The Effect Of MiR-326 On Proliferation,Migration And Apoptosis Of Non-small Cell Lung Cancer And Its Regulation Of CCND1 Signaling Pathway

Non-small-cell lung cancer(NSCLC)is one of the most common human cancers,and the mortality rate ranks first among tumors for male.non-small cell lung cancer(NSCLC)accounts for about 85%of lung cancer cases.When clinical symptoms appear,it has developed into the middle or late stage.The traditional treatment methods are not ideal,because the survival time is limited,and the cost is high.Early lung cancer often manifests as asymptomatic pulmonary nodules.Even X-ray chest radiography is difficult to accurately diagnose,and tumors can be found only when the lesion is large.Low-dose CT can significantly improve the detection rate of early lung cancer,but it is not suitable for routine physical examination.At present,there is no specific molecular biomarker for lung cancer diagnosis,so most patients with lung cancer have reached the advanced stage at the time of treatment.Early detection and early treatment of lung cancer can significantly improve the survival rate and quality of life of patients.The data show that the 10-year survival rate of stage I lung cancer patients is 88%,and the 5-year survival rate of stage III/IV lung cancer patients is only about 15%.It can be seen that early screening of the NSCLC population is necessary.Through the research on lung cancer genes and their mechanism of action,the implementation of precise molecular targeted therapy has become an important research area.Therefore,by exploring the mechanism of NSCLC development and development at the molecular level,searching for molecules with higher specificity and sensitivity as diagnostic markers or potential therapeutic targets will provide a new strategy for early diagnosis and treatment of NSCLC,which has great potential public health significance.MiRNAs are a class of evolutionarily conserved short-chain RNAs,containing about 22 nucleotides,which work in the way that induce mRNAs degradation or inhibit the translation of mRNAs by binding to the mRNAs 3'-UTR of the target site,resulting in a decrease in the protein levels of the target genes.MiRNAs involve in a variety of biological processes,which include cell proliferation,differentiation,invasion,migration and disease prognosis.MiRNAs play important roles in different types of cancer.A single miRNA can target multiple mRNAs simultaneously,while a single mRNA can also be regulated by multiple miRNAs at the same time.Because of the large number of miRNAs present in the cell,the style of two-way regulation forms a complex intracellular network that participates in many physiological processes and diseases in the body.In recent years,it has been found that many miRNAs are deregulated in NSCLC by studying the miRNAs expression profiles in NSCLC tissue.Recent studies have shown that these specific miRNAs are closely related to the pathogenesis,metastasis,recurrence and prognosis of NSCLC,and may serve as potential biomarkers for the diagnosis and treatment of tumors.Great progress has been made in the study of the mechanism of action of miRNAs in NSCLC.Studies have shown that many miRNAs have important promoting or inhibiting effects on the development of NSCLC.Based on the specific expression of miR-326 in NSCLC tissues,our study mainly researches its biological function in NSCLC both in vitro and in vivo and explore its impact on the development of NSCLC,in order to provide reliable theoretical basis for lung cancer diagnosis and therapy.Part I The expression of miR-326 in lung cancer tissues and NSCLC cell linesObjective:To verify the differential expression of miR-326 in miRNA high-throughput sequencing data of lung cancer tissues,and to detect miR-326 in human non-small cell lung cancer(NSCLC)cancer tissues and adjacent to cancer by RT-PCR Differences in expression in tissues and verification of miR-326 expression in normal human embryonic lung fibroblast cell line HELF and human NSCLC cell lines A549,SPC-A-1,H1299,SK-MES-1 and 95-D.Methods:miR-326 was a miRNA with significant differential expression from the high-throughput sequencing data of miRNAs in this study.High-throughput sequencing showed low expression in lung cancer tissues.Using qRT-PCR technology,we measured the expression of miR-326 in 39 lung cancer and paracancerous tissues from the Department of Oncology,Central South Hospital,Wuhan University.The expression of miR-326 in human embryonic lung fibroblast cell line HELF and human NSCLC cell lines A549,SPC-A-1,H1299,SK-MES-1 and 95-D was also verified.Result:By comparing the expression of miR-326 in lung cancer and adjacent tissues,the results showed that the expression of miR-326 in lung cancer tissues was significantly different from that in adjacent tissues,and the difference