Objective To analyze the expression level and clinicopathological significance of ultra-conserved RNA-339 (uc.339) in cervical squamous cell carcinoma (CSCC), and to explore the effect and related mechanism of uc.339 on proliferation of cervical cancer cells.
Methods Clinical features of 102 CSCC specimens were collected. The expression level of uc.339 in the CSCC tissues was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and the correlations between the uc.339 and the pathological characteristics were analyzed. MTT assay and clone formation assay were used to analyze the effect of uc.339 on proliferation of cells. Bioinformatics was use to predict the downstream target gene of uc.339; the qRT-PCR was used to verify the effect of uc.339 on the target gene microRNA-339-5P (miR-339-5P); the double luciferase report experiment was used to verify the regulatory effect of uc.339 on miR-339-5P; the rescue reversion experiment was used to verify the effects of uc.339 on regulation of miR-339-5P and the proliferation of cervical cancer cells.
Results The result of qRT-PCR showed that the expression level of uc.339 in the CSCC tissues was significantly higher than that in the normal cervical tissues adjacent to cancer (P < 0.05). The expression of uc.339 was significantly related to the tumor size (Pearson′s R=7.909) and pathological grading (Pearson′s R=-4.859) (P < 0.05), but had no significant correlations with the patient′s age, human papillomavirus (HPV) infection, lymph node metastasis and TNM staging (P>0.05). MTT assay indicated that over-expression of uc.339 was able to promote the proliferation of cervical cancer cells, while decline in the expression of uc.339 was able to inhibit the proliferation of cells (P < 0.05). The cell clone formation experiment showed that the number of clone formation in cervical cancer cells overexpressing uc.339 was significantly higher than that in the Scramble controls, and the number of clone formation in cervical cancer cells with reduced uc.339 expression was significantly lower than that in the si-control (P < 0.05). Bioinformatics predict showed that miR-339-5P was the target gene of uc.339; after over-expression of uc.339 in cervical cancer cells, the expression level of miR-339-5P decreased significantly, while the expression level of miR-339-5P increased significantly after reducing uc.339 expression (P < 0.05). Double luciferase report experiment confirmed that uc.339 was able to specifically bind to miR-339-5P, and negatively regulate the expression of miR-339-5P. The result of rescue experiment suggested that over-expression of miR-339-5P was able to eliminate promoting effect of uc.339 on proliferation of cervical cancer, and reducing the expression of miR-339-5P was able to further stimulate the proliferation activity of cervical cancer cells.
Conclusion The uc.339 is up-regulated in CSCC, which may be a new oncogenic gene. The uc.339 can promote the proliferation of CSCC cells by inhibiting the expression of miR-339-5P.
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