| Background and Objective:Lung cancer is the most common malignant tumor in the world,and it is also the most dangerous malignant tumor to human health and life.The incidence and mortality of lung cancer in China are increasing year by year.In recent years,along with to encourage smoking cessation,screening of high-risk population and the implementation of measures to boost the process of diagnosis and treatment,a growing number of early lung cancer can be detected early,but there are still quite a number of patients with lung cancer diagnosis is advanced or even late,distant metastasis have occurred,studies have shown that the 5-year overall survival(OS)of advanced non-small cell lung cancer(NSCLC)was less than 20%.The invasion and metastasis of lung cancer are the sign and characteristic of malignant lung cancer,and they are also the direct cause of failure of treatment and death of patients.Therefore,to investigate the changes of signal transduction pathways related to invasion and metastasis of lung cancer will not only help to reveal the molecular mechanism of invasion and metastasis of lung cancer,but also provide new targets and pathways for blocking the signal transduction of invasion and metastasis of lung cancer and reversing the phenotype of invasion and metastasis of lung cancer.Invasion and metastasis of lung cancer are complex biological phenomenon,which involves multiple factors,multiple genes,multiple signal pathways and multiple stages.In recent years,with the development of molecular biology techniques and theories,a class of tumor metastasis suppressor genes has been discovered,which are related to tumor metastasis.The inactivation,mutation or abnormal expression of tumor metastasis suppressor genes lead to abnormal regulation of tumor metastasis phenotype,and eventually lead to tumor metastasis.In addition,recent studies have found that some miRNAs were also involved in the regulation of invasion and metastasis in lung cancer.However,up to now,the understanding of the molecular mechanism of lung cancer invasion and metastasis is still very limited.It is urgent to study the regulation process,signal regulation pathway and molecular mechanism of lung cancer invasion and metastasis.microRNAs(miRNAs)are a class of endogenous and noncoding single stranded small RNAs that widely exist in animal cells,plant cells and viruses.Their length are about 21-25 nucleotides.Since the first miRNA was discovered in 1993,more than 2500 miRNAs have been discovered,accounting for only about 1% of protein-coding genes,but they could regulate about 30% of protein-coding genes in humans and participated in a variety of biological processes,which were closely related to human cell proliferation,differentiation,apoptosis and the occurrence and development of diseases.miRNAs can bind to the 3’-untranslated region(UTR)binding sites of their target genes,and then bind to the RNA-induced silencing complex(RISC)to promote the degradation of messenger RNAs(m RNAs)or hinder their translation,thereby regulating the expression of their downstream genes in a positive or negative way,thus affecting the biological behavior of normal cells or tumor cells.In recent years,research results suggested that miRNAs played a crucial regulatory role in the occurrence and development of lung cancer.A large number of studies have confirmed that miRNAs played an important role in the occurrence,differentiation,proliferation,apoptosis,invasion and metastasis of lung cancer cells through various regulatory mechanisms.Our previous work found that miR-4778-5p was highly expressed in high metastatic potential lung cancer cell lines,which may be involved in the regulation of invasion and metastasis of lung cancer.However,so far,what role does miR-4778-5p play in the invasion and metastasis of lung cancer? Is it inhibition or promotion of lung cancer metastasis? What is the pathway of miR-4778-5p regulating invasion and metastasis of lung cancer? Through what signaling pathway is involved in the regulating invasion and metastasis of lung cancer? And the molecular mechanism of regulating the invasion and metastasis of lung cancer have not been reported at home and abroad.In order to explore and answer the above scientific questions,we studied the expression of miR-4778-5p between human bronchial epithelial cell line Beas-2B and lung cancer cell lines with different histological types and metastatic potentials.The effect of miR-4778-5p on the biological behavior of lung cancer cells in vitro was studied by overexpression or down-regulation of miR-4778-5p.The stable expression cell line of miR-4778-5p was established to study the effect of miR-4778-5p on tumorigenicity and metastasis of lung cancer xenografts in nude mice.The downstream target genes of miR-4778-5p and their effects on downstream target genes were predicted by Targestscan,Miranda,Pictar software and