| HMGA1, which locates in nuclear, is a sort of non-histon protein. It highly expresses during embryonic period and is very low or undetectable in normal cells. Recently, it is found that the expression of HMGA1 is very high in malignant tumors and whose high expression correlates with the development and metastasis of tumor cells. Although the relationship of HMGA1 with tumor is broadly studied by researchers around the world, there are still a lot of questions remain unclear. Working as a sort of constructional transcription factor, HMGA1 can regulates the expression of many genes associated with tumor activation and metastasis, whether it has something to do with tumor immune inhibition is unknown. In the meantime, the mechanism of HMGA1 regulating the growth and metastasis of tumor cells is still unknown. In this study, we silenced HMGA1 through RNAi in SMMC-7721 cells, and studied the biological changes of tumor cells, and at the same time we studied the alteration of immune inhibition after the silence of HMGA1 .The aim of this research is to assess the possibility of selecting HMGA1 as a promising target for tumor therapy.RT-PCR and immunocytochemistry were adopted to detect the expression of HMGA1 in different cell lines including tumor cells and normal cells. Plasmid for RNAi was constructed to silence HMGA1 and the plasmid was transfected into SMMC-7721 cell line to silence HMGA1. Transfection rate was assessed under laser confocal microscope and semi-quantity RT-PCR was used to detect the silencing efficiency of the constructed RNAi plasmid.The stable HMGA1 gene silencing cell clone was selected under the pressure of G418. FACS was used in detecting the transfection efficiency in stable transfected cells.Semi-quantity RT-PCR and Western blot were applied to detect the silencing of HMGA1 in stable transfection group so that to assess the success of stable HMGA1 silencing cell line construction.Under the technical plateform of HMGA1 gene silencing cell line, we studied the growth and immunosuppression changes of tumor cells without the high expression of HMGA1 by MTT method, plate clone formation test, FACS detection of cell cycle and apoptosis, gene expression level detection through semi-quantity RT-PCR,et al.MMP-2/9 gene expression level and their activity were also assyed in this experiment. The immnosuppression was also assayed by detecting the peripheral blood cell proliferation uner the cell culture supernatant in different cell groups and the mechanism of this immunosuppression was explored through the detection of mRNA levels of tumor derived immunosuppressing factors by semi-RT-PCR. The results were as followings: (1) HMGA1 was highly expressed in 8 tumor cell lines and was undetectable in 2 normal cell lines. This experiment suggests that the high expression of HMGA1 in tumor cells is a common character.In this experiment, we reported the detection of HMGA1 in hepatocellular carcinoma cell lines and stomache cell lines for the first time. (2) HMGA1 expression(0.34±0.11) was effectively silenced in RNAi cells, while the expression of HMGA1 in plasmid control(negative control for short, 1.54±0.15) and SMMC-7721 control(control for short, 1.48±0.14) cells were still highly expressed, with the transfection rate as more than 40% and the suppression rate was 80%.This experiment reveals that the construction of RNAi plasmid targeting HMGA1 is successful. (3)FACS analysis showed that the transfection rate, both in RNAi group and in negative control group,were more than 90% and the mRNA level of HMGA1 in RNAi group(0.28±0.09) was greatly droped compared with control and negative control group (1.51±0.19 and 1.47±0.17 respectively),with the suppressing rate of 82% and the same suppression was also found in Western blot analysis.This showed that the stable construction of HMGA1 gene silencing cell line was successfully setup.(4)The detection of cell proliferation through MTT showed: in the first 5 days, all cells in the three groups grow without difference,while feom the sixth day,cells in RNAi group(0.94±0.13) proliferared significantly slower than that in negative control and control groups(1.21±0.07 and 1.21±0.04 respectively,p<0.05).There was no notable difference found between the two control groups(p>0.05).From then on until the 8th day, when the MTT experiment finished, the cell proliferation was slower than control groups.