MiR-100-3p Suppresses Lung Cancer Cell Proliferation And Inhibits The G1/S Transition By Targeting CDC25A

[Background]Lung cancer,also known as bronchial lung cancer,is one of the most common malignancies and is the highest mortality rate of malignant tumors all over the word.About 50% of lung cancer patients have been in the progress stage and the prognosis is poor.Therefore,the studies of molecular mechanisms of lung cancer have been the focus of basic and clinical researchers.Many studies have shown that the development of lung cancer is associated with genetic or epigenetic changes that lead to abnormal gene expression profiles.Recently,the role of micro RNA(miRNA)in the development of lung cancer has attracted the attention of researchers.miRNA consists of about 22 nucleotides(nt)and regulates the expression of the gene by pairing with the complementary nucleotide sequence in the 3'-noncoding region(3'-UTR)of the target mRNA after transcription.At present,more than 2042 mature miRNAs have been identified in humans.These miRNAs constitute a network that can regulate the expression of more than 30% of the cellular protein.So far,studies have shown that miRNAs involve a wide range of cellular processes,including development,cell proliferation,differentiation and apoptosis.Approximately 50% of the miRNA genes are located in fragile and genomic regions,which are frequently amplified or deleted in cancer.Therefore,abnormal expression of miRNA plays an important role in the development and progression of lung cancer.Our preliminary experiments have shown that miR-100-3p is low-expressed in lung cancer and plays an important biological role in the malignant progression of lung cancer cells.At the same time,we found a significant change in cell cycle after altering the expression of miR-100-3p in lung cancer cell lines,which aroused our concern.Cell cycle is an important physiological process of cells,which is a necessary process of cell proliferation and differentiation.And the disorder of proliferation is an important manifestation of cell canceration.Numerous regulatory factors and exact signaling pathways are involved in the regulation of cell cycle.CDC25 A is a cell cycle control protein,and its overexpression is associated with the occurrence of many tumors,which has attracted the attention of many reaserchers.It has been reported that CDC25 A is highly expressed in a variety of cancer cell lines or tumor tissues including lung cancer and is associated with tumorigenesis and poor prognosis.Recently,it has been found that miRNAs such as miR-let7,miR-21 and miR-449 a can suppress cell proliferation or induce apoptosis by negatively regulating CDC25 A in cancer cells.Based on above,we questioned whether miR-100-3p could affect the proliferation of lung cancer cells by regulating cell cycle? Does CDC25 A have participated in this process?In this study,we further discussed the role of miR-100-3p in the regulation of cell cycle and proliferation of lung cancer cells and explored the possible mechanism by determining the directly target gene of miR-100-3p.We hoped to provide more molecular basis and therapeutic targets for the development of lung cancer.[Methods] 1.In the previous experiment,we successfully established A549 cell lines stably expressing miR-100-3p inhibitor & negative control and L18 cell lines stably expressing miR-100-3p mimic & negative control.The cell cycle changes of the lung cancer cell lines were detected by flow cytometry(FCM).2.MTT assay,plate cloning and tumor formation in nude mice were used to detect the effect of miR-100-3p on proliferation of the A549 and L18 cell lines in vitro and in vivo.3.Biological predictive software such as Targetscan and Miranda predicted that miR-100-3p and CDC25 A mRNA3'UTR regions were complementary.Next,the double luciferase reporter gene assay was used to identify the relationship between miR-100-3p and CDC25 A mRNA3'UTR regions.4.Immunohistochemistry(IHC)was used to detect the expression of CDC25 A in cancer tissues and corresponding adjacent tissues of lung cancer patients.5.The expression of CDC25 A mRNA were detected by RT-PCR as well as the CDC25 A and downstream cycle-related protein were detected by Western blot.6.Construction of CDC25 A interference plasmid and overexpression plasmid.We interfered with the expression of CDC25 A in the A549 cells which had been inhibited miR-100-3p and raised the expression of CDC25 A in L18 cells which had been overexpressed miR-100-3p.Then we detected cell cycle,cell proliferation and the expression of CDC25 A and its downstream cycle-related proteins.[Result]1.The effect of miR-100-3p on the cell cycle of lung cancer cells: Flow cytometry showed that in A549 cells,which had been inhibited miR-100-3p,the percentage of G1 phase was significantly lower and the proportion of S phase was significantly increased than in the control group.The result showed that the cell cycle was promoted from G1 phase to S phase.In L18 cells,which had been overexpressed miR-100-3p,the proportion of G1 was significantly higher and the proportion of S phase were significantly decreased than in the control group.G1 phase arrest might occurred in the experiment group.2.The effect of miR-100-3p on the proliferation of lung cancer cells: the proliferation ability and clonogenic ability of A549 cells transfected with miR-100-3p inhibitor were increased,and the tumor growth rate were also raised in tumourigenicity assay using a tumor xenograft model compared with control group(P <0.001).The proliferation rate and clonogenic ability of L18 cells transfected with miR-100-3p mimic were significantly lower,and the tomur growth rate were significantly lower than the control group.3.Bioinformatics analysis revealed that the CDC25 A mRNA3'UTR region 28-32 had a7-base seed sequence that was fully complementary to miR-100-3p;constructed CDC25 A @ psi-CHECK2-Report-Luc plasmids and miR-100-3p mimic/inhibitor was transferred into 293 T cells.The fluorescence intensity was significantly enhanced compared with the control group,proving that the mRNA which code CDC25 A was a target gene of miR-100-3p.4.Immunohistochemical results showed that the expression of CDC25 A in cancer tissues of lung cancer patients with low expression of miR-100-3p was significantly higher than that in the paracancerous tissues.5.Along with inhibiting or overexpressing miR-100-3p level in the lung cancer cells,CDC25 A and its downstream-related protein expression performed increased or decreased correspondingly.6.Transfection of CDC25 A interference plasmids or overexpression plasmids can partially neutralize or downregulate miR-100-3p induced cell cycle,cell proliferation,and changes in CDC25A-related cyclin events.[Conclusion]1.miR-100-3p negatively regulates the cell cycle and inhibits the growth of lung cancer cells.2.miR-100-3p inhibits the G1/S transition by targeting CDC25 A in lung cancer cells.