The Mechanisms Of LncRNA HAS2-AS1 Promotes Serous Ovarian Cancer Progression By Targeting MiR-186-5p/Twist1 Axis

BackgroundOvarian cancer,one of the most common malignant tumor of the female reproductive system with high morbidity and mortality rate,has become a major factor that affect women’s lives and health.Serous ovarian cancer accounts for approximately 70%of all epithelial ovarian cancer,of which over 90%are high-grade serous ovarian cancer,the most malignant subtype.Although clinical treatment strategies have been developed in recent years,post-operative recurrence,poor prognosis and chemotherapy resistance still have some adverse impacts on patients’long-term survival and quality of life.Therefore,we urgently need to further investigate the mechanisms involved in the development of plasma ovarian cancer to provide a possible theoretical basis for treatment and disease prediction.LncRNA(long-non coding RNA),which is longer than 200nt and cannot encode proteins,can be involved in the malignant biology of many neoplasms and plays an important role in neoplasms development.HAS2-AS1(hyaluronan synthase 2 antisense RNA 1)is a tumor-associated lncRNA involved in the regulation of neoplasms malignant biological behaviors such as aberrant cell proliferation,invasive metastasis,epithelial mesenchymal transition and drug resistance,and is associated with poor patient prognosis,playing an oncogene role in a variety of neoplasms.The aim of this study was to investigate the biological role of lncRNA,HAS2-AS1 in the development of serous ovarian cancer and its possible regulatory mechanism as a competing endogenous RNA(ceRNA),providing potential directions for the early diagnosis and treatment strategy of serous ovarian cancer.Materials and Methods1.To verify the expression of HAS2-AS1 in serous ovarian cancer tissues.Clinical tissue samples of high-grade serous ovarian cancer(HGSOC)were collected,and normal fallopian tube tissue(FT)was used as a control.The FT and HGSOC tissues were examined using real-time quantitative qRT-PCR to analyze and compare the differences in HAS2-AS1 expression between the two groups of specimens.2.To investigate the biological function of HAS2-AS1 in serous ovarian cancerA2780 and UWB1.289 ovarian cancer cells were selected to construct HAS2-AS1 overexpression and low expression cell lines.To verify the efficiency of overexpression and silencing of HAS2-AS1,and to clarify the effect of HAS2-AS1 on the biological function of ovarian cancer cells by cell function assay:MTT assay and plate cloning assay were performed to verify the role of HAS2-AS1 in the growth and proliferation of ovarian cancer cells;Transwell assay was performed to detect the effect of HAS2-AS1 on the invasion and metastasis of ovarian cancer cells;Western Blot was used to verify the effect of HAS2-AS1 on the expression of proteins in the EMT process.3.To investigate the effect of HAS2-AS1 on cisplatin resistance in ovarian cancer cellsHAS2-AS1 expression levels in A2780 and drug-resistant A2780/DDP were detected by qRT-PCR.Overexpression and underexpression of HAS2-AS1 on cisplatin sensitivity of ovarian cancer cells were analyzed by MTT and plate clone formation assay.4.To verify the interaction between HAS2-AS1 and miR-186-5pBioinformatics analysis was performed to predict the downstream miRNA molecules of HAS2-AS1,and miR-186-5p was found to be a potential target of HAS2-AS1.Dual-luciferase reporter assay was performed to verify the interaction between miR-186-5p and HAS2-AS1.qRT-PCR was used to detect the change of miR-186-5p expression level after silencing HAS2-AS1,To verify the difference of miR-186-5p expression in HGSOC tissues and FT tissues,Person correlation analysis was performed to verify the correlation between HAS2-AS1 and miR-186-5p expression in tumor tissues.5.To explore the biological function of miR-186-5p in serous ovarian cancerTransfection of miR-186-5p mimics/NC in A2780 and UWB 1.289 overexpression efficiency was verified by qRT-PCR;the role of miR-186-5p in the growth and proliferation of ovarian cancer cells was verified by MTT assay;Transwell assay was performed to detect the effect of miR-186-5p on the invasion of ovarian cancer cells.The effect of miR-186-5p on the expression of signature protein during EMT was verified by WB;the effect of miR-186-5p on cellular cisplatin resistance was verified by MTT in cells stimulated with a gradient concentration of cisplatin.6.To validate the miR-186-5p downstream target gene Twist1Bioinformatics analysis predicted that miR-186-5p has a binding site to the 3’UTR region of Twist1.The interaction between miR-186-5p and Twist1 was verified by Dual luciferase reporter assay.qRT-PCR and Western Blot assays were performed to detect changes in Twist1 mRNA and protein expression after overexpression and inhibition of miR-186-5p,HAS2-AS1,respectively.7.To validate the HAS2-AS1/miR-186-5p/Twist1 axis by rescue experimentsThree cell lines were constructed by transfecting shNC,shHAS2-AS1,shHAS2-AS1 and miR-186-5p inhibitor in A2780 and UWB1.289,respectively.The expression of miR-186-5p and Twist1 at the RNA level in the three groups of cells was detected by qRT-PCR;the effect on Twistl protein expression was detected by Western Blot.The effects on the proliferation,invasive and metastatic ability and drug resistance of ovarian cancer cells were detected by MTT,plate cloning assay,Transwell assay,and cisplatin gradient concentration dosing assay to complete the exploration of the mechanism of HAS2-AS1/miR-186-5p/Twist1 axis regulating the biological function of ovarian cancer.Results1.The