Research On Specific HER-2 SiRNA In Ovarian Carcinoma Cell Line

Introduction and ObjectivesThere are 60% ~ 90% epithelial ovarian tumor in ovarian primary malignant tumors. The morbidity of ovarian neoplasms is in the rank of the third in female genital malignant tumor, but the first mortality. The cytoreductive surgery and cisplatin - based chemotherapy is the basic treatment principle. But the tumor cells show drug resistance which influence the curative effect and lead to the failure of chemotherapy, the five - year — survival is only 20% -30%. The HER -2 gene is a member of the epidermal growth factor receptor (EGFR) family and encodes a 185kDa protein with tyrosine kinase activity. The amplification or enhanced transcription of the HER - 2 gene is associated with the oncogenesis or increases severity types of cancers. The HER - 2 protein can dimerize with other members of the EGFR family, and it is thought that overexpression of the HER - 2 gene product provides mitogenic signals to tumor cells resulting in increased growth potential and rendering them resistant to apoptotic factors. Increased expression of the HER - 2 is common in cancer and correlates with neo-plastic progression. Experimental and clinical data have indicated that HER - 2 overexpression correlated with various critical processes in the development, maintenance , and spread of malignant tumors. The reduction of HER - 2 leads to a failure in downstream signal cascades, and subsequently blocks the routes to gene activation or more direct modulators of mitogenesis and other cancer promoting phenotypes. Blockade of HER - 2 signaling pathway, therefore, represents a new perspective on the development of novel and selective anticancer therapies. RNA interference (RNAi) is a powerful tool for the analysis of gene function in many organisms by introducing double - stranded RNA ( dsRNA) intocells and leading to the sequence - specific degradation of endogenous RNAs that match the dsRNA. Small interfering RNA(siRNA) have been successfully used to silence a myriad of genes in plant and animal cells. The planning of targeted sequence is the key step of RNAi experiment. There are five methods in the execution of RNAi in current research. The chemically synthesized siRNA can effectively decrease the expression of targeted gene, but the expensive cost restricts its widely application. Vector mediated RNAi is suitable for the research after specific sequences are screened and identified, while the procedure of screening is comparatively complicated. Cock - tail and siRNA expression frame methods cant be applied in the screening of targeted sequence. Considering above factors, we conducted T7 transcription in vitro to acquire siRNA, a-voiding shortcomings of other methods. The first part: The screeing and identification of target site of HER - 2 gene by T7 transcription in vitro system. The second part: To investigate the effects of RNA interference targeting HER - 2 gene on the biological traits of human ovarian carcinoma. The third part;The change of chemsensitivity to cisplatin after silencing of HER - 2 gene in SKOV -3 cell line.Meterials and MethodsThe first part: T7 transcription in vitro system was firstly stablished in our work, applying GAPDH as the positive subject. Lipofectamine 2000 mediated transient transfection was conducted to transmit the siRNA into SKOV -3 cells. Three pairs of specific targeted sequence were selected in the coding region of HER - 2 mRNA. Transfection of HER - 2 siRNA was conducted with Lipofectamine 2000 in ovarian carcinoma cell line SKOV -3. The HER -2 gene expression was assessed by real - time PCR and Western Blotting assays. The second part: The antiproliferation ability of ovarian carcinoma cells was tested by MTT experiment. The antiapoptosis effects of sequence specific siRNA were assessed with flowcytometry and the change of morphology was assessed by TUNEL. Changes of invasive and chemotactic mobile capacity of SKOV -3 cells were measured by polycarbonates coated with or without Matrigal. The expres-sion of c - fos^c - jun^NF - kBnP53 protein were assessed by Western blot. The third part;The chemosensitivity of transfected cells to cisplatin was measured by MTT. The apoptosis percent was tested after HER - 2 siRNA plus cisplatin on SKOV - 3 by Annexin - V. And the apoposis related protein expression was tested by Western blot.ResultsThe first part: 1. Western blot results show that the expression of GAPDH protein was decreased in specific transfected cells and with the siRNA dose increased, the expression of GAPDH protein was decreased. This result proved our system of in vitro T7 Ixanscription was feasible in siRNA mediated RNAi. With extend of transfection time, a descending trend was also found in the expression level of HER - 2 mRNA by real - time PCR. Results also show that HER - 2 siRNA fl[ can silence the mRNA expression by 70% and in a time and dose dependent manner. So, we use transfected with HER - 2 siRNA M as specific interference group.The second part: (1) Specific group cells were slower than normal and nonspecific groups in the rate of cell proliferation( P <0. 01). (2) The apoptosis percentage of SKOV - 3 cells markedly increased 48h after transfected with HER - 2 siRNA, and increased in a time - depended manner. The apoptosis rate of transfected HER -2 siRNA SKOV -3 cells on 6th day was (53. 16 ±0. 96)% , which was the highest, whereas the control group was (4. 07 ±0. 32)%. The apoptosis percentage of interference group was higher than that of nonspecific and control groups(P <0. 001). No significant difference in apoptosis rate was found between the control group and nonspecific group(P >0. 05). (3) From the point of morphology, cytoplasm concentration, thickening mottling in nucleus and magnitude diversity to form apoptotic bydy were detected in the specific interference group by TUNEL technology. There was a significant difference between specific interference group and other groups (P<0.001). (4) The expression of c - fos, c - jun, NF - kB protein decreased and the expression of P53 protein was increased in specific interference group. (5 ) The invasion andchemotactic mobile capacity of SKOV - 3 cells are decreased after trancfect HER -2 siRNA into the cell line.The third part: (1) Mter three groups exposed to cisplatin, the cell survival rate descreased with close of cisplatin increased. There was a significant difference between specific interference group (58.13 ±4.67)% and nonspecific group(65.28 ±5.73)% N control group(68.46 ±2.83)% , when began to be given l|xg/mL cisplain(P <0. 001). No significant difference in cell survival rate was found between control group and nonspecific group (P >0. 05). (2) The apoptosis rate of specific interference puls cisplatin group was higher than other groups (P<0.001). (3) The expression of antiapoptotic protein Bel - 2, Survivin, XIAP were lower than cisplatin group, and the expression of apoptotic protein Smac was higher than cisplatin group. There was a significant difference between two groups ( P > 0. 05 ).ConclusionsThe first part: (1) With the siRNA dose increased, the expression of GAP-DH protein was decreased. This result proved our system of in vitro T7 transcription was feasible in siRNA mediated RNAi. (2) HER -2 siRNA Iff can silence the mRNA expression by 70% and in a time and dose dependent manner. SiRNA synthesized by in vitro transcription effectively suppressed HER - 2 expression.The second part: (1) HER - 2 siRNA can inhibited cell proliferation and induced cell apoptosis significantly. (2) HER -2 siRNA can inhibited cell invasion and chemotactic mobile capacity of SKOV - 3 cells. ( 3 ) These results showed that HER - 2 siRNA mainly influence the expression of c - fos, c - jun, P53 and NF - kB in the HER -2 signal pathway.The third part: (1) HER - 2 siRNA can induced cell apoptosis significantly and increased the sensitivity of SKOV -3 to cisplatin. (2)The mechnism of induce cell apoptosis by HER - 2 siRNA plus cisplatin may be related with the expression of Bel - 2, Survivin and XIAP protein descteased and the expression of Smac protein increased. The successful application of HER -2 siRNA plus cis-? 8 ?platin in HER - 2 overexpressing cells may extend the list of available therapeutic modalities in the treatment of human ovarian cancer.