RNA-binding protein MEX3D promotes cervical carcinoma tumorigenesis by destabilizing TSC22D1 mRNA

Abstract

RNA-binding proteins (RBPs) have been related to cancer development. Their functions in cervical cancer, however, are virtually unknown. One of these proteins, Mex-3 RNA-binding family member D (MEX3D), has been recently found to exhibit oncogenic properties in a variety of cancer types. In this present study, the functional roles and the regulatory mechanisms underlying MEX3D were examined in cervical cancer. The detection of MEX3D mRNA expression levels in cervical tissues was performed using reverse transcription-quantitative PCR. For functional analysis, for detecting apoptosis and cell proliferation in cervical cancer cells, the Cell Counting Kit-8, colony formation, and flow cytometry were utilized (SiHa and CaSki). The potential mechanisms of MEX3D were assessed and elucidated utilizing western blot analysis, RNA pull-down, RNA immunoprecipitation, and mRNA stability assays. For verification of MEX3D role in vivo, mouse xenograft models were established. When compared to normal cervical tissues, MEX3D expression was observed to be higher in cervical cancer tissues. MEX3D expression was increased in human papillomavirus (HPV) 16 positive cervical cancer tissues and positively regulated by HPV16 E7. When MEX3D expression was knocked down in cervical cancer cells, cell proliferation was decreased, colony formation was inhibited, and apoptosis was promoted. Furthermore, in a mouse xenograft model, knocking down MEX3D expression reduced cervical cancer tumor growth. In addition, MEX3D acted as an RBP to reduce TSC22 domain family protein 1 (TSC22D1) mRNA stability by directly binding to TSC22D1 mRNA. The findings revealed that MEX3D is upregulated by HPV16 E7 and has a crucial oncogenic in cervical cancer development via sponging TSC22D1 for destabilizing its mRNA levels. According to the findings of this study, MEX3D may be a potential therapeutic target for treating cervical cancer patients.

Fig. 1. MEX3D expression is elevated in cervical cancer tissues.

A RT-qPCR validation of MEX3D mRNA in seven cervical cancer tissues and seven normal cervical tissues. B MEX3D mRNA expression levels in 25 cervical normal epithelium samples and 38 cervical cancer tissue samples. C Representative IHC images of MEX3D expression of the normal cervix, LSIL, HSIL, and cervical cancer tissues. D IHC scores analyses of MEX3D protein expression in normal cervix (n = 30), LSIL (n = 147), HSIL (n = 129), and cervical cancer tissues (n = 159). E IHC scores analyses of MEX3D protein expression in HPV16-negtive normal cervix (n = 30), HPV16-positive HSIL (n = 20), and HPV16-positive cervical cancer (n = 43). NS not significant; *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 2. MEX3D stimulates cervical cancer cells proliferation whereas inhibits its apoptosis.

A Western blot analysis was utilized for estimating MEX3D protein expression levels in CaSki and SiHa cells after being transfected with a negative control siRNA or two MEX3D-specific siRNAs. B The growth curve in CaSki and SiHa cells was estimated through CCK8 assays after being transfected with a negative control siRNA or two MEX3D-specific siRNAs. C Knockdown of MEX3D with two MEX3D-specific siRNAs suppressed cell viability shown by colony formation in CaSki and SiHa cells. D Apoptosis level in CaSki and SiHa cells after being transfected with two MEX3D-specific siRNAs or a negative control siRNA. E Western blot was utilized for determining the levels of MEX3D overexpression in CaSki and SiHa cells utilizing a constructed and control plasmid. F–H MEX3D overexpression stimulated cellular proliferation (F, G) whereas the suppressed rate of cellular apoptosis in CaSki and SiHa cells (H). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. MEX3D is related to TSC22D1 RNA levels and regulates TSC22D1 mRNA stability in cervical cancer cells.

A Venny diagram of transcripts stabilized and bound by MEX3D. Transcripts bound by MEX3D in the RIP sequencing data and transcripts destabilized upon MEX3D knockdown in CaSki and SiHa cells. B The mRNA levels of seven genes were determined utilizing qRT-PCR in SiHA cells overexpressing MEX3D. When comparing the anti-flag sample to the IgG sample, TSC22D1 mRNA was substantially enriched. C TSC22D1 mRNA was found to bind to MEX3D and confirmed by RIP assay both in CaSki and SiHa cells. D, E For validating the interactions of MEX3D with TSC22D1, RNA pull-down and RNA-fluorescent in situ hybridization assays were done. F, G Downregulation of MEX3D expression in CaSki and SiHa cells elevated TSC22D1 mRNA and protein levels. H To inhibit RNA synthesis, actinomycin-D was added. TSC22DQ mRNA levels in CaSki and SiHa cells with MEX3D were knockdown or not. *P < 0.05, ***P < 0.001, and ****P < 0.0001.

Fig. 4. TSC22D1 knockdown attenuates MEX3D-mediated tumorigenesis.

A TSC22D1 knockdown significantly by siRNAs in protein levels in CaSki and SiHa cells. B–D TSC22D1 knockdown stimulated cellular proliferation, whereas the suppressed rate of cellular apoptosis. E–G TSC22D1 knockdown mitigated the decreased cell proliferation and increased cellular apoptotic rate mediated by MEX3D knockdown. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5. MEX3D expression knockdown reduces the growth of xenograft tumor.

Non-SCID mice were injected with SiHa/NC-shRNA and SiHa/MEX3D-shRNA cells subcutaneously. Tumor volume was estimated every 7 days, and all mice were euthanized under general anesthesia 9 weeks following injection. A SiHa/MEX3D-shRNA cells grow at a significantly slower rate than SiHa/NC-shRNA cells. B Representative images of mice euthanized at week nine following injection (n = 6). C Macro images of subcutaneous tumors. D Subcutaneous tumor weights in naked mice using sh-MEX3D or a negative control. E H&E, Ki-67, MEX3D, and TSC22D1 protein expression in xenograft tumors were analyzed by IHC. ****P < 0.0001.

Fig. 6. HPV16 E7 stimulates MEX3D expression in cervical cancer cells.

A Levels of MEX3D mRNA expression were analyzed by qRT-PCR in SiHa and CaSki cells underwent transfection with two E7 siRNAs and si-NC. B Protein levels of MEX3D, E7 and pRb were determined following transfection with two E7 siRNAs and si-NC. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 7. This article schematic diagram.

Sufficient quantities of endogenous MEX3D generated during cervical cancer tumorigenesis are demonstrated by schematic diagram. HPV16 E7 activates MEX3D expression in cervical cancer cells. MEX3D binds to the TSC22D1 mRNA and destabilizes TSC22D1 mRNA and promoting proliferation and reducing apoptosis of cervical cancer.