Oxymatrine induces anti-tumor response in cervical cancer by modulating circ_0008460/miR-197-3p/ribonucleotide reductase subunit M2 (RRM2)

Abstract

Oxymatrine (OMT) has exhibited an anti-cancer role in human cancers, including cervical cancer (CC). The dysregulated circular RNAs (circRNAs) are key regulators in cancer biology, and circ_0008460 was upregulated in CC. This study was performed to investigate the circRNA-based molecular mechanism for OMT in CC. RNA detection for circ_0008460, microRNA-197-3p (miR-197-3p), or ribonucleotide reductase subunit M2 (RRM2) was completed using reverse transcription-quantitative polymerase chain reaction assay. Cell behaviors were assessed by Cell Counting Kit-8 assay for cell viability, colony formation assay or Edu assay for cell proliferation, flow cytometry for cell apoptosis, and wound healing assay/transwell assay for migration/invasion. Protein expression examination was conducted using western blot. Dual-luciferase reporter assay and RNA pull-down assay were applied to confirm target binding. Tumor xenograft assay was performed for OMT research in vivo. OMT induced circ_0008460 downregulation in CC cells. OMT-induced inhibitory effects on cell growth, migration, and invasion but promoting effect on cell apoptosis were attenuated by circ_0008460. Circ_0008460 directly interacted with miR-197-3p, and OMT inhibited malignant behaviors of CC cells via mediating circ_0008460/miR-197-3p axis. RRM2 acted as a target for miR-197-3p and circ_0008460 affected the RRM2 level through absorbing miR-197-3p. OMT upregulated miR-197-3p to inhibit RRM2 expression to impede CC cell development. CC tumorigenesis was suppressed by OMT via targeting circ_0008460/miR-197-3p/RRM2 axis in vivo. These results suggested that OMT restrained CC cell progression in vitro and tumor growth in vivo by downregulating circ_0008460 to mediate miR-197-3p/RRM2 axis.

Keywords: Circ_0008460; RRM2; cervical cancer; miR-197-3p; oxymatrine.

Graphical abstract

Figure 1.

OMT reduced circ_0008460 expression in CC cells. (a-b) Circ_0008460 level was assayed via RT-qPCR in CC tissues (a) and CaSki/SiHa cells (b). (c-d) Linear WHSC1 and circ_0008460 levels were detected using RT-qPCR after RNase R treatment for total RNA. (e-f) Circ_0008460 expression was examined through RT-qPCR in control, 2 mg/mL, 4 mg/mL, and 6 mg/mL OMT groups of CaSki and SiHa cells. *P < 0.05.

Figure 2.

Circ_0008460 abrogated OMT-induced inhibition of CC cell malignant behaviors. (a) RT-qPCR was applied for assessing the overexpression efficiency of oe-circ_0008460. (b-k) CaSki and SiHa cells were divided into control, OMT (6 mg/mL), OMT+vector, and OMT+oe-circ_0008460 groups. (b) CCK-8 assay was used for determining cell viability. (c-d) Colony formation assay (c) and Edu assay (d) were employed for examining proliferation ability. (e) Flow cytometry was exploited for measuring the apoptosis rate. (f-g) Western blot was applied for assaying protein levels of apoptosis-related markers. (h-i) Wound healing assay was used for detecting cell migration. (j-k) Transwell assay was employed for evaluating cell migration (j) and invasion (k). *P < 0.05.

Figure 3.

Circ_0008460 served as a natural sponge for miR-197-3p. (a) Venn Diagram analysis of miRNA targets from starbase and circinteractome. (b-c) The miRNA targets for circ_0008460 were screened by pull-down assay. (d) Circ_0008460 and miR-197-3p binding prediction in starbase. (e) The efficacy of miR-197-3p mimic was assessed via RT-qPCR. (f-h) Target binding between circ_0008460 and miR-197-3p was confirmed through dual-luciferase reporter assay (f-g) and RNA pull-down assay (h). (i-k) RT-qPCR was performed for miR-197-3p level quantification in CC tissues (i), CaSki/SiHa cells (j), and 6 mg/mL OMT-treated CC cells (k). NC: normal control, *P < 0.05.

Figure 4.

OMT inhibited CC cell progression by regulating circ_0008460/miR-197-3p axis. CaSki and SiHa cells were treated with control, OMT (6 mg/mL), OMT+vector, OMT+oe-circ_0008460, OMT+oe-circ_0008460+ miR-NC, and OMT+oe-circ_0008460 + miR-197-3p. (a) Cell viability was measured using CCK-8 assay. (b-c) Cell proliferation was evaluated via colony formation assay (b) and Edu assay (c). (d-f) Cell apoptosis was analyzed through flow cytometry (d) and western blot (e-f). (g-j) Cell motility was assessed by wound healing assay (g-h) and transwell assay (i-j). NC: normal control, *P < 0.05.

Figure 5.

Circ_0008460 regulated RRM2 expression via sponging miR-197-3p. (a) RRM2 3ʹUTR sequence was predicted to have binding sites with miR-197-3p sequence in starbase. (b-c) Dual-luciferase reporter assay was used for interaction analysis between miR-197-3p and RRM2 3ʹUTR in CaSki and SiHa cells. (d-e) RT-qPCR and western blot were conducted for level examination of RRM2 after miR-NC or miR-197-3p transfection. (f-k) RRM2 mRNA and protein levels were determined in CC samples (f-g), CaSki/SiHa cells (h-i), and OMT-exposed CC cells (j-k) using RT-qPCR and western blot. (l-m) RRM2 mRNA and protein detection was performed via RT-qPCR and western blot in vector, oe-circ_0008460, oe-circ_0008460 + mi-NC, and oe-circ_0008460+ miR-197-3p groups. NC: normal control, *P < 0.05.

Figure 6.

OMT exhibited anti-cancer function in CC cells by upregulating miR-197-3p to downregulate RRM2. (a-b) Transfection efficiencies of anti-miR-197-3p (a) and si-RRM2 (b) were examined by RT-qPCR and western blot, respectively. (c-l) 6 mg/mL-treated CaSki and SiHa cells were transfected with anti-miR-197-3p, anti-miR-197-3p+si-RRM2, or relative controls. (c) CCK-8 assay was performed for viability detection. (d-e) The examination of cell proliferation was conducted using colony formation assay (d) and Edu assay (e). (f-h) Cell apoptosis analysis was implemented through flow cytometry (f) and western blot (g-h). (i-l) Wound healing assay (i-j) and transwell assay (k-l) were performed to assess cell motility. NC: normal control, *P < 0.05.

Figure 7.

OMT inhibited tumor growth of CC by downregulating circ_0008460 to mediate miR-197-3p/RRM2 axis in vivo. CaSki xenograft models of lenti-NC, OMT+lenti-NC, and OMT+lenti-circ_0008460 were constructed in mice. (a-b) Tumor volume (a) and weight (b) in nude mice. (c) Images of tumor tissues. (d-e) Circ_0008460 (d) and miR-197-3p (e) levels were assayed through RT-qPCR. (f-g) Western blot and IHC assays were used for protein determination of RRM2 (f) and Ki67 (g) in tumors. NC: normal control, *P < 0.05.