Effects Of SMP30 Transcripts On Biological Behavior Of Hepatoma Cells And The Research On Related Mechanisms

BACKGROUND: There are three alternative spliceosomes in senescence marker protein 30(SMP30),including the full-length(900 bp),exon 4-del eted transcript(684 bp),and exon 4,5 deleted transcripts(552bp).The 900 bp transcript expresses the protein SMP30-a,the 684 bp transcript expresse s the protein SMP30-b,and the 552 bp transcript expresses the protein SM P30-c.SMP30-a has low expression in hepatocarcinoma tissues and inhibit s the proliferation and invasion of hepatoma cells.There are few studies on SMP30-b and SMP30-c.OBJECTIVE: In this study,we constructed SMP30 lentiviral expression v ectors for different transcripts,and overexpressed them in hepatoma cells to study the effect of different protein subtypes of SMP30 on the biologic al behavior of hepatoma cells.METHODS:(1)The eukaryotic expression vectors of three transcripts of SMP30 were constructed and transiently transfected into 293 T cells.Weste rn blot experiments were performed to determine whether all three transcri pts of SMP30 could be expressed in eukaryotic cells.(2)To construct the lentiviral expression vector for three transcripts of SMP30,and the hepatoma cells SK-Hep-1 were infected with the lentivirus carrying the target gene and the lentivirus with no-loading respectively.After screening by pu romycin,the over-expressing cell lines of SMP30 transcripts were construc ted..(3)Quantitative Real-time PCR(qRT-PCR)and Western Blot assays were used to detect the expression levels of three transcripts of SMP30 at transcriptional and protein levels,respectively.(4)Transwell invasion and migration assay was used to detect the invasion and migration ability of hepatocellular carcinoma cells.(5)The CCK-8 assay detects the prolifera tion of hepatoma cells.(6)To detect the changes in cell cycle and apopt osis by the flow cytometry.(7)To detect the changes in cell cycle-relate d proteins by western blot.RESULTS:(1)After three transcripts of SMP30 were overexpressed in 293 T cells,Western blot analysis showed that the three transcripts could be expressed in protein level.(2)Successfully constructed SK-Hep-1-LV5,S K-Hep-1-SMP30-a,SK-Hep-1-SMP30-b,and SK-Hep-1-SMP30-c stable tra nsgenic cell lines after screening by puromycin.(3)The expressions of thr ee transcripts of SMP30 were significantly up-regulated at mRNA and pro tein levels(P<0.001).CCK8 cell proliferation assay results showed that th e proliferative capacity of SK-Hep-SMP30-b and SK-Hep1-SMP30-c cells was inhibited(P<0.01),and the change of SK-Hep-1-SMP30-a cell prolifer ation ability is not significant(P>0.05).(4)Transwell invasion and m igration experiments showed that the migration and invasion ability of SK-Hep-1-SMP30-a,SK-Hep-1-SMP30-b,and SK-Hep-1-SMP30-c cells were reduced significantly(p<0.01).(5)The results of cell cycle experiments showed that the S phase of SK-Hep1-SMP30-b and SK-Hep1-SMP30-c c ells were increased significantly,and S phase arrest occurred(P<0.05),while SK-Hep1-SMP30-a cell cycle is not obviouly changed.(6)Apoptosis experiments showed that the diffrerences of apoptosis among SK-Hep-1-S MP30-a,SK-Hep-1-SMP30-b and SK-Hep-1-SMP30-c cells were not signif icant(P>0.05).(7)Cyclin-related proteins expression: CyclinA2 and Cyclin D3 were down-regulated in SK-Hep1-SMP30-a、SK-Hep1-SMP30-b and S K-Hep1-SMP30-c,while the cyclin-dependent kinase inhibitor P21 Waf1/Ci P1 was up-regulated in the three groups(P<0.05).Meanwhile,the expressi on of CyclinD1、the cyclin-dependent kinase CDK4 、CDK6 and The cyc lin-dependent kinase inhibitor P18 INK4KC、P27 KiP1 were not significan tly changed in the three groups(P>0.05).The expression of the cyclindependent kinase CDK2 was reduced only in SK-Hep-SMP30-c cells(P<0.05),but did not show significant changes in SK-Hep1-SMP30-a and SKHep1-SMP30-b cells.CONCLUSION:(1)The three transcripts of SMP30 can inhibit the prolif eration of hepatoma cells by down-regulating CyclinA2,CyclinD3 and upr egulating the expression of cyclin-dependent kinase inhibitor P21 Waf1/Ci P1.(2)Three transcripts of SMP30 can inhibit the invasion and migration o f SK-Hep1 cells but have no obvious inhibitory effect on apoptosis.(3)T he strongest inhibitory effect of SMP30-c on SK-Hep1 cells may be relate d to its ability to down-regulate the expression of cyclin-dependent kinase CDK2.