Long non-coding RNA tumor protein 53 target gene 1 promotes cervical cancer development via regulating microRNA-33a-5p to target forkhead box K2

Abstract

Long non-coding RNA tumor protein 53 target gene 1 (TP53TG1) has been unraveled to exert regulatory effects on cancer progression, while the regulatory function of TP53TG1 on cervical cancer (CC) via regulating microRNA (miR)-33a-5p/Forkhead box K2 (FOXK2) axis remains rarely explored. This study aims to unearth the regulatory mechanism of TP53TG1/miR-33a-5p/FOXK2 axis in CC. The CC clinical samples were collected, and CC cells were cultured. TP53TG1, miR-33a-5p and FOXK2 levels were examined in CC tissues and cells. The CC cells were transfected with high- or low-expressed TP53TG1, FOXK2 or miR-33a-5p to determine the changes of CC cell biological activities and the status of phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. The tumorigenesis in nude mice was conducted. The relationship among TP53TG1, miR-33a-5p and FOXK2 was validated. TP53TG1 and FOXK2 expression levels were increased and miR-33a-5p expression level was reduced in CC cells and tissues. The silenced TP53TG1 or FOXK2, or elevated miR-33a-5p decelerated the CC cell development and restrained the activation of PI3K/AKT/mTOR signaling pathway. The depleted FOXK2 or elevated miR-33a-5p reversed the effects of decreased TP53TG1 on CC cell progression. TP53TG1 sponged miR-33a-5p, which targeted FOXK2. The experiment in vivo validated the outcomes of the experiment in vitro. TP53TG1 accelerates the CC development via regulating miR-33a-5p to target FOXK2 with the involvement of PI3K/AKT/mTOR signaling pathway. This study provides novel theory basis and distinct therapeutic targets for CC treatment.

Keywords: Cervical cancer; MicroRNA-33a-5p; biological development; forkhead box K2; long non-coding RNA tumor protein 53 target gene 1; phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway.

Figure 1.

TP53TG1 is elevated in CC tissues and cells and reduced TP53TG1 represses cell growth and promotes apoptosis of CC cells. (a), TP53TG1 level in CC tissues and normal tissues (n = 45) was detected by RT-qPCR; (b), TP53TG1 level in CC cell lines and normal cervical epithelial cells (HcerEpic) was examined by RT-qPCR; (c), TP53TG1 expression in HeLa cells after the transfection with sh-TP53TG1 or oe-TP53TG1 was assessed by RT-qPCR; (d), cell proliferation after transfection with sh-TP53TG1 or oe-TP53TG1 was assessed by CCK-8 assay; (e), cell migration after transfection with sh-TP53TG1 or oe-TP53TG1 was detected by the scratch test; (f), cell invasion after transfection with sh-TP53TG1 or oe-TP53TG1 was determined by Transwell assay; (g), cell apoptosis after transfection with sh-TP53TG1 or oe-TP53TG1 was assessed by flow cytometry. (h), the change of tumor size and volume after transfection with sh-TP53TG1 or oe-TP53TG1 in nude mice. The data in the figure were all measurement data, and the values were represented by mean ± standard deviation, the t-test was used for the comparison between two groups, ANOVA was adopted for the comparison among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after ANOVA; * P < 0.05 vs. HcerEpic cells; # P < 0.05 vs. the TP53TG1-NC group; n = 6; the cell experiment was repeated at least three times independently.

Figure 2.

TP53TG1 targets miR-33a-5p. (a), the binding site for TP53TG1 and miR-33a-5p was predicted by the bioinformatic website; (b), the targeting relationship between miR-33a-5p and TP53TG1 was validated by luciferase reporter gene assay; (c), the effects of TP53TG1 on miR-33a-5p enrichment were detected by RNA pull-down assay; (d), the correlation between TP53TG1 and miR-33a-5p in CC tissues was assessed by linear regression analysis; The data in the figure were all measurement data, and the values were represented by mean ± standard deviation, the t-test was used for the comparison between two groups; P < 0.05 was regarded as the indicator of statistical significance. The cell experiment was repeated at least three times independently.

Figure 3.