(5) Clone formation tests showed that cells in the negative control and control groups growed well.Both the number of clone formation and the clone formation ratio in negative control and control groups (351.25±32.69 and 358.75±31.81, 70.25±6.54% and 71.75±6.36%,respectively) were significantly higher compared with RNAi group(145.25±12.09 and 29.05%±2.11 respectively).No significant difference was found between the two control groups(p>0.05). (6)The analysis of cell cycle through FACS showed that:the percentage of G0-G1 phase in RNAi group(75.21±2.35%)was significantly higher compare with negative control and control(37.98±3.02% and 39.23±3.63 % respectively , p<0.01) , while the percentage of G2-S(24.79±2.35%) was significantly lower than that in the two control groups(62.03±3.01% and 60.77±3.63% respectively ,p<0.01);The apoptosis peak appeared before G1 phage in RNAi group, and the apoptosis cell account for 29.46±3.04% ,significantly higher than that in negative control and control group (1.96±0.76% and 2.04±0.70% respectively,p<0.01 )(.7)The cell cycle protein mRNAs and apoptosis protein mRNAs detected through semi-quantity showed that: P57 mRNA leveling RNAi group(0.90±0.16) significantly higher than control and negative control groups(0.69±0.15 and 0.54±0.13 respectively,p<0.05),while no significant difference was found when compared with that of HL7702cells (0.95±0.15,p<0.05).Cyclin G1 mRNA level in RNAi group(0.47±0.10)was remarkably lower compare with control and negative control group(s1.52±0.22 and 1.47±0.18 respectively, p<0.01),and no significant difference was found compared with that in HL7702 (0.49±0.17,p>0.05). CDK4 mRNA level in RNAi group(0.32±0.12)was remarkably lower than that in control and negative control groups(1.13±0.12 and 1.13±0.18 respectively , p<0.01 ) , when compared with that in HL7702cells,the difference was not notable(0.37±0.13,p>0.05);CDK6 mRNA level detection showed that the mRNA level in controland negative control groups(1.52±0.10 and 1.42±0.15 respectively) was notably higher compare with RNAi group(1.09±0.13,p<0.05),while no significant difference was found between RNAi group and HL7702cell groups(1.05±0.16,p>0.05)。Bcl-2 gene expression was found in control and negative control groups,while it was undetectable in RNAi group and HL7702 cells.In our experiment,the Bax mRNA was unedtactable in the two control groups but was found in RNAi group and HL7702 cells,and its level in RNAi group(1.19±0.20) was significantly higher than in HL7702 cells(0.74±0.15,p<0.05).(8)Gelatinase spectrum analysis showed that :the gelatinase activity in control and negative control group could be detected while it could not be detected in RNAi group. MMP-2 and MMP-9 mRNA were detectable by RT-PCR in two control groups but not in RNAi group and HL7702 cells.(9)Claudin-6 expression level in RNAi group(1.12±0.23)was significantly higher than that in control and negative control groups(0.63±0.13 and 0.61±0.18 respectively,p<0.05),no notable difference was found comparing with that in HL7702 cells(1.16±0.25,p>0.05).(10)Assay of PBMC proliferation under different cell groups in different tumor culture supernatants concentration showed that at the minimum dilution ratio of 1:8 the growth of PBMC could be inhibited by the tumor cell supernantant,so this ratio was selected for next PHA stimulation experiment. At 1:8 dilution of cell culture aupernatant,the PBMC natural proliferation in RNAi group(0.44±0.03)was significantly higher than control and negative control(0.28±0.03 and 0.32±0.03,p<0.05),no notable difference was found when compared with normal culture(0.42±0.02,p>0.05);Under 75μl/mlPHA stimulation,PBMC growth in RNAi group(0.88±0.01) was notably higher than control and negative control(0.54±0.04 and 0.60±0.01 respectively, p<0.01),while compared with normal cell culture the difference was not remarkable(0.89±0.01,p>0.05).These results showed that HMGA1 silencing could release the growth suppression of tumor cellto PBMC.(11)Detection of IL-10,TGF-β,and Fas L mRNA level by RT-PCRshowed that these three genes were not detectable in RNAi group and HL7702 cells,but in control groups they were detectable. VEGF expression was detectable in all the four groups,but in RNAi group its expression level(0.36±0.19) was notably lower than in control and negative control groups(0.91±0.10 and 0.78±0.12 respectively,p<0.05,),while compared with HL7702 cellsit was not significantly different(0.25±0.04,p>0.05).The gene expression level of IL-2Rаin RNAi group(0.32±0.11) was significantly lower than that in control and normal control groups(0.97±0.14 and 1.09±0.17 respectively,p<0.05),while compared with that in HL-7702 cells,the difference was not notable(0.25±0.19,p>0.05).In conclusion, HMGA1 is the key molecule in controlling tumor cell growth and immunosuppression. Silencing the expression of HMGA1 gene can inhibit the proliferation and metastasis of tumor cells, promote tumor cell apoptosis and reverse the tumor immunosuppression. Our study has laid a theoretical and experimental foundation for selecting HMGA1 as therapeutic target in tumor therapy.
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