| Background and objective Lung cancer is one of the common tumors with high mortality rate, and almost 80% is non-small cell lung cancer(NSCLC). Although the further improvement in diagnosis and treatment, 30%-40% patients are still with poor prognosis because of metastasis and diffusion of tumor. It will help us understand the pathogenesis of tumor and find new therapeutic method to study the potential mechanisms about the occurrence and development of tumorMicro RNAs(mi RNAs) are non-coding, single-stranded and small RNAs, approximately 22 nucleotides in length. It is almost expressed in all eucaryote cells, and highly conserved between different species and evolution. mi RNAs can bind to complimentary sequences in the 3′UTRs of m RNAs of target gene and negatively regulate the post-transcriptional and/or translational process of target gene, which futher affect the biological behavior of cells including proliferation, differentiation, cell cycle and apoptosis. These phenomenons also show mi RNAs, as both oncogenes and tumor suppressors, plays roles in the occurrence and development of tumor. Increasingly, abnormal expression of mi RNAs have been found in a variety of malignant tumors, including leukemia, colonic carcinoma, lung cancer, breast cancer, brain cancer and prostate cancer and so on.Further discussions are needed to detect and explore the expression level of mi R-495 in NSCLC tissues and the relevant mechanism about biological function and mechanism regulation.Metastasis-associated protein 3(MTA3), a key co-regulatory protein, belongs to the metastasis-associated protein family, which also includes MTA1 and MTA2. Evidence suggests that MTA3 is upregulated in several cancer types, including carcinoma of the esophagus, breast, lung, uterus, and nasopharynx, and is associated with tumor progression and metastasis by interacting with various cell signaling pathways. On the basis of the result of Agilent mi RNA microarray, we found that MTA3 was a potential target of mi R-495 by bioinformatic analysis. According to the research of target gene in lung cancer, our study firstly confirm mi R-495 MTA3 expression and further determine the role of mi R-495 in regulating the proliferation, migration and cell cycle progression of NSCLC cells to discover its potential molecular mechanism in NSCLC.The research includes two parts. The first part is altered mi RNA expression and MTA3 expression in NSCLC tissues and correlation with clinicopathological characteristics of NSCLC patients.The second part is effect of mi R-495 on the proliferation, migration and cell cycle progression of the NSCLC cell line calu-3 and investigation about mechanism of mi R-495 as a tumor suppressor.Part One mi R-495 and MTA3 expression in NSCLC tissues andanalysis of correlation with clinicopathological characteristics of NSCLC patients Methods 1. Clinical sample collection:72paired NSCLC and adjacent normal lungtissueswere collected. 2. Agilent mi RNA microarray was used to analyze the mi RNA expression profile in3 paired NSCLC and adjacent normal lung tissues 3. The expression levels of mi R-495 were validated by q RT-PCR, and MTA3expression was also detected by q RT-PCR and western blot in 72 pairedNSCLCand adjacent normal lung tissues. Spearman relativity analysis was usedto analyze the correlation between mi R-495 and MTA3 expression. 4. According to mi R-495 and MTA3 expression levels in NSCLC tissues wereassociated with clinicopathological characteristics of NSCLC patients. 5. SPSS 21.0 was used to analyze all the dates. The ANVOA was used toanalyzethe comparison among more groups. The LSD-t was used to analyze thecomparison two groups. The student’s t-test was used to analyze measurementdata. The relation of two variables was analyzed by the Spearman correlationanalysis. α=0.05 is considered as significance. Results 1. The result of Agilent mi RNA microarray showed 56 differentially more thantriple expressed mi RNAscomparing to adjacent normal lung tissues including 26mi RNAs up-regulated and 30 mi RNAs down-regulated. 2. Compared to adjacent normal lung tissues, mi R-495 expression is significantlyup-regulated but MTA3 expression down-regulated in NSCLC tissues(P<0.05).Spearman correlation analysis showed both had an inverse correlation. 3. Mi R-495 and MTA3 expression in NSCLC tissues were associated with theoccurrence of lymph node metastases, differentiation and TNM stage(P < 0.05).There was no statistically significant difference between their expression andeither age, gender or pathological types(P> 0.05).Part Two Effect of mi R-495 on the biological behavior of the NSCLC cell line Methods 1. q RT-PCR and western blot were used to detect the expression of mi R-495 andMTA3 in NSCLC cell line A549,Calu-3,H460,H1299,H1650 and SPC-A-1. 