Mechanism Investigation Of Long Non-coding RNA SNHG3 Induced Occurrence And Development Of Non-small Cell Lung Cancer

Objective:As a subset of long non-coding RNAs(lncRNAs),small nucleolar RNA host genes(SNHGs)play critical roles in tumor progression reportedly.lncRNA SNHGs are abnormally expressed in a variety of tumors and their expression level is closely related to the prognosis of tumor patients.Therefore,the detailed mechanism is worthy of further study.lncRNA SNHG3(small nucleolar RNA host gene 3)can promote the proliferation and migration of non-small cell lung cancer(NSCLC)cells,but the specific molecular mechanism and its correlation with the clinic and prognosis of NSCLC patients are still unclear.The purpose of this study is to clarify the function and potential mechanism of lncRNA SNHG3 in the development and occurrence of NSCLC,to determine whether lncRNA SNHG3 can be used as a prognostic marker in patients with NSCLC,and to provide a new target for clinical treatment of NSCLC.Methods:1.The expression level of lncRNA SNHG3 in NSCLC tissues and adjacent normal tissues was compared using TCGA database,and the correlation between the expression level of lncRNA SNHG3 and the survival rate and tumor recurrence rate of NSCLC patients was analyzed.2.The effect of lncRNA SNHG3 on the proliferation and invasion of non-small cell lung cancer cells was studied by overexpression and knockdown of lncRNA SNHG and cellular experiments.3.The miRNAs that may bind to lncRNA SNHG3 were identified using the starBasev2 predictive tool,and the correlation between miRNAs and lncRNA SNHG3 expression,as well as the correlation between miRNAs and survival rate and tumor metastasis in patients with non-small cell lung cancer was analyzed by Pearson.4.The biological role of lncRNA SNHG3 and miRNA in regulating proliferation of non-small cell lung cancer cells was investigated using MTT and Transwell invasion assay.5.Bioinformatics tools were used to predict the target genes of the miRNA.The expression of target genes in NSCLC tissues and adjacent normal tissues was analyzed,and the correlation between the target gene level and the miRNA level,as well as the correlation between the target gene level and the survival rate and tumor metastasis of NSCLC patients were analyzed.6.The binding sites of target gene 3’UTR to miRNA were predicted by software,and the regulation of lncRNA SNHG3 and miRNA on target gene was determined by cellular experiments.7.The effect of lncRNA SNHG3 on regulating growth of non-small cell lung cancer was determined using nude-mouse transplanted tumor model.Results:1.The expression level of lncRNA SNHG3 in NSCLC samples was analyzed from the TCGA cohort and data showed that lncRNA SNHG3 expression was dramatically upregulated in NSCLC tissues compared with match normal tissues(n=58).LncRNA SNHG3 expression was also significantly increased in NSCLC tissues(n=515)compared with unpaired normal tissues(n=58).In the light of lncRNA SNHG3 expression levels,the patients’ survival time and survival status,a cutoff value(5.737)of lncRNA SNHG3 was gained in NSCLC patients,and the patients were divided into high and low SNHG3 expression groups.As indicated in Table 1,high lncRNA SNHG3 expression was associated with lymph node infiltration in NSCLC patients.Kaplan-Meier analysis showed that the patients with high lncRNA SNHG3 expression harbored a lower survival rate and a higher recurrence rate as compared with those with low lncRNA SNHG3 expression.Univariate and Multivariate Cox regression analysis demonstrated that high expression of lncRNA SNHG3 was an independent prognosis marker of poor survival and tumor recurrence in NSCLC patients.2.We measured the expression of lncRNA SNHG3 in NSCLC cell lines and found that lncRNA SNHG3 harbored an increased expression in SPC-A1 cell line but a reduced expression in A549 cell line as compared with the BEAS-2B cell line.The overexpressed efficiencies of lncRNA SNHG3 plasmids in A549 or knockdown efficiencies of si-SNHG3 in SPC-A1 were determined by qRT-PCR analysis.We found that forced expression of lncRNA SNHG3 facilitated cell proliferation and invasion as compared with the control group,but knockdown of lncRNA SNHG3 had the opposite effects.Western blot analysis showed that lncRNA SNHG3 increased the expression of PCNA and MMP2 in A549 cells,but knockdown of lncRNA SNHG3 reduced their expression in SPC-A1 cells.3.We utilized the starBasev2.0 prediction tools