| Objective:Lung cancer is a common malignant tumor in clinic,with high anti-morbidity and high mortality as the main features.Non-small cell lung cancer is the main pathological type of lung cancer,accounting for 85% of all types of lung cancer;LncRNA long-chain non-coding RNA is a type of non-coding RNA with a length of more than 200 nucleotides.They interact with proteins,RNA and DNA.Function to regulate gene expression at the transcriptional and post-transcriptional levels,participate in transcriptional activation,genomic imprinting and chromosomal modification,and many other biological processes,as well as processes such as cell development,proliferation,differentiation,apoptosis,migration,invasion,and metastasis.Participation in chemotherapy resistance is related to targeted therapy.LncRNA MIR503 HG is 786 bp LncRNA,and is involved in the process of tumor development.In lymphoma-related research,LncRNA MIR503 HG induces miR-503 through miR-503 / Smurf2 / TGFBR axis and inhibits Smurf2 to stabilize the expression of TGFBR tumor growth factor receptor,and ultimately promotes the growth and proliferation of lymphoma cells.Silencing LncRNA MIR503 HG leads to defects in angiogenesis,and the lack of LncRNA MIR503 HG in endothelial cells affects the cell cycle and inhibits the formation of capillaries.The isolated model of heart tissue proves that LncRNA MIR503 HG has potential clinical significance for the structural integrity of blood vessels.Although there have been many studies on the correlation between LncRNA MIR503 HG and cancer,there is no report on the biological function and regulation mechanism of LncRNA MIR503 HG in lung cancer.In summary,in order to study the regulatory mechanism of LncRNA MIR503 HG involved in the proliferation and apoptosis of lung cancer,this study examined the expression of LncRNA MIR503 HG in lung cancer patients and normal lung tissue adjacent to the cancer,and at the same time,the overall level of LncRNA MIR503 HG was explored by regulating its downstream The mechanism by which miRNAs function.Methods: 1.Randomly select 29 pairs of fresh lung cancer tissues and adjacent normal lung tissue samples collected from the first affiliated hospital,4 lung cancer cell linesA549,NCI-H1299,NCI-H1975,NCI-H2170 and human normal bronchial epithelial cell lines BEAS-2B,Real-time PCR to detect the relative expression of LncRNA MIR503 HG,analyze the correlation between LncRNA MIR503 HG and lung cancer,and select 2 lung cancer cells with relatively high expression levels for cell transfection experiment 2.Silence After the expression of LncRNA MIR503 HG,MTT method and Ki67 immunofluorescence staining were used to detect cell proliferation activity,flow cytometry was used to detect cell cycle,flow cytometry and immunofluorescence TUNEL were used to detect cell apoptosis,and Western blot was used to detect cell cycle and apoptosis-related protein expression.3.Real-time PCR detection of LncRNA MIR503 HG,miR-489-3p and miR-625-5p in lung cancer tissues and adjacent normal lung tissue samples,lung cancer cell lines A549,NCI-H1299,NCI-H1975,NCI-H2170 and humans The relative expression level of the normal bronchial epithelial cell line BEAS-2B,the relationship between the expression level of LncRNA MIR503 HG and the expression level of miR-489-3p and miR-625-5p was analyzed.4.A549 and NCI-H2170 cells were selected as the research objects.After silencing the two cells with LncRNA MIR503 HG shRNA,Real-time PCR was used to detect the relative expression of miR-489-3p and miR-625-5p.Bioinformatics software was used to predict the target binding site of LncRNA MIR503 HG and its target miRNAs,and the dual luciferase reporting system was used to verify the targeting relationship between LncRNA MIR503 HG and miR-489-3p and miR-625-5p.5.A549 cells were selected as the research object,which were divided into A549 group,NC mimic group and miRNAs mimic group.After grouping,MTT method was used to detect cell proliferation activity,and flow cytometry was used to detect cell apoptosis.6.A549 cells were selected as the research object,divided into LncRNA MIR503 HG shRNA group,LncRNA