**Downregulation of miR-17~92 Expression Increase Paclitaxel Sensitivity in Human Ovarian Carcinoma SKOV3-TR30 Cells via BIM Instead of PTEN**

#### **Ting Shuang <sup>1</sup> , Chunxue Shi 1,2,† , Shuang Chang 1,† , Min Wang 1, \* and Cui Hong Bai <sup>1</sup>**


*Received: 24 December 2012; in revised form: 23 January 2013 / Accepted: 4 February 2013 / Published: 8 February 2013*

**Abstract:** To better understand the molecular mechanisms of paclitaxel resistance in ovarian carcinoma, we evaluated the expression of miRNAs using miRNA microarray between human ovarian carcinoma SKOV3 cells and paclitaxel resistant SKOV3-TR30 cells. Results showed that 69 miRNAs were upregulated while 102 miRNAs were downregulated in SKOV3-TR30 cells. Using real-time PCR, we further clarified that miR-17~92 was overexpressed in SKOV3-TR30 cells compared with that in SKOV3 cells. We then established stable virally transduced SKOV3-TR30-m-PTIP-Sponge all SKOV3-TR30 cells and its vector-only control SKOV3-TR30-m-PTIP-GFP cells. Real time-PCR revealed that SKOV3-TR30-m-PTIP-Sponge all cells expressed approximately 6.18-fold lower levels of miR-17~92 compared with the control group. Decreased expression of miR-17~92 resulted in cell cycle arrest in the G2/M phase and growth inhibition. After the transduction, the BIM protein level was increased in SKOV3-TR30 cells and luciferase reporter assays revealed that miR-17~92 binds directly to the 3'-UTR of BIM. Results of luciferase reporter assays accompanied with Western Blot showed that although miR-17~92 binds directly to the 3'-UTR of PTEN, the PTEN protein expression level was upregulated slightly while the result is of no statistical significance. Our results showed that miR-17~92 could be a causal factor of the downregulation of BIM in SKOV3-TR30 cells and thus induce the paclitaxel resistance in SKOV3-TR30 cells.

**Keywords:** miR-17~92 cluster; transduction; ovarian carcinoma; paclitaxel resistance; PTEN; BIM

#### **1. Introduction**

Ovarian carcinoma is one of the most common gynecological malignant tumors. Its incidence is ranked second in malignant tumors of the female reproductive system and is gradually increasing in recent years. The most recommended treatment worldwide is surgery, along with paclitaxel and platinum–based (first-line chemotherapy) adjuvant chemotherapy; however, its mortality rate is still about 70%, which is the highest in gynecological malignancies [1]. One of the most significant reasons for such high morality rate is that about 30%~40% of patients with ovarian carcinoma are resistant to chemotherapy and, moreover, 60% of first-line chemotherapy-sensitive patients are resistant to chemotherapy after six months. Therefore, clarification of the exact mechanism of resistance and resistance reversal of ovarian carcinoma has become extremely urgent and important research topic.

MicroRNAs (miRNAs) contain 18–24 nucleotides and are small, non-coding RNAs that post transcriptionally regulate gene expression through translational repression and mRNA degradation [2]. Recent research has shown the tumor suppressant and oncogenic potential of a number of miRNAs, underscoring their importance in human cancer therapeutic and diagnostic applications [3–7].

BIM is a member of the BH3-only family of pro-apoptotic proteins and is expressed in a wide variety of tissues. BIM can initiate apoptosis by directly activating Bax through interaction with the Bcl-2/Bax herterodimer complex, which can further induces mitochondrial cell death [8,9]. It plays a critical role in tumor cell biology, including the regulation of tumorigenesis through activities as a tumor suppressor, tumor metastasis, and tumor cell survival. Therefore, it has gradually become an interesting target for cancer chemotherapy. PTEN plays a well-established role in the negative regulation of the PI3K pathway, which is frequently activated in several cancer types, including ovarian cancer. PTEN loss of function occurs in a wide spectrum of human cancers through mutations, deletions and transcriptional silencing. The expression of BIM and PTEN is highly regulated by its transcriptional and post-translational levels.

