CirSLC7A6 may affect the chemoresistance of ovarian cancer through regulation of miR-2682-5p and downstream gene SLC7A6

To investigate differentially expressed circRNAs in chemotherapy-sensitive and -resistant cells, high-throughput transcriptomic sequencing and bioinformatics analysis were performed. Results of the present study demonstrated that the expression of CirSLC7A6 was significantly increased in A2780/CDDP cells (Fig. 1A). Furthermore, CirSLC7A6 may be involved in the regulation of miR-2682-5p, and enriched biological functions mainly included cell proliferation, metabolism and adhesion (Fig. 1B-D). As shown in Fig. 1E, results of the RT-qPCR analysis demonstrated that CirSLC7A6 was markedly upregulated in A2780/CDDP cells, compared with A2780 cells (P < 0.05). MiR-2682-5p was downregulated in A2780/CDDP cells compared with that in A2780 cells (P < 0.05). Results of the present study demonstrated that the expression of CirSLC7A6 and miR-2682-5p were consistent with the results obtained from high-throughput transcriptomic sequencing. Moreover, results of the present study demonstrated that SLC7A6 was highly expressed in A2780/CDDP cells compared to A2780 cells (P < 0.05), which was consistent with the expression of CirSLC7A6. However, the opposite was found for the expression of miR-2682-5p. Therefore, CirSLC7A6 was identified as a molecular target for subsequent research on the chemoresistance of ovarian cancer. We hypothesized that CirSLC7A6 possesses a regulatory association with miR-2682-5p, and SLC7A6 may act as a downstream regulatory gene.

Fig. 1

CirSLC7A6 may affect the chemoresistance of ovarian cancer through regulating miR-2682-5p and downstream gene SLC7A6. A Network of circRNAs and genes in A2780 and A2780/CDDP cells. B GO and KEGG pathway analysis. C Heatmap. D Binding sites of CirSLC7A6 and miR-2682-5p. Reverse transcription-quantitative PCR was carried out to detect the expression of CirSLC7A6, miR-2682-5p and SLC7A6 in (E) A2780 and A2780/CDDP cells, (F) A2780/CDDP shGFP and A2780/CDDP shCirSLC7A6 cells. And (G) A2780 Vector and A2780 CirSLC7A6 cells. circRNA, circular RNA; miRNA, microRNA; SLC7, solute carrier family 7; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes

To verify the regulatory association between CirSLC7A6 and miR-2682-5p, and to further explore the mechanism of CirSLC7A6 regulating the chemoresistance of ovarian cancer, CirSLC7A6 was silenced in A2780/CDDP cells and overexpressed in A2780 cells. Transfection efficiency was verified using RT-qPCR. As shown in Fig. 1F, the expression of miR-2682-5p was significantly increased, while the expression of SLC7A6 was significantly decreased following knockdown of CirSLC7A6 (P < 0.05). However, overexpressing CirSLC7A6 exhibited the opposite effect on the expression of miR-2682-5p and SLC7A6 (P < 0.05; Fig. 1G). These results suggested that there is a regulatory association between CirSLC7A6 and miR-2682-5p, and SLC7A6 is a downstream regulatory gene. CirSLC7A6 may affect the uptake of amino acids, resulting in the chemoresistance of ovarian cancer cells via suppression of miR-2682-5p and activation of SLC7A6.

CirSLC7A6 knockdown increases the anti-proliferative and pro-apoptotic effects of combined cisplatin and baicalein in resistant ovarian cancer cells

To investigate whether baicalein suppresses the proliferation and promotes the apoptosis of ovarian cancer cells via the regulation of CirSLC7A6/miR-2682-5p/SLC7A6, a CCK-8 assay and flow cytometry were performed. The sensitivity of normal ovarian epithelial cells (T29) to baicalein was lower than that of ovarian cancer cells (Fig. 2B-D), suggesting that baicalein exerted a lower cytotoxicity in normal ovarian epithelial cells.

