MicroRNA-153 Inhibits The Cell Proliferation And Invasion Ability Of Non-small Cell Lung Cancer Cells By Targeting COX-2

BackgroundLung cancer is one of the most common malignant tumors in the world,and has become the leading cause of death in the city's population.It can be divided into non small cell lung cancer and small cell lung cancer,non-small cell lung cancer accounted for about 80%of all lung cancer,including adenocarcinoma,squamous cell carcinoma,large cell carcinoma,etc.In spite of the improvement of the existing methods of diagnosis,treatment and targeted drug development,the recurrence rate and mortality rate were high,and the rate of 5-year survival is less than 15%.Metastasis of lung cancer is one of the main causes of death in patients.Although great progress has been made in the mechanism of metastasis of lung cancer,it is not very clear till now.In the recent time,more and more studies have shown that epigenetic factors such as microRNA(miRNA)are not only involved in the regulation of proliferation of malignant tumor cells,but also have an important role in tumor invasion and metastasis.Therefore,the role of miRNA in non-small cell lung cancer has been paid more and more attention.It has become a hotspot in recent years.MicroRNAs are endogenous small RNA molecules of 20-25 nucleotides length,regulating gene expression in the post-transcriptional level by inhibiting transcription.MicroRNAs are small single-stranded RNAs that repress mRNA translation and trigger mRNA degradation.Aberrant miRNA levels in cancer,with some miRNAs reported to actively regulate tumorigenesis.miRNAs play the roles of oncogenic miRNAs(oncomiRs),tumorsuppressive miRNAs(anti-oncomiRs)and miRNA regulators in some cancer.Recent studies have indicated that microRNA-153(miR-153)plays an important role in many kinds of tumors,but the effect is not very consistent.In the prostate cancer,miRNAs play the role of oncogenes,while in glioblastoma,epithelial cancer and leukemia in the inhibition of tumor growth,miRNAs play the role of tumor suppressor genes.ObjectiveThis study aimed to elucidate the clinical features of non-small cell lung cancer patients and the expression level of miR-153.We also established a lung cancer cell line A549/miR-153 and SPC-A-1/miR-153 which can express miRNA-153 stably and studied on how miRNA-153 affects A549 lung cancer cells on the biological function of A549 lung cancer cells and exploring its possible mechanisms.And discussed the potential use of miRNAs as biomarkers in the diagnosis of lung cancer and as prognostic indicators of this disease.Part? Expression of miR-153 and COX-2 in non-small cell lung cancer tissues and their correlation with clinicopathological characteristicsMethods1.137 NSCLC samples were retrieved from the Hospital.RNA was extracted from formalin-fixed tissues,using TRIzol reagent,PureLinkTM FFPE RNA Isolation Kit and mirVana PARIS kit.2.The expression patterns of miR-153 in 137 pairs of human lung cancer tissues and adjacent normal lung tissues were analyzed by using qRT-PCR.The relationship between miR-153 expression and clinicopathological parameters were examined by chi-square test.Kaplan-Meier method and the log-rank test were used to determine the difference in overall survival(OS)rates between two groups.For quantitative real-time PCR,the miRNA-specific TaqMan MicroRNA Assays(Applied Biosystems)for miR-153 was used as described by the manufacturer.U6 snRNA was used as an endogenous control for miRNA detection.The expression of miR-153 was quantified by measuring cycle threshold(Ct)values and normalized using the 2-??Ct method relative to U6 snRNA.3.The expression of miR-153 and COX-2 mRNA in lung cancer and its adjacent tissues was detected by real-time fluorescence quantitative PCR.The correlation between the expression of miR-153 and COX-2 mRNA in lung cancer and its adjacent tissues was analyzed.Results1.The expression of miR-153 was reduced significantly,compared with adjacent normal lung tissues(P<0.05).We observed that the expression level of miR-153 was positively correlated with the clinical stage(P=0.005),lymph node status(P=0.014),distant metastasis(P=0.004),and differentiated degree(P<0.001)in NSCLC patients.According to the Kaplan-Meier survival analysis,the patients with low miR-153 expression exhibited evidently poorer overall survival rates than those with high miR-153 expression(P<0.05).Multivariate analysis showed that the expression of miR-153 was an independent and significant factor associated with poor OS rates(P=0.002).2.The expression of miR-153 was reduced significantly,compared with adjacent normal lung tissues(0.24±0.17 vs 1.00±0.00,P<0.05).The expression of COX-2 mRNA was upregulated significantly,compared with adjacent normal lung tissues(2.08±0.17 vs 1.00±0.00,P<0.05).According to Spearman correlation analysis,The expression levels of miR-153 was negatively correlated with the expression of COX-2 mRNA in lung cancer tissues(r=-0.727).Part II Upregulation of miR-153 expression inhibited the proliferation and invasion of non-small cell lung cancer