luciferase double reporter genes.RT-PCR and Western blot were used to detect the expression of GSK-3β m RNA and GSK-3βprotein in normal human bronchial epithelial cell line Beas-2B and lung cancer cell lines with different histological types and metastatic potentials.To investigate whether miR-4778-5p affects the invasion and metastasis of lung cancer cells by regulating the expression of GSK-3β gene,and to preliminarily explore its mechanism in invasion and metastasis of lung cancer cells.To investigate the effects of GSK-3β gene supplementation on the biological behavior changes of lung cancer cells induced by miR-4778-5p overexpression and its molecular mechanisms.Materials and Methods:1.RT-PCR was used to verify the expression of miR-4778-5p in human normal bronchial epithelial cell line Beas-2B and different lung cancer cell lines(human lung adenocarcinoma cell line A549,human neuroendocrine cancer cell line NCI-H1299,human large cell lung cancer cell line NCI-H460,human low metastatic potential large cell lung cancer cell line NL9980,human high metastatic potential large cell lung cancer cell line L9981,human low metastatic potential giant cell lung cancer cell line 95 C,human high metastatic potential giant cell lung cancer cell line 95 D,human lung squamous cell line YTMLC-90 and human lung adenocarcinoma cancer cell line NCI-H1975.2.The transient transfection was used to construct miR-4778-5p overexpressed and miR-4778-5p down-regulated human lung cancer cell lines with different metastatic potential,including human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics transfected with miR-4778-5p-mimics,human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor,human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,human low metastatic potential giant cell lung cancer cell line95C-miR-4778-5p-mimics transfected with miR-4778-5p-mimics,human low metastatic potential giant cell lung cancer cell line 95C-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,human high metastatic potential giant cell lung cancer cell line 95D-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor and human high metastatic potential giant cell lung cancer cell line 95D-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector.3.The RT-PCR was used to detect the expression levels and differences of miR-4778-5p in above 8 cell lines.4.The CCK-8 test,wound healing test and transwell test were used to detect the ability of proliferation,migration and invasion of the above 8 lung cancer cell lines.5.The gene stable transfection technique was used to construct the human low metastasis potential large cell lung cancer cell line NL9980-luc-lv-miR-4778-5p stably transfected with miR-4778-5p and the human low metastasis potential large cell lung cancer cell line NL9980-luc-lv-con stably transfected with miR-4778-5p empty vector.6.The lung cancer xenograft models were established in nude mice with the human low metastatic large cell lung cancer cell line NL9980-luc-lv-miR-4778-5p by stably transfected with miR-4778-5p and the human low metastatic large cell lung cancer cell line NL9980-luc-lv-con stably transfected with miR-4778-5p empty vector.In vivo imaging was used to detect the tumorigenicity,distant metastasis and differences of NL9980-luc-lv-miR-4778-5p and NL9980-luc-lv-con in nude mice.7.Targest Scan,Miranda and Pic Tar software were used to predict the downstream target genes of miR-4778-5p and the influencing mechanism of downstream effects of miR-4778-5p on the target genes.8.Double luciferase reporter gene detection was used to verify whether GSK-3β was regulated by miR-4778-5p.9.RT-PCR was used detected the expressive level and difference of miR-4778-5p among the human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics transfected with miR-4778-5p-mimics,human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor,human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,human low metastatic potential giant cell lung cancer cell line95C-miR-4778-5p-mimics transfected with miR-4778-5p-mimics,human low metastatic potential giant cell lung cancer cell line 95C-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,human high metastatic potential giant cell lung cancer cell line 95D-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor,and human high metastatic potential giant cell lung cancer cell line 95D-miR-4778-5p-inhibitor-con transfected with 95D-miR-4778-5p empty vector.The effects of miR-4778-5p on m RNA and protein expression levels of downstream target gene GSK-3β were detected by RT-PCR and western blot.10.The 3.1-GSK-3β and 3.1-con plasmids were amplified by gene amplification.The human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics transfected with miR-4778-5p-mimics,the human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-3.1-GSK-3β transfected with miR-4778-5p-mimics and 3.1-GSK-3β,the human low