Experimental Study On The Effect Of LINC01614 On The Biological Behavior And Cisplatin Resistance Of Lung Cancer A549 Cells

Part OneObjective: To investigate the effects of LINC01614 on proliferation,invasion,cell cycle and apoptosis of non-small cell lung cancer A549 cells.Methods: 1.Bioinformatics analysis:the TCGA database was used to analyze the expression of LINC01614 on non-small cell lung cancer,while correlating the effect of LINC01614 expression on survival as well as clinicopathological features.2.Real-time fluorescent quantitative PCR(q RT-PCR)assay was performed to detect the difference in LINC01614 expression between non-small cell lung cancer A549 cells and human BEAS-2B expression differences in normal lung epithelial cells.3.Using liposome transfection,an si RNA specifically targeting the LINC01614 gene(si-LINC01614)and a negative control sequence si-RNA(si-Negative Control,si-NC)were transfected into A549 cells to construct the si-LINC01614 cell group and si-NC cell group,respectively.4.The transfection effect of si-LINC01614 was verified by q RT-PCR assay.5.Cell Counting Kit(CCK)assay was performed to detect the effect of silencing LINC01614 on the proliferation ability of A549 cells.6.Using Incu Cyte S3 to monitor the effect of silencing LINC01614 on the growth state of A549 cells.7.Using transwell cell invasion assay to detect the effect of silencing LINC01614 on the invasive ability of A549 cells.8.Using flow cytometry assay to detect the effect of silencing LINC01614 on the cell cycle of A549 cells.9.Using flow cytometry to detect the effect of silencing LINC01614 on apoptosis of A549 cells.SPSS22.0 was used for statistical analysis of the data.Results: 1.Analysis of the biological information of LINC01614 showed that the expression of LINC01614 was significantly upregulated in non-small cell lung cancer tissues compared with normal tissues(P < 0.05);the overall survival of patients in the group with high LINC01614 expression was shorter compared with those in the group with low LINC01614 expression(P < 0.05);The expression of LINC01614 did not correlate with gender,age,tumor size and lymphatic metastasis(P >0.05),and the expression of LINC01614 had significant correlation with distant metastasis and tumor stage(P < 0.05).2.q RT-PCR results showed that the expression of LINC01614 was significantly upregulated on A549 cells compared with BEAS-2B cells(P < 0.05).3.q RT-PCR results showed that the expression of LINC01614 was significantly downregulated in the si-LINC01614 cell group compared with the A549 cell group as well as the si-NC cell group(P < 0.05,respectively).4.The monitoring results of CCK assay and Incu Cyte S3 showed that the proliferation ability of A549 cells transfected with si-LINC01614 was significantly inhibited compared with the A549 cell group and si-NC cell group(P <0.05,respectively).5.The results of invasion assay showed that compared with the A549 cell group and si-NC cell group,the silent LINC01614 A549 cells with silencing of LINC01614 had a significantly lower number of invasive cells compared with the A549 cell group and si-NC cell group(P < 0.05,respectively).6.Flow cytometry results showed that the G0/G1 phase was prolonged and the S phase was shortened in the si-LINC01614 cell group compared with the A549 cell group and si-NC cell group(P < 0.05,respectively).7.Flow cytometry assays showed that compared with the si-NC cell group,silencing of A549 cells with LINC01614 had decreased apoptosis rate(P < 0.05).Conclusion: LINC01614 promotes proliferation,invasion,and apoptosis of non-small cell lung cancer A549 cells.Part Two Objective: To investigate the biological role of LINC01614 in non-small cell lung cancer A549 cells and its drug resistance related mechanism.Methods:1.Sequencing of LINC01614 knockdown A549 cells constructed using CRISPR/Cas9 technology was applied to explore the effect of LINC01614 on transcriptome genes in A549 cells.2.q RT-PCR was used to validate the transcriptome differential genes MCAM and ABCC3 at the gene level.3.Western blot was used to validate the chemotherapy resistance-related gene MCAM at the protein level.4.CCK assay was used to detect the change of drug sensitivity of A549 cells to cisplatin after knockdown of LINC01614 under the effect of different concentrations of cisplatin.Results: 1.Differential gene expression analysis of transcriptome data showed that knockdown of LINC01614 resulted in 2713 DEGs,including 1626 up-regulated genes and1087 down-regulated genes.2.GO functional enrichment analysis showed that DEGs were associated with intracellular signaling,cell adhesion,etc.3.KEGG pathway enrichment analysis showed that DEGs were associated with Wnt signaling pathway,TGF-β signaling pathway,and Rap1 signaling pathway,etc.4.Drug resistance-associated gene ABCC3 was selected from DEGs for validation with MCAM: q RT-PCR results showed that knockdown of LINC01614 significantly down-regulated the expression of MCAM on A549 cells(P < 0.05)and ABCC3 on A549 cells(P < 0.05).5.Western blot assay showed that the protein expression of chemoresistance-associated gene MCAM was significantly decreased in A549 cells after knockdown of LINC01614(P < 0.05).6.The results of CCK assay showed that the IC50 of A549 cells to cisplatin was significantly increased after knockdown of LINC01614(P< 0.05).Conclusion:1.LINC01614 may be related to cell proliferation,invasion and apoptosis pathways.2.MCAM expression at both gene and protein levels in A549 cells was significantly decreased after knockdown of LINC01614.3.Resistance of A549 cells to cisplatin was reduced after knockdown of LINC01614.