The Potential Role And Mechanism Of MiR-339-5p In NSCLC(Non-Small Cell Lung Cancer)

Background and ObjectiveAccording to the global cancer statistics published in 2018,the incidence and mortality rate of lung cancer remain the highest all over the world.Lung cancer in our country shows the same trend as the other countries.It is reported that Xuanwei area in Yunnan Province demonstrated highest incidence and mortality rate in the world,indicating significant regional specificity.In some places of Xuanwei,the mortality rate of female lung adenocarcinoma ranks the first place in China.Among which the mortality rate of non-smoking female patients is 20 times the average level in China,ranking the highest in the world.This gives us an important warning sign in the prevention and treatment of lung cancer in China.Our previous study discovered that the high incidence and mortality of lung cancer in Xuanwei area was related to the indoor pollution caused by the use of local special coal combustion,and the unique indoor pollution environment may change microRNAs spectrum in some ways,leading to the occurrence and development of lung cancer in Xuanwei.In our previous study,we detected 155 differentially expressed microRNAs in 24 lung cancer tissues of Xuanwei by microarray.Among them,microRNA-339-5p is one of the up-regulated microRNAs.According to some researches,the expression of microRNA-339-5p in most tumors was lower than that in corresponding normal tissues,while there’s only a report related to lung cancer.However,the expression of microRNA-339-5p in lung cancer was inconsistent and the mechanism of microRNA-339-5p hasn’t been further studied.By the analysis of the data in TCGA,we found that the expression of microRNA-3 39-5p was up-regulated in lung adenocarcinoma and squamous cell carcinoma.In our previous work,we also found the expression of microRNA-339-5p was up-regulated in lung cancer cells.All the above elicit our several thoughts:Does the expression of microRNA-339-5p differ between lung cancer and normal lung tissues?Is there any discrepancy of expression between Xuanwei lung cancer and non-Xuanwei lung cancer?If the answer is yes,does this microRNA play a role in the occurrence and development of lung cancer?What about the mechanism?In this study,we detected the expression of microRNA-339-5p in non-small cell lung cancer(NSCLC)tissues and serum,and analyzed its relationship with pathological and clinical factors and its diagnosis value.Besides,we explored the effect of down-regulation of microRNA-339-5p expression on cell proliferation,migration,invasion and tumorigenesis in nude mice through in vivo and in vitro experiments.We also preliminarily studied the target genes and signaling pathways of microRNA-339-5p.In all,our study may provide a theoretical basis for the pathogenesis and prevention of lung cancer.Section Ⅰ Expression of microRNA-339-5p in non-small cell lung cancer(NSCLC)and its clinical significanceObjective:To explore the role of miRNA-339-5p and clinical significance in non-small cell lung cancer,we analyzed the expression of microRNA-339-5p in TAGA database and tissue chip data,tissue of non-small cell lung cancer patients(cancer tissues,adjacent cancers)and serum samples(lung cancer patients,non-cancer patients)from Xuanwei and non-Xuanwei areas,and the relationship between miRNA-339-5p and clinical factors.Methods:1.To investigate the expression of miR-339-5p in Xuanwei lung adenocarcinoma and normal lung tissues by analyzing tissue chip data.2.To investigate the expression of microRNA-339-5p in non-small cell lung cancer(NSCLC)and normal lung tissues by analyzing TCGA database data.3.The expression of microRNA-339-5p in 63 cases(30 Xuanwei and 33 non-Xuanwei)of non-small cell lung cancer(NSCLC)and their distal normal lung tissues was detected by qPCR.Then the expression of microRNA-339-5p was analyzed in cancer and normal tissues.And the correlation between expression level of microRNA-339-5p and the clinicopathological characteristics(age,sex,smoking,region,histological type,lymph node metastasis and staging)of the patients was analyzed.Finally,the diagnostic value of microRNA-339-5p in lung cancer was analyzed.4.Quantitative polymerase chain reaction(qPCR)was used to detect the expression of miRNA-339-5p in 104 serum samples of NSCLC and 50 serum samples