Based On The Follicle-stimulating Hormone Receptor-mediated Targeted Therapy Of Ovarian Cancer Experiments

Ovarian cancer is the leading cause of death amongst all gynecological cancers, the 5-year survival rate is only 30%. Patients are always in later stages of ovarian cancer when they are first diagnosed. The lack of early detection and effective treatment makes it more subtle to handle to doctors and medical science researchers. Recently, the newly arrived targeted gene therapy seems to have a better solution to this difficulty. In this paper, we used CXCL1_RNAi-P as a target gene and loaded it with a ligand conjugated gene carrier to find a proper way to against ovarian cancer.In the first part of this paper, we examined the CXCL1 mRNA expression in ovarian carcinoma cell line (ES-2 and SKOV-3) and checked the CXCL1 knockdown effect on CXCL1 mRNA expression and CXCL1 protein expression by tansfecting siRNA into cells. The chemically synthesized siRNA is expensive, especially when specific lable is needed. Thus, we used 4 previously synthesized siRNAs to silence the CXCL mRNA expression and protein expression by performing PT PCR and ELISA assay to find the most effective siRNA target sequence.In the second part, we constructed an RNAi plasmid system by integrating the most efficient siRNA target sequence into a linear plasmid in which it has a GFP reporter gene. Sequencing test indicated that the correct target sequence was successfully cloned into the plasmid. Then we examined the tranfection efficiency and CXCL1 knockdown effects of the RNAi plasmid by observing GFP expression and conducting a semi quantitative PCR. The experimental results showed that the transfection efficiency of the RNAi plasmid was 61.2% and the CXCL1 mRNA expression inhibition efficiency was 54.1%.In order to improve specificity of delivering RNAi plasmid (CXCL1_RNAi-P) into ovarian clear cell carcinoma ES-2 cell line. We created an RNAi plasmid carrier in the third part of this paper; in which FSH peptide was conjugated to a pegylated PEI based system. FSH peptide is able to specifically bind to and be recognised by FSHR on the surface of the ES-2 cell line, we assumed these features could be used to enhance transfection efficiency. We first used a difunctional PEG derivatives (MAL-PEG-NHS) containing both maleimide (MAL) and N-hydroxysuccinimidyl ester (NHS) groups. The NHS groups were selectively conjugated to amine groups of FSH peptide. Then the MAL groups were fully conjugated with the primary amine groups of PEI. Due to the disadvantages of the PEI as a DNA carrier, such as toxicity or DNA solubility etc, we improved the system by grafting 0.5%,1%and 2%FSH peptide conjugated PEG (F-PEG-MAL) to PEI (0.5%,1% and 2% F-PEG-PEI). The F-PEG-PEIs were mixed with RNAi to form complexes at different N/P ratio. We examined particle size, zeta potential, gel ratardation, loading efficiency, cell toxicity and transfection efficiency by the according assays to find optimal complex.2% F-PEG-PEI /DNA complex at N/P:25 (F-NP) showed a proper particle size and zeta potential, 100% loading efficiency, low toxicity and 68.9% tansfection efficiency. Then we used RT PCR and ELISA assay to test the CXCL1 silencing effect of this RNAi system. The results showed that F-NP had a CXCL1 mRNA expression and protein expression inhibition efficiency at 59.1% and 52.7%.In the last part, we examined the biological effects of the F-NP based RNAi system, including cell proliferation, cell mobility and cell invasive ability, by using CCK-8 assay, cell migration test and cell invasion experiment. We found that silencing CXCL1 express in ES-2 cell line can lead to inhibit cancer cell proliferation, migration and invasion. It suggests that CXCL1 might be the key player in ovarian cancer development, growth and matastasis. And with FSH conjugated RNAi system had a better performance at all aspects due to its specific affinity to the ES-2 cell line. The cell proliferation of F-NP group was inhibited at the rate of 37.9%(96h), the inhibitory effect is evident compare to non FSH conjugated system(C-NP)(P<0.01). In the cell migration and cell invasion test, the F-NP also showed significant inhibitory effect compare to the C-NP group(P<0.01).Finally, the F-NP RNAi system we created in this study is capable of delivering RNAi drugs more efficiently into FSHR positive cell lines to knockdown the target gene and to inhibit tumour cell growth and development. In addition, it suggests that this system might have a good performance in related in vivo applications. Our study might be considered as a strong experimental tool of new targeted gene therapy against ovarian cancer.