cellsMethods1.Construct miRNA-153 lentivirus vectors and empty vectors and establish cell line A549/miR-153 and SPC-A-1/miR-153 which stably express miR-153 by transfecting miRNA-153 lentivirus vector into A549 and SPC-A-1 cells and control cell A549/control and SPC-A-1/control by transfecting empty vectors into A549 and SPC-A-1 cells.And the expression level of miR-153 in A549/miR-153,SPC-A-1/miR-153 and A549/control cells,SPC-A-1/control were detected by real-time PCR.2.The proliferation capacity differences among the three cell groups(A549,A549/miR-153,A549/control,SPC-A-1,SPC-A-1/miR-153 and SPC-A-1/control))were investigated by cell growth curve created by MTT assays.3.After transfected 36h,useTranswell migration assay to investigate.Cell invasion and metastasisResults1.Construct A-153 lentivirus vectors and empty vectors successfully and stab cell line A549/miR-153 which stably express miR-153 and control cell A549/control which was transfected with empty lentivirus vector by transfecting miRNA-153 lentivirusand empty vectors into A549 cells which was transfected with empty lentivirus vectors.And the expression level of miR-153 in A549/miR-153 and A549/control cells were detected by real-time PCR.The relative expression level of miR-153 in A549 as 1,the expression level of miR-153 in cells of A549/control and A549/miR-153 is(1.07±0.04)and(8.49±0.16).The relative expression level of miR-153 in A549/miR-153 group increased significantly compared with A549 and A549/control group.2.The proliferation capacity differences among the three cell groups(A549,A549/miR-153 and A549/control)were investigated by cell growth curve created by MTT assays.The proliferation and invasion ability of lung cancer cells were significantly inhibited by exogenous overexpression of miR-153.The A490 of the cells in each group were measured by MTT assay at 0,1,2,3,4 and 5 days respectively.The results showed that the proliferation ability of A549/miR-153 cells was decreased.There are similar findings in SPC-A-1 cell line.These results indicated that overexpression of miR-153 could inhibit the proliferation of A549 and SPC-A-1 cells.3.To examine what an role the miR-153 play on the lung cancer cells' invasion,we used Transwell chamber method to detect cell invasiveness.Transwell invasion test showed that the number of cells in A549/control and A549/miR-153 group was 79±1.35,53±1.22 respectively.There are similar findings fo SPC-A-1 cell line.The results showed that exogenous overexpression of miR-153 can inhibit the invasion ability of A549 and SPC-A-1 cells and the difference was statistically significant(P<0.01).These results suggest that the overexpression of miR-153 can significantly inhibit the proliferation and invasion of non-small cell lung cancer cells.Part III Molecular mechanisms of miR-153 inhibiting the proliferation and invasion in non-small cell lung cancer cells by targeting COX-2Methods1.Screen out potential miR-153 target gene COX-2 through the use of bioinformatics software and considering preliminary experimental results of biological functions.2.Detect the changes of COX-2 after transfecting miR-153 in A549?A549/control?A549/miR-153 and SPC-A-1?SPC-A-1/control.SPC-A-1/miR-153 cells by Real time PCR and Westem blot3.A549 and SPC-A-1 cells were transiently transfected by COX-2-si-RNA.The proliferation ability of cell was tested by MTT assay.Invasive ability of A549/si-control,A549/si-RNA,SPC-A-1/si-control and SPC-A-1/si-RNA cells were investigated by Transwell assay.4.To investigate the molecular mechanism of miR-153 inhibiting the proliferation of A549 and SPC-A-1 cells,Western blot was used to detect the expression of Bax and Bcl-2 in A549/miR-153,A549/control,A549/si-COX-2,A549/si-control,SPC-A-1/miR-153,SPC-A-1/control,SPC-A-1/si-COX-2 and SPC-A-1/si-control cells.5.To investigate the molecular mechanism of miR-153 inhibiting the invasion of A549 and SPC-A-1 cells,Western blot was used to detect the expression of MMP2,MMP9 in A549/miR-153,A549/control,A549/si-COX-2,A549/si-control,SPC-A-1/miR-153,SPC-A-1/control,SPC-A-1/si-COX-2 and SPC-A-1/si-control cells.6.Elisa assay was used to detect the effect of miR-153 on PGE2 in cell culture supernatant in A549/miR-153,A549/si-COX-2 SPC-A-1/miR-153,SPC-A-1/si-COX-2 and their respective control cells.7.A dual luciferase reportor construct with or without 3'UTR of COX-2 was transfected into 293 cells in the presence of a miR-153 minic,and Firfly and Renila luciferase activities were measured after 24h.Results1.Screen out potential miR-153's target gene COX-2 by using bioinformatics software and considering preliminary experimental results of biological functions.2.Real time PCR and Westem blot were used to detect the changes of COX-2 after transfecting miR-153.miR-153 can inhibit the expression of COX-2 in lung cancer cell line A549 and SPC-A-1.The relative expression of COX-2 in A549 as 1,the relative expression of COX-2 in A549/control group and A549/miR-153 group was(1.03±0.02)?