Expression Of MNAT1 In NSCLC And Its Effect On Cell Biological Behavior

Objective1.To investigate the expression level and significance of MNAT1 in non-small cell lung cancer(NSCLC)tissues.2.Observe the effect of MNAT1 on proliferation,cycle,apoptosis and migration of NSCLC cells.3.To detect the effect of MNAT1 on apoptosis and cell cycle related proteins in NSCLC cells and to explore the possible mechanism.Method1.Expression of MNAT1 in NSCLC tissues and NSCLC cells(1)bioinformatics database were used to analyze the expression differences of MNAT1 in NSCLC tissues and normal lung tissues.(2)48 cases of NSCLC tissue and matched normal tissue samples were collected in Nanfang Hospital in recent two years.The expression level of MNAT1 was detected by immunohistochemistry,and the relationship between MN AT1 expression and clinicopathological features of NSCLC was analyzed.(3)mRNA expression levels of MNAT1 in NSCLC cell lines spc-a-1,H322,A549,H446,H460 and H1299 were detected by rt-pcr,and protein expression levels of MNAT1 in NSCLC cell lines were detected by western bolt.2.Constructing and Confirming the MNAT1 low expression Cell Line and the MNAT1 overexpression Cell Line.(1)combined with rt-pcr and western blot results of NSCLC cell lines,we selectA549 and H322 cell lines with high MNAT1 expression to transfer siRNA and verify its effect.we selectH1299 with low MNAT1 expressionfor eukaryotic overexpression vector transfection to overexpress the target gene MNAT1,and verify its effect.(2)Construction of lentivirus vector stably interfered by MNAT1 and stable transfection of NSCLC cell lines A549 and H322.After transfection,the fluorescence rate of the cells was observed under fluorescence microscope,and the stable efficiency was verified by PCR fluorescence quantitative assay and western blot assay.3.Effect of MNAT1 on proliferation,cycle,apoptosis and migration of NSCLC cells.(1)MTT assay and plate cloning assay were used to detect the proliferation of NSCLC cells in vitro.(2)the established stable interfering cell lines A549-shMNAT1 and A549-shNC were injected into the subcutaneous of nude mice,and the subcutaneous tumorigenesis of nude mice was observed every day after two weeks,and the tumor size was detected with vernier calipers every three days.(3)flow cytometry was used to detect and analyze the effect of low expression and overexpression of MNAT1 on cell cycle.(4)flow cytometry was used to detect and analyze the effect of low MNAT1 expression and overexpression on apoptosis.(5)the migration ability of NSCLC cells was detected by scratch test and transwell test.4.Effect of MNAT1 on apoptosis and cell cycle related proteins in NSCLC cells and preliminary discussion.Western bolt assay was used to detect the effect of MNAT1 on apoptosis and cyclin-related proteins in NSCLC cells.The potential pathways were also discussed5.All the data obtained were statistically analyzed using SPSS20.0 software.Independent sample t test was used to compare the independent groups,which was expressed as mean standard deviation of 3 independent experiments.The expression of MNAT1 in NSCLC tissues and its clinicopathological parameters were evaluated by chi-square test.Result1.MNAT1 was up-regulated in NSCLC tissues,and was differentially expressed in NSCLC cell lines.(1)Bioinformatics database analysis showed that MNAT1 expression in NSCLC tissues was higher than that in normal lung tissues.The immunohistochemical results were consistent with the database results,and the expression level of MNAT1 in NSCLC tissues was higher than that in normal lung tissues(P<0.001).(2)Among the clinical parameters,the expression level of MNAT1 in NSCLC tissues was not correlated with patients' age,gender,smoking status,tumor pathological type,tumor size,single or multiple tumors,tumor site,EGFR mutation or no(P>0.05).However,the expression level of MNAT1 was related to the metastasis of NSCLC(P<0.05).2.MNAT1 low expression cell line and MNAT1 overexpression cell line were constructed and verified.(1)siRNA interfered with the construction and validation of cell lines.We successfully screened and selected the si-MNAT1#2 fragment with the highest interference efficiency,and constructed instantaneous transfected cell lines h322-si-MNAT1,h322-si-nc,a549-si-MNAT1 and a549-si-nc.WB verified that the interference efficiency was successful(p<0.001).