Fig. 2

CirSLC7A6 knockdown increases the anti-proliferative and pro-apoptotic effects of the combined treatment of cisplatin and baicalein on resistant ovarian cancer cells. A Chemical structure of baicalein. B Baicalein inhibits the proliferation of T29 cells. C Cisplatin inhibits the proliferation of six ovarian cancer cell types. D Baicalein inhibits the proliferation of six ovarian cancer cell types. E Comparison of the inhibition of six ovarian cancer cell types treated with the same drug. F Comparison of the inhibition of six ovarian cancer cell types treated with different drugs. G Comparison of the apoptosis rates of six ovarian cancer cell types treated with the same drug. H Comparison of the apoptosis rate of A2780 cells treated with different drugs. I Comparison of the apoptosis rate of A2780/CDDP cells treated with different drugs. J Comparison of the apoptosis rate of A2780/CDDP shGFP cells treated with different drugs. K Comparison of the apoptosis rate of A2780/CDDP shCirSLC7A6 cells treated with different drugs. L Comparison of the apoptosis rate of A2780 Vector cells treated with different drugs. M Comparison of the apoptosis rate of A2780 CirSLC7A6 cells treated with different drugs. circRNA, circular RNA; SLC7, solute carrier family 7; shRNA, short hairpin RNA

Results of the CCK-8 assay and flow cytometry (Fig. 2E-M) demonstrated that the sensitivity of A2780 cells to cisplatin was higher than that of A2780/CDDP cells, and cisplatin effectively attenuated the proliferation and promotes the apoptosis of A2780 cells. However, CirSLC7A6 knockdown did not increase the anti-proliferation and pro-apoptotic effects of cisplatin in A2780/CDDP cells, which indicated that knockdown of CirSLC7A6 may not improve cisplatin sensitivity. In addition, overexpression of CirSLC7A6 reduced the anti-proliferative and pro-apoptotic effects of cisplatin in A2780 cells and reduced cisplatin sensitivity. Baicalein exhibited similar anti-proliferative effects on A2780 and A2780/CDDP cells; however, the pro-apoptotic effects were higher in A2780/CDDP cells. CirSLC7A6 knockdown enhanced the anti-proliferative and pro-apoptotic effects of baicalein in A2780/CDDP cells. Overexpression of CirSLC7A6 attenuated the anti-proliferative and pro-apoptotic effects of baicalein in A2780 cells. Compared with A2780 cells, a combination of cisplatin and baicalein exhibited stronger anti-proliferative effects and weaker pro-apoptotic effects in A2780/CDDP cells. Knockdown of CirSLC7A6 significantly promoted the anti-proliferative and pro-apoptotic effects of the combination of cisplatin and baicalein in A2780/CDDP cells. Overexpression of CirSLC7A6 exerted the opposite effect in A2780 cells. A combination of cisplatin and baicalein was superior to either cisplatin or baicalein treatment alone in suppressing cell proliferation and inducing cell apoptosis in A2780 and A2780/CDDP cells. These results suggested that baicalein may inhibit cell proliferation in coordination with cisplatin. CirSLC7A6 knockdown markedly increased the chemosensitivity of baicalein, and significantly improved the anti-proliferative and pro-apoptotic effects of the combination of cisplatin and baicalein in A2780/CDDP cells. However, overexpression of CirSLC7A6 significantly reduced the chemosensitivity of baicalein.

Baicalein suppresses the metastasis of resistant ovarian cancer via the regulation of CirSLC7A6

To investigate whether baicalein inhibits cell metastasis in ovarian cancer, wound healing and Transwell assays were conducted. As shown in Fig. 3A, there was no significant difference in cell mobility between A2780 and A2780/CDDP cells (P > 0.05). Similarly, there was no significant difference in cell mobility between A2780/CDDP shCirSLC7A6 cells and A2780/CDDP shGFP cells (P > 0.05). There was no significant difference in cell mobility between A2780 CirSLC7A6 cells and A2780 Vector cells (P > 0.05). These results suggested that CirSLC7A6 expression exhibited no significant effects on the migration of ovarian cancer cells.