metastatic potential giant cell lung cancer cell line95C-miR-4778-5p-mimics transfected with miR-4778-5p-mimics,the human low metastatic potential giant cell lung cancer cell line 95C-miR-4778-5p-mimics transfected with the miR-4778-5p-mimics empty vector,and the human low metastatic potential giant cell lung cancer cell line 95C-miR-4778-5p-mimics-3.1-GSK-3β transfected with miR-4778-5p-mimics-3.1-GSK-3β were constructed by transient transfection technique.11.Western blot was used to detect the expression level and differences of GSK-3β among the above 6 lung cancer cell lines.12.The CCK-8 test,wound healing test and transwell test were used to detect the ability and differences of proliferation,migration and invasion among the above6 lung cancer cell lines.Results:1.The expression levels of miR-4778-5p among normal bronchial epithelial cell line Beas-2B,human lung adenocarcinoma cell line A549,human neuroendocrine lung cancer cell line NCI-H1299,human low metastatic potential large cell lung cancer cell line NL9980,human high metastatic potential large cell lung cancer cell line L9981,human low metastatic potential giant cell lung cancer cell line 95 C,human high metastatic potential giant cell lung cancer cell line 95 D,human lung squamous cell line YTMLC-90 and human lung adenocarcinoma cell line NCI-H1975 were significantly different by F test(P=0.000).Pairwise comparison showed that the expression level of miR-4778-5p in the normal bronchial epithelial cell line Beas-2B was significantly lower than that in A549,NCI-H1299,NCI-H460,NL9980,L9981,95 C,95D,YTMLC-90 and NCI-H1975cells(P=0.000).The expression level of miR-4778-5p in A549 cells was significantly higher than that in NCI-H1299,NCI-H460,NL9980,95 C and YTMLC-90 cells(P=0.000-0.001),the expression level of miR-4778-5p in A549 cells was significantly lower than that in 95 D and NCI-H1975 cells,and there was no significant difference of expression level of miR-4778-5p between A549 and L9981 cells(P=0.322).The expression level of miR-4778-5p in NCI-H1299 cells was significantly higher than that in NCI-H460,NL9980,95 C and YTMLC-90 cells(P=0.000),the expression level of miR-4778-5p in NCI-H1299 cells was significantly lower than that in L9981,95 D and NCI-H1975 cells.The expression level of miR-4778-5p in NCI-H460 cells was significantly lower than that in L9981,95 D and NCI-H1975 cells(P=0.000),the expression level of miR-4778-5p in NCI-H460 cells was significantly higher than that in 95 C and YTMLC-90 cells(P=0.040-0.045),and there was no significant difference of expression level of miR-4778-5p between NCI-H460 and NL9980 cells(P=0.747).The expression level of miR-4778-5p in NL9980 cells was significantly lower than that in L9981,95 D and NCI-H1975 cells(P=0.000),the expression level of miR-4778-5p in NL9980 cells was significantly higher than that in 95 C and YTMLC-90 cells(P=0.023-0.026).The expression level of miR-4778-5p in L9981 cells was significantly higher than that in 95 C and YTMLC-90 cells(P=0.000),the expression level of miR-4778-5p in L9981 cells was significantly lower than that in 95 D and NCI-H1975 cells(P=0.000).The expression level of miR-4778-5p in 95 C cells was significantly lower than that in 95 D and NCI-H1975 cells(P=0.000),and there was no significant difference of expression level of miR-4778-5p between 95 C and YTMLC-90 cells(P=0.942).The expression level of miR-4778-5p in 95 D cells was significantly higher than that in YTMLC-90 and NCI-H1975 cells(P=0.000).The expression level of miR-4778-5p in YTMLC-90 cells was significantly lower than that in NCI-H1975 cells(P=0.000).2.Compared with the human low metastatic potential large cell lung cancer cell line NL9980,the expression level of miR-4778-5p in human high metastatic potential large cell lung cancer cell line L9981 was increased significantly(P<0.001).Compared with the human low metastatic potential giant cell lung cancer cell line 95 C,the expression level of miR-4778-5p in human high metastatic potential giant cell lung cancer cell line 95 D was increased significantly(P<0.01).3.Compared with the human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the expression of miR-4778-5p in human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was increased significantly(P<0.01).Compared with the human low metastatic potential giant cell lung cancer cell line95C-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the expression of miR-4778-5p in human low metastatic potential giant cell lung cancer cell line 95C-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was increased significantly(P<0.001).4.Compared with the human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the expression of miR-4778-5p in human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor was decreased significantly(P<0.001).Compared with the human high metastatic potential giant cell lung cancer cell