Long Non-Coding RNA OIP5-AS1 Promotes Malignant Phenotype And Adriamycin Resistance Of Breast Cancer By Targeting SOX2 Via MicroRNA-129-5p

Background and objectiveBreast cancer is a serious threat to women’s health.The statistical analysis of cancer in China suggests that one year in 2015,the number of new cases of breast cancer accounted for 15%of all new cancers in women.Statistical analysis of cancer in the United States in 2018 shows that the incidence of breast cancer is the first in women’s cancer incidence.Although breast cancer treatments are varied and developed,the mortality rate still ranges from 20%to 40%in patients treated according to the National Comprehensive Cancer Network guidelines,due to local recurrence and distant metastasis.Therefore,the development of breast cancer has been the attention focus of clinicians and scientists all over the world.Nowadays,RNA research has become a hot topic in life science.More than 98%of the genome is non-coding RNA(ncRNA)that does not have the function of protein coding.long non-coding RNA(lncRNA)is a ncRNA with a length longer than 200nt.More and more studies have shown that lncRNA mainly regulated gene regulation from epigenetic,transcriptional and post-transcriptional levels,and is involved in regulating physiological and pathological processes of cells including tumorigenesis.Studies have shown that lncRNA OIP5-AS1 is involved in the development of glioma,lung cancer and liver cancer.However,its expression pattern and biological function in breast cancer are not clear,and further studies are needed.This study researched the expression pattern of OIP5-AS1 in breast cancer and conducted the further study on the biological function of OIP5-AS1 in breast cancer in the level of cell and animal experiments.Moreover,we clarified that OIP5-AS1 promotes progression of breast cancer by targeting SOX2 via microRNA-129-5p(mIR129-5p).Our study provided a new potential target for breast cancer research.Materials and Methods1.Real-time quantitative PCR(qRT-PCR)method was used to detect the difference expression of OIP5-AS1 and miR-129-5p in breast cancer tissue and paired adjacent tissue samples,and the correlation between the expression level of OIP5-AS1 and miR129-5p with the clinicopathological features of the breast cancer patients was analyzed.2.The qRT-PCR method was used to detect the expression of OIP5-AS1 in different breast cancer cell lines and MDA-MB-231 and SK-BR-3 cell lines was chosen as the appropriate cell lines for the functional study.The methods of CCK-8 assay,colony formation assay,Transwell assay,wound healing assay and flow cytometry were used to detect the proliferation,colony formation ability,invasion ability,migration ability and anti-apoptotic ability after downregulation of OIP5-AS1.3.MDA-MB-231 in groups of sh-NC or sh-OIP5-AS1 was injected subcutaneously to nude mice for the formation of xenografts so that the function of OIP5-AS1 in breast cacer was studied in vivo.4.The qRT-PCR method was used to detect the expression of miR-129-5p in different breast cancer cell lines and MDA-MB-231 and SK-BR-3 cell lines was chosen as the appropriate cell lines for the functional study.The methods of CCK-8 assay,colony formation assay,Transwell assay,wound healing assay and flow cytometry were used to detect the proliferation,colony formation ability,invasion ability,migration ability and anti-apoptotic ability after upregulation of miR-129-5p.5.Luciferase reporter assay,qRT-PCR and western Blot assay were used to verify the relationship among the three of OIP5-AS1,miR-129-5p and SOX2.6.A rescue experiment was set up in the breast cancer cell line:sh-NC,sh-OIP5-AS 1,shOIP5-AS1+anti-miR-129-5p,sh-OIP5-AS1+SOX2,The methods of CCK-8 assay,colony formation assay,Transwell assay,wound healing assay and flow cytometry were used to detect the proliferation,colony formation ability,invasion ability,migration ability and anti-apoptotic ability in different groups of cells.7.qRT-PCR method was applied to detect the expression of OIP5-AS1 in Adriamycin resistance breast cancer cells and corresponding parental breast cancer cells.And Adriamycin resistance cells of MDA-MB-231(MDA-MB-231/ADR)was chosen for the functional experiments.CCK-8 assay was used to detected the cell viability of cells at different adriamycin concentrations after upregulation of OIP5-AS1,and the change of IC50 of Adriamycin was calculated.Flow cytometry assay was applied to detect the change of apoptosis level of MDA-MB-231/ADR cell lines after upregulation of OIP5AS1.Results1.Compared with normal breast tissues,the expression of OIP5-AS1 in breast cancer tissues was significantly upregulated and the expression of miR-129-5p in breast cancer tissues was significantly downregulated.High expression of OIP5-AS1 is associated with tumor size,lymph node metastasis,pathological grading and clinical staging while low expression of miR-129-5p is associated with pathological grading and clinical staging.2.After downregulation of OIP5-AS1 expression in breast cancer cell lines MDA-MB231 and SK-BR-3,the proliferation,colony formation,migration and invasion ability was inhibited and apoptosis rate was increased.Meanwhile,downregulation of OIP5AS1 expression can effectively inhibit the growth of subcutaneous implants in vivo.3.After upregulation of miR-129-5p expression in breast cancer cell lines MDA-MB-231 and SK-BR-3,the proliferation,colony formation,migration and invasion ability was inhibited and apoptosis rate was increased.4.There was binding sites of miR-129-5p existing in the sequence of OIP5-AS1,miR129-5p combined with the binding site and inhibit the activity of its luciferase.OIP5AS1 promoted the malignant phenotype of breast cancer by targeting SOX2 via functioning as a ceRNA of miR-129-5p.5.Downregulation of OIP5-AS1 expression in Adriamycin resistance breast cancer can effectively inhibit the cell viability at the same concentration of Adriamycin,IC50 of Adriamycin in sh-OIP5-AS 1 group was significantly lower than the group of sh-NC.Downregulation of OIP5-AS1 can increase the apoptosis rate of Adriamycin resistance breast cancer cell lines.ConclusionOur study explored the expression of lncRNA OIP5-AS1 in breast cancer and its promoting role in breast cancer malignant phenotype.Detection of clinical sample showed a high expression of OIP5-AS1 in breast cancer tissue samples and mechanism research showed that OIP5-AS1 promoted malignant phenotype of breast by targeting SOX2 via sponging miR-129-5p.What’s more,we showed a promote role of OIP5AS1 in Adriamycin resistance in breast cancer.Collectively,OIP5-AS1 is associated with the progression of breast cancer and Adriamycin resistance,and is expected to be a molecular marker for the diagnosis and treatment of breast cancer patients.