| Background Ovarian cancer is one of the common malignancies in the female reproductive system.Although the incidence is lower than that of cervical cancer and endometrial cancer,the mortality rate is the highest,which seriously threatens the health of women.The symptoms of onset of ovarian cancer are hidden.As the disease progresses,typical symptoms of the tumor will appear later,and 70%-80% of patients are already advanced at the time of initial diagnosis.Ovarian cancer progresses rapidly,with a five-year survival rate of only 20%-36.1%.At present,the main treatment is to maximize surgical resection of tumor tissue and then receive platinum/paclitaxel chemotherapy.Because the traditional treatment is not effective and the recurrence rate is high,exploring the molecular mechanism of ovarian cancer gene therapy to obtain effective therapeutic targets and finding new therapeutic methods is an important research to improve the prognosis and recurrence rate of ovarian cancer patients.Question.Recent studies have shown that functional small-molecule ds RNAs can not only inhibit gene expression,but also activate relevant gene transcription,ie RNA activation(RNAa),by targeting gene promoters or non-coding transcription.Small activation RNAs(sa RNAs)are ds RNAs with small RNA-activated molecules that have the opposite function of RNA interference.Compared with traditional RNAi,the se of RNAa does not need to consider whether a certain tumor has a specific oncogene as a target gene,opening up new ideas for scientists to overcome cancer treatment difficulties.It has also been reported that the combination of asymmetric small molecules with a targeting site may be expected to reduce the off-target rate and increase the efficiency of the action.On the other hand,the p21 gene is a cyclin-dependent kinase inhibitor downstream of the p53 gene.As a negative regulator,it is involved in reducing the replication and accumulation of damaged DNA,thereby exerting a role in suppressing cancer.Many new drugs and radiation therapy are used in cancer treatment and are closely related to p21 protein.Here,we designed asymmetrically designed small-molecule activated RNA and chemical modification experiments for the tumor suppressor gene p21,which has a lot of research support,to investigate the inhibitory effect of ovarian cancer cells.Objective 1.Through in vitro cell experiments,the functional ds RNA synthesized was tested to regulate the up-regulation of p21 at the transcriptional level of ovarian cancer cell A2780.2.In vitro detection of the selected asymmetric small activated dsp21-RNA(15/21)and chemically modified S-VEsa RNA and S-CHOsa RNA upregulate the expression of p21 in ovarian cancer cell A2780 after transcription Effects on genes;3.Observe the change of biological behavior of ovarian cancer cell A2780 such as proliferation,colony formation,cell cycle,invasive ability,etc.4.A preliminary evaluation of the therapeutic effect of asymmetric small activating RNA targeting p21 on ovarian cancer cells via the antisense strand.Methods 1.According to the previously reported small-activated RNA(ds P21-322)that is targeted to the upstream region of the p21 promoter and is designed as a structurally asymmetrical small RNA with inconsistent double-stranded base numbers: sa RNA(dsp21-RNA(15/ 21),dsp21-RNA(15/21))and the sa RNA: vitamins modified with 5′-end of AQ-VEsa RNA and S-VEsa RNA,and cholesterol-modified 5 ′-end of AQ-S-CHOsa RNA and S-CHOsa RNA;2.Small-molecule ds P21-322 and its modified sa RNA were transiently co-transfectedinto ovarian cancer cells A2780 by cell,and the expression of p21 in RNA level was detected by quantitative PCR,and the p21 protein was monitored by immunohistochemistry.Expression level growth,screening for sa RNA that can effectively upregulate the target gene;3.The sa RNA:dsp21-RNA(15/21)which can effectively up-regulate p21 in ovarian cancer cells,S-VEsa RNA and S-CHOsa RNA were transfected into ovarian cancer cells A2780 by transient cotransfection of cells.The effects of dsp21-RNA(15/21),S-VEsa RNA,and S-CHOsa RNA on the expression of p21-related genes(cyclin D,CD4 and CD6)were examined by quantitative PCR and Western blotting.3.Observe the growth of ovarian cancer cells after the up-regulation of p21 expression.Observe the change of cell clone inoculation survival rate by cell clone formation experiment.Calculate the cell survival rate by CCK8 absorbance measurement;4.The effect of changes in p21 levels on the cell cycle of ovarian cancer cells A2780 was detected by flow cytometry(FCM);5.Transwell assay was used to detect the change of invasion ability of ovarian cancer cells after transfection of dsp21-RNA(15/21),S-VEsa RNA and S-CHOsa RNA.6.Statistical analysis was performed using SPSS 19.0 software.The results were expressed as ±s.Fisher’s exact test and Student’s t test were used to compare the data of the two groups.Variance analysis and LSD test were used to compare data between the two groups.The test level was α=0.05.P < 0.05 was considered statistically significant.Results 1.Compared with the negative control,ds P21-322 can effectively upregulate the RNA level and protein level of the tumor suppressor gene p21 expression in ovarian cancer cell A2780;truncated asymmetric small molecule dsp21-RNA(15/21)and Both chemically modified S-VEsa RNA and S-CHOsa RNA could effectively up-regulate the expression of p21 in tumor suppressor gene of RNA and protein in ovarian cancer cell A2780.2.Western blot results showed that the expression of E-cadherin protein in ovarian cancer cell A2780 after transfection of dsp21-RNA(15/21),S-VEsa RNA and S-CHOsa RNA,but the expression of cyclin D,CD4 and CD6 Level down;3.Ovarian cancer cells can be seen clearly under the microscope,compared with the negative control group,cell growth is poor,proliferation slows down,apoptosis increases;cell clone formation test results suggest that p21 expression upregulated ovarian cancer cell inoculation survival rate Decreased,the number of clones formed decreased;CCK8 showed that the survival rate of ovarian cancer cells decreased in the positive group;4.The up-regulation of p21 expression caused ovarian cancer cells to stay in the G0/G1 phase of the cell cycle.5.Overexpression of p21 can make the number of ovarian cancer cells passing through the cell less,that is,reduced invasive ability.Conclusions 1.Ds P21-322 and its asymmetrically modified sa RNA: dsp21-RNA(15/21)and chemically modified S-VEsa RNA and S-CHOsa RNA can upregulate the expression of p21 in ovarian cancer cell A2780 at the transcriptional level.Antisense strand plays a leading role.2.Small activated RNAs and chemically modified small activated RNAs that were truncated to asymmetric double-strands not only inhibited the expression of p21,but also affected the expression of p21-related genes and altered biological activity.3.It was verified that p21 as an anti-oncogene can make ovarian cancer cells grow and proliferate.The survival rate and cell cycle of cloning inoculation are inhibited,the apoptosis rate is increased and the invasion ability of cells is weakened.Provide some reference for the treatment of ovarian cancer. |
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