MicroRNA-30e Functions as a Tumor Suppressor in Cervical Carcinoma Cells through Targeting GALNT7

Abstract

Cervical cancer is the third most common cancer in women worldwide. However, the underlying mechanism of occurrence and development of cervical cancer is obscure. In this study, we observed that miR-30e was downregulated in clinical cervical cancer tissues and cervical cancer cells. Next, overexpression of miR-30e reduced the cervical cancer cell growth through MTT, colony formation, EdU, and Transwell assay in SiHa and Caski cells. Subsequently, UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7) was identified as a potential miR-30e target by bioinformatics analysis. Moreover, we showed that miR-30e was able to bind to the 3'UTR of GALNT7 by luciferase reporter assay. In addition, the mRNA and protein levels of GALNT7 in cervical cancer cells were downregulated by miR-30e. And we validated that downregulation of GALNT7 repressed the proliferation of SiHa and Caski cells by MTT, colony formation, and Transwell assay. We identified that the restoration of GALNT7 expression was able to counteract the effect of miR-30e on cell proliferation of cervical cancer cells. Furthermore, we found that the expression levels of GALNT7 were frequently upregulated and negatively correlative to those of miR-30e in cervical cancer tissues. In addition, we validated that restoration of GALNT7 rescued the miR-30e-suppressed growth of cervical cancer xenografts in vivo. In conclusion, the current results suggest that miR-30e may function as tumor suppressors in cervical cancer through downregulation of GALNT7. Both miR-30e and its novel target, GALNT7, may play an important role in the process of cervical cancer.

Figure 1

Expression of miR-30e mRNA in cervical tissues and cell lines. (A) qRT-PCR of miR-30e relative to U6 expression in cervical cancer and normal tissues. (B) qRT-PCR of miR-30e relative to U6 expression in immortalized cervical epithelial cell line and cervical cancer cell lines. *P < .05, **P < .01, Student's t test. The experiment was repeated at least three times.

Figure 2

MiR-30e represses proliferation and colony formation of SiHa and Caski cells. (A) miR-30e mimics or NC was transfected into the cells, and qRT-PCR was performed to determine miR miR-30e expression. (B) The effects of overexpression or knockdown of miR-30e on SiHa cell growth viability. (C) The effects of overexpression or knockdown of miR-30e on Caski cell growth viability. (D) Effect of miR-30e on cell proliferation was determined by colony formation assays in SiHa and Caski cells. (E, F) Effect of miR-30e on cell proliferation was determined by EdU assays in SiHa and Caski cells, respectively (scale bar = 100 μm). (G, H) Cell invasion. *P < .05, **P < .01, Student's t test. The experiment was repeated at least three times.

Figure 3

MiR-30e directly targets GALNT7 and inhibits its expression. (A and B) A schematic of the bioinformatics predicted seed region in the 3′UTR of GALNT7, as well as mutated 3′UTR used in this study. (C and D) Effect of miR-30e mimics or anti–miR-30e on the luciferase activity of the plasmid GALNT7–3′UTR and GALNT7–3′UTR-mut in SiHa cells. (E and F) qRT-PCR analysis the efficiency of overexpression miR-30e and Western blot shows suppression of the GALNT7 protein by miR-30e mimics. **P < .01, Student's t test.

Figure 4

Knockdown of GALNT7 restrains the cervical cancer cells proliferation and colony formation. (A) qRT-PCR analysis the GALNT7 mRNA expression level in SiHa and Caski cells after treatment with si-GALNT7. (B) Western blot shows the GALNT7 protein expression level in SiHa and Caski cells after treatment with si-GALNT7. (C and D) Cell growth was measured by MTT assay at different time intervals in SiHa and Caski cells, respectively. (E and F) Effect of si-GALNT7 on cell proliferation was determined by colony formation assays in SiHa and Caski cells, respectively. (G and H) Cell invasion. *P < .05, **P < .01, Student's t test. The experiment was repeated at least three times.

Figure 5

GALNT7 rescues miR-30e–induced cellular phenotypes in cervical cancer cells. (A) Cells were co-transfected with the GALNT7 vector with or without miR-30e mimics. (B) The GALNT7 protein level was measured by Western blot at 48 hours after transfection. (C and D) MTT assay shows the effect of above-mentioned treatment in SiHa and Caski cells, respectively. (E and F) Colony formation assay was used to evaluate the cell viability in SiHa and Caski cells, respectively. (G and H) Cell invasion. *P < .05, **P < .01, Student's t test. The experiment was repeated at least three times.

Figure 6

Restoration of GALNT7 can rescue the miR-30e–suppressed growth of cervical cancer xenografts in vivo. (A) The image of tumors in nude mice (n = 6). (B) The tumor growth curve of cervical cells injected into female nude mice is shown. (C) The average tumor weight of each group is shown. (D) Immunohistochemical staining for Ki67 in tumor tissues; scale bar, 50 μm. ** P < .01, Student's t test.

Figure 7

MiR-30e is negatively correlated to GALNT7 in cervical cancer tissues. (A) GALNT7 mRNA expression levels were analyzed by qRT-PCR in cervical cancer tissues and adjacent normal tissues. (B) Kaplan-Meier analysis of overall survival in cervical cancer patients. (C) Correlation of miR-30e levels with GALNT7 mRNA levels was examined by qRT-PCR analysis in 30 cases of clinical cervical cancer tissues (Pearson's correlation coefficient, r = −0.586). **P < .01, Student's t test.