Our study has got the Ethics board approval Committee of Wuhan No.1 Hospital (approval number: wj6911) following the Declaration of Helsinki. Each participant signed an informed consent for inclusion before they participated in the study. 38 cervical cancer patients who underwent surgical section in Wuhan No.1 Hospital between December 2016 and April 2020 were enrolled (Table 1). All patients did not receive any chemotherapy or adjuvant therapy before surgical section. Tumor tissues and non-tumor adjacent ($>$2 cm away from tumor tissues) were collection under surgical treatment and preserved in –80 ${}^{\circ }$C until use.

Human cervical cell line Hela, L-OHP resistant cervical Hela/L-OHP cell line, human normal cervical epithelial cell line H8, and HEK-293 cells were from the Chinese Academy of Sciences (Shanghai). All cells received culture in Dulbecco’s Modified Eagle Medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (Gibco, Grand Island, NY, USA) in a 5% CO${}_2$ humidified incubator at room temperature.

After preparing cell suspensions, we added 5, 10, 15, 20, 25, and 30 $\mu$M concentrations of L-OHP (Sigma-Aldrich, San Francisco, CA, USA) cell medium. Cells were treated with L-OHP for 48 hours and changed into fresh medium. Hela/L-OHP cells were cultured with the 50% maximal inhibitory concentration (IC50) of transfected cells against L-OHP for 3 hours to assess the chemical sensitivity of the drug. After treatment with drug, cells were subjected to further experiments.

miR-34a-5p mimics, mimics-NC, miR-34a-5p inhibitor, and inhibitor-NC were purchased from Taitool Bioscience (Shanghai, China). si-NC, si-MDM4, ov-NC, and ov-MDM4 were designed and synthesized by Bio-Rad (Hercules, CA, USA). Then, Hela/L-OHP cells were transfected with miR-34a-5p mimics, mimics-NC, miR-34a-5p inhibitor, inhibitor-NC, si-NC, si-MDM4, ov-NC, and ov-MDM4 by using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) as per instructions.

Total RNA extraction from cells was done via TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Afterward, cDNA was synthesized from RNA by using a Reverse Transcription Kit (Invitrogen, Carlsbad, CA, USA) following the protocol. Then, miScript SYBR Green PCR Kit (Qiagen, Shanghai, China) and ABI 7500 Fast PCR system (Thermo Fisher Scientific, Waltham, MA, USA) were applied to conduct PCR analysis. The cycling conditions included 95 ${}^{\circ }$C for 10 minutes, 40 cycles of 94 ${}^{\circ }$C for 10 seconds, and 62 ${}^{\circ }$C for 20 seconds. Relative expressions of miR-34a-5p and MDM4 were normalized based on U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as analysis standardization via 2${}^{-\mathrm{\Delta }\mathrm{\Delta }CT}$ method, respectively. Primers sequences were presented in the following:

The Cell Counting Kit-8 (Beyotime Biotechnology, Shanghai, China) and was used to assess the cultured cell viability and proliferation. 1 $\times$ 10${}^4$ cells/well cells were seeded in 96-well plates and cultured at 5% CO${}_2$, 37 ${}^{\circ }$C. At 0, 12, 24, and 48 hours, cells were processed with 10 $\mu$L CCK-8 reagent and cultured for another 4 hours avoid lights. Finally, a microplate reader (PerkinElmer, Waltham, MA, USA). was utilized to measure the OD value of each sample at 450 nm.

Cell apoptosis rates were determined via Apoptosis Assay Kit (Takara, Shiga, Japan) according to instructions. After inoculation in 6-well plates for 24 hours, cells were collected, washed with cold phosphate buffer saline (PBS), and double-stained with Annexin V-fluorescein isothiocyanate (0.25 mg/mL) and propidium iodide (PI) for 15 minutes at room temperature in the dark. Finally, stained cells were measured by a Flow Cytometry system (BD Bioscience, San Jose, CA, USA) to analyze with FCS Express V4 software (Glendale, CA, USA).

