RNA Interference Targeting HDAC1Sensitizes Ovarian Cancer SKOV3/DDP Cells To Cisplatin

【Background】 Mulitidrug resistance（MDR）of tumor cells is an important elementwhich leads to ineffectiveness of chemotherapy. The molecular mechanisms of MDRare very complex. MDR are mediated by P-gp, MRP, BCRP, LRP and othertransporters, may be related with GST, Topo II, PKC and other enzymes, or may beconnected with apoptosis-related genes expression.HDAC1is a kind of histone deacetylases. It could catalyze the deacetylation ofhistone, modify the structure of chromosome and regulate the expression of genes. It’shighly expressed in majority of tumor cells and closely related with the pathogensis oftumor. Previous studies have found that HDAC1was highly expressed inSKOV3/DDP cells. It’s still unclear whether HDAC1is involved in the MDR ofovarian cancer. We raised the question whether the sensitivity of SKOV3/DDP cells toDDP and the expression of P-gp, Bcl-2, Bax and other MDR-related genes could beinfluenced by silencing HDAC1. Therefore, we conducted the present study on theseissues.The present study is divided into the following three parts:Chapter one: the resistance detection of SKOV3/DDP【Aim】 To identify the tolerance of SKOV3/DDP cells to cisplatin and determinewhether we should use it as the cell model of resistance cell in tumor.【Methods】 We observed the morphological differences between SKOV3cells andSKOV3/DDP cells and the influence of DDP to the growing of SKOV3cells andSKOV3/DDP cells. The growth inhibition rates of cells to different concentrations ofDDP were detected by MTT to calculate the resistance index of SKOV3/DDP cells.【Results】There is a significant morphological difference between SKOV3cells andSKOV3/DDP cells. They have different tolerance to DDP, and the resistance index of SKOV3/DDP cells to DDP is3.15. We would treat SKOV3/DDP cells as ovariancancer model of drug-resistance to do some research on reversal of tumor resistance.Chapter two: the identification of interference efficiency ofshRNA HDAC1recombinant plasmid【Aim】 The recombinant plasmids were synthesized in company and the plasmidwith the highest interference efficiency to SKOV3/DDP cells was selected.【Methods】 We choosed target sequences interfering HDAC1mRNAand send themto company to synthesis recombinant plasmids, including the interfering recombinantplasmids “shRNA HDAC1-1” and “shRNA HDAC1-2”, and the correspondingnegative control plasmid “shRNA control”. The recombinant plasmids were identifiedusing restriction endonuclease and nucleic acid sequencing. The recombinantplasmids were transferred to SKOV3/DDP cells using liposome transfection method.Total RNA was extracted and RT-PCR was done to detect the expression of HDAC1mRNA in each group.【Results】 The identification results of restriction endonuclease and nucleic acidsequencing showed that all recombinant plasmids were eligible. The relativeexpression of HDAC1mRNA in SKOV3/DDP cells transferred with “shRNAHDAC1-2” was lower than that transferred with “shRNA HDAC1-1”, and thedifference has statistical significance. We established empirical study of using“shRNA HDAC1-2” plasmid to silence HDAC1, since this plasmid had higherinterference efficiency.Chapter three: the influence of shRNA HDAC1recombinant plasmid to SKOV3/DDP cells【Aim】 To detect the influence of recombinant plasmid interfering HDAC1toSKOV3/DDP cells【Methods】 The mRNA levels of MDR, Bcl-2and Bax in SKOV3/DDP cellstransferred with the interfering recombinant plasmids were measured by usingreal-time PCR. The effects on apoptosis were examined by using flow cytometry. Thesensitivity of cells to DDP were measured by using MTT method. 【Results】 The mRNA levels of MDR and Bcl-2were significantly reduced inSKOV3/DDP cells transferred with the interfering recombinant plasmid comparedwith the negative control plasmid. The mRNA levels of Bax showed no significantchange between all groups. Apoptotic and necrotic cells were increased. Thesensitivity of SKOV3/DDP cells transferred with the interfering recombinant plasmidto DDP was increased and the resistance index reduced to2.04.【Conclusions】1. RNAi targeting HDAC1sensitized SKOV3/DDP cells to cisplatin, which may berelated to the reduction of the level of MDR1/P-gp mRNA. HDAC1may play a role inreversing the MDR of ovarian cancer cells.2. RNAi targeting HDAC1induced apotosis of SKOV3/DDP cells, which may beconnected with the reduction of the level of Bcl-2mRNA...