The Potential Role And Mechanism Of Hsa-mir-135b-5p In Non-small Cell Lung Cancer In Xuanwei Area

Background and ObjectiveLung cancer is the most common malignancy and is a leading cause of mortality worldwide.Lung cancer have become the most common incident cancer(509,000)and the leading cause of cancer(610;000)in men,and the second common incident cancer(224,000)and the leading cause of cancer(178,000)in women in China.Broad sense of Xuanwei area included Xuanwei,Fuyuan,Qilin and Zhanyi et al.,which is located in the northeastern of Yunnan provience where is rich for late Permian coal.The incidence of lung cancer rate in Xuanwei area is among the highest in rural China,also in the world.The Xuanwei lung cancer has its unique features and remarkable regional characteristics.Our previous study suggested that burning "smoky coal" may cause indoor air pollution and that was the major reason why the female lung cancer incidence rates in Xuanwei was such high.Burning "smoky coal" may release plenty of carcinogenic substance such as polycyclic aromatic hydrocarbons(PAHs),silica nanoparticles and PM2.5.Long-term,low dose exposure to these unique carcinogenic substances may result in unique miRNA profile changed,which promote the development and progression of lung cancer in Xuanwei.Our previous work detected the miRNA profiles in 24 female lung adenocarcinoma and paired nontumor tissues in Xuanwei by miRNA array analysis(U1202224).155 differentially expressed(?2-fold change)miRNAs were identified(65 upregulated and 90 downregulated).Among these differentially expressed miRNAs,miR-135b-5p(miR-135b)was one of the most upregulated miRNAs(FC=5.70,P=0.006).Using another 10 non-Xuanwei NSCLC as control,the microarray analysis also suggested that the expression of miR-135b in Xuanwei NSCLC was higher than non-Xuanwei(FC=2.75,P=0.019).The potential role of miR-135b in Xuanwei NSCLC and its potential mechanism are still unclear.Our present study would detect the expression of miRNA in tissue and also in serum in NSCLC.Then we would explore how miR-135b affected the biological behavior(proliferation,migration,invasion and colony formation)of lung cancer cell lines by reducing the expression of miR-135b.The potential target of miR-135b was also evaluated.At last,in vivo experiment was done to evaluate the tumour suppressing effect of miR-135b.The present study may lay the theoretical foundation in the development,prevention and treatment of Xuanwei lung cancer.Section I:The expression of miR-135b in NSCLC and its clinical significance Objective:To explore the potential role of miR-135b in NSCLC and its clinical significance,the miR-135b expression were detected in microarray data,TCGA data set,samle tissues and serum.Methods:(1)Previous microarray data was analyzed to explore the expression level of miR-135b in Xuanwei NSCLC;also the differential expression of miR-135b in Xuanwei and non Xuanwei subjects was also analyzed.(2)The expression level of miR-13 5b between NSCLC and normal lung tissue was analyzed by TCGA data set.(3)The expression level miR-135b of 70 NSCLC(Xuanwei subjects,37 non-Xuanwei subjects)and paired nontumor tissues was detected by qPCR.The expression difference of miR-135b between Xuanwei and non-Xuanwei subjects was analyzed.The asociation between miR-135b expression and the clinicopathological features of NSCLC was also analyzed.(4)The serum miR-135b level in 104 NSCLC and 50 cancer-free controls was detected to explore whether miR-13 5b was suitable to serve as serum biomarker in NSCLC.Results:(1)Previous microarray analysis suggested,miR-135b was significantly upregulated in 24 Xuanwei NSCLC when compared with paired nontumor tissues(FC=5.695,P=0.0059,FDR=0.0269).Another comparison was done between 24 Xuanwei and 10 non-Xuanwei NSCLC,we found miR-135b was still highly expressed in Xuanwei NSCLC when compared with non-Xuanwei subjects(FC= 2.748,P=0.0193,FDR= 0.2682).(2)Using TCGA data,we found miR-135b was upregulated in both LUAD(FC=6.913,P=4.00R10-30)and LUSC(FC=2.910,P=2.101 R10-11).(3)Among 70 NSCLC,when compared with paired nontumor tissues,miR-135b was upregulated in 56 cases and only downregulated in 14 patients.The miR-135b expression level in tumor tissues was significantly higher than nontumor tissues(0.0497±0.0120 vs.0.0254±0.0077,P=0.0004).The expression level of miR-135b was higher in Xuanwei than non-Xuanwei NSCLC(6.944±0.871 vs.3.695 ± 0.495,P=0.0023).The similar results was also found in LUAD patients(6.849±0.892 vs.4.134 ±0.612,P=0.0331).The clinicopathological characteristics analysis suggested high miR-135b expression was associated with advanced stage(P=0.030)and positive lymph node metastasis(P=0.007).(4)There was no difference between serum miR-135b expression in NSCLC and cancer-free controls.The AUC of serum miR-135b was 59.1%.Conclusion:(1)miR-135b was upregulated in NSCLC tissues.High miR-135b expression was associated with advanced stage and positive lymph node metastasis.(2)The expression level of miR-135b was higher in Xuanwei NSCLC when compared with non-Xuanwei NSCLC.(3)The expression level of serum miR-135b was not significant between NSCLC and cancer-free controls.miR-135b may be not suitable to serve as serum biomarker in NSCLC.Section ?:The effect of miR-135b on the biological behavior in NSCLC cell lineObjective:This part aimed to evaluate the effect of miR-135b on the biological behavior(proliferation,migration,invasion and colony formation)in A549 and XWLC-05 cell lines by reducing the expression of miR-135b.Methods:(1)The expression level of miR-135b in five NSCLC cell lines(A549,XWLC-05,NCI-H1975,GLC and YTLMC)and normal bronchial epithelial cell line BEAS-2B was detected by qPCR.