line95D-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the expression of miR-4778-5p in human high metastatic potential giant cell lung cancer cell line 95D-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor was decreased significantly(P<0.05).5.Compared with the human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the proliferation ability in human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was enhanced significantly(P<0.05).Compared with the human low metastatic potential giant cell lung cancer cell line95C-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the proliferation ability in human low metastatic potential giant cell lung cancer cell line 95C-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was enhanced significantly(P<0.001).6.Compared with the human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the migration ability in human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was enhanced significantly(P<0.001).Compared with the human low metastatic potential giant cell lung cancer cell line95C-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the migration ability in human low metastatic potential giant cell lung cancer cell line 95C-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was enhanced significantly(P<0.001).7.Compared with the human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the invasive ability in human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was enhanced significantly(P<0.001).Compared with the human low metastatic potential giant cell lung cancer cell line95C-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the invasive ability in human low metastatic potential giant cell lung cancer cell line95C-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was enhanced significantly(P<0.01).8.Compared with the human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the proliferation ability in human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor was weaken significantly(P<0.05).Compared with the human high metastatic potential giant cell lung cancer cell line95D-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the proliferation ability in human high metastatic potential giant cell lung cancer cell line 95D-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor was weaken significantly(P<0.001).9.Compared with the human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the migration ability in human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor was weaken significantly(P<0.01).Compared with the human high metastatic potential giant cell lung cancer cell line95D-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the migration ability in human high metastatic potential giant cell lung cancer cell line 95D-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor were weaken significantly(P<0.01).10.Compared with the human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the invasive ability in human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor was weaken significantly(P<0.001).Compared with the human high metastatic potential giant cell lung cancer cell line95D-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the invasive ability in human high metastatic potential giant cell lung cancer cell line 95D-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor was weaken significantly(P<0.01).11.The RT-PCR result showed that the expression level of miR-4778-5p in NL9980-luc-lv-miR-4778-5p cells stable overexpressed with miR-4778-5p was significantly higher than that in NL9980-luc-lv-con cells stable overexpressed with LV-con(P<0.001).12.In the xenograft tumor model of lung cancer,a small subcutaneous xenograft tumor was observed in the right inguinal region of nude mice at the 3rd week.During the period of 4-8 weeks,the xenograft tumor in nude mice showed logarithmic growth,and the volume of xenograft in the human low metastasis potential large cell lung cancer cell line NL9980-luc-lv-miR-4778-5p stably overexpressed miR-4778-5p was significantly greater than that in the human low metastasis potential large cell lung cancer cell line NL9980-luc-lv-con stably overexpressed miR-4778-5p empty vector(P<0.001).13.The NL9980-luc-lv-miR-4778-5p cells stable overexpressed with miR-4778-5p resulted in obvious heterotopic metastasis in the lungs of nude mices.The fluorescence intensity of lung tissue in NL9980-luc-lv-miR-4778-5p group was significantly stronger than that in NL9980-luc-lv-con group.The number of lung metastasis in NL9980-luc-lv-miR-4778-5p group was significantly more than that in NL9980-luc-lv-con group(P<0.001).14.The weight of transplanted tumors of NL9980-luc-lv-miR-4778-5p group was significantly heavier than that of NL9980-luc-lv-con group(P<0.001).15.The results of dual luciferase reporter gene assay showed that the luciferase activity in p IR-GSK-3β-miR-4778-5p-mimic group was significantly