to screen 12 miRNAs which may bind to lncRNA SNHG3,among which miR-340-5p/-135a-5p/139-5p had a dramatical decrease in NSCLC tissues as compared with the adjacent normal tissues.Pearson correlation analysis demonstrated that miR-340-5p rather than miR-135a-5p/139-5p possessed a negative correlation with lncRNA SNHG3 expression in NSCLC tissues.A cutoff value(3.687)of miR-340-5p was obtained and divided the patients into high and low miR-340-5p expression groups.Low expression of miR-340-5p was positively associated with distant metastases in patients with NSCLC.Kaplan-Meier analysis revealed that miR-340-5p expression level was positively correlates to survival of NSCLC patients.4.The binding sites between miR-340-5p and SNHG3 were identified.We co-transfected WT or Mut SNHG33’UTR with miR-340-5p mimic into A549 or miR-340-5p inhibitor into SPC-A1 cells.The results from luciferase activity analysis showed that miR-340-5p weakened the luciferase activity of WT SNHG3 in A549 cells,but its inhibitor had an opposite effect.miR-340-5p mimic or inhibitor had no effects on that of Mut SNHG3 compared to the control group.The overexpressed efficiency of miR-340-5p mimic in A549 or the knockdown efficiency of miR-340-5p inhibitor in SPC-A1 was determined by qRT-PCR analysis.In addition,ectopic lncRNA SNHG3 expression inhibited the expression of miR-340-5p,while silencing SNHG3 increased miR-340-5p expression.Moreover,after co-transfection with SNHG3 plasmids and miR-340-5p mimic into A549 or si-SNHG3 and miR-340-5p inhibitor into SPC-A1 cells,we found that overexpression of miR-340-5p suppressed the proliferation and attenuated the proliferation promoting effect of SNHG3 on A549 cell line,but miR-340-5p inhibitor displayed an opposite effect.5.We then screened 8 targets of miR-340-5p using the four prediction tools,among which HOXA10,KRIT1,SERBP1 and PDIK1L indicate an extreme upregulation in NSCLC tissues.Pearson correlation analysis indicated that HOXA10 rather than KRIT1,SERBP1 and PDIKIL exhibited a negative correlation with miR-340-5p in NSCLC tissues.We gained a cutoff value(3.304)of HOXA10 in NSCLC,and divided the NSCLC patients into high and low HOXA10 expression groups.We found that HOXA10 expression was associated with distant metastases in NSCLC.Kaplan-Meier analysis indicated that the expression level of HOXA10 negatively correlates to survival of NSCLC patients.Multivariate Cox regression analysis unveiled that high expression of HOXA10 was an independent prognosis marker for poor survival of patients with NSCLC.6.The binding sites between miR-340-5p and WT or Mut HOXA10 3’UTR were identified.To verify whether miR-340-5p can bind to HOXAIO 3’UTR,we co-transfected WT or Mut HOXA10 3’UTR and miR-340-5p mimic or its inhibitor into A549 or SPC-A1 cells and found that miR-340-5p reduced the luciferase activity of WT HOXA10 3’UTR in A549 cells,but its inhibitor showed an opposite effect.They exerted no effects on that of Mut HOXA10 3’UTR as compared to the control group.Additionally,after co-transfection with SNHG3 plasmids and miR-340-5p mimic or si-SNHG3 and miR-340-5p inhibitor in A549 or SPC-A1 cells,qRT-PCR and Western blot analysis indicated that miR-340-5p downregulated HOXAIO expression and attenuated lncRNA SNHG3-induced HOXAIO expression in A549 cells,but miR-340-5p inhibitor indicated an opposite effect.7.We compared the tumor size between control and si-SNHG3 groups.We found that the tumor growth was inhibited in si-SNHG3 transfected SPC-Al cells as compared with the control group.Both the tumor weight and volume were decreased in si-SNHG3 group compared to control group.HE staining demonstrated that the number of tumor cells in si-SNHG3 group was also decreased compared to control group,and IHC analysis indicated that Ki-67 index was lowered by silencing lncRNA SNHG3 as compared with the control group.Conclusion:1.Elevated expression of lncRNA SNHG3 was an independent prognosis marker for poor survival of patients with NSCLC.2.lncRNA SNHG3 enhanced the proliferation and invasion of NSCLC cells3.Low expression of miR-340-5p was associated with poor survival in NSCLC patients.4.lncRNA SNHG3 regulated NSCLC cell proliferation by the mediation of miR-340-5p.5.High expression of HOXA10 was an independent prognosis marker for poor survival of patients with NSCLC.6.miR-340-5p reversed LncRNA SNHG3-induced HOXA10 expression in NSCLC cells.7.Knockdown of lncRNA SNHG3 suppressed the tumorigenesis of NSCLC.