Effect Of Gene Silencing HMGA1on Growth And Apoptosis Of Prostate Cancer

In1973, HMGA1(High mobility group protein) was first discovered in bovinethymus cells by Goodwin and his colleages. It is a class of small nucleus proteins,which has strong water-soluble and show high mobility in polyacrylamide gelelectrophoresis.,It can participate in the process of a variety of basic celldifferentiation including transcriptional regulation, embryogenesis, transformation,cell cycle regulation, differentiation, apoptosis, aging, viral integration and DNArepair. HMGA1is highly expressed in normal embryonic cells, especially in the stageof pregnancy when a number of important organs developed, whereas less or littleexpressed in adult tissues. However, many studies have shown that the expression ofHMGA1gene is an important factor of inducing malignant transformation of normalcells and the expression of HMGA1is positively correlated the degree of malignancy.Our previous studies found that HMGA1was highly expressed in a variety of tumorcells including human prostate cancer cells which consistented with the literature.Small interfering RNA (siRNA) which constitute of21-23nucleotides plays the roleof RNA interference through mediating the degradation of homologous complementspecific mRNA sequences, RNAi plays the effect of gene silencing after transcription,more accurately, modification or translation of the mRNA level showed a specialsequence of post-transcriptional gene silencing (PTGS). Until now, RNAi technologyhas been widely used in biomedical research of cancer and various diseases. It mayprovide experimental evidence for the biomedical effect of HMGA1in prostate cancercell through detecting the growth and apoptosis of prostate cancer cell PC-3M aftersilencing HMGA1gene We chose HMGA1as interference target and transfected pU6mRFPsiRNA-HMGA1recombinant plasmid into human prostate cancer cells PC-3M, thendetected the growth and apoptosis of prostate cancer cell PC-3M after silencingHMGA1gene, so it may provide experimental evidence for the biomedical effect ofHMGA1in prostate cancer cell.Methods:1. The mRNA and protein expression of HMGA1were examined by RT-PCR andimmunocytochemistry in PC-3M cells;2. The amplification and restriction enzyme digestion of PU6mRFPsiRNA-HMGA1recombinant plasmid;3.We transfected pU6mRFPsiRNA-HMGA1recombinant plasmid to PC-3M cellswhich has high level expression of HMGA1using liposome transient transfectionmethod and observed the efficiency of transfection by inverted fluorescentmicroscope;4. The mRNA and protein expression of HMGA1were examined by RT-PCR、immunocytochemistry and Western blot in PC-3M cells afterpU6mRFPsiRNA-HMGA1recombinant plasmid transfected;5The PC-3M cells proliferation activity was detected by MTT assay afterpU6mRFPsiRNA-HMGA1recombinant plasmid transfected;6. The PC-3M cells colony-forming ability was detected by Plate cloning assay afterpU6mRFPsiRNA-HMGA1recombinant plasmid transfected;7. The apoptosis of PC-3M cells by FACS(fluorescence activated cell sorting) afterpU6mRFPsiRNA-HMGA1recombinant plasmid transfected;8. The expression of tumor cell apoptosis-related factors include Bcl-2, Bax, p53,DR3and immune suppression associated factor include TGF-β,IL-10, VEGF, FasLwere detected by RT-PCR in mRNA levels. Results:1. HMGA1mRNA and protein were highly expressed in PC-3M cells by RT-PCR andimmunocytochemistry methods.2. PU6mRFPsiRNA-HMGA1recombinant plasmid was consistent with the expectedfragment size by restriction enzyme digestion.3. The red fluorescence can be observed by inverted fluorescence microscope inPC-3M cells after PU6mRFPsiRNA-HMGA1recombinant plasmid transientlytransfected after48hours and transfection efficiency is about50%-60%.4. The mRNA and protein expression of HMGA1in pU6mRFPsiRNA-HMGA1RNAi group were reduced compared with the control group and pU6mRFP emptyvector control group by RT-PCR,Westernblotting and Immunofluorescencecytochemistry methods.5. The cell proliferation activity was decreased in pU6mRFPsiRNA-HMGA1RNAigroup compared with the control group and pU6mRFP empty vector controlgroup by MTT assay(p <0.05).6. The colony formation ability was decreased in pU6mRFPsiRNA-HMGA1RNAigroup compared with the control group and pU6mRFP empty vector controlgroup by Flat cloning experiments.7. The percentage of apoptotic cells (61.9%) was significantly increased inpU6mRFPsiRNA-HMGA1RNAi group compared with the control group (4.5%)and pU6mRFP empty vector control group (4.49%) by Flow cytometry.8. The mRNA expression of Bcl-2, DR3, VEGF, FasL, TGF-β, IL-10were decreasedin pU6mRFPsiRNA-HMGA1RNAi group compared with the control group andpU6mRFP empty vector control group, whereas the mRNA expression of p53and Bax were increased.Conclusion:1. Silencing HMGA1gene can inhibit the mRNA and protein expression of HMGA1in prostate cancer PC-3M cells. .2. Silencing HMGA1gene can inhibit the proliferation activity and colony formingability in prostate cancer PC-3M cells.3. Silencing HMGA1gene can promote apoptosis of PC-3M cells.4. Silencing HMGA1gene can promote the mRNA expression of apoptosis-relatedfactors p53and Bax and inhibit the mRNA expression of the antiapoptotic factorBcl-2and DR3in PC-3M cells.5.. Silencing HMGA1gene can inhibit the mRNA expression of immunosuppressionrelated factors VEGF, FasL, TGF-β and IL-10in PC-3M cells.