SPARCL1 Expression Regulation Mechanism And Its Function In Ovarian Cancer Cisplatin-Resistant Cells

Objetcive: Studies of SPARCL1 associated with ovarian cancer or tumor resistance are weak,and reports related to drug resistance regulation of ovarian cancer are rare.Based on bioinformatics methods,SPARCL1 was used to explore the regulation of tumor progression and drug resistance in ovarian cancer.The effetcs of SPARCL1 on the biological function of ovarian cancer SKOV3/DDP cells were investigated through in vitro experiments.Then the miRNAs involved in the regulation of SPARCL1 expression were screened and analyzed in the ovary.Expression Changes and Their Regulation Mechanisms in Drug Resistance.Methods: Bioinformatics methods such as biological process/pathway annotation,gene-protein interaction,etc.were performed by Coremine medical and GeneMANIA;gene expression information and related clinical data were extracted by databases such as Oncomine and TCGA.SKOV3/DDP cells overexpressing SPARCL1 were constructed using lentiviral vetcors.The expression of SPARCL1 was detetced by RT-q PCR and Western blot.The IC50 and growth curve were detetced by CCK8 assay.Cell proliferation was detetced by plate colony and scratch assay.Flow cytometry Cell cycle and apoptosis.Bioinformatics techniques such as TargetScan,microRNA.org,and microT-CDS databases were used to predict mi RNAs of target SPARCL1 and screened for miRNAs.Results:1.Bioinformatics analysis results: SPARCL1 down-regulated in ovarian cancer,through a variety of ways to regulate ovarian cancer drug resistance,such as cell proliferation,cell cycle,cell migration,apoptosis and angiogenesis.There is a diretc interaction between SPARCL1 and 6 genes(TNF,FOS,ABCB1,STAT3,WWOX,PMS2).It can be concluded that SPARCL1 is closely related to ovarian cancer drug resistance through various pathways.The difference in SPARCL1 expression correlates with the histological grade of ovarian cancer.2.Validation in vitro: The SPARCL1 overexpression virus was stably transfetced into SKOV3/DDP.After overexpression of SPARCL1,the IC50 of SKOV3/DDP against cisplatin decreased significantly.The SKOV3/DDP cell group had a statistically significant difference in the growth rate between SKOV3/DDP-OE and SKOV3/DDP and SKOV3/DDP-EV groups at 48h(P<0.05).Under the condition of 7.5 μM cisplatin dosing,the colony forming ability of SPARCL1-overexpressing SKOV3/DDP cells was significantly reduced(P<0.05).Scratch healing ability of SPARCL1-overexpressing SKOV3/DDP cells was also significantly reduced.Flow cytometry showed that overexpression of SPARCL1 blocked SKOV3/DDP cell cycle in G0/G1 phase,but there was no significant difference in cell apoptosis.3.To regulate the microRNA screening and validation results of SPARCL1: Based on the prediction software,three microRNAs that can potentially regulate the expression of SPARCL1 were screened and further confirmed by the dual luciferase reporter system that miR-448 could negatively target the SPARCL1 gene.Conclusion:1.As a potential tumor suppressor gene,SPARCL1 influences drug resistance in ovarian cancer through biological methods such as cell proliferation,cell cycle,and angiogenesis,and interacts diretcly with 6 genes to influence the progression of drug resistance in ovarian cancer.2.Over-expression of SPARCL1 significantly increased the sensitivity of drug-resistant cells to cisplatin and inhibited cell proliferation,colony formation and cell migration,and arrested the cell cycle.3.miR-448 can target the SPARCL1 gene and play a regulatory role in the predicted locus(Position 62-68/211-218).