of cancer-free controls to explore whether miRNA-339-5p was suitable to serve as a serum marker for the diagnosis of NSCLC.Results:1.Previous data from Xuanwei lung adenocarcinoma microarray showed that 155 microRNAs were differentially expressed(65 up-regulated and 90 down-regulated)compared with normal lung tissues,of which microRNAs-339-5p were up-regulated(FC=2.0189,p=0.0001,FDR=0.00017).2.TCGA database data showed that the expression of miRNA-339-5p was up-regulated in lung adenocarcinoma(Fold Change=2.334,P=2.618×10-11<0.001)and lung squamous cell carcinoma(Fold Change=1.589,P=4.323×10-6<0.001).3.In 63 samples(30 Xuanwei region and 33 non-Xuanwei region)of NSCLC,the expression of miRNA-339-5p in lung cancer tissues was significantly higher than that in matched normal lung tissues(P=0.007<0.01).According to the analysis of relationship between clinicopathological characteristics and miRNA-339-5p expression,the lymph node metastasis rate was higher in the patients expressed high level of miRNA-339-5p(P=0.02<0.05),but there was no significant correlation between the expression of miiRNA-339-5p and age,sex,smoking status,region,histological type and stage(P>0.05).The ROC(Receiver Operation Characteristic Curve)analysis of microRNA-339-5p in lung cancer tissues was performed using MedCalc software.When the cutoff value was 0.1195,microRNA-339-5p achieved the highest diagnostic efficiency,with a sensitivity of 55.6%,specificity of 74.6,and AUC(Area Under the Curve)0.639.4.There was no significant difference in the expression of serum miRNA-339-5p in non-small cell lung cancer and non-cancer control group(P=0.07>0.05).Conclusions:1.miR-339-5p is highly expressed in non-small cell lung cancer tissues and is associated with lymph node metastasis.There was no significant difference in the expression of miR-339-5p between Xuanwei non-small cell lung cancer and non-Xuanwei non-small cell lung cancer.2.microRNA-339-5p has only a low accuracy in the diagnosis of lung cancer,which has a certain reference value for the diagnosis of lung cancer.3.There was no significant difference in the expression of miR-339-5p in serum of NSCLC and non-cancer group,hence it isn’t suitable as serum markers in the diagnosis of NSCLC.Section Ⅱ Effect of miR-339-5p on biological behavior of non-small cell lung cancer(NSCLC)cell linesObjective:To investigate the effects of down-regulation of microRNA-339-5p on proliferation,migration and invasion of SPC-A1 and XWLC-05 non-small cell lung cancer cell lines.Methods:1.The expression levels of microRNA-339-5p in normal bronchial epithelial cell line(BEAS-2B)and lung cancer cell lines(SPC-A1,A549,XWLC-05,YTLMC and GLC)were detected by qPCR,and lung cancer cell lines with high expression of microRNA-339-5p were screened.2.miR-339-5p inhibitor was transfected into SPC-A1 and XWLC-05 cells,after determining the efficiency of transfection,stable transfected cells were selected for proliferation,migration and invasion ability experiments.Results:1.Compared with the expression level of microRNA-339-5p in BEAS-2B cell line,microRNA-339-5p was highly expressed in lung cancer cell lines(SPC-A1,A549,XWLC-05,YTLMC and GLC)(p<0.05),and the expression of miR-339-5p was the highest in SPC-A1 cell line.2.After stable transfection of miR-339-5p inhibitor,the expression of miR-339-5p in SPC-A1 and XWLC-05 cells decreased significantly compared with normal and negative control groups(p<0.05).3.Functional experiments of lung cancer cells:(1)Down-regulation of miR-339-5p inhibited the proliferation of SPC-A1 and XWLC-05 cells(P<0.05).(2)Downregulation of miR-339-5p could inhibit the migration of SPC-A1 and XWLC-05 cells(P<0.05).(3)Downregulation of miR-339-5p could inhibit the invasive ability of SPC-A1 and XWLC-05 cell lines(P<0.05).Conclusion:The expression of miR-339-5p in lung cancer cell lines SPC-A1,A549,XWLC-05,YTLMC and GLC was higher than that of BEAS-2B,and the expression of miR-339-5p in lung cancer cell line SPC-A1 was the highest.Decreased miR-339-5p inhibited the proliferation,migration and invasion of lung cancer cell lines SPC-A1 and XWLC-05.miR-339-5p might play an important role in serving as an oncogene in the development of non-small cell lung cancer.Section Ⅲ The effect of microRNA-339-5p on the proliferation of non-small cell lung cancer(NSCLC)in vivo studyObjective:To