MiR-33a-5p is depleted in CC tissues, and miR-33a-5p elevation hinders HeLa cell growth and tumorigenesis whereas reduced miR-33a-5p has the opposite impacts on CC cells. (a), miR-33a-5p expression in CC tissues and normal tissues (n = 45) was detected by RT-qPCR; (b), miR-33a-5p expression after transfection with miR-33a-5p mimic or miR-33a-5p inhibitor was examined by RT-qPCR; (c), cell proliferation after transfection with miR-33a-5p mimic or miR-33a-5p inhibitor was assessed by CCK-8 assay; (d), cell migration after transfection with miR-33a-5p mimic or miR-33a-5p inhibitor was detected by scratch test; (e), cell invasion after transfection with miR-33a-5p mimic or miR-33a-5p inhibitor was determined by Transwell assay; (f), cell apoptosis after transfection with miR-33a-5p mimic or miR-33a-5p inhibitor was assessed by flow cytometry. The data in the figure were all measurement data, and the values were represented by mean ± standard deviation, the t-test was used for the comparison between two groups, ANOVA was adopted for the comparison among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after ANOVA; * P < 0.05 vs. the miR-33a-5p NC group. the cell experiment was repeated at least three times independently.

Figure 4.

FOXK2 is targeted by miR-33a-5p. (a), the binding site of miR-33a-5p and FOXK2 was predicted by bioinformatic website; (b), the targeting relationship between miR-33a-5p and FOXK2 was verified by luciferase reporter gene assay; (c/d), the effects of the up- or down-regulation of miR-33a-5p on FOXK2 expression were examined by RT-qPCR and Western blot assay; (e), the correlation between FOXK2 and miR-33a-5p in CC tissues was assessed by linear regression analysis. The data in the figure were all measurement data, and the values were represented by mean ± standard deviation, the t-test was used for the comparison between two groups; ANOVA was adopted for the comparison among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after ANOVA; * P< 0.05 vs. the miR-33a-5p NC group

Figure 5.

Inhibition of FOXK2 represses the development of CC cells. (a), FOXK2 expression in CC tissues and normal tissues (n = 45) was detected by RT-qPCR; (b/c), FOXK2 level after transfection with sh-FOXK2 or oe-FOXK2 was examined by RT-qPCR and Western blot assay; (d), cell proliferation after transfection with sh-FOXK2 or oe-FOXK2 was assessed by CCK-8 assay; (e), cell migration after transfection with sh-FOXK2 or oe-FOXK2 was detected by scratch test; (f), cell invasion after transfection with sh-FOXK2 or oe-FOXK2 was determined by Transwell assay; (g), cell apoptosis after transfection with sh-FOXK2 or oe-FOXK2 was assessed by flow cytometry. The data in the figure were all measurement data, and the values were represented by mean ± standard deviation, the t-test was used for the comparison between two groups; ANOVA was adopted for the comparison among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after ANOVA; * P < 0.05 vs. the FOXK2-NC group.  the cell experiment was repeated at least three times independently.

Figure 6.

Reduced TP53TG1 inhibits the development of CC via up-regulating miR-33a-5p to suppress FOXK2. (a/b), miR-33a-5p and FOXK2 level in HeLa cells after transfection with sh-TP53TG1 or oe-TP53TG1 was detected by RT-qPCR and Western blot assay; (c), cell proliferation after transfection with sh-TP53TG1 + miR-33a-5p inhibitor or sh-TP53TG1 + oe-FOXK2 was assessed by CCK-8 assay; (d), cell migration after transfection with sh-TP53TG1 + miR-33a-5p inhibitor or sh-TP53TG1 + oe-FOXK2 was detected by scratch test; (e), cell invasion after transfection with sh-TP53TG1 + miR-33a-5p inhibitor or sh-TP53TG1 + oe-FOXK2 was determined by Transwell assay; (f), cell apoptosis after transfection with sh-TP53TG1 + miR-33a-5p inhibitor or sh-TP53TG1 + oe-FOXK2 was assessed by flow cytometry. The data in the figure were all measurement data, and the values were represented by mean ± standard deviation; ANOVA was adopted for the comparison among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after ANOVA; in panel 6a and 6b, * P < 0.05 vs. the sh-TP53TG1-NC group; # P < 0.05 vs. the sh-TP53TG1 + FOXK2-NC group; $ P < 0.05 vs. the sh-TP53TG1 + miR-33a-5p-NC group.

Figure 7.

TP53TG1/miR-33a-5p/FOXK2 activates the PI3K/AKT/mTOR signaling pathway via promoting related protein activity. (a-b), the phosphorylation levels of PI3K, AKT and mTOR in HeLa cells were detected by Western blot assay. The data in the figure were all measurement data, and the values were represented by mean ± standard deviation, one-way ANOVA was adopted for the comparison among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA; * P < 0.05 vs. the TP53TG1-NC group; # P < 0.05 vs. the miR-33a-5p-NC group; & P < 0.05 vs. the FOXK2-NC group; ^ P < 0.05 vs. the sh-TP53TG1 + miR-33a-5p-NC group; % P < 0.05 vs. the sh-TP53TG1 + FOXK2-NC group.