LncRNA‐RMRP promotes nucleus pulposus cell proliferation through regulating miR‐206 expression

Abstract Long noncoding RNAs (LncRNAs) are involved in the pathogenesis of intervertebral disc degeneration (IDD). However, the biological function and expression of RMRP were still unclear. In our study, we showed that RMRP expression was up‐regulated in degenerated NP tissues compared to normal NP samples, and higher RMRP expression was associated with the disc degeneration grade. Further studies indicated that ectopic expression of RMRP enhanced NP cell growth and also enhanced the expression of ki‐67, PCNA and cyclin D1 in the NP cell. Moreover, overexpression of RMRP promoted the expression of Type II collagen and aggrecan and suppressed the expression of MMP13 and ADAMTS4. In addition, we found that the expression of miR‐206 was down‐regulated in degenerated NP tissues compared to normal NP samples, and lower miR‐206 expression was correlated with the disc degeneration grade. Interestingly, we indicated that miR‐206 expression in NP tