Silencing of circular RNA_0000326 inhibits cervical cancer cell proliferation, migration and invasion by boosting microRNA-338-3p-dependent down-regulation of CDK4

Abstract

Cervical cancer is one of the leading causes of cancer-related death among women, which is attributed partly by limited treatment options. Recent studies have provided in-depth explanations regarding the role of circular RNA in cancers. We aimed to investigate the role of circular RNA_0000326 in cervical cancer. Bioinformatics analysis revealed a high circ_0000326 expression in cervical cancer. Cervical cancer cells and tissues were also observed to have elevated levels of circ_0000326 and the upregulation of circ_0000326 depended on the stage of cancer. Transfection with siRNA of circ_0000326 resulted in the inhibition of proliferation, migration and cell cycle of cancer cells. Interestingly, we confirmed that circ_0000326 served as a sponge for microRNA-338-3p and that the miRNA bound to Cyclin-dependent kinase 4. In the presence of microRNA-338-3p mimic or silencing of circ_0000326, Cyclin-dependent kinase 4 expression was decreased. Transfection with microRNA-338-3p mimic inhibited cell clone formation and proliferation. Moreover, in vivo experiment revealed that the injection of shRNA-circ_0000326 lentivirus suppressed tumor growth and decreased Cyclin-dependent kinase 4 expression. Taken altogether, our results showed that circ_0000326 exerted oncogenic effects on cervical cancer by upregulating Cyclin-dependent kinase 4 via sponging microRNA-338-3p. This systematic investigation on circ_0000326 could provide further insight into cervical cancer.

Keywords: cervical cancer; circ_0000326; circular RNA; cyclin-dependent kinase 4; microRNA-338-3p.

Figure 1

Circ_0000326 is upregulated in cervical cancer. (A) Circ_0000326 expression in cervical cancer analyzed through GSE102686 (circRNA_000543 circBase stands for circ_0000326). (B) Circ_0000326 expression in 60 paired cervical cancer tissues and adjacent normal tissues determined by RT-qPCR. (C) Correlation between circ_0000326 expression and T classifications (T1-T4) in 60 cases of cervical cancer tissues. * p < 0.05 vs. normal tissues. Data were expressed as mean ± standard deviation and analyzed by paired t test. n = 60.

Figure 2

Circ_0000326 promotes proliferation of cervical cancer tissues. (A) Circ_0000326 expression in cervical cancer cell lines upon transfection with shRNA#1, shRNA#2, and shRNA#3. * p < 0.05 vs. the HaCaT cell line or NC. (B) The relative expression of TCONS_l2_00004572 and circ_0000326 after RNase R digestion, * p < 0.05 vs. MOCK. (C) Relative expression of circ_0000326 in nuclear and cytoplasm. * p < 0.05 vs. Cytoplasm. (D) Subcellular localization of circ_0000326 determined by FISH (× 400). (E) Clone formation assay of circ_0000326-silenced cervical cancer cells. (F) The number of cell clones of circ_0000326-silenced cervical cancer cells in clone formation assay. (G) The proliferation SiHa and Hela cell after silence of circ_0000326 determined by EdU assay (× 200). (H) EDU-positive cell rate of SiHa and Hela cells. (I) The cell viability of circ_0000326 silenced SiHa and Hela cells detected by CCK-8. (J) Protein expression of cell cycle-associated proteins (cyclinD1, P21 and P27) in cells after treatment of circ_0000326 determined by Western blot analysis. From figure (D–J), * p < 0.05 vs. NC treatment. Data were expressed as mean ± standard deviation. The data between two groups were analyzed by unpaired t-test with independent sample while the data among multiple groups was analyzed by ANOVA followed by Tukey’ s post hoc test. Data at different time points were analyzed by Two-Way ANOVA.

Figure 3

Silence of circ_0000326 impairs cervical cancer cell migration and invasion. (A) Cervical cancer cell migration and invasion in HeLa and SiHa cell lines after silence of circ_0000326 detected by Transwell assay (× 200). (B) Number of migrating and invasive cells in HeLa and SiHa cell lines after silence of circ_0000326. (C) The influence of circ_0000326c silencing on the expression of MMP-2 and MMP-9 detected using Western blot analysis. * p < 0.05 vs. SiHa and Hela cells treated with NC. Data were expressed as mean ± standard deviation and analyzed by unpaired t test. Experiments were conducted 3 times independently.