(0.48±0.03)detected by RT-PCR.Compared with A549/control,COX-2 mRNA was significantly down-regulated in A549/miR-153 cells(p<0.05).There has similar findings of SPC-A-1 cell line.3.RNAi technology was used to interfere the expression level of COX-2 in lung cancer cells,and the growth status of the cells was detected by MTT method for 5 days before and after the interference.A549 and SPC-A-1 cells were transiently transfected by COX-2-si-RNA.The expression levels of COX-2 was significantly decreased in A549/si-COX-2 group compared to control cells.Silence of COX-2 can inhibit the invasion and proliferation of A549 and SPC-A-1 cells.The results of MTT showed that the proliferation ability of A549/si-COX-2 cells was decreased.Transwell invasion test showed that the number of A549/si-control and A549/si-COX-2 cells through the filter membrane was(85± 1.63)and(50±0.65).Compared with A549/si-control,the number of A549/si-COX-2 cells passing through the filter membrane decreased significantly,and the difference was statistically significant(p<0.05).There are similar findings of SPC-A-1 cell line.These results suggest that COX-2 silencing can inhibit the invasion and proliferation of A549 and SPC-A-1 cells.It is speculated that miR-153 may inhibit the proliferation and invasion of A549 and SPC-A-1 cells and may be related to the inhibition of COX-2.4.In order to further study the molecular mechanism of miR-153 inhibiting the proliferation of A549 cells,the expression levels of Bcl-2 and Bax in the cells of A549/miR-153,A549/si-COX-2 and control group were further detected.The expression of Bcl-2 and Bax in A549-control as 1.The results showed that the relative expression of Bcl-2 and Bax to A549-control in A549-miR-153,A549/si-control and A549/siCOX-2 were(0.207 ± 0.015),(1.017 ± 0.023),(0.207 ? 0.015);the relative expression of Bax respectively(2.013 ± 0.085)and(1.05 ± 0.01),(2.043 ± 0.076).The expression of Bax in A549/miR-153 and A549/si-COX-2 cells was significantly higher than that in the corresponding control group,and the expression of Bcl-2 in A549/miR-153 and A549/si-COX-2 was significantly lower than that in the corresponding control group(p<0.01).The ratio of Bcl-2 to Bax(Bcl-2/Bax)in A549/miR-153 and A549/si-COX-2 was significantly lower than that of the corresponding control group(p<0.01).There has similar findings of SPC-A-1 cell line.The results suggest that miR-153 can inhibit the proliferation of tumor cells by inhibiting the expression of apoptosis inhibitory protein Bcl-2 and up regulating the expression of apoptosis inducing protein Bax by inhibiting the expression of COX-2.5.In order to further study the molecular mechanism of miR-153 inhibiting the invasion of A549 cells,the expression levels of MMP-2 and MMP-9 in the cells of A549/miR-153,A549/si-COX-2 and their control group were detected.The basalexpression in A549-control as 1,the relative expression of MMP-2 expression levels were(0.33 ± 0.015),(0.95 ± 0.031),(0.36 ± 0.026)in A549-miR-153,A549/si-control and A549/siCOX-2 group;The relative expression of MMP-9 were(0.12 ± 0.015)and(1.07±0.032),(0.43±0.076)respectively in A549-miR-153,A549/si-control and A549/siCOX-2 group.The expression of MMP-2 and MMP-9 in A549/miR-153 and A549/si-COX-2 were significantly lower than those in the control group(p<0.01).There has similar findings of SPC-A-1 cell line.The results suggest that miR-153 can inhibit the invasion and metastasis of A549 and SPC-A-1 lung cancer cells by inhibiting the expression of MMP-2 and MMP-9 through targeting inhibition of COX-2.6.Elisa assay was used to detect the effect of miR-153 on PGE2 in cell culture supernatant in A549/miR-153,A549/si-COX-2 SPC-A-1/miR-153,SPC-A-1/si-COX-2 and their respective control cells.Compared with the corresponding control group,the concentration of PGE2 in the' supernatant of A549/miR-153,A549/si-COX-2,SPC-A-1/miR-153 and SPC-A-1/si-COX-2 groups decreased obviously.Therefore,it is speculated that miR-153 inhibits the proliferation and invasion of A549 and SPC-A-1 cells in vitro by inhibiting COX-2 and further reducing the production of PGE2.7.A dual 1uciferase reportor construct with or without 3'UTR of COX-2 was transfected into 293 cells in the presence of a miR-153 minic,and Firfly and Renila luciferase activities were measured after 24h.Double luciferase reporter gene results showed that miR-153 inhibited the expression of COX-2 directly.Conclusions1.The expression of miR-153 is lower in non-small cell lung cancer.Decreased expression of miR-153 might be a potential unfavorable prognostic factor for patients with NSCLC.The expression levels of miR-153 was negatively correlated with the expression of COX-2 mRNA in lung cancer tissues.2.A549/miR-153 cell line and SPC-A-1/miR-153 with miR-153 overexpression and its control cell line A549/control and SPC-A-1/miR-153 were established.Upregulation of miR-153 in lung cancer cell lines in vitro can inhibit cell proliferation and invasion.3.miR-153 as a tumor suppressor decreases cell proliferation and cell migration ability in human lung cancer cells A549 and SPC-A-1 by targeting COX-2 through down-regulating PGE2 then down-regulating Bcl-2?MMP-2 and MMP-9...