investigate the effect of down-regulation of microRNA-339-5p on tumorigenesis in nude mice.Methods:1.SPC-A1 cells were transfected with lentiviruses to construct stable transfected cell lines with low expression of microRNA-339-5p and negative control.2.microRNA-339-5p inhibitor,negative control(NC)and blank control cell lines were inoculated subaxillary in nude mice.The general vital signs of nude mice were observed and the size of tumors was measured.Nude mice were executed at 37th days to measure the size and weight of the tumors.The tumors were made into paraffin blocks,HE staining was performed and the expression of Ki-67 was detected by immunohistochemistry(IHC).Results:1.After the lentivirus vector inhibitor was transfected with SPC-A1,the expression of miR-339-5p in inhibitor group was significantly lower than that in NC group,and the transfection efficiency of cells was more than 50%under fluorescence microscope,and the expression of miR-339-5p in the miR-339-5p low expression group(miR-339-5p inhibitor)was downregulated than that in the negative control group(NC).2.There was no significant difference in body weight among the three groups.After 24 days,the volume of tumors in the miR-339-5p low expression group(miR-339-5p inhibitor)was smaller than that in the negative control group(NC)and the blank control group(Control)(P<0.05),after the execution of nude mice,the weight of tumors in the miR-339-5p low expression group(miR-339-5p inhibitor)was lower than that in the negative control group(NC)and the blank control group(Control)(P<0.05).3.T he expression of Ki-67 in miR-339-5p inhibitor group was higher than that of NC group and control group(P<0.05).Conclusion:Down-regulation of the expression of microRNA-339-5p could significantly inhibit the tumorigenic ability of non-small cell lung cancer(NSCLC)cells in nude mice,a decrease in the Ki-67 index(IRS)was observed after down-regulation of miR-339-5p.SectionⅣ Study on BCL6 as a direct target of miR-339-5p Objective:To investigate whether BCL6 is the target gene of microRNA-339-5p in non-small cell lung cancer,and to analyze whether microRNA-339-5p and BCL6 is associated with prognosis.Methods:1.Bioinformatics methods was carried out to predicts the target genes of microRNA-339-5p.2.Luciferase reporter gene assay was used to verify whether BCL6 is a direct target gene of microRNA-339-5p.3.The expression change of target gene of SPC-A1 in lung cancer cell lines was detected by Western blot after down-regulation of microRNA-339-5p.4.QPCR was used to detect the expression of BCL6 in non-small cell lung cancer(NSCLC)tissues and its paired distal normal lung tissues of 63 samples.Regression analysis was used to analyze the correlation between BCL6 and the expression of microRNA-339-5p.5.GEPIA was used to analyze the expression of BCL6 in non-small cell lung cancer(NSCLC)and normal lung tissues.6.KM plotter was used to analyze the relationship of the expression of BCL6 with the prognosis of non-small cell lung cancer(NSCLC).Results:1.Bioinformatics predicts that BCL6 is a target gene of microRNA-339-5p.2.Luciferase assay indicated that BCL6 is a direct target gene of microRNA-339-5p.3.After down-regulation of microRNA-339-5p in SPC-A1 cells,Western blot showed that the expression of BCL6 was up-regulated.4.Compared with the distal normal lung tissue,BCL6 expression was lower in non-small cell lung cancer(NSCLC)(P<0.05),and was negatively correlated with the expression of microRNA-339-5p(R=0.236,P=0.044<0.05).5.GEPIA analysis showed that the expression of BCL6 in lung adenocarcinoma and lung squamous cell carcinoma was lower than that in normal lung tissues.6.KM plotter analysis showed that the overall survival(OS)in the overexpressed BCL6 was longer(HR=0.71,95%CI:0.6-0.84,P=6.2×10-5<0.001),and the prolonged trend of progression-survival(PFS)in the overexpressed BCL6(HR=0.77,95%CI:0.58)-1.01),but there was no statistical difference(P=0.056>0.05).Conclusion:BCL6 is a direct target gene of miR-339-5p,BCL6 is low expressed in non-small cell lung cancer(NSCLC),and is related to the prognosis of non-small cell lung cancer(NSCLC),which might inhibit the occurrence and development of non-small cell lung cancer,miR-339-5p may regulate the occurrence and development of non-small cell lung cancer(NSCLC)by regulating the expression of BCL6.