Figure 4

miR-338-3p binds to circ_0000326. (A) The potential binding site between circ_0000326 and miR-338-3p predicted by database. (B) The binding relationship between circ_0000326 and miR-338-3p detected by dual-luciferase reporter gene assay. * p < 0.05 vs. NC. (C) RTq-PCR analysis of miR-338-3p following RNA pull-down assays with circ-0000326 probes in SiHa and Hela cells. * p < 0.05. (D) Circ_0000326 co-precipitation with Ago2 using RIP, * p < 0.05 vs. IgG. (E) RTq-PCR analysis of miR-338-3p expression after sh-circ-0000326, * p < 0.05 vs. NC. Data were expressed as mean ± standard deviation and analyzed by unpaired t test.

Figure 5

Circ_0000326 promotes CDK4 expression by functioning as a sponge of miR-338-3p. (A) CDK4 expression in cervical cancer tissues predicted by TCGA database. (B) Representative images of immunohistochemistry (× 200) and positive-CDK4 ratio. (C) RT-qPCR analysis of CDK4 expression in 60 paired cervical cancer tissues and adjacent normal tissues. (D) The binding relationship between CDK4 and miR-338-3p detected using dual-luciferase reporter assay. (E, F) CDK4 mRNA expression in SiHa and Hela cells after alteration of miR-338-3p and silencing of circ_0000326 measured by RT-qPCR. (G–I) CDK4 protein expression in SiHa and Hela cells after treatment of circ_0000326 detected by Western blot analysis. Data were expressed as mean ± standard deviation. The data between two groups were analyzed by unpaired t-test with independent sample while the data among multiple groups was analyzed by ANOVA followed by Tukey’ s post hoc test.

Figure 6

Circ_0000326 promotes cervical cancer cell proliferation by targeting miR-338-3p. (A) Representative images of clone formation assay upon treatment with NC, miR-338-3p-mimic, miR-338-3p-inhibitor or sh-circ_0000326 + miR-338-3p-inhibitor. (B) Cervical cancer cell proliferation measured by EdU (× 200). (C) Cervical cancer cell proliferation rates measured by EdU. (D) OD value of cervical cancer cells determined by CCK-8. (E) The protein expression of cell cycle-related gene using Western blot analysis. * p < 0.05. vs. SiHa and Hela cells treated with NC. Data were expressed as mean ± standard deviation. The data between two groups were analyzed by unpaired t-test with independent sample while the data among multiple groups was analyzed by ANOVA. Data at different time points were analyzed by Two-Way ANOVA.

Figure 7

Circ_0000326 promotes cervical cancer cell migration and invasion by targeting miR-338-3p. (A) Cervical cancer cell migration and invasion in HeLa and SiHa cell lines after miR-338-3p mimic or miR-338-3p inhibitor detected by Transwell assay (× 200). (B) Number of migrating and invasive cells in HeLa and SiHa cell lines after silence of circ_0000326. (C) Western blot analysis of MMP-2 and MMP-9 after treatment with miR-338-3p mimic or miR-338-3p inhibitor. Data were expressed as mean ± standard deviation and the data among multiple groups was analyzed by ANOVA.

Figure 8

Silencing of Circ_0000326 inhibits tumor growth. (A) Effect of intratumoral LV-sh-circ_0000326 on tumor growth in BALB/c nude mice. (B) Representative images of the xenograft tumors in each group 3 weeks after subcutaneous implantation of SiHa cells stably infected with sh-circ_0000326 lentivirus or control lentivirus (LV-NC). (C) Effect of sh-circ_0000326 on tumor weight 3 weeks after subcutaneous injection of SIHA cells stably infected lv-sh-circ_0000326 or LV-NC. (D) Tumor sections were stained for CDK4 (in brown) and quantified by counting CDK4 positive staining area (pixels) (× 200). * p < 0.05. Data were expressed as mean ± standard deviation and verified by t-test, n = 10.