expression level of HAS2-AS1 was higher in HGSOC tissue specimens(p<0.001)than in fallopian tube tissues elevated HAS2-AS1 expression correlated with overall clinical survival time in patients(p=0.011).2.The results of cell function assay showed that HAS2-AS1 was able to promote abnormal growth of ovarian cancer in A2780 and UWB 1.289 cells by MTT assay compared to negative control,and the plate cloning assay showed that the ability of cell clone formation was significantly enhanced after overexpression of HAS2-AS1;Transwell assay results showed that after HAS2-AS1 overexpression,the number of A2780 and UWB 1.289 ovarian cancer cells moving down transwell chambers were significantly increased,and the invasion and migration ability of the cells were significantly enhanced,and the opposite cellular results were observed after interference with HAS2-AS1;the signature molecules of the EMT process(E-Cad,N-Cad,ZEB1,Vimentin)were changed,and interference with HAS2-AS1 expression,the expression of epithelial marker E-Cad was upregulated and the expression of mesenchymal markers N-Cad,ZEB1 and Vimentin was decreased,with opposite results after overexpression of HAS2-AS1.3.Drug resistance assays showed that HAS2-AS1 expression was elevated approximately 1.8-fold in A2780/DDP-resistant cells compared to the A2780 cell line.Gradient dosing results showed that silencing HAS2-AS1 expression resulted in decreased cell survival and increased cell sensitivity to cisplatin.Cellular activity increased after overexpression of HAS2-AS1 and cisplatin resistance increased.4.Bioinformatics analysis showed that HAS2-AS1 and miR-186-5p have complementary binding sites.Dual luciferase reporter assay showed that overexpression of miR-186-5p attenuated the fluorescence intensity of wild-type pmirGLO-HAS2-AS1 plasmid,and the fluorescence intensity of the plasmid did not change significantly after mutating the binding site,indicating that there was an interaction between miR-186-5p and HAS2-AS1.qRT-PCR assay results showed that inhibition of HAS2-AS1 expression was able to increase miR-186-5p expression level,and HAS2-AS1 could negatively regulate the expression of miR-186-5p.The expression of miR-186-5p was abnormally reduced in high-grade plasma ovarian cancer tissue specimens(p=0.0176),and the Person correlation showed that HAS2-AS1 was negatively correlated with miR-186-5p expression in tumor tissues.5.The results of miR-186-5p cell function assay showed that overexpression of miR-186-5p inhibited cell proliferation,invasive metastatic ability,and increased cell cisplatin sensitivity.6.Bioinformatics analysis revealed that the miR-186-5p seed sequence has a binding site to the 3’UTR region of Twist1 mRNA.Dual luciferase reporter assays showed that the miR-186-5p mimics group was able to significantly inhibit luciferase activity after transfection with wild-type Twist1 sequences,while the mutant plasmids showed no difference in the value of fluorescence intensity change,indicating that miR-186-5p was able to bind to Twist1 at the predicted site.Silencing of HAS2-AS1 expression or overexpression of miR-186-5p was able to significantly inhibit Twistl mRNA and protein expression levels;overexpression of HAS2-AS1 or inhibition of miR-186-5p was followed by an increase in Twistl expression levels,indicating a positive correlation between HAS2-AS1 and Twistl,and miR-186-5p negatively regulated the expression of Twist1.7.Rescue rescue experiments showed that cotransfection of shHAS2-AS1 and miR-186-5p inhibitor partially reversed the inhibition of proliferation and invasive migration ability of ovarian cancer cells induced by silencing HAS2-AS1,resulting in reduced sensitivity and increased resistance to cisplatin.qRT-PCR and Western blot results showed that Inhibition of HAS2-AS1 expression could lead to a decrease in Twistl expression at the mRNA and protein levels,and this inhibition was negatively regulated by miR-186-5p.It indicates that HAS2-AS1 can regulate Twist1 expression through competitive binding of miR-186-5p and promote the development of serous ovarian cancerConclusions1.The expression of HAS2-AS1 is commonly elevated in serous ovarian cancer and correlates with poor prognosis.2.HAS2-AS1 promotes abnormal proliferation and invasive metastasis of serous ovarian cancer and causes cisplatin resistance.Targeting blockade of HAS2-AS1 negatively regulates the malignant biological behavior of serous ovarian cancer.3.HAS2-AS1 regulates Twist1 expression through competitive binding of miR-186-5p and promotes the development of serous ovarian cancer.Innovations and significance of the research1.The study investigated the biological function of HAS2-AS1 in serous ovarian cancer,and demonstrated that HAS2-AS1 can promote the proliferation and growth of ovarian cancer,promote the invasive metastasis of ovarian cancer through the EMT process,and cause cisplatin resistance in ovarian cancer.2.The study clarified the role of HAS2-AS1/miR-186-5p/Twist1 regulatory axis in the development of serous ovarian cancer.Shortcomings of the research1.Although the study found the expression characteristics of HAS2-AS1 in ovarian cancer tissue samples,the practical application value of HAS2-AS1 needs to be expanded and verified in relation to clinical examples.2.The study did not construct a tumor transplantation model in nude mice,if we can conduct subcutaneous tumor formation and cisplatin intraperitoneal perfusion experiments in nude mice to verify the behavioral characteristics of HAS2-AS1 in vivo,it will provide a more solid theoretical basis for the experiments.