(2)miR-135b inhibitor was transfected in A549 and XWLC-05 cell lines.The transfection efficiency was evaluated by qPCR.The proliferation,migration,invasion and colony formation assay was conducted.Results:(1)When compared with BEAS-2B,miR-135b was upregulated in all five NSCLC cell lines(A549,XWLC-05,NCI-H1975,GLC and YTLMC).(2)miR-135b inhibitor was successfully transfected in A549 and XWLC-05 cell lines.The qPCR analysis suggested transfection with miR-135b inhibitor(Anti-miR-135b)would significantly reduce the expression of miR-135b when compare with normal control or negative control.(3)The cell viability was decreased in 48,72 and 96 hours in A549,and 72 and 96 hours in XWLC-05 after reduced miR-135b expression.(4)After transfected with miR-135b inhibitor,the migration and invasion ability of NSCLC cell lines(A549 and XWLC-05)was inhibited in 48 hour.(5)After transfected with miR-135b inhibitor,the clone-forming capacity of A549 and XWLC-05 was inhibited.Conclusion:miR-135b was upregulated in NSCLC cell lines.The proliferation,migration,invasion and colony formation abilities were inhibited after reduced miR-135b expression.miR-135b may play a role of oncomirs,and could promote the development and progression of NSCLC.Section ?:The research of STAT6 as the direct target of miR-135bObjective:This part aimed to explore whether STAT6 is the direct target of miR-135b.Methods:(1)The potential target gene of miR-135b was predicted by bioinformatics method.(2)The Dual luciferase reporter assay system was applied to verify whether STAT6 is the direct target of miR-135b.qPCR and Western blot were applied to detect the STAT6 expression on mRNA and protein level in A549 and XWLC-05 after reduced miR-13 5b expression.(3)The expression level of STAT6 in NSCLC and its paired nontumor tissues was detected by qPCR.The relationship between STAT6 expression and its relationship between clinicopathological characteristics were analyzed.Regression analysis was also conducted to explore the asscoation between miR-13 5b and STAT6 expression.(4)The expression of STAT6 level was detected in five NSCLC cell lines(A549,XWLC-05,NCI-H1975,GLC and YTLMC)and normal bronchial epithelial cell line BEAS-2B by qPCR.(5)GEPIA and KM plotter based on online microarray datasets were applied to explore the expression difference of STAT6 in NSCLC and normal lung tissues,and the prognostic role of STAT6 in NSCLC.Results:(1)Bioinformatics prediction suggested that STAT6 was one of the potential targets genes of miR-135b.(2)The Dual luciferase reporter assay system verified that STAT6 is the direct target of miR-135b.The qPCR and Western blot analysis suggested that the expression level of STAT6 on mRNA and protein level was upregulated in A549 and XWLC-05 after reduced the miR-13 5b expression.(3)STAT6 was downregulated in NSCLC when compared with paired nontumor tissues.miR-135b expression was negatively related with STAT6 expression(R=-0.025,P=0.005).No significant association was found between STAT6 expression and the clinicopathological characteristics of NSCLC.(4)When compared with BEAS-2B,except for YTMLC(P>0.05),miR-135b was downregulated in A549,XWLC-05,NCI-H1975 and GLC.(5)GEPIA analysis suggested that STAT6 was downregulated in both LUAD and LUSC when compared with normal lung tissues.KM plotter suggested that high STAT 6 expression was associated with longer OS(HR=0.69,95%CI:0.60-0.78,P=7.7×10-9).Although the trend of high STAT6 may be associated longer PFS was observed(HR=0.85,95%CI:0.70-1.03),the statistic difference was not significant(P=0.088).Conclusion:STAT6 is the direct target of miR-135b.STAT6 was downregulated in NSCLC and associated with the prognosis of NSCLC.It may serve as a tumor suppressor gene in the development of NSCLC.Oncomirs miR-135b may promote the development and progression of NSCLC by regulating the expression of STAT6.Section IV:In vivo study of the effect of miR-135b on tumor proliferation in NSCLCObjective:This part aimed to explore the effect of miR-135b on turnor formation in nude mice.Methods:(1)Lentivirus was stably transfected in A549 to construct reduced miR-135b and negative control cell line.(2)Three group A549(Anti-miR-135b,NC and Control)were cultured and then injected subcutaneously into the flank of BALB/c node mice(8 for each groups).The general physical state,the weight of mice and the tumor volume were routinely observed and measured.After 37 days,all BALB/c node mice were killed.We measured the tumor volumes and weights of mice.qPCR and IHC analyses were done to detected the STAT6 expression in tumor nodes.Results:(1)There was no difference between weights of mice in three groups.The tumor volume in Anti-miR-135b group was significantly smaller than Control and NC groups after 20 days.The tumor weight in Anti-miR-135b group was significantly lower than Control and NC groups.(2)Both qPCR and IHC analyses suggested that the STAT6 expression level in Anti-miR-135b group was higher than control and NC groups.Conclusion:Reduced miR-135b expression could inhibit tumor formation and growth in nude mice.Reduced miR-135b expression was associated with higher STAT6 expression in tumor node tissues.