Fig. 3

Baicalein suppresses the migration and invasion of resistant ovarian cancer cells through regulating CirSLC7A6. A Intercellular comparison of the migration rate. B Intracellular comparison in A2780 and A2780/CDDP cells. C Intracellular comparison in A2780/CDDP shGFP and A2780/CDDP shCirSLC7A6 cells. D Intracellular comparison in A2780 Vector and A2780 CirSLC7A6 cells. E Intercellular comparison of cell invasion. F Intracellular comparison in A2780 and A2780/CDDP cells. G Intracellular comparison in A2780/CDDP shGFP and A2780/CDDP shCirSLC7A6 cells. H Intracellular comparison in A2780 Vector and A2780 CirSLC7A6 cells. circRNA, circular RNA; SLC7, solute carrier family 7; shRNA, short hairpin RNA

As shown in Fig. 3B, in A2780 cells, the cell migration of the control group was higher than that of all treatment groups (P < 0.05). The cell mobility of the cisplatin group was higher than that of the baicalein group (P < 0.05). There was no statistical difference between the baicalein group and the group treated with a combination of cisplatin and baicalein (P > 0.05). These results indicated that the inhibitory effects of baicalein were stronger than cisplatin; however, a combination of the two drugs did not inhibit the migration of A2780 cells. In A2780/CDDP cells, cell migration of the control group was higher than that of all treatment groups (P < 0.05). Cell mobility of the cisplatin group was higher than that of the baicalein group (P < 0.05). In addition, cell migration in the baicalein group was higher than the group treated with a combination of both drugs (P < 0.05). These results revealed that baicalein may inhibit the migration of A2780/CDDP cells more effectively than cisplatin. Cisplatin and baicalein potentially synergically inhibit the migration of A2780/CDDP cells.

As shown in Fig. 3C, in A2780/CDDP shCirSLC7A6 cells, migration of the control group was higher than that of all treatment groups (P < 0.05). The cell mobility of the cisplatin group was significantly higher than that of the baicalein group (P < 0.05). Cell migration of the baicalein group was higher than that of the group treated with a combination of both drugs (P < 0.05). These results indicated that migration inhibition is significantly enhanced following treatment with a combination of both drugs, compared with groups treated with either baicalein or cisplatin alone in A2780/CDDP shCirSLC7A6 cells.

As shown in Fig. 3D, in A2780 CirSLC7A6 cells, migration in the control group was 12.6%, which was higher than that of all treatment groups (P < 0.05). However, there was no statistical difference among cisplatin, baicalein and the combined treatment groups (P > 0.05). These results indicated that a combination of both drugs may not inhibit the migration of A2780 CirSLC7A6 cells.

As shown in Fig. 3E, A2780 cells were more invasive than A2780/CDDP cells (P < 0.05), and the invasion of A2780/CDDP shCirSLC7A6 cells was significantly increased, compared with A2780/CDDP shGFP cells (P < 0.05). However, the invasion of A2780 CirSLC7A6 cells was significantly decreased compared with A2780 Vector cells (P < 0.05). The results demonstrated that invasion of A2780/CDDP cells is lower than that of A2780 cells. CirSLC7A6 knockdown enhanced the invasion of A2780/CDDP cells, while overexpression of CirSLC7A6 attenuated the invasion of A2780 CirSLC7A6 cells.

As shown in Fig. 3F, in A2780 cells, invasion of the control group was higher than that of all treatment groups (P < 0.05). In addition, invasion of the cisplatin group was lower than that of the baicalein group (P < 0.05), while invasion of groups treated with either cisplatin or baicalein alone was higher than that of the group treated with a combination of both drugs (P < 0.05). Treatment of both cisplatin and baicalein in combination inhibited the invasion at a higher level, than either drug alone. In A2780/CDDP cells, the invasion of the control group was higher than that of all treatment groups (P < 0.05), but there was no statistical difference among treatment groups (P > 0.05). Cisplatin and baicalein exerted similar inhibitory effects on the invasion of A2780/CDDP cells, and a combination of both drugs did not synergically inhibit cell invasion.

As shown in Fig. 3G, in A2780/CDDP shCirSLC7A6 cells, invasion of the control group was higher than that of all treatment groups (P < 0.05), and invasion of the cisplatin group was lower than that of the baicalein group (P < 0.05). In addition, invasion in groups treated with either cisplatin or baicalein alone was significantly higher than that of the group treated with a combination of both drugs (P < 0.05). The results demonstrated that CirSLC7A6 knockdown enhanced the synergistic inhibitory effects of cisplatin and baicalein on the invasion of A2780/CDDP cells.

