Pathological significance of circular RNA-101748 expression in ovarian cancer and its effect on proliferation

Objective To investigate the pathological significance of circular RNA-101748(circ-101748) expression in serous ovarian cancer (SOC) and the effect of regulating microRNA(miR)-340 expression on ovarian cancer cell proliferation.

Methods A total of 69 cases of SOC were collected. The positive expression of circ-101748 in serous ovarian cancer was detected by RNA in situ hybridization (RISH), and the relationship between circ-101748 positive expression and the pathological characteristics of SOC was statistically analyzed; Methyl Thiazolyl Tetrazolium(MTT) proliferation activity assay was used to detect the effect of circ-101748 on the proliferation of ovarian cancer cells; bioinformatics predicts the circ-101748 downstream microRNA and microRNA downstream target protein; quantitative real-time polymerase chain reaction (qRT-PCR) and double luciferase report experiments were used to verify the regulatory effect of circ-101748 on miR-340 and that of miR-340 on Ki-67; the rescue test was used to confirm the effects of the circ-101748/miR-340 signal axis on regulating Ki-67 and the proliferation of ovarian cancer cells.

Results RISH results showed that circ-101748 positive staining was brown yellow, located in the cytoplasm and nucleus; the number of positive circ-101748 cases was 73.91%(51/69), which was higher than 11.59% (8/69) in adjacent normal tissues, and the difference was statistically significant (P < 0.05). In SOC, the positive expression of circ-101748 in SOC was closely correlated with tumor size (χ²=9.763, P < 0.05) and tumor stage (χ²=9.656, P < 0.05), but not correlated with age, pathological grade or lymph node metastasis (P>0.05). MMT assay showed that the proliferative activity of ovarian cancer cells increased after overexpression of circ-101748, while the proliferative activity of ovarian cancer cells decreased significantly after decreased expression of circ-101748 (P>0.05). It was predicted by circBANK that miR-340 was the target gene of circ-101748; the result of qRT-PCR showed that the expression level of miR-340 decreased significantly after overexpression of circ-101748 in ovarian cancer cells (P < 0.05), and the expression level of miR-340 increased significantly after decreasing the expression of circ-101748 (P < 0.05); the double luciferase report experiment confirmed that circ-101748 could specifically bind to miR-340 and negatively regulate the expression of miR-340. The proliferation protein Ki-67 was predicted to be the downstream target gene of miR-340 by TargetScan and miRbase. Further double luciferase report experiment verified that the wild type Ki-67 3'untranslated region could reduce the luciferase activity of miR-340, thus miR-340 could inhibit the expression of Ki-67 protein through Ki-67 3'UTR. The results of rescue experiment showed that overexpression of miR-340 could eliminate the effect of circ-101748 on the expression of Ki-67 in ovarian cancer cells, while overexpression of circ-101748 or reduction of miR-340 could further stimulate the expression of Ki-67 in ovarian cancer cells; the circ-101748 could directly inhibit the expression of miR-340, while miR-340 could directly inhibit the expression of Ki-67. Circ-101748/miR-340/Ki-67 signaling pathway played a significant role in promoting the growth and proliferation of ovarian cancer.

Conclusion Circ-101748 is an oncogene in ovarian cancer, which can promote the proliferation and growth of ovarian cancer cells by regulating miR-340/Ki-67 signaling axis.