2. Mi R-495 agomir and mi R-495 scramble were respectively synthetized andtransfected into calu-3 cells using LipofectamineTM2000. 3. q RT-PCR was used to detect the change of mi R-495 expression in every group. 4. CCK8 assay was used to detectthe change of calu-3 cellproliferationin everygroup. 5. The experiments about nude mice were used to detect the growth oftransplantation tumor and expression of MTA3 protein in every group. 6. FCM assay was used to detect the change of calu-3 cellcycle and cell cyclerelated proteins in every group. 7. Transwell assay and Wound healing assay were used to detect the change ofcalu-3invasion and metastasis. 8. Bioinformatic analysis was used to predict the target gene of mi R-495. Thewild-type and mutant 3’ UTR segments of MTA3 were constructed intopmir GLO, which were cotransfected into calu-3 cell along with mi R-495 agomiror scramble. Luciferase reporter assays were performed to determine if MTA3 isregulated by mi R-495. 9. pc DNA3.1-MTA3 was constructed and respectively cotransfected into calu-3 cellalong with mi R-495 agomir or scramble.Restore experiments were performed toanalyze the interaction mechanism between mi R-495 and MTA3. 10. Related experiments were performed to compare the effects of si RNA-MTA3 andmi R-495 up-regulation on calu-3 cell proliferation,invasion and cellcycle. 11. SPSS 21.0 was used to analyze all the dates. The ANVOA was used to analyzethe comparison among more groups. The LSD-t was used to analyze thecomparison two groups. The student’s t-test was used to analyze measurementdata. The relation of two variables was analyzed by the Spearman correlationanalysis. α=0.05 is considered as significance. Results 1. Mi R-495 and MTA3 expression in six lung cancer cell lines(A549, Calu-3, H460,H1299, H1650 and SPC-A-1). Low expression of mi R-495 was observed inA549, Calu-3, H460, H1650 and SPC-A-1 cells, but was highly expressed inH1299 cells(P<0.05). MTA3 m RNA and protein expression were significantlyhigher in H1299 lung cancer cells compared with A549, Calu-3, H460, H1650and SPC-A-1 cells(P<0.05). 2. Mi R-495 expression was significantly up-regulated in mi R-495 group, whichillustrated it was satisfied to transfect mi R-495 agomir into calu-3 cells. 3. According to the results of the CCK8 and colony formation assay, theproliferation of calu-3 transfected with mi R-495 agomir was markedly decreasedwhen compared to the control groups(NC and Mock group)(P<0.05). 4. The experiment results of nude mice found that the significant inhibition in tumorgrowth and volume was observed in the mi R-495 group(P<0.05). 5. According to the results of the FCM assay, the percentage of calu-3 cells inG0/G1 phase increased visibly in mi R-495 group, compared with NC and Mockgroup, while the percentage in S phase was reduced(P<0.05). Western blotanalysis showed that cyclin A, cyclin D1 and phospho-Rb protein expression wasdecreased in calu-3 cells in mi R-495 group. 6. The results of transwell and wound healing assays found the invasion andmetastasis of calu-3 cells in mi R-495 group was significantly lower(P<0.05). 7. The results of bioinformatic analysis and luciferase reporter assays showedMTA3 is regulated by mi R-495. 8. The restore experiments found that MAT3 without 3’UTR of wasn’t regulated bymi R-495, and its function can offset the roles of mi R-495 on the biologicalbehavior of the NSCLC cell line calu-3. 9. The experiments about si RNA-MTA3 and mi R-495 up-regulation on calu-3 cellsindicated that mi R-495 can bind to MTA3 m RNA 3’ UTR and significantlydecreased MTA3 protein expression, which leaded to the inhibition ofproliferation, invasion and cell cycle. Conclusion 1. In NSCLC tissues, Agilent mi RNA microarray showed 56 differentially morethan triple expressed mi RNAs comparing to adjacent normal lung tissuesincluding 26 mi RNAs up-regulated and 30 mi RNAs down-regulated. Mi R-495was down-regulated in NSCLC tissues and associated with the occurrence oflymph node metastases, differentiation and TNM stage. Spearman correlationanalysis showed mi R-495 had an inverse correlation with MTA3. 2. In NSCLC cell line calu-3, up-regulation of mi R-495 inversely regulated MTA3expression by binding to MTA3 m RNA 3’ UTR, which can suppress cellproliferation, migration and cell cycle. Our findings suggest that mi R-495 mayplay an important role in regulating the carcinogenesis and development ofhuman NSCLC, and may be a potential therapeutic target in NSCLC. |
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