As shown in Fig. 3H, in A2780 CirSLC7A6 cells, invasion of the control group was higher than that of the cisplatin group (P < 0.05), and invasion of the control group was lower than that of the baicalein or combined treatment groups (P < 0.05). Invasion of the baicalein group was significantly higher than that of the combined treatment group (P < 0.05). These results suggested that overexpression of CirSLC7A6 attenuates the inhibitory effects of baicalein and a combination of both drugs on the invasion of A2780 cells.

CirSLC7A6 knockdown enhances the synergistic inhibitory effects of cisplatin and baicalein on tumor growth in ovarian cancer

To further verify the chemosensitivity of baicalein through the regulation of CirSLC7A6/miR-2682-5p/SLC7A6 in ovarian cancer cells, tumor xenografts were established. Consistent with the previous results of the present study, as shown in Fig. 4A, in the tumor model subcutaneously transplanted with A2780 cells, the tumor weight of the control group was higher than that of the treatment groups (P < 0.05). The tumor weight of the cisplatin group was significantly lower than that of the baicalein group (P < 0.05). The tumor weight of the combined treatment group was lower than that of either the cisplatin or baicalein groups (P < 0.05). In addition, the tumor volume of the control group was larger than that of the treatment groups (P < 0.05). The tumor volume of the cisplatin group was smaller than that of the baicalein group (P < 0.05). The tumor volume of the combined treatment group was significantly lower than that of either the cisplatin or baicalein groups (P < 0.05). These results suggested that cisplatin exhibited an improved inhibition on tumor growth than baicalein. A combination of cisplatin and baicalein synergically suppressed tumor growth.

Fig. 4

CirSLC7A6 knockdown enhances the synergistic inhibitory effects of cisplatin and baicalein on the tumor growth of ovarian cancer. A Tumors subcutaneously transplanted with A2780 cells. B Tumors subcutaneously transplanted with A2780/CDDP cells. C Tumors subcutaneously transplanted with A2780/CDDP shGFP cells. D Tumors subcutaneously transplanted with A2780/CDDP shCirSLC7A6 cells. E–F Cisplatin plus baicalein causes no injury in the liver or kidney of mice transplanted with A2780/CDDP shCirSLC7A6 cells. circRNA, circular RNA; SLC7, solute carrier family 7; shRNA, short hairpin RNA

As shown in Fig. 4B, in the tumor model subcutaneously transplanted with A2780/CDDP cells, the tumor weight of the control group was higher than that of the treatment groups (P < 0.05). There was no statistical difference in tumor weight between the cisplatin and baicalein groups (P > 0.05). The tumor weight of the combined treatment group was significantly lower than that of either the cisplatin or baicalein groups (P < 0.05). In addition, the tumor volume of the control group was larger than that of the treatment groups (P < 0.05). There was no significant difference in tumor volume between the cisplatin and baicalein groups (P > 0.05). The tumor volume of the combined treatment group was significantly smaller than that of either the cisplatin or baicalein groups (P < 0.05). These data indicated that cisplatin and baicalein exerted similar inhibitory effects on tumor growth. The combination of cisplatin and baicalein synergically inhibited tumor growth.

As shown in Fig. 4D, in the tumor model subcutaneously transplanted with A2780/CDDP shCirSLC7A6 cells, the tumor weight of the control group was higher than that of the treatment groups (P < 0.05). The tumor weight of the cisplatin group was higher than that of the baicalein group (P < 0.05). The tumor weight of the combined treatment group was significantly lower than that of the cisplatin and baicalein groups (P < 0.05). In addition, the tumor volume of the control group was larger than that of the treatment groups (P < 0.05), but there was no significant difference in tumor volume between the cisplatin and baicalein groups (P > 0.05). The tumor volume of the combined treatment group was significantly lower than that of the cisplatin and baicalein groups (P < 0.05). Baicalein exhibited a higher inhibition on tumor growth than cisplatin. The combination of cisplatin and baicalein exerted significantly synergistic effects.