decreased due to the overexpression of miR-4778-5p in human low metastasis potential large cell lung cancer cell line NL9980 and human low metastasis potential giant cell lung cancer cell line 95 C transfected with miR-4778-5p(P<0.05,P<0.05,respectively).16.The expression level of GSK-3β m RNA among normal human bronchial epithelial cell line Beas-2B and A549,NCI-H1299,NCI-H460,NL9980,L9981,95 C,95D,YTMLC-90 and NCI-H1975 cell lines were significant difference by F test(P=0.000).The results of pairwise comparison showed that the expression level of GSK-3β m RNA in Beas-2B cells was significantly higher than in A549,NCI-H1299,NCI-H460,NL9980,L9981,95 C,95D,NCI-H1975 and YTMLC-90cells(P=0.000-0.020).The expression level of GSK-3β m RNA in A549 cells was significantly higher than in NCI-H1299,NCI-H460,L9981 and 95 D cells(P=0.000),there was no significant difference of expression level of GSK-3β m RNA between A549 and NL9980,95 C,YTMLC-90 and NCI-H1975 cells(P=0.158-0.707).The expression level of GSK-3β m RNA in NCI-H1299 cells was significantly lower than in NL9980,L9981,95 C,95D,YTMLC-90 and NCI-H1975 cells(P=0.000),there was no significant difference of expression level of GSK-3β m RNA between NCI-H1299 cells and NCI-H460 cells(P=0.502).The expression level of GSK-3βm RNA in NCI-H460 cells was significantly lower than in NL9980,L9981,95 C,95D,YTMLC-90 and NCI-H1975 cells(P=0.000).The expression level of GSK-3βm RNA in NL9980 cells was significantly higher than in L9981 and 95 D cells(P=0.000),there was no significant difference of expression level of GSK-3β m RNA between NL9980 cells and 95 C,YTMLC-90 and NCI-H1975 cells(P=0.311-0.963).The expression level of GSK-3β m RNA in L9981 cells was significantly lower than in 95 C,YTMLC-90 and NCI-H1975 cells(P=0.000),the expression level of GSK-3β m RNA in L9981 cells was significantly higher than in 95 D cells(P=0.002).The expression level of GSK-3β m RNA in 95 C cells was significantly higher than in95 D cells(P=0.000),there was no significant difference of expression level of GSK-3β m RNA between 95 C cells and YTMLC-90,NCI-H1975 cells(P=0.350-0.897).The expression level of GSK-3β m RNA in 95 D cells was significantly lower than in YTMLC-90 and NCI-H1975 cells(P=0.000).There was no significant difference of expression level of GSK-3β m RNA between YTMLC-90 and NCI-H1975 cells(P=0.290).17.The expression level of GSK-3β protein among human normal bronchial epithelial cell line Beas-2B cells,A549,NCI-H1299,NCI-H460,NL9980,L9981,95 C,95D,NCI-H1975 and YTMLC-90 cells were highly significant different by F test(P=0.000).The results of pairwise comparison showed that the expression level of GSK-3β protein in Beas-2B cells was highly significant higher than in A549,NCI-H1299,NCI-H460,NL9980,L9981,95 C,95D,YTMLC-90 and NCI-H1975cells(P=0.000).The expression level of GSK-3β protein in A549 cells was significantly lower than in NCI-H1299 and NL9980 cells(P=0.000-0.011),the expression level of GSK-3β protein in A549 cells was significantly higher than in L9981,95 C and 95 D cells(P=0.000),there was no significant difference of the GSK-3β expression between NCI-H460,YTMLC-90 and NCI-H1975 cells(P=0.080-0.148).The expression level of GSK-3β protein in NCI-H1299 cells was significantly higher than in NCI-H460,L9981,95 C,95D,YTMLC-90 and NCI-H1975 cells(P=0.000),the expression level of GSK-3β protein in NCI-H1299 cells was significantly lower than in NL9980 cells(P=0.025).The expression level of GSK-3β protein in NCI-H460 cells was significantly lower than in NL9980 cells(P=0.005),the expression level of GSK-3β protein in NCI-H460 cells was significantly higher than in L9981,95 C and 95 D cells(P=0.000-0.006),there was no significant difference of the GSK-3β expression between NCI-H460 and YTMLC-90 and NCI-H1975 cells(P=0.738-0.850).The expression level of GSK-3βprotein in NL9980 cells was significantly higher than in L9981,95 C,95D,YTMLC-90 and NCI-H1975 cells(P=0.000).The expression level of GSK-3βprotein in L9981 cells was significantly lower than in YTMLC-90 and NCI-H1975cells(P=0.007-0.010),there was no significant difference of the GSK-3β expression between L9981 cells and 95 C and 95 D cells(P=0.068-0.903).The expression level of GSK-3β protein in 95 C cells was significantly higher than in 95 D cells(P=0.033),the expression level of GSK-3β protein in 95 C cells was significantly lower than in YTMLC-90 and NCI-H1975 cells(P=0.010-0.013).The expression level of GSK-3βprotein in 95 D cells was significantly lower than in YTMLC-90 and NCI-H1975cells(P=0.000).There was no significant difference of the GSK-3β expression between YTMLC-90 and NCI-H1975 cells(P=0.884).18.Compared with the human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the expression level of GSK-3β m RNA in human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was downregulated significantly(P<0.001).Compared with the