Inhibiting microRNA-301b suppresses cell growth and targets RNF38 in cervical carcinoma

2.1 Cells, tissues, and culture

The normal cell line HcerEpic, and cervical carcinoma cell lines were purchased from ATCC, Manassas. All the cells were cultured in RPMI 1640 medium (Corning) with 1% penicillin/streptomycin and 10% FBS.

The paracancerous normal tissues and tumor tissues were collected from Binzhou Central Hospital. Clinicopathologic features of the patients were as shown in Table 1. Moreover, the clinical staging and clinicopathological classifications were determined according to the International Federation of Obstetrics and Gynecology (FIGO). The use of human tissues in this study was approved by the Institutional Review Board of Binzhou Central Hospital.

2.2 Quantitative real-time polymerase chain reaction

To evaluate miR-301b expression, quantitative real-time polymerase chain reaction (qRT-PCR) was used as described previously.¹⁴ The primers specific for miR-301b were synthesized from GeneChem (Shanghai, China). The MiRNeasy Mini Kit (QIAGEN, Germany) was used to extract total RNA, and the Quantscript RT Kit (QIAGEN, Hilden, Germany) was used to reverse transcribe the miRNA bulge-loop. The miR-301b expression was quantified using a TaqMan MicroRNA assay (Thermo Fisher Scientific) and normalized to U6 RNA levels.

To evaluate RNF38 expression, qRT-PCR was used with SYBR Green qPCR Master Mix (Novoprotein, China). The primers used were as follows: RNF38, forward 5′-TTCCTTCTTATCGGTTCA-3′ and reverse 5′-AACTCGTGGTTACAGGGT-3′; GAPDH, forward 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse 5′-TGGTGAAGACGCCAGTGGA-3′.

2.3 MiRNA, siRNAs, plasmid construction, and transfection

The miR-301b inhibitors, inhibitor-NC, miR-301b mimics, and mimic-NC were synthesized from GeneChem (Shanghai, China). The siRNAs against RNF38 were obtained from Ribobio, Guangzhou, China. The human RNF38 gene and its mutant (RING-H2 domain was deleted) were amplified by PCR, and cloned into pcDNA3.1 vector with a Myc tag. Cervical carcinoma cells were transfected with 100 nM miRNAs or siRNAs by Lipofectamine RNAiMAX (Invitrogen), and plasmids were transfected into cervical carcinoma cells by Lipofectamine2000 (Invitrogen) according to the manufacturer's instruction.

2.4 Cell growth and viability

Viable cells of cervical carcinoma cells were measured by CCK-8 according to the manufacturer's instructions (Bimake).

2.5 Luciferase assay

The fragments containing predicted miR-301b binding sites or the mutants were amplified by PCR and subcloned into pGL3 vector. Then, the plasmids along with inhibitor-NC or miR-301b inhibitors were transfected into HeLa cells for 48 hours, followed by luciferase assay using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instruction.

2.6 Western blot

To detect the protein levels, western blot was carried out as described previously.¹⁵ The primary antibodies against cyclin D1, CDK4, p53, and GAPDH were purchased from CST, Danvers, Massachusetts. Anti-RNF38 and anti-myc tag antibodies were purchased from Santa Cruz Biotechnology, California.

2.7 Statistical analysis

To compare the differences between two groups, Student's t test was used, and all statistical tests were two-sided. P value less than .05 was considered statistically significant.

To analyze the overall survival of cervical carcinoma patients with low or high miR-301b expression, Kaplan-Meier plotter based on pan-cancer miRNA database was used.¹⁶ To analyze the expression of RNF38 in cervical squamous cell carcinoma and endocervical adenocarcinoma tumor tissues and normal tissues, GEPIA database matched TCGA normal and GTEx data was used online (http://gepia2.cancer-pku.cn/#analysis). Correlation analysis was performed by using GraphPad Prism 7 software. MicroRNA.org (http://www.microrna.org) was used to predict miR-301b targets.

3.1 MiR-301b is elevated in cervical carcinoma, and predicts a negative index for cervical carcinoma patients

To evaluate miR-301b expression in cervical carcinoma, paracancerous and tumor tissues of cervical carcinoma were collected. As shown in Figure 1A,B, miR-301b was elevated in cervical carcinoma tumor tissues compared with the paracancerous tissues. Then, four cervical carcinoma cell lines and one human normal cervical epithelial cell line were also prepared. As shown in Figure 1C, miR-301b was also found upregulated in cervical carcinoma cell lines compared with the normal cervical epithelial cell line. Additionally, the Kaplan-Meier plotter showed cervical carcinoma patients with high miR-301b expression had a shorter overall survival (Figure 1D). These data indicated miR-301b was a negative index for cervical carcinoma patients.

3.2 Silence of miR-301b inhibits cervical carcinoma cell growth

Subsequently, the miR-301b inhibitors were used to knock down miR-301b in cervical carcinoma cells, and the results showed miR-301b inhibitors could significantly reduce miR-301b expression in HeLa and CaSKi cells (Figure 2A). Moreover, silence of miR-301b markedly suppressed the cell growth in both of HeLa and CaSKi cells (Figure 2B,C). Additionally, knockdown of miR-301b markedly downregulated cyclin D1 and CDK4 expression, and upregulated p53 expression, which further indicated miR-301b inhibitors suppressed cell growth by regulating cell cycle progression (Figure 2D).

3.3 MiR-301b targets RNF38, and inhibits RNF38 expression in cervical carcinoma

Our further studies found, miR-301b was predicted to bind to the 3′UTR of RNF38 (Figure 3A). To elucidate it, luciferase assay was then carried out. As shown in Figure 3B, miR-301b mimics significantly decreased wild-type RNF38 3′UTR-driven luciferase activity, but miR-301b inhibitors significantly upregulated the luciferase activity. However, both of miR-301b mimics and inhibitors had no effect on the mutated RNF38 3′UTR-driven luciferase activity, which further indicated miR-301b targeted RNF38 (Figure 3B). To further confirm it, HeLa and CaSKi cells were treated with miR-301b inhibitors or mimics. As shown in Figure 3C,D, knockdown of miR-301b markedly upregulated RNF38 expression in cervical carcinoma cells, but overexpression of miR-301b significantly decreased RNF38 expression, which further suggested RNF38 was a novel target of miR-301b in cervical carcinoma cells.

3.4 RNF38 is downregulated in cervical carcinoma, and negatively associated with miR-301b

It has been shown RNF38 was involved in the tumorigenesis of several cancers,^{12, 13} but its function in cervical carcinoma was not investigated. First, we investigated the public cancer database, and it showed RNF38 was significantly downregulated in cervical squamous cell carcinoma and endocervical adenocarcinoma tumor tissues compared with the normal tissues (Figure 4A). To further confirm it, paracancerous and tumor tissues of cervical carcinoma were collected. As shown in Figure 4B,C, RNF38 was significantly downregulated in the tumor tissues of cervical carcinoma compared with the paracancerous tissues. What is more, miR-301b expression in cervical carcinoma was negatively correlated with RNF38 expression (Figures 3 and 4D).

Moreover, overexpressed RNF38 significantly inhibited cell growth in both of HeLa and CaSKi cells, but not the mutant of RNF38, which indicated that RNF38 exerted its function through its E3 activity (Figure 5A,B). In contrast, knockdown of RNF38 could enhance cell viability (Figure 5C). However, overexpression of miR-301b significantly suppressed RNF38-induced cell growth inhibition in cervical carcinoma cells (Figure 5D,E). These results further suggested RNF38 was a target of miR-301b.