LncRNA-UCA1 enhances MMP-13 expression by inhibiting miR-204-5p in human chondrocytes

Osteoarthritis (OA) is a common degenerative disease characterized by degeneration of articular cartilage. Increasing studies showed that long noncoding RNAs (lncRNAs) play important roles in the cartilage damage. However, little is known about the role of UCA1 in the osteoarthritis. The expression level of UCA1 was upregulated in the OA cartilage. Overexpression of UCA1 suppressed the miR-204-5p expression in the chondrocytes. The expression of miR-204-5p was downregulated in the OA cartilage. Moreover, the expression of miR-204-5p was negatively correlated with the UCA1 expression in the OA cartilage. Elevated expression of UCA1 promoted the chondrocytes cell proliferation and overexpression of miR-204-5p suppressed chondrocytes cell proliferation. In addition, overexpression of UCA1 decreased the expression of the type II collagen and type IV collagen expression in the chondrocytes. Elevated expression of miR-204-5p promoted the type II collagen and type IV collagen expression in the chondrocytes. We idetified MMP-13 was a direct target gene of miR-204-5p in the chondrocytes. Overexpression of UCA1 enhanced the MMP-13 expression in the chondrocytes. Elevated expression of UCA1 regulated the chondrocytes cell proliferation and collagen expression through inhibiting the miR-204-5p expression.These results suggested that UCA1 played as an important regulator of survival and matrix synthesis of chondrocytes partly through suppressing the miR-204-5p expression.


INTRODUCTION
Osteoarthritis (OA) is a degenerative disease of the joints and is regarded by tenderness, pain, crepitus, limited movement, which is the most prevalent cause of mobility-associated disability [1][2][3][4]. The pathogenesis of OA is multifactorial and involves the interaction of several factors [5,6]. The etiology of OA is also complex such as failure of nutrient supply, genetic predisposition, trauma and abnormal mechanical loading [7][8][9][10][11].
In this study, we sought to the expression of UCA1 in the OA cartilage and normal cartilage. We showed that the expression level of UCA1 was upregulated in the OA cartilage. Overexpression of UCA1 suppressed the miR-204-5p expression and enhanced the MMP-13 expression in the chondrocytes.

The expression of UCA1 was upregulated in the OA cartilage
We firstly determined the expression of UCA1 in the OA cartilage and normal cartilage. As shown in the Figure 1, the expression level of UCA1 was highest in the moderate and severe group compared to in the normal cartilage and mild OA cartilage. The data indicated that the expression level of UCA1 was upregulated in the OA cartilage.

The expression of miR-204-5p was downregulated in the OA cartilage
The expression of UCA1 was significantly upregulated in the chondrocytes after treated with pcDNA-UCA1 ( Figure 2A). Overexpression of UCA1 suppressed the miR-204-5p expression in the chondrocytes ( Figure  2B). We then determined the expression of miR-204-5p in the OA cartilage and normal cartilage. As shown in the Figure 2C, the expression level of miR-204-5p was lowest in the moderate and severe group compared to in the normal cartilage and mild OA cartilage. The data indicated that the expression level of miR-204-5p was downregulated in the OA cartilage. Moreover, the expression of miR-204-5p was negatively correlated with the UCA1 expression in the OA cartilage ( Figure 2D).
Overexpression of UCA1 promoted the MMP-13 expression in the chondrocytes ( Figure 3F).

Overexpression of UCA1 decreased the expression of the type II collagen and type IV collagen
Overexpression of UCA1 suppressed the type II collagen ( Figure 5A) and type IV collagen ( Figure 5B) expression in the chondrocytes. In addition, elevated expression of miR-204-5p promoted the type II collagen ( Figure 5C) and type IV collagen ( Figure 5D) expression in the chondrocytes.