(2)construction and validation of overexpressed plasmid vectors.MNAT1 overexpressed plasmid vector was purchased,and was verified by sequencing to have no sequence mutation or deletion,and was transfected into cell line H1299,rt-pcr and verified by western bolt.MNAT1 was successfully overexpressed in H1299 cell line.(3)construction of MNAT1 stable interference carrier.MNAT1 stable interfering lentivirus vector was purchased,and the target gene was successfully inserted by sequencing detection.Stable transfection of A549 and H322 NSCLC cell lines.The cell fluorescence rate,fluorescence quantitative PCR and western blot were observed to verify the efficiency after stabilization.Cell lines h322-shMNAT1,h322-shNC and a549-shMNAT1 and a549-shNC were successfully constructed,and the expression in the interference group was lower than that in the NC group(P<0.001).3.Effect of MNAT1 on proliferation,cycle,apoptosis and migration of NSCLC cells(1)the effect of MNAT1 on the proliferation of NSCLC cells in vitro MTT assay showed that the low expression of MNAT1 inhibited the cell growth rate of NSCLC(P<0.005).Overexpression of MNAT1 increased cell growth rate(P<0.05).The results of plate clonal formation experiment were consistent with the results of MTT experiment,and the number of NSCLC cell clonal formation in the experimental group was significantly reduced after MNAT1 was inhibited(P<0.01).After overexpression of MNAT1,the number of NSCLC cell clones in the experimental group increased significantly(P<0.01).(2)the effect of MNAT1 on the proliferation of NSCLC cells in vivo.After subcutaneous tumor formation in nude mice,tumor size was measured,tumor body was stripped,and immunohistochemistry was performed to detect the expression of ki-67 in tumor tissue.It was found that the tumor tissue size of the MNAT1 knockdown group was smaller than that of the control group in the tumor bodies formed by nude mice,and the difference was statistically significant(P<0.05).The expression of ki-67 in MNAT1 knockdown group was also decreased(P<0.05).(3)the effect of MNAT1 on the cycle of NSCLC cells.Flow cytology results showed that compared with the control group,the percentage of cells in G1 phase increased significantly after MNAT1 expression decreased(P<0.01),while the percentage of cells in S phase decreased(P<0.05).After overexpression of MNAT1,the percentage of G1 cells decreased(P<0.05)and the percentage of S cells increased(P<0.05),indicating that MNAT1 expression was involved in the process of NSCLC cell cycle.Low expression of MNAT1 can make cell cycle stagnate in G1 phase,and overexpression of MNAT1 can make cell cycle accelerate.(4)the effect of MNAT1 on apoptosis of NSCLC cells.Flow cytology results showed that compared with the control group,the percentage of early and late apoptotic cells increased after MNAT1 expression decreased(P<0.05).(5)the effect of MNAT1 on the migration ability of NSCLC cells.In the scratch experiment,the results showed thatcompared with the control group,the low expression of MNAT1 significantly decreased the cell migration ability(P<0.05),while the overexpression of MNAT1 enhanced the cell migration ability(P<0.05).In the Transwell experiment,the results showed that the low expression of MNAT1 significantly decreased the cell migration ability compared with the control group(P<0.001).However,overexpression of MNAT1 enhanced the migration ability of cells(P<0.05).4.To detect the effect of MNAT1 on apoptosis and cyclin-related proteins of NSCLC cells and explore the possible mechanismThe expression of MNAT1 increases the phosphorylation of Akt,inhibits the expression of cyclin-related proteins p21 and p27,increases the expression of cyclinD1,decreases the expression of apoptosis-related protein Bax,and increases the expression of cleavedcaspase-3.MNAT1 may affect the cycle and apoptosis-related proteins by affecting the phosphorylation of Akt,thus accelerating the process of cell cycle and inhibiting apoptosis.Conclusion1.MNAT1 overexpression in NSCLC tissues compare with normal lung tissues,and associate with lymph node metastasis.2.The expression of MNAT1 in NSCLC cells can promote its proliferation,accelerate the process of cell cycle,reduce apoptosis and promote cell migration.3.MNAT1 may affect the cycle and apoptosis-related proteins by affecting the phosphorylation of Akt,thus accelerating the process of cell cycle and inhibiting apoptosis.