MiR-96 enhances cellular proliferation and tumorigenicity of human cervical carcinoma cells through PTPN9

Abstract

Up to date, the cervical cancer remains to be one of the leading gynecological malignancies worldwide. MicroRNAs (miRNAs) play critical roles in the process of tumor initiation and progression. However, miR-96 has rarely been investigated in human cervical carcinoma. We aimed to investigate the biological function and underlying molecular mechanism of miR-96 in human cervical carcinoma. MiR-96 levels were determined by qRT-PCR. Protein tyrosine phosphatase, non-receptor type 9 (PTPN9) mRNA and protein levels were investigated by qRT-PCR and western blotting. The cellular proliferation in cervical cells was monitored by CyQuant assay. Soft agar assay was employed to determine the tumorigenicity. 3' UTR luciferase assay was used to validate the target gene of miR-96. SPSS was used to analyze statistical significance in different treatment. MiR-96 was dramatically upregulated in human cervical tumor tissues. Overexpression of miR-96 was found to significantly promote the cellular proliferation and tumorigenicity of cervical cells. Furthermore, we showed that PTPN9 was a direct target gene of miR-96 and had opposite effect to those of miR-96 on cervical cells. MiR-96 may promote the cellular proliferation and tumorigenicity of cervical cells by silencing PTPN9. Our study highlights an importantly regulatory role of miR-96 and suggests that an appropriate manipulation of miR-96 may be a new treatment of human cervical carcinoma in the future.

Keywords: Cervical carcinoma; MiR-96; PTPN9; Proliferation.

Fig. 1

MiR-96 expression in cervical cancer tissues. Taqman qRT-PCR detection of miR-96 expression in 12 cases of cancer tissue and paired non-tumor tissues. U6 was employed as an internal loading control. *P < 0.05 versus non-tumor tissues.

Fig. 2

miR-96 promote the cellular proliferation and tumorigenicity. (A) CyQuant assay was used to monitor the cellular proliferation. The HeLa cells were transfected with miR-NC or mir-96. CyQuant assay was performed as indicated time. *p < .05 versus Mock, **p < .05 versus miR-NC; (B) Soft agar assay was employed to determine the tumorigenicity in untransfected cells (Mock) and miR-96 transfected cells. Scale bar = 50 μm.

Fig. 3

PTPN9 is the direct target of miR-96. (A) Taqman qRT-PCR detection of PTPN9 expression in 12 cases of cancer tissue and paired non-tumor tissues. β-Actin was used as an internal reference. *P < .05 versus non-tumor tissues; (B) western blot was used to detect the PTPN9 protein expression from 6 randomly chosen non-tumorous tissues or tumor tissues, respectively. β-Actin was used as a loading control. (C) 3′ UTR luciferase reporter assay. The HeLa cells were cotransfected with PTPN9 3′ UTR reporter with miR-NC or mir-96. Luciferase assays were performed after 48 h. The β-galactosidase activity was used for normalization. *p < .05 versus Mock, **p < .05 versus miR-NC; (D) relative PTPN9 mRNA expression level was analyzed by Taqman qRT-PCR in HeLa cells transfected with miR-NC or miR-96. *p < .05 versus Mock, ** p < .05 versus miR-NC; (E) Western blot analysis forPTPN9 in the HeLa cells after transfected with miR-96. Mock was used as a negative control.

Fig. 4

PTPN9 overexpression repressed the cellular proliferation and tumorigenicity. (A) Taqman qRT-PCR was used to analyze PTPN9 mRNA levels after PTPN9 overexpression. *p < .05 versus Mock, **p < .05 versus plasmid-NC; (B) Western blot was employed to detect PTPN9 protein expression after PTPN9 overexpression; (C) After 48 h of transfection, the untransfected (Mock) and transfected cells were cultured for the indicated time. The cellular proliferation was determined by CyQuant assay. *p < .05 versus Mock, **p < .05 versus plasmid-NC; (D) Soft agar assay was employed to detect the tumorigenicity in Mock and miR-96 transfected cells. Scale bar = 50 μm.