MIR503 HG shRNA + NC inhibitor group,LncRNA MIR503 HG shRNA+miR-489-3p inhibitor group,and LncRNA MIR503 HG shRNA+miR-625-5p inhibitor group,and grouped together After that,MTT method was used to detect cell proliferation activity,flow cytometry was used to detect cell apoptosis,and Western blot was used to detect cell cycle and apoptosis-related protein expression.Results: 1.The expression level of lncRNA MIR503 HG in non-small cell lung cancer tissues was significantly higher than that in normal adjacent tissues(P <0.01).Theexpression level of lncRNA MIR503 HG in non-small cell lung cancer cell lines is higher than that in human normal bronchial epithelial cells.2.A549 and NCI-H2170 with relatively high lncRNA MIR503 HG were used for lncRNA MIR503 HG shRNA transfection.MTT results showed that compared with the control group,the cell proliferation ability of the MIR503 HG shRNA group was significantly reduced(P <0.05),Ki67 immunofluorescence staining results Same trend as MTT results.The results of cell cycle experiments showed that compared with the control group,the number of cells in the G1 and S phases of the MIR503 HG shRNA group decreased significantly(P <0.01),and the number of cells in the G2 phase increased significantly(P <0.01).The results of apoptosis showed that compared with the control group,the level of apoptosis in the lncRNA MIR503 HG shRNA group increased significantly(P <0.01),and the results of the immunofluorescence TUNEL experiment were the same as the results of apoptosis.Western blot results showed that the expression levels of cycle-related proteins PCNA,cyclinD1 and cyclinE in lncRNA MIR503 HG shRNA group decreased,the expression levels of p16 and p21 increased,and apoptosis-related proteins Bax,Bad,cleaved-caspase-3,cleaved-caspase-9,Bax expression increased,Bcl-2 and Survivin expression decreased.3.Real-time PCR showed that compared with normal lung tissue adjacent to cancer,the expression levels of miR-489-3p and miR-625-5p in non-small cell lung cancer tissues were reduced,and the difference was statistically significant(P<0.01)Compared with human normal bronchial epithelial cell line BEAS-2B,the expression levels of miR-489-3p and miR-625-5p were reduced in A549 and NCI-H2170 cell lines.The results showed that compared with the control group,the expression levels of miR-489-3p and miR-625-5p in the lncRNA MIR503 HG shRNA group were significantly increased(P <0.01).The results of the dual Luciferase reporting system showed that LncRNA MIR503 HG can target and bind to miR-489-3p or miR-625-5p.5.MTT results showed that compared with the control group,the cell proliferation ability of miR-489-3p mimic group was significantly reduced(P <0.05),the cell proliferation ability of miR-625-5p mimic group was significantly reduced(P <0.01),and the cells were withered The death results showed that,compared with the control group,the level of apoptosis in the miRNAs mimic group was significantly increased(P <0.01).6.MTT results showed that compared with the LncRNA MIR503 HG shRNA group,the cellproliferation ability of the LncRNA MIR503 HG shRNA + miR-489-3p inhibitor group was significantly improved(P <0.05),and the cell proliferation ability of the LncRNA MIR503 HG shRNA + miR-625-5p inhibitor group Significantly improved(P <0.01).Apoptosis results showed that compared with the LncRNA MIR503 HG shRNA group,the apoptosis level of the LncRNA MIR503 HG shRNA + miRNAs inhibitor group was significantly reduced(P <0.01).Western blot results showed that compared with the LncRNA MIR503 HG shRNA group,the expression levels of cycle-related proteins PCNA,cyclinD1 and cyclinE increased,and the expression levels of p16 and p21 proteins decreased in the LncRNA MIR503 HG shRNA + miRNAs inhibitor group;apoptosis-related proteins Bcl-2,Survivin expression increased while Bax,Bad,cleaved-caspase-3,cleaved-caspase-9 expression decreased.Conclusions:LncRNA MIR503 HG is highly expressed in non-small cell lung cancer;LncRNA MIR503 HG can target miR-489-3p and miR-625-5p to regulate lung cancer proliferation and apoptosis. |
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