| Objective:More and more evidence shows that micro RNA expression disorders play an important role in the development of tumors.Many reports have shown that mi R-125a-5p is down-regulated in many tumor tissues,but its role in the development and progression of lung cancer is still unclear.Therefore,the current study aimed to observe the expression of miR-125a-5p in lung cancer tissues and lung cancer cell lines,explore its effects on the biological characteristics of lung cancer cells and analyze its possible mechanism.Regulating the expression of miR-125a-5p is expected to benefit treatment strategy for the patients with lung cancer.Methods:1.In all,18 fresh lung cancer tissues and adjacent normal lung tissues were collected from patients with lung cancer during the surgery in our hospital and were immediately frozen in liquid nitrogen after surgery for subsequent experiments.Before surgery,all the patients had no radiotherapy and chemotherapy,no other treatment history,and no inflammatory diseases.The study protocol was approved by the Ethics Committee of The Affiliated Hospital of Nantong University,Nantong,China.The expression of miR-125a-5p mRNA in 18 tissues and adjacent tissues was detected by qRT-PCR.2.Human bronchial epithelial cells(HBE),as well as lung cancer cell lines 95-D,A549,HCC827 and NCI-H1299 were cultured in vitro.The expression of mi R-125a-5p mRNA in HBE,95-D,A549,HCC827 and NCI-H1299 cells was detected by qRT-PCR.3.Transfection of miR-125a-5p mimic or mi R-125a-5p inhibitor in A549 cells was used to overexpress or down-regulate the expression of mi R-125a-5p.The proliferation,migration and apoptosis of the transfected cells were detected by MTT,immunohistochemistry,flow cytometry and Transwell culture system.4.The wild type(WT)of STAT3 luciferase reporter plasmid(STAT3WT-luc)and the mutation(MU)type of STAT3 luciferase reporter plasmid(STAT3MU-luc)were constructed.STAT3WT-luc or STAT3MU-luc and miR-125a-5p mimic or negative control were transfected into A549 cells.After 48 h of transfection,the cells were collected.Dual-luciferase reporter gene system was adopted in this experiment.Western blot was used to detect the expression of STAT3 protein in A549 cells transfected with miR-125a-5p mimic.5.The STAT3 recombinant expression plasmid,pcDNA3.1-STAT3,was constructed.pcDNA3.1-STAT3 and miR-125a-5p mimic or negative control were transfected into A549cells.The proliferation,migration and apoptosis of the transfected cells were detected by MTT,immunohistochemistry,flow cytometry and Transwell culture system.Results:1.mi R-125a-5p mRNA expression in lung cancer was significantly down-regulated.The expression of mir-125a-5p mRNA in 18 cases of cancer tissues and adjacent tissues was detected by qRT-PCR.The expression of miR-125a-5p mRNA was down-regulated in cancer tissues compared with adjacent tissues,and the difference was statistically significant(p<0.01).2.miR-125a-5p mRNA expression in lung cancer cell lines was significantly down-regulated.The expression of mi R-125a-5p mRNA in HBE,95-D,A549,HCC827and NCI-H1299 cells was detected by qRT-PCR.The expression of mi R-125a-5p mRNA in lung cancer cell lines 95-D,A549,HCC827 and NCI-H1299 was lower than HBE,the difference was statistically significant(p<0.01).There was no significant difference in the expression of mir-125a-5p mRNA among lung cancer cell lines 95-D,A549,HCC827 and NCI-H1299(p>0.05).3.miR-125a-5p expression was observed after transfection with miR-125a-5p mimic or mi R-125a-5p inhibitor in A549 cells.The expression of mi R-125a-5p mRNA in transfected A549 cells was detected by qRT-PCR.The results showed that the expression of miR-125a-5p mRNA in A549 cells transfected with miR-125a-5p mimic was significantly higher than that in normal control group and negative control group,The difference was statistically significant(p<0.01).The expression of mi R-125a-5p mRNA in A549 cells transfected with mi R-125a-5p inhibitor was significantly lower than that in normal control group and negative control group(p<0.01).4.mi R-125a-5p inhibited A549 cell viability.The viability of transfected A549 cells was analyzed by MTT.The viability of A549 cells in miR-125a-5p mimic transfection group was lower than that in normal control group and negative control group(p<0.01).The viability of A549 cells transfected with