Study On Expression And Function Of ADAM10and Co-expression Plasmidbased SiRNA-ADAM10and GRIM-19in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most frequently occurring tumoursworldwide, affecting more than50,000people each year. Primary liver cancer is the fifth mostcommon malignancy in men and the ninth in women. As therapeutic effect of this cancer isstill not good, new treatments are urgently needed.Thus, there is a need to understand themolecular mechanism of this cancer and to develop sensitive and specific molecular markersand novel therapies. A disintegrin and metalloproteases (ADAMs) comprise a family of Type Itransmembrane proteins containing integrin and metalloproteinase domain which are widelyexpressed in various tissues and cells. A disintegrin and metalloproteinase10(ADAM10) is animportant member of the ADAM family. Previou studies have shown that ADAM10highlyexpresses in a variety of tumors and promots proliferation, migration and invasion of thetumor cells, however, studies on ADAM10in HCC is still rare.This study contains three parts:1. Protein and transcript level expression of ADAM10and its relationship with pathological features of HCC and its prognostic value were evaluated.2. Effects of silencing ADAM10on tumor growth and proliferation, migration and invasion ofof HCC cells in vitro and in vivo were evaluated.3. According to results of the first two parts,effect of co-expression plasmid-based ADAM10-specific siRNA and GRIM-19on tumorgrowth and proliferation, migration and invasion of of HCC cells in vitro and in vivo wereevaluatedThe main results were as followed:1. Expression of ADAM10and its relationship with pathological features ofHCCTissues of98patients with HCC were collected to be used to investigate the protein andtranscript level expression of ADAM10and its relationship with pathological features of HCCusing immunohistochemistry and real time PCR. Results showed that74samples stained withpositive for ADAM10and only samples from24patients stained with negative for ADAM10.Proportion of positive in tumors above5cm was90.63%, and that of Metastasis IV group was100%. Results of immunostaining were not significantly relavant with gender and age of patients(P＞0.05). TNM stage(P＜0.05) and tumour differentiation(P＜0.01)significantlyaffected results of immunostaining. Results of Real time PCR showed that transcript levelexpression of ADAM10in tissues of HCC was significantly higher than that in non-canceroustissue samples(P＜0.01). Patients with positive staining have the lower survival index thanthose with negative staining(P＜0.01).2. Effects of silencing ADAM10on tumor growth and proliferation,migration and invasion of of HCC cellsRecombinant shRNA expression vector carrying ADAM10was constructed and theninfected into human HCC cell line HepG2. Results of MTT colorimetric and TUNEL assayshowed that downregulation of ADAM10by RNAi could inhibit cell proliferation andpromote cell apoptosis. Results of western blot showed that downregulation of ADAM10could significantly inhibit expression of Survivin and Bcl-2and the phosphorylation of AKTand PI3K in HepG2cells. Downregulation of ADAM10could significantly inhibit migrationand invasion of HepG2cells. Results of tumor growth in vivo showed that downregulation ofADAM10could significantly inhibit tumor growth.3. Effect of co-expression of ADAM10-specific siRNA and GRIM19on HCCpSi-ADAM10-GRIM-19(Co-expressed-ADAM10-specific siRNA and GRIM19plasmid), plasmid pSi-ADAM10(encoding siRNA specific to ADAM10) and plasmidpGRIM-19(containing GRIM19coding region) were constructed and transfected into HepG2cells. Results of real-time PCR and western blot fully convinced the transfection. Compared tothe controls either ADAM10-specific siRNA or GRIM-19alone, pSi-ADAM10-GRIM19inHepG2cancer cells significantly inhibited cell proliferation and lead to the higher percentageof cells in G1phase and lower percentage of cells in S phase by decreaing expression ofcyclinD1and increasing of P21(P<0.05). pSi-ADAM10-GRIM19could promote th apotosisof HCC cells by increasing the activity of caspase-3, caspase-8and caspases-9and decreasingexpression of Bcl-2and Survivin. Treating with pSi-ADAM10-GRIM-19plasmid significantlyincreased the inhibitory effect on the migration and invasion of HepG2cells by inhibitingexpression of MMP-2and MMP-9. Results of tumor growth in vivo showed thatpSi-ADAM10-GRIM-19plasmid could derease expression of ADAM10and increasing expression of GRIM-19and induce cell apotosis and tumor growth(P<0.05). Our resultsdemonstrated that simultaneous expression of ADAM10-specific siRNA and GRIM-19(pSi-ADAM10-GRIM-19) in HepG2tumor cells significantly inhibited cell proliferation,cycle, migration and invasion and induced cell apoptosis in vitro, as well as suppressed tumorgrowth in mice model, when compared to the controls, pSi-ADAM10or pGRIM-19alone.In summary, ADAM10is strongly expressed in a large proportion of HCC cases.Downregulation of ADAM10expression using RNA silencing significantly suppressed theproliferation, cell migration and cell invasion in vitro, and tumor growth in vivo. Co-expressedADAM10-specific siRNA and GRIM19synergistically and more effectively suppressed HCCtumor growth and cell proliferation, cell migration and cell invasion in vitro...