group,sh-LINC00355 group,miR-195 mimic,miR-195 inhibitor,miR-195inhibitor+ sh-LINC00355 group.The expressions of LINC00355,miR-195 and CCNE1 were detected by RT-PCR.Western Blot was used to detect the expression of proliferation,cycle and apoptosis related proteins in each group.12.The proliferation of cells in each group after transfection was detected by EDU test,clonal formation test,cell cycle test and nude mouse tumorigenesis test,apoptosis of cells in each group was detected by flow cytometry.13.SPSS22.0 statistical analysis software was used to analyze the experimental data,and P<0.05 indicated that the results were statistically significant.Results: 1.Bioinformatics predicted the targeting relationship of LINC00355/ miR-195/CCNE1 and analyzed the expressions of the three.GSE43458 data of lung adenocarcinoma genome-wide chip GSE43458 were downloaded from the pubmed GEO public database,and CCNE1 was the most significantly differentially expressed mRNA.The TCGA database was further used to confirm that CCNE1 was highly expressed in lung adenocarcinoma and correlated with poor prognosis.Bioinformatics predicted that miR-195 could be used as the target miRNA interacting with CCNE1,and further predicted that long-chain non-coding RNA LINC00355 could bind to miR-195 in a targeted way.Moreover,TCGA database analysis indicated that LINC00355 was highly expressed in lung adenocarcinoma,while miR-195 was poorly expressed.Correlation analysis showed that miR-195 expression level was negatively correlated with CCNE1 and LINC00355.2.LINC00355 expression was verified in lung adenocarcinoma tissues and cell lines.The postoperative histological specimens of 103 cases of lung adenocarcinoma were collected from Liaoning Cancer Hospital,and the expression of LINC00355 was detected byRT-PCR.Compared with adjacent normal lung tissues,the expression level of LINC00355 in lung adenocarcinoma tissues was significantly increased,which was consistent with the results in chip GSE43458.In addition,the expression of LINC00355 in lung adenocarcinoma was not significantly correlated with gender,age,smoking history,but with tumor diameter,lymph node metastasis and TNM stage.RT-PCR was used to detect the expression levels of LINC00355,miR-195 and CCNE1 in lung cancer cell lines and normal lung epithelial cells.The results showed that the expression levels of LINC00355 and CCNE1 in lung cancer cell lines were higher than those in HBE normal lung epithelial cells,and the expression levels of miR-195 in lung cancer cell lines were lower than those in HBE normal lung epithelial cells.3.Down-regulation of LINC00355 can inhibit the proliferation of lung adenocarcinoma cells.The results of EDU assay showed that the proliferation of sh-LINC00355 cells was slower than that of NC group.The results of cell clonal formation experiment showed that the ability of cell clonal formation in sh-LINC00355 group was significantly decreased compared with the NC group.Cell cycle detection by flow cytometry suggested G1/G0-S cell cycle arrest in A549 cells after LINC00355 knockdown.Annexin V FITC/PI double staining was used to analyze the apoptosis of knockdown LINC00355 cells.The results showed that compared with the control group,the proportion of early apoptosis of sh-LINC00355 cells was significantly increased.Western blot showed that the levels of CDK6,PCNA,Cyclin-D1,and Bcl-2 in lung adenocarcinoma cells were significantly reduced after LINC00355 down-regulated,while Bax expression was significantly up-regulated.4.Verification of LINC00355/miR-195/CCNE1 targeting relationship.FISH experiments in A549 cells confirmed that LINC00355 mainly existed in cytoplasm,and bioinformatics predicted that miR-195 had a common binding site with LINC00355 and CCNE1.Luciferase reporter gene results indicated that compared with luciferase reporter vector containing mutant LINC00355 and CCNE1 fragments,overexpression of miR-195 significantly decreased luciferase activity in wild-type vector group,suggesting a direct targeting relationship between miR-195 and LINC00355 and CCNE1.RNA pull-down experiment showed that compared with mut-miR-195,LINC00355 binding of wt-miR-195 was significantly increased,which again verified that miR-195 andLINC00355 could bind directly.The RIP experimental results suggested that the expression levels of LINC00355 and miR-195 in the Ago2 immune complex were significantly higher than those in the IgG group,suggesting that LINC00355 and miR-195 may exist in the same RISC complex in non-small cell lung cancer cells.5.LINC00355 negatively regulated miR-195 and positively regulated CCNE1,and the regulation of CCNE1 was dependent on miR-195.To further verify the regulatory relationship between LINC00355,CCNE1 and miR-195,we first used RT-PCR to detect the expressions of LINC00355,CCNE1 and miR-195 in Blank group,NC group,miR-195 mimic group,miR-195 inhibitor group,sh-LINC00355 group and miR-195 inhibitor + sh-LINC00355 group,respectively.It suggested that interference with LINC00355 could up-regulate miR-195 expression and down-regulate CCNE1 expression.In addition,the six groups of cells in cell clone formation,EDU experiment,cell apoptosis,cell cycle experiment,tumor formation in nude mice experiment and Western Blot experiment,the results of which showed that down-regulated LINC00355 or up-regulated miR-195 can inhibit the proliferation of A549 cells,and miR-195 could reverse the effect of LINC00355 on proliferation of A549 cells.The above experimental results confirmed that LINC00355 negatively regulated miR-195 and positively regulated CCNE1,and the regulation of LINC00355 on CCNE1 was dependent on miR-195.Conclusion: 1.LINC00355 is highly expressed in lung adenocarcinoma and is significantly associated with poor prognosis;2.In vivo and in vitro experiments have proved that LINC00355 can promote the proliferation of lung adenocarcinoma cells;3.MiR-195 has a direct targeting relationship with LINC00355 and CCNE1;4.LINC00355 negatively regulates miR-195 and positively regulates CCNE1 and regulation of CCNE1 by LINC00355 dependend on miR-195.Therefore,the above experimental results confirmed that LINC00355 promoted the proliferation of lung adenocarcinoma cells by down-regulating miR-195 and up-regulating CCNE1 expression. |
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