Wild and mutant MDM4 were subcloned into pGL4 luciferase vector (Promega, Madison, WI, USA) to construct WT-MDM4 and mut-MDM4 following the protocol. Then, HEK-293 cells and Hela/L-OHP cells received transfection with miR-34a-5p mimics or mimics-NC together with WT-MDM4 or mut-MDM4 via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Luciferase activity measurement was done via Firefly Luciferase Assay Kit (Promega, Madison, WI, USA) and normalized to Renilla luciferase activity.

Total proteins received separation with Radio-Immunoprecipitation assay (RIPA) lysis buffer and determined by BCA protein kit (Pierce, Rockford, IL, USA). Then, 25 $\mu$g proteins were loaded by 12% sulfate polyacrylamide gel electropheresis (SDS-PAGE) and transferred onto Polyvinylidene fluoride (PVDF) films. Following bloc in 5% skim milk for 50 min, films were probed with primary antibodies (rabbit anti-MDM4, 1:1000, ab16058, rabbit anti-GAPDH, 1:1000, ab9485, Abcam, Cambridge, MA, USA) at 4 ${}^{\circ }$C overnight to incubate with HPR-conjugated secondary antibody at room temperature for 1 hour. Finally, bands were detected via enhanced ECL kit (Pierce, Rockford, IL, USA), and GAPDH served as the internal control.

All experiments were repeatedly done three times. Data presentation were depicted as mean $\pm$ SD. GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA) was run to analyze all the data. Significance between two or more experimental groups was analyzed using Wilcoxon’s test or one-way ANOVA with Tukey’s post hoc test. P 0.05 reminded statistical significance.

To detect the mechanisms of miR-34a-5p in cervical cancer, we detect miR-34a-5p expression in cervical cancer tissues, human cervical epithelial cell H8 and cervical cancer cell line Hela. As depicted in Fig. 1A, miR-34a-5p was visually diminished in tumor tissues vs. non-tumor tissues, which had a similarly decrease in Hela cells compared with H8 cells (Fig. 1B). Furthermore, compared with Hela cells, miR-34a-5p was also down-regulated in L-OHP resistant cervical cancer cell line Hela/L-OHP cells (Fig. 1C). IC50 of L-OHP in Hela and Hela/L-OHP cells were 10 and 20 $\mu$M, respectively (Fig. 1D). Finally, after exposing Hela/L-OHP cells to different concentrations of L-OHP, expressions of miR-34a-5p were detected accordingly. As demonstrated in Fig. 1E, miR-34a-5p decreased gradually in Hela/L-OHP with the increase of L-OHP dose.

Fig. 1.

miR-34a-5p was significantly down-regulated in Hela and Hela/L-OHP cell lines. (A) miR-34a-5p expression in tumor tissues and non-tumor tissues from 38 cervical cancer patients. ${}^{**}$P = 0.0072, tumor tissues vs. non-tumor tissues. (B) miR-34a-5p in C-33A and Hela cell lines. ${}^{**}$P = 0.0068, Hela vs. C-33A. (C) miR-34a-5p in Hela and Hela/L-OHP cells. ${}^{**}$P = 0.0057, Hela/L-OHP vs. Hela. (D) IC50 of miR-34a-5p in Hela and Hela/L-OHP cells measured by CCK-8 assay. (E) Changes of miR-34a-5p levels under different concentrations of L-OHP in Hela/L-OHP cells. ${}^{*}$P = 0.038, ${}^{**}$P = 0.0082, ${}^{***}$P 0.001 vs. 0 $\mu$M group.

