The Study Of The Mechanism Of Lnc RNA UCA1/miR-193a Signal Pathway Regulating The Proliferation,Migration And Invasion Of NSCLC Cells

Objective: To explore the effects of UCA1/miR-193 a signal pathway on the proliferation,migration and invasion of NSCLC cells,and to study its mechanism.Methods:Part one: A549 and H1299 cells were used as the research object without any intervention treatment.MTT method was used to detect the proliferation ability of two groups of cells under the same cultivation conditions and at the same time(1d-2d-3d-4d-5d).The ability of cell migration and invasion was detected by scratch and Transwell.QRT-PCR was used to detect the gene expression of UCA1 and miR-193 a in the cells.The protein expressions of CDK6,EGFR,KIF7 and KRAS were detected by Western blot.Part two: The siRNA target sequence of UCA1 was screened first,and A549 cells were co-transfected with siRNA-Lipofectamine 2000 complex.According to the different siRNA sequence,A549 cells were divided into siRNA-1 group,siRNA-2 group and siRNA-3 group.QRT-PCR was used to detect the expression of UCA1 gene in the three groups.A549 cells were then divided into A549-NC group and siRNA-1 group,and H1299 cells were divided into H1299-NC group and H1299-overexpression group.MTT,scratches,and Transwell were used to detect cell proliferation,migration,and invasion ability,q RT-PCR detection of UCA1,miR-193 a gene expression in each group of cells.Double luciferase reporter assay was used to verify that miR-193 a is the target gene of UCA1;Western blot was used to detect the expression of CDK6,EGFR,KIF7 and KRAS.Part three: MiR-193 a mimic and miR-193 a inhibitor were transfected into A549 and H1299 cell lines by Lipofectamine 2000 transfection reagent.A549 cells were divided into mimic NC group and miR-193 a mimic group,H1299 cells were divided into inhibitor NC group and miR-193 a inhibitor group.The proliferation,migration and invasion of the cells were detected by MTT,scratch and Transwell,respectively.QRT-PCR was used to detect the gene level of UCA1 in cells,and Western blot was used to detect the expression of CDK6,EGFR,KIF7 and KRAS proteins in cells.Results:Part one: In the detection of cell absorbance value,the absorbance value of A549 cells is lower than that of H1299 cells.In the cell scratch test,the wound healing ability of A549 cells was significantly lower than that of H1299 cells after 24 hours;and in a single field of view,the number of invasion of A549 cells was significantly less than that of H1299 cells.UCA1 expression was lower in A549 cells than in H1299 cells;miR-193 a expression was higher in A549 cells than in H1299 cells;protein expression of CDK6,EGFR,KIF7,and KRAS in A549 cells was lower than in H1299 cells.Part two: Among the three interference sequences,the relative expression of UCA1 gene in siRNA-1 group was the lowest,and the absorbance,wound healing ability and invasion number of A549 cells in siRNA-1 group were significantly lower than those in A549-NC group.Compared with H1299-NC group,the absorbance value,wound healing ability and cell invasion number of H1299-overexpression group were significantly higher than those of H1299-NC group.QRT-PCR results showed that the expression of miR-193 a gene was significantly up-regulated in A549 cells which inhibited the expression of UCA1,while the expression of miR-193 a in H1299 cells was significantly down-regulated after increasing the expression of UCA1.Double fluorescence reporter gene assay showed that the luciferase activity of cells co-transfected with Lnc UCA1-3’UTR WT+miR-193a-5p mimics was significantly decreased,while the luciferase activity of cells in Lnc UCA1-3’UTR MUT+miR-193a-5p mimics group and Lnc UCA1-3’UTR WT+ miRNA NC group was restored.The results of protein detection showed that the expression of CDK6,EGFR,KIF7 and KRAS in A549 cells was inhibited after inhibiting the expression of UCA1,while the overexpression of UCA1 upregulated the expression of CDK6 and other proteins in H1299 cells.Part three: In the detection of cell absorbance value,the miR-193 a mimic significantly decreased the cell absorbance value,and miR-193 a inhibitor could increase the cell absorbance value.The miR-193 a mimic weakens the wound healing ability,while miR-193 a inhibitor can improve the wound healing ability of cells.The number of cell invasion in single visual field in miR-193 a mimic group was significantly lower than that in NC group,and that in miR-193 a inhibitor group was significantly higher than that in NC group.In addition,miR-193 a mimic could inhibit the expression of UCA1 gene in A549 cells,while miR-193 a inhibitor could up-regulate the expression of UCA1 in H1299 cells.The miR-193 a mimic could down-regulate the protein expression of CDK6,EGFR,KIF7 and KRAS,while miR-193 a inhibitor could significantly up-regulate the expression of CDK6 and other proteins.Conclusion:1.UCA1/miR-193 a signaling pathway molecules are differentially expressed in NSCLC cells,resulting in different proliferation,migration,and invasion capabilities of NSCLC cells.2.UCA1 may play the role of oncogene in NSCLC cells,and overexpression of UCA1 can promote the proliferation,migration and invasion of NSCLC cells.miR-193 a plays a similar role as a tumor suppressor gene.Overexpression of miR-193 a can inhibit the proliferation,migration,and invasion of NSCLC cells;miR-193 a is a direct target of UCA1,and their expression in NSCLC cells is negatively correlated.3.UCA1/miR-193 a signal pathway is involved in the regulation of malignant biological behaviors such as proliferation,migration and invasion of NSCLC cells.