To identify the toxicity of cisplatin and baicalein on the liver and kidney, H&E staining was performed. H&E staining of the liver (Fig. 4E) demonstrated that in the tumor model subcutaneously transplanted with A2780 cells, there was no congestion in the control or cisplatin groups. Notably, congestion occurred in the baicalein and combined treatment groups, and it was more severe in the combined treatment group, suggesting that the combined treatment of cisplatin and baicalein may increase toxicity to the liver. In the tumor model subcutaneously transplanted with A2780/CDDP cells, congestion in treatment groups was more severe than in the control group. Congestion in the baicalein group was more severe than that of the combined treatment group, which indicated that the toxicity of the combined treatment of cisplatin and baicalein in the liver may be weakened. In the tumor model subcutaneously transplanted with A2780/CDDP shCirSLC7A6 cells, congestion in the treatment groups was more severe than in the control group. There was no significant difference among treatment groups, indicating that the combined treatment did not increase liver injury.

H&E staining of the kidney (Fig. 4F) demonstrated that in the tumor model subcutaneously transplanted with A2780 cells, there was no renal tubular expansion in the control or baicalein groups. Notably, renal tubular expansion occurred in the cisplatin and combined treatment groups. These findings demonstrated that baicalein was less toxic on the kidney than cisplatin or a combination of cisplatin and baicalein. However, the combined treatment of cisplatin and baicalein did not increase renal toxicity. In the tumor model subcutaneously transplanted with A2780/CDDP cells, there was no renal tubular expansion in the control group. Notably, renal tubular expansion in the cisplatin and combined treatment groups was more severe than that in the baicalein group. These findings demonstrated that cisplatin and a combination of cisplatin and baicalein exerted increased toxicity in the kidney. However, the combined treatment of both drugs did not increase renal toxicity. In the tumor model subcutaneously transplanted with A2780/CDDP shCirSLC7A6 cells, renal tubular expansion was only found in the cisplatin group, indicating that the toxicity of cisplatin in the kidney was greater than that of either baicalein or a combined treatment of cisplatin and baicalein. Notably, baicalein alleviated the renal toxicity of cisplatin.

Baicalein inhibits the proliferation and metastasis of resistant ovarian cancer cells through regulation of CirSLC7A6

RT-qPCR and immunohistochemistry were used to detect the expression of CirSLC7A6, miR-2682-5p and SLC7A6 in tumor tissues. As shown in Fig. 5A, results of the RT-qPCR analysis indicated that the expression of CirSLC7A6 and SLC7A6 in the A2780/CDDP cell xenograft were notably higher than those in the A2780 cell xenograft; however, miR-2682-5p expression was decreased (P < 0.05). The expression levels of CirSLC7A6 and SLC7A6 in the A2780/CDDP shCirSLC7A6 cell xenograft were significantly lower than those in the A2780/CDDP shGFP cell xenograft; however, miR-2682-5p was increased (P < 0.05). As shown in Fig. 5B, in the A2780, A2780/CDDP and A2780/CDDP shCirSLC7A6 cell xenografts, baicalein and the combined treatment of both drugs reduced the expression of CirSLC7A6 and SLC7A6, and increased the expression of miR-2682-5p (P < 0.05). Notably, cisplatin promoted the expression of CirSLC7A6 and SLC7A6, and inhibited the expression of miR-2682-5p in the A2780/CDDP cell xenograft (P < 0.05).

Fig. 5

Reverse transcription-quantitative PCR indicated that the expression levels of CirSLC7A6 were significantly decreased, and a combination of cisplatin and baicalein downregulated the expression of CirSLC7A6 in the A2780/CDDP shCirSLC7A6 cell xenograft. A Intercellular comparison. B Intracellular comparison. C-D Immunohistochemical results demonstrated that the expression levels of SLC7A6 were significantly decreased and the combined treatment of cisplatin and baicalein downregulated the expression of SLC7A6 in the A2780/CDDP shCirSLC7A6 cell xenograft. circRNA, circular RNA; SLC7, solute carrier family 7; shRNA, short hairpin RNA

As shown in Fig. 5(C-D), results of the immunohistochemical analysis demonstrated that the expression of SLC7A6 was significantly higher in the A2780/CDDP cell xenograft than that in the A2780 cell xenograft (P < 0.05). The expression of SLC7A6 was markedly lower in the A2780/CDDP shCirSLC7A6 cell xenograft than in the A2780/CDDP shGFP cell xenograft (P < 0.05). In the A2780, A2780/CDDP and A2780/CDDP shCirSLC7A6 cell xenograft, the combined treatment of both drugs suppressed the expression of SLC7A6 (P < 0.05). Animal experiments further confirmed that CirSLC7A6 may regulate the expression of miR-2682-5p and SLC7A6, and CirSLC7A6 knockdown enhanced the inhibitory effects of the combined treatment of both drugs on SLC7A6.