We transfected miR-34a-5p mimics and miR-34a-5p inhibitor in Hela/L-OHP cells, and confirmed the transfection efficacy subsequently. miR-34a-5p was significantly increased in the mimics group while down-regulated in the inhibitor group, indicating the transfection was successful in Fig. 2A. Then, Fig. 2B revealed over-expressing miR-34a-5p could suppress Hela/L-OHP cell viability while miR-34a-5p inhibitor presented opposite effects. On the contrary, the apoptosis results in Fig. 2C–H unveiled that miR-34a-5p mimics promote cell apoptosis of Hela/L-OHP while miR-34a-5p silencing presented opposite effects. Collectively, these results suggested miR-34a-5p promotes the chemosensitivity of cervical cancer cells to L-OHP.

Fig. 2.

miR-34a-5p increased the chemosensitivity of cervical cancer cells to L-OHP. (A) Transfection efficacy of miR-34a-5p. ${}^{**}$P = 0.0064, mimics vs. mimics-NC; ${}^{\mathrm{\#}}$P = 0.045, inhibitor vs. inhibitor-NC. (B) Cell viability in Hela/L-OHP cells after transfection with miR-34a-5p mimics or miR-34a-5p inhibitor. ${}^{**}$P = 0.0057, mimics vs. mimics-NC; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$P 0.001, inhibitor vs. inhibitor-NC. (C–H) Cell apoptosis in Hela/L-OHP cells after evaluated by flow cytometry analysis. ${}^{***}$P 0.001, mimics vs. mimics-NC; ${}^{\mathrm{\#}\mathrm{\#}}$P = 0.0084, inhibitor vs. inhibitor-NC.

Furthermore, we exploited underlying roles of MDM4 in regulating Hela/L-OHP cell development. As shown in Fig. 3A, MDM4 prominently increased in tumor tissues from cervical cancer patients compared with non-tumor tissues. Meanwhile, in Fig. 3B,C, MDM4 was remarkably increased in Hela cells and Hela/L-OHP cells compared with H8 or Hela cells. These results implied that MDM4 was implicated in the carcinogenesis of cervical cancer. Afterward, we found that the MDM4 level was gradually increased after adding L-OHP concentration in a dose-dependent manner (Fig. 3D). We constructed MDM4 silencing or overexpressing Hela/L-OHP cell lines. As depicted in Fig. 3E, MDM4 was markedly increased in ov-MDM4 while reduced in the si-MDM4 group, suggesting the transfection was successful. Cell viability and apoptosis results in Fig. 3F–L demonstrated that MDM4 overexpressing promoted Hela/L-OHP cell viability but inhibit apoptosis; however, MDM4 silencing presented the opposite effects on Hela/L-OHP cell progression.

Fig. 3.

MDM4 was up-regulated in Hela/L-OHP cells and suppressed the chemosensitivity of cervical cancer cells to L-OHP. (A) Expression level of MDM4 in tumor tissues and non-tumor adjacent tissues from cervical cancer patients. ${}^{**}$P = 0.0059, tumor tissues vs. non-tumor tissues. (B) MDM4 was elevated in Hela cells, vs. C-33A cells. ${}^{**}$P = 0.0043, Hela vs. C-33A. (C) Highly expressed MDM4 was found in Hela/L-OHP cells. ${}^{**}$P = 0.0062, Hela/L-OHP vs. Hela. (D) Expression levels of MDM4 after adding different doses of L-OHP in Hela/L-OHP cells. ${}^{*}$P = 0.042, ${}^{**}$P = 0.0036, ${}^{***}$P 0.001 vs. 0 $\mu$M group. (E) Transfection efficiency of MDM4. (F) CCK-8 assay revealed the Hela/L-OHP cell viability after transfection. ${}^{**}$P = 0.0021, ov-MDM4 vs. ov-NC; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$P = 0.0033, si-MDM4 vs. si-NC. (G–L) Cell apoptosis in Hela/L-OHP cells. ${}^{***}$P 0.001, ov-MDM4 vs. ov-NC; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$P = 0.0041, si-MDM4 vs. si-NC.