miR-125a-5p inhibitor was significantly higher than that of normal control group and negative control group(p<0.01).5.miR-125a-5p inhibited A549 cell proliferation.The transfected A549 cell proliferation was detected by BrdU immunohistochemistry.The number of BrdU~+cells of miR-125a-5p mimic transfection group were significantly lower than those in the normal control group and the negative control group(p<0.01).The number of BrdU~+cells in miR-125a-5p inhibitor group was significantly higher than those in normal control group and negative control group(p<0.01).6.Apoptosis of A549 cells induced by miR-125a-5p.The number of apoptotic cells of miR-125a-5p mimic transfected group was significantly higher than those of normal control group and negative control group(p<0.01).The number of apoptotic cells of miR-125a-5p inhibitor group was significantly lower than those of blank control group and negative control group(p<0.01).7.miR-125a-5p inhibited the invasion and migration of A549 cells.Transwell culture system showed that the number of migrated cells in miR-125a-5p mimic transfected group was significantly lower than that in normal control group and negative control group(p<0.01).The number of cells transfected with mi R-125a-5p inhibitor was significantly higher than that of the normal control group and the negative control group(p<0.01).8.STAT3 was a target gene of miR-125a-5p.The result of luciferase reporter assay showed that the activity of luciferase was significantly decreased after transfection with miR-125a-5p mimic in STAT3 3’UTR-WT luciferase reporter assay,and the difference was statistically significant(p<0.01).Compared with the negative control group,the activity of luciferase did not change significantly after transfection with mi R-125a-5p mimic in STAT3 3’UTR-MU luciferase reporter assay(p>0.05).9.miR-125a-5p down-regulated STAT3 expression in A549 cells.The expression of STAT3 protein in transfected A549 cells was detected by Western blot.The results showed that the expression of STAT3 protein in miR-125a-5p mimic transfection group was significantly lower than that in normal control group and negative control group(p<0.01).10.Overexpression of STAT3 could reverse the biological characteristics changes of lung carcinoma cell induced by miR-125a-5p overexpression.Western blot analysis showed that STAT3 protein expression in pcDNA3.1-STAT3 group was significantly higher than that in normal control group,negative control group and pcDNA3.1-STAT3+miR-125a-5p mimic group,the difference was statistically significant(p<0.01).There was no significant difference between the other three groups(P>0.05).The results of MTT assay showed that the cellular viability of pcDNA3.1-STAT3 group was significantly higher than that of normal control group,negative control group and pcDNA3.1-STAT3+miR-125a-5p mimicr group,the difference was statistically significant(p<0.01).There was no significant difference between the other three groups(p>0.05).Compared with the blank control group,the negative control group and the pcDNA3.1-STAT3+miR-125a-5p mimic group,the apoptotic cells of pcDNA3.1-STAT3group were significantly decreased,the difference was statistically significant(p<0.01).There was no significant difference among the other three groups(p>0.05).The results of Transwell culture system showed that the number of migrated cells in pc DNA3.1-STAT3group was significantly higher than that in blank control group,negative control group and pcDNA3.1-STAT3+miR-125a-5p mimicr group(p<0.01).The difference among the other three groups was not statistically significant(p>0.05).Conclusions:1.The expression of miR-125a-5p was down-regulated in lung cancer tissues and corresponding lung cancer cell lines.2.Overexpression of miR-125a-5p in lung cancer cell lines can inhibit the viability,proliferation and migration of lung cancer cells,and induce the apoptosis of lung cancer cells.3.miR-125a-5p can target to STAT3 3’UTR and down-regulate the expression of STAT3 in lung cancer cells,thus exerting its biological function.4.The overexpression of STAT3 in lung cancer cells can reverse the biological characteristics changes caused by overexpression of miR-125a-5p,promote the viability,proliferation and migration ability of lung cancer cells,and inhibit the apoptosis of lung cancer cells. |
PDF Full Text Request