Effect And Mechanism Of SiRNA LSINCT5 Combined With Cisplatin On Ovarian Cancer Cells

Background Ovarian cancer is one of the three most common malignant tumors of the female reproductive organ,its mortality rate ranks first in gynecological malignancies,which have become a major cancer threat to women’s lives and health.Clinical routine treatment of ovarian cancer is a complex treatment,which is based on the surgery combined with platinum-based chemotherapy and radiotherapy.Although we have taken such an effective and positive treatment,the recurrence and metastasis rate is still very high.The production of drug resistance and the more serious side effects also make the patient’s treatment effect and long-term survival rate unsatisfactory.Therefore,continuous exploration of new anti-tumor methods is the key to the treatment of malignant ovarian tumor.At present,many emerging treatment methods(cytokine therapy,molecular targeted therapy)have achieved good clinical efficacy.Long chain non encoding RNA is a class of RNA molecules that do not encoding proteins,and the length of the transcript is more than 200 nucleotides.Current research shows that lncRNA is involved in a variety of important regulatory processes,which are included with X chromosome silencing,genomic imprinting,chromatin modification,transcriptional activation,transcriptional interference,nuclear transport and so on.It affects the occurrence and development of tumor,and has a certain relationship with the invasion,metastasis and prognosis of patients.Long stress-induced non-coding transcript5 is one of the long non-coding RNA.Some studies have indicated that the expression of LSINCT5 in ovarian cancer was significantly increased,LSINCT5 could promote the proliferation and activation of malignant tumor cells.But the literature on the relationship between LSINCT5 and ovarian cancer has not been reported at home and abroad.Objective Previous studies have demonstrated that the expression of LSINCT5 in ovarian cancer 5tissue is increased,and it can inhibit the proliferation,migration,invasion and other biological behavior of ovarian cancer cells by interfering with its expression.On the basis of previous studies,this study will lay the foundation for the gene targeted therapy combined with chemotherapy for ovarian cancer by evaluating the effect of the silencing LSINCT5 gene combined with cisplatin on ovarian cancer SKOV3 cells and investigating the significance of small interfering RNA gene combined with cisplatin and the mechanism of its anti-tumor effect in the treatment of epithelial ovarian cancer..Methods 1.Epithelial ovarian cancer SKOV3 cells were divided into 5 groups according to different treatment factors.((1)Control group(2)NC-siRNA group(3)Cisplatin group(4)LSINCT5-siRNA+Cisplatin group(5)LSINCT5-siRNA group)2.LSINCT5 gene was transferred into ovarian cancer SKOV3 cells by small interfering technology.LSINCT5-siRNA combined with cisplatin act on ovarian cancer cells.3.Using CCK-8 to detect the toxic effects of different concentrations of cisplatin(0、1.5、3、4.5、6、12μg/ml)on ovarian cancer cells at different time periods(24h、48h、72h).The appropriate time and drug concentration were selected as the factors of drug treatment in the following experiments.4.CCK-8 was used to detect the inhibitory rate of 48 h in ovarian cancer cells with different concentrations of cisplatin(0、1.5、3、4.5、6、12μg/ml)after LSINCT5-siRNA were transfected for 24 h.5.Observe the growth and morphological changes of the cells under the microscope.6.Apoptosis rate of different treatment groups were detected by flow cytometry.7.RT-PCR was used to detect the level of LSINCT5 gene in ovarian cancer SKOV3 cells after transfection with LSINCT5-siRNA,and the expression level of Caspase3 gene and Caspase9 gene were detected in 5 different treatment groups of ovarian cancer SKOV3 cells.8.Blot Western was used to detect the expression levels of Caspase3 protein and Caspase 9 protein in ovarian cancer SKOV3 cells of 5 different treatment groups.Results 1.Under the fluorescence microscope,the transfection efficiency of LSINCT5-siRNA on ovarian cancer SKOV3 cells was about 80%.2.RT-PCR results showed that the relative expression of LSINCT5 in the LSINCT5-siRNA group was significantly lower than that in the NC-siRNA group(P <0.05)after the transfection of ovarian cancer with 48 h.It was suggested that small interfering RNA could effectively down the expression level of LSINCT5 gene in ovarian cancer cells.3.According to the results of CCK-8,we selected the following experimental drug treatment factors were: cisplatin concentration in 3μg/ml,48 h.The cell inhibition rate was 30.5%,which was more suitable.4.The cells of each experimental group were observed under light microscope.The number of ovarian cancer cells in simple LSINCT5-siRNA group and simple cisplatin group were decreased in different degrees,and cellular morphology have varying degrees of change.In LSINCT5-siRNA combined with cisplatin group,the growth of cells was sparse,the structure of cells was disorder and lack polarity.Cells were bifurcate and morphology of “horn”.5.The CCK-8 results showed that the silencing LSINCT5 gene can significantly improve the sensitivity of ovarian cancer cells to cisplatin.6.The results of flow cytometry showed that the apoptosis rate of transfected LSINCT5-siRNA combined with cisplatin group was significantly higher than that of simple LSINCT5-siRNA group and simple cisplatin group,(P<0.05).The transfected LSINCT5-siRNA can help cisplatin to promote the apoptosis of ovarian cancer cells.7.From the gene level,the results of RT-PCR verified that the expression levels of Caspase3 gene(2.217±0.193)and Caspase9 gene(2.019±0.113)which are associated with apoptosis were significantly increased in LSINCT5-siRNA combined with cisplatin group.The difference was statistically significant.8.From the protein level,the result of blot Western verified that the expression levels of Caspase3 protein(1.005±0.066)and Caspase9 protein(1.245±0.050)which are associated with apoptosis were significantly increased in LSINCT5-siRNA combined with cisplatin group.This shows that LSINCT5-siRNA combined with cisplatin was able to enhance the apoptotic effect of ovarian cancer SKOV3 cells.Conclusion 1.Silencing LSINCT5 increases the sensitivity of ovarian cancer cells to cisplatin.Effective down-regulation of LSINCT5 combined with cisplatin significantly inhibits the growth and proliferation of ovarian cancer cells and promotes the apoptosis of ovarian cancer cells.2.LSINCT5-siRNA combined with cisplatin can enhance the effect of the treatment of ovarian cancer.The mechanism of action may be related to the down-regulation ofLSINCT5 and activation of caspase9 pathway and Caspase3 pathway,which is related to the cascade effect of cell apoptosis,and the specific signaling pathway is still needed to be further studied.This study established a solid experimental basis for the gene targeted therapy combined with chemotherapy for ovarian cancer.