Through TargetScan online tool, we hypothesized that MDM4 was a potential target of miR-34a-5p (Fig. 4A). Afterward, the dual-luciferase reporter assay confirmed co-transfection with miR-34a-5p mimics reduce the luciferase activity in WT-MDM4 groups; however, there were no significant differences in Mut-MDM4 groups (Fig. 4B,C). Furthermore, transfection with miR-34a-5p mimics could impede MDM4 expression; whereas, co-transfection with ov-MDM4 could partially reverse the decline of MDM4 induced by miR-34a-5p mimics. Similarly, miR-34a-5p inhibitor could increase MDM4 expression in Hela/L-OHP cells; however, the increase could be partially counteracted by co-transfection si-MDM4 (Fig. 4D,E). These results suggested miR-34a-5p directly targeted MDM4.

Fig. 4.

miR-34a-5p directly targeted MDM4. (A) Potential binding sites of miR-34a-5p and MDM4. (B,C) Dual-luciferase reporter assay. ${}^{**}$P = 0.0022, miR-mimics vs. miR-NC. (D) Expression levels of MDM4 after transfection with miR-34a-5p mimics or miR-34a-5p inhibitor. ${}^{***}$P 0.001, mimics vs. control; ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$P 0.001, inhibitor vs. control. (E) MDM4 expressions after co-transfection with miR-34a-5p and MDM4. ${}^{**}$P = 0.0016, mimics vs. control; ${}^{\mathrm{\#}}$P = 0.036, mimics + ov-MDM4 vs. mimics; P = 0.0035, inhibitor vs. control; ${}^{\$}$P = 0.027, inhibitor + si-MDM4 vs. inhibitor.

After confirming the targeted relationship between miR-34a-5p and MDM4, we further elucidated the role of miR-34a-5p/MDM4 axis in Hela/L-OHP cell progression. miR-34a-5p mimics decrease Hela/L-OHP cell viability but promote cell apoptosis in Fig. 5A–G; however, the effects of miR-34a-5p over-expressing on Hela/L-OHP cell development could be counteracted by co-transfection with ov-MDM4. Meanwhile, miR-34a-5p silencing could promote Hela/L-OHP viability and suppress apoptosis; whereas, the MDM4 knockdown could counteract impacts of the miR-34a-5p inhibitor on Hela/L-OHP cells. Finally, in Fig. 5H,I, we found compared with the control group, protein expression of MDM4 showed a decrease in miR-34a-5p mimics group, while increase in the miR-34a-5p inhibitor group. However, ov-MDM4 or si-MDM4 partially antagonized miR-34a-5p analogs or inhibitor-induced differences.

Fig. 5.

miR-34a-5p enhanced the chemosensitivity of cervical cancer cells to L-OHP by targeting MDM4. (A) CCK-8 assays unveiled the Hela/L-OHP cell viability. ${}^{**}$P = 0.0037, mimics vs. control; ${}^{\mathrm{\#}}$P = 0.028, mimics + ov-MDM4 vs. mimics; P 0.001, inhibitor vs. control; ${}^{\$}$P = 0.043, inhibitor + si-MDM4 vs. inhibitor. (B–G) Cell apoptosis measurement done by flow cytometry assay. ${}^{**}$P = 0.0049, mimics vs. control; ${}^{\mathrm{\#}}$P = 0.025, mimics + ov-MDM4 vs. mimics; P = 0.017, inhibitor vs. control; ${}^{\$}$P = 0.015, inhibitor + si-MDM4 vs. inhibitor. (H,I) Protein expressions of MDM4 after co-transfection with miR-34a-5p and MDM4. ${}^{**}$P = 0.0053, mimics vs. control; ${}^{\mathrm{\#}}$P = 0.038, mimics + ov-MDM4 vs. mimics; P = 0.0064, inhibitor vs. control; ${}^{\$}$P = 0.041, inhibitor + si-MDM4 vs. inhibitor.