Overexpression of miR-17~92 has been observed in lymphomas and solid tumors [10] and is related to cell proliferation. The gene cluster of miR-17~92 resides with intron 3 of cl3orf25 non-protein-coding gene at 13q31.3 gene [8,11]. Studies by Lewis, BP, and others [12] have shown that miR-17~92 may play a role in PTEN and BIM. In recent years, more and more studies have reported that abnormal expression of PTEN and BIM has participated in the formation of tumor drug-resistance [13–15].

Yet it is not clear if miR-17~92 gene cluster has an impact on the paclitaxel resistance of ovarian carcinoma through affecting the expression of the BIM or PTEN protein. Furthermore, the effect of both BIM and PTEN on paclitaxel resistant in ovarian cancer cells has not been thoroughly researched; particularly, the mechamism involved in their expression regulation has barely been studied in ovarian carcinoma chemoresistance.

In this study, we investigated whether BIM or PTEN gene was post-transcriptionally regulated by miR-17~92 and the contribution of miR-17~92 to BIM or PTEN protein levels in SKOV3-TR30 cells. In addition, we also investigated the impact of miR-17~92 on SKOV3-TR30 cell proliferation and cell cycle. We aim to get a better understanding of the molecular mechanisms of paclitaxel resistance in SKOV3-TR30 cells, to provide a clue for further investigation of the function of miR-17~92 and its target genes, and their correlation in ovarian carcinoma paclitaxel resistance.

### **2. Results**

### *2.1. MiRNA Expression Profiling*

A microarray platform was used to analyze and compare the pattern of miRNA expression between the parental SKOV3 cell line and its counterpart paclitaxel resistant SKOV3-TR30 cell line. The expression profiles of 171 miRNAs changed significantly: 69 upregulated miRNAs (miR-17, miR-19b, miR-92-1) and 102 downregulated miRNAs (miR-134, miR-34, miR-196b) in SKOV3-TR30 cells as compared with SKOV3 cells (Figure 1).

**Figure 1.** Analysis of miRNAs Expression Profile of Sensitive and Drug-resistant Human Ovarian carcinoma Cells. Data were analyzed by the CLUSTER analysis software CLUSTER 3.0. Yellow means high expression, blue represents the low expression.

#### *2.2. miR-17~92 Was Overexpressed in SKOV3-TR30 Cells Compared with SKOV3 Cells*

We next studied the expression level of miR-17~92 in SKOV3 and SKOV3-TR30 cells using real-time PCR. Amplification Curve and Melting Curve of miR-17~92 and internal control β-actin see Figure 2. The expression level of miR-17~92 in the SKOV3-TR30 cells is indicated as "1" as control group. Compared to SKOV3-TR30 cells, the expression level of miRNA-17~92 in SKOV3 cells is 0.557, and the difference is of statistically significant (*t* = 9.193,*p <* 0.05) (Table 1).

The result revealed that the expression level of miR-17~92 was markedly increased in paclitaxel resistant SKOV3-TR30 cells compared with paclitaxel sensitive SKOV3 cells.

**Figure 2.** Amplification Curve and Melting Curve of miR-17~92 Expression in SKOV3, SKOV3-TR30, SKOV3-TR30-m-PTIP-Sponge all cells and the SKOV3-TR30-m-PTIP-GFP cells Detected by real-time PCR. (**a**) Amplification Curve of Expression of miR-17~92; (**b**) Melting Curve of Expression of miR-17~92.

**Table 1.** Expression level of miR-17~92 in Different Groups.


\* represents compared with the expression level of miRNA-17~92 in SKOV3, the different expression level of miRNA-17~92 in SKOV3-TR30, SKOV3-TR30-m-PTIP-GFP and SKOV3-TR30-m-PTIP-Sponge all is of great significance, *p* < 0.05.