was statistically significant(p<0.05).The expression of miR-326 in lung cancer was significantly lower than that in adjacent tissues.At the same time,in vitro cell experiments showed that the expression of miR-326 in normal lung cell lines was significantly higher than that in lung cancer cells,consistent with the expression trend in tissues,and consistent with high-throughput sequencing results.Conclusion:miR-326 is lowly expressed in non-small cell lung cancer tissues and lung cancer cell lines.Part II The function of miR-326 in human lung cancer in vitro and in vivoObjective:To clarify the biological function of miR-326 in human NSCLC cells and its effect on tumorigenesis of NSCLC cells in nude mice.Methods:The synthetic miR-326 mimics and inhibitors and their negative controls were used to transiently transfect human NSCLC cell lines A549 and SPC-A-1 to explore the potential biological functions of miR-326 in NSCLC cells.The effects of miR-326 on the proliferation of NSCLC cells were detected by CCK-8,BrdU and plate cloning.The effect of miR-326 on the apoptosis of NSCLC cells was detected by flow cytometry.The migration of NSCLC cells was observed by scratch and Transwell experiments.And changes in the ability to invade.The effect of miR-326 on the tumorigenic ability of NSCLC cells in vivo was examined by nude mice tumor formation assay.Results:In the cell function test,CCK-8 assay showed that the OD value of the miR-326 overexpression group was significantly lower than that of the control group at 24h,48h,72h and 96h,and the OD value of the miR-326 group was significantly increased.High;BrdU results show that miR-326 overexpression can significantly reduce the proportion of red blood cells,reduce the DNA synthesis of NSCLC cells,that is,inhibit cell proliferation.The inhibition of miR-326 can increase the proportion of red blood cells,suggesting that the synthesis of DNA is increased and the proliferation of cells is enhanced.The apoptosis results detected by flow cytometry show that miR-326 overexpression can significantly increase the early stage of lung cancer cells.The proportion of dead cells,and interference with miR-326 can significantly reduce this ratio;the results of scratch test showed that miR-326 overexpression can significantly inhibit the migration ability of A549 and SPC-A-1 in NSCLC cells,while inhibiting the expression of miR-326.Significantly enhance the migration ability of the two NSCLC cells;Transwell migration and invasion assay results show that inhibition of miR-326 expression can significantly enhance cell migration and invasion,and miR-326 overexpression can significantly reduce the migration and invasion of NSCLC cells.Tumor formation in nude mice showed that miR-326 overexpression significantly reduced the volume of tumor tissue in vivo and decreased its weight,indicating that miR-326 can inhibit the tumorigenic ability of subcutaneous A549 cells in nude mice.Therefore,miR-326 is in vivo.It also significantly inhibited the proliferation of lung cancer cells.Conclusion:miR-326 can significantly inhibit the proliferation,invasion and migration of A549 and SPC-A-1 in NSCLC cells and induce apoptosis,and it also has a significant inhibitory effect on the proliferation of NSCLC in vivo.Therefore,miR-326 is a miRNA that inhibits the development and progression of cancer.Part III The miR-326 targets to regulate the expression of CCND1 in NSCLC to play a role in tumor suppressionObjective:To investigate the effect of miR-326 on the regulation of CCND1 in lung cancer cells and its effect on the proliferation of lung cancer cells.Methods:Bioinformatics was used to predict and validate the target CCND1 of miR-326,and the possibility of binding of miR-326 to CCND1 was verified by the luciferase reporter gene.Using synthetic miR-326 mimics and inhibitors to alter the levels of miR-326 and CCND1 in lung cancer cell lines,qPCR and Western blot were used to verify that miR-326 exerts a tumor suppressor effect by inhibiting CCND1 expression.In addition,we used invasion,migration and colony formation assays to examine the effects of CCND1 on proliferation and metastasis of lung cancer cells.We then examined the effects of miR-326 on the expression of casepase-3,casepase-7,cyclin D2,p21,p57,MMP7,MMP9,and cleaved casepase-3 in tumor biological behavior-related proteins.Results:miR-326 was able to target the 3'-UTR region of CCND1.Overexpression of miR-326 significantly inhibited the expression of CCND1 in cells,resulting in decreased expression of Cyclin D1 protein.In addition,miR-326 can affect the expression of casepase-3,casepase-7,cyclin D2,p21,p57,MMP7,MMP9 and cleaved casepase-3 in lung cancer cells.Conclusion:miR-326 can affect the proliferation,apoptosis,invasion and metastasis of lung cancer cells by regulating CCND1 signaling pathway.