Baicalein attenuates PI3K/Akt/mTOR and Erk/p38 MAPK pathways and sensitizes cisplatin via the regulation of CirSLC7A6 in resistant ovarian cancer cells

To further elucidate the role of baicalein in the cisplatin sensitivity of resistant ovarian cancer cells, western blot analysis was carried out to measure the protein expression levels of PI3K-p85α, P-Akt, Akt, P-mTOR, mTOR, P-Erk, Erk, P-p38, p38 and Bcl-2 (Fig. 6A-C). These results demonstrated that knockdown of CirSLC7A6 potentially inhibits the phosphorylation of Akt, mTOR, Erk and p38, and reduces the activity of Bcl-2, thereby inhibiting cell proliferation and promoting cell apoptosis.

Fig. 6

Baicalein inhibits the proliferation of resistant ovarian cancer cells through regulating CirSLC7A6. A Protein bands. B The quantitative histograms of the intercellular comparison between A2780 and A2780/CDDP cells. C The quantitative histograms of the intercellular comparison between A2780/CDDP shGFP and A2780/CDDP shCirSLC7A6 cells. D The quantitative histograms of the intracellular comparison in A2780 and A2780/CDDP cells. E The quantitative histograms of the intracellular comparison in A2780/CDDP shGFP and A2780/CDDP shCirSLC7A6 cells. Baicalein inhibits the metastasis of resistant ovarian cancer cells through regulating CirSLC7A6. F Protein bands. G The quantitative histograms of the intercellular comparison. H The quantitative histograms of the intracellular comparison. circRNA, circular RNA; SLC7, solute carrier family 7; shRNA, short hairpin RNA

As shown in Fig. 6D, in A2780 cells, all treatment options inhibit the phosphorylation of mTOR and decrease the expression of Bcl-2, thereby suppressing proliferation and promoting apoptosis of A2780 cells. In A2780/CDDP cells, all treatment options inhibited the phosphorylation of Akt, mTOR, Erk and p38, and decreased the activity of Bcl-2, thereby inhibiting proliferation and promoting apoptosis of A2780/CDDP cells.

As shown in Fig. 6E, in A2780/CDDP shCirSLC7A6 cells, cisplatin not only inhibits the activation of PI3K/Akt/mTOR signaling, but also inhibits the expression of P-Erk, P-p38 and Bcl-2. On the other hand, baicalein reduced the phosphorylation of mTOR and Erk, and reduced the overall expression of Bcl-2. By contrast, the combined treatment of cisplatin and baicalein exerted anti-tumor effects through inhibition of the phosphorylation of Akt, mTOR and Erk, and the expression of Bcl-2.

These findings suggested that CirSLC7A6 knockdown inhibits the phosphorylation of Akt, mTOR, Erk and p38, and reduces the expression of Bcl-2; however, it does not increase the sensitivity of ovarian cancer cells to cisplatin. The mechanism of resistance to cisplatin is complex. Although CirSLC7A6/miR-2682-5p/SLC7A6 and downstream pathways are suppressed, compensation mechanisms may be activated. CirSLC7A6 knockdown alone did not improve the sensitivity of cisplatin. A combination of both cisplatin and baicalein may inhibit the CirSLC7A6/miR-2682-5p/SLC7A6 pathway and block the compensation of amino acid transportation. This may increase the intracellular cisplatin concentration; thus, inhibiting the phosphorylation of Akt, mTOR and Erk, and decreasing the level of Bcl-2, leading to ovarian cancer cells apoptosis.

To investigate the molecular mechanism underlying baicalein-regulated cell metastasis, western blot analysis was carried out to determine the protein expression levels of MMP2 (Fig. 6F-H). These results indicated that CirSLC7A6 knockdown inhibits the expression of MMP2. The combination of both cisplatin and baicalein further significantly reduced the protein expression level of MMP2, and subsequently suppressed the metastasis of ovarian cancer cells.