human low metastatic potential giant cell lung cancer cell line95C-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the expression level of GSK-3β m RNA in human low metastatic potential giant cell lung cancer cell line 95C-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was downregulated significantly(P<0.05).19.Compared with the human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the expression level of GSK-3β m RNA in human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor was upregulated significantly(P<0.001).Compared with the human high metastatic potential giant cell lung cancer cell line95D-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the expression level of GSK-3β m RNA in human high metastatic potential giant cell lung cancer cell line 95D-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor was upregulated significantly(P<0.01).20.Compared with the human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the expression level of GSK-3β protein in human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was downregulated significantly(P<0.001),and the expression level of β-catenin was upregulated significantly(P<0.001).Compared with the human low metastatic potential giant cell lung cancer cell line95C-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the expression level of GSK-3β protein in human low metastatic potential giant cell lung cancer cell line 95C-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was downregulated significantly(P<0.001),and the expression level of β-catenin was upregulated significantly(P<0.001).21.Compared with the human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the expression level of GSK-3β protein in human high metastatic potential large cell lung cancer cell line L9981-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor was upnregulated significantly(P<0.001),and the expression level of β-catenin was downregulated significantly(P<0.001).Compared with the human high metastatic potential giant cell lung cancer cell line95D-miR-4778-5p-inhibitor-con transfected with miR-4778-5p-inhibitor empty vector,the expression level of GSK-3β protein in human high metastatic potential giant cell lung cancer cell line 95D-miR-4778-5p-inhibitor transfected with miR-4778-5p-inhibitor was upnregulated significantly(P<0.01),and the expression level of β-catenin was downregulated significantly(P<0.01).22.Compared with NL9980-miR-4778-5p-mimics-con cells,the expression level of GSK-3β protein in NL9980-miR-4778-5p-mimics cells was downregulated significantly(P<0.001),and the expression level of β-catenin was upregulated significantly(P<0.001).After overexpressing GSK-3β gene,compared with NL9980-miR-4778-5p-mimics cells,the expression level of GSK-3β protein in NL9980-miR-4778-5p-mimics-3.1-GSK-3β cells was upregulated significantly(P<0.05),and the expression level of β-catenin was downregulated significantly(P<0.01).23.Compared with 95C-miR-4778-5p-mimics-con cells,the expression level of GSK-3β protein in 95C-miR-4778-5p-mimics cells was downregulated significantly(P<0.001),and the expression level of β-catenin was upregulated significantly(P<0.01).After overexpressing GSK-3β gene,compared with95C-miR-4778-5p-mimics cells,the expression level of GSK-3β protein in95C-miR-4778-5p-mimics-3.1-GSK-3β cells was upregulated significantly(P<0.001),and the expression level of β-catenin was downregulated significantly(P<0.01).24.Compared with the human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the proliferation ability in human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was enhanced significantly(P<0.001).After overexpressing GSK-3β gene,compared with NL9980-miR-4778-5p-mimics cells,the proliferation ability in NL9980-miR-4778-5p-mimics-3.1-GSK-3β cells transfected with miR-4778-5p-mimics and 3.1-GSK-3β was significantly weakened(P<0.001).25.Compared with the human low metastatic potential giant cell lung cancer cell line 95C-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the proliferation ability in human low metastatic potential giant cell lung cancer cell line 95C-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was enhanced significantly(P<0.001).After overexpressing GSK-3β gene,compared with 95C-miR-4778-5p-mimics cells,the proliferation ability in 95C-miR-4778-5p-mimics-3.1-GSK-3β cells transfected with miR-4778-5p-mimics and 3.1-GSK-3β was significantly weakened(P<0.001).26.Compared with the human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics-con transfected with miR-4778-5p-mimics empty vector,the migration ability in human low metastatic potential large cell lung cancer cell line NL9980-miR-4778-5p-mimics transfected with miR-4778-5p-mimics was enhanced significantly(P<0.01).After overexpressing GSK-3β gene,compared with NL9980-miR-4778-5p-mimics cells,the mi... |
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