DISCUSSION
In our study, we found that the expression level of UCA1 was upregulated in the OA cartilage. Overexpression of UCA1 suppressed the miR-204-5p expression in the chondrocytes. The expression of miR-204-5p was downregulated in the OA cartilage. Moreover, the expression of miR-204-5p was negatively correlated with the UCA1 expression in the OA www.impactjournals.com/oncotarget  Previous studies demonstrated that lncRNA UCA1 played important roles in the development of tumors such as renal cell carcinoma, gastric cancer, colon cancer, hepatocellular carcinoma and osteosarcoma [34][35][36][37][38]. For example, Jiao et al. [39]. demonstrated that UCA1 expression was upregulated in the esophageal cancer tissues and ecoptic expression of UCA1 increased esophageal cancer cell proliferation by regulating the miR-204 and Sox4 expression. Chen et al. [40]. showed that UCA1 expression level was upregulated in the pancreatic cancer samples and the konckdown expression  [41]. demonstrated that UCA1 expression level was higher in the lymph node metastasis samples than in the endometrial cancer tissues and the proliferative endometrium. Inhibition of UCA1 suppressed the endometrial cancer cell migration and invasion. However, the role of UCA1 in OA is still unknown. In this study, we found that the expression level of UCA1 was highest in the moderate and severe group compared to in the normal cartilage and mild OA cartilage. The data indicated that the expression level of UCA1 was upregulated in the OA cartilage. Elevated expression of UCA1 promoted the chondrocytes cell proliferation and overexpression of miR-204-5p suppressed chondrocytes cell proliferation. There results suggested that UCA1 play crucial roles in pathogenesis and progression of OA.
It has showed that lncRNAs act as regulators for miRNAs expression regulation. Previous studies showed that UCA1 promoted gemcitabine/cisplatin resistance by CREB regulating miR-196a-5p expression in the bladder cancer cells [28]. Fang et al. [34]. demonstrated that UCA1 iIncreased multi-drug resistance of gastric cancer through downregulating miR-27b expression. Bian et al. showed that UCA1 enhanced the 5-fluorouracil resistance and cell proliferation in the colorectal cancer through regulating the miR-204-5p expression. In line with this, we demonstrated that overexpression of UCA1 suppressed the miR-204-5p expression in the chondrocytes. The expression of miR-204-5p was downregulated in the OA cartilage. Moreover, the expression of miR-204-5p was negatively correlated with the UCA1 expression in the OA cartilage. Elevated expression of miR-204-5p promoted the type II collagen and type IV collagen expression in the chondrocytes. Moreover, we idetified MMP-13 was a direct target gene of miR-204-5p in the chondrocytes. Previous data demonstrated that MMP-13 was at low expression level in the articular cartilage during physiologic ECM turnover and was upregulated in human OA. MMP-13 can degrade the expressions of type 2 collagen and aggrecan. Furthermore, we showed that elevated expression of UCA1 regulated the chondrocytes cell proliferation and collagen expression through inhibiting the miR-204-5p expression. In conclusion, we indicated that expression level of UCA1 was upregulated in the OA cartilage. Overexpression of UCA1 suppressed the miR-204-5p expression and enhanced the MMP-13 expression in the chondrocytes. These data provide the possibility of UCA1/ miR-204-5p as therapeutic targets for the treatment of OA.

Tissue samples
Normal knee cartilage samples were collected from patitents which were underwent the amputation due to trauma with no OA or rheumatoid arthritis history. Cartilage tissues of OA were obtained from OA patients who underwent knee replacement surgery. The diagnosed of these patients were followed to the criteria of American College of Rheumatology. This study was approved by Research Ethics Committee of the affiliated hospital of jining medical university and complied with Declaration of Helsinki. The written informed consent was collected from each participant. All cartilage tissues were divided into three groups following to the Kellgren/Lawrence Criterion: normal cartilage (K/L, Grade 0) for the normal group, low grade of OA cartilage (K/L Grade I and II) for the mild group, and high grade of OA cartilage (K/L, Grade III and IV) for the moderate and severe group.

Cell culture and cell transfection
The human chondrocytes cell line C28/I2 (immortalized juvenile costal chondrocytes cell line) was purchased from ATCC (American Type Culture  Collection, USA) and was cultured in the DMEM with fetal bovine serum (FBS), penicillin, and streptomycin. LncRNA UCA1 and control vector, miR-204-5p mimic and scramble were purchased from GenePharma (Shanghai, China). Cell transfection was performed by using the Lipofectamine 2000 (Invitrogen, USA) following to manufacturer's information.

Western blot analysis
Cell lysate was prepared by using the RIPA buffer and the protein concentration was measured by BCA protein kit (Pierce, Rockford, IL). Total protein was isolated through 12% SDS-PAGE and transferred to the nitrocellulose membrane. Membrane was blocked with 5 % nonfat milk and incubated overnight with the primary antibodies (MMP-13 and GAPDH, Sigma). After washing in TBST, the membrane was incubated HRP-conjugated secondary antibodies. The membrane was detected with ECL (enhanced chemiluminescence).

Cell proliferation
Cells were cultured in the 96-well plate and continued to culture for 0, 24, 48 and 72 hours, respectively. Cell proliferation was measured by Cell Counting Kit 8 (CCK-8, Dojindo, Japan) following to the manufacturer's information. The end product was determined spectrophotometrically at the wavelength of 450 nm.

Luciferase reporter assay
The amplified DNA sequences were cloned to the pmiR-RB-REPORT™ Vector to form MMP-13 3′UTR (WT) and mutated MMP-133′UTR (Mut) luciferase vectors. For the reporter assay, cells were plated in the 96-well plate and were co-transfected with miR-204-5p mimic and scramble and MMP-13 3′UTR (WT) or mutated MMP-133′UTR (Mut) luciferase vectors. After 48 hours, the luciferase activity was determined by using Dual Luciferase Assay System (Promega, Madison, WI, USA) following to the manufacturer's information.

Statistical analysis
Value was shown as means ± (standard deviation) SD. The statistical assay was performed by using SPSS 17.0 (IBM Corporation, USA). Student t test was used to assess the significant difference between two groups and the differences between more than two groups were tested by one-way ANOVA. P < 0.05 was thought to be statistically significant.

ACKNOWLEDGMENTS AND FUNDING
This work was supported by natural science foundation of shandong province (Grant Numbers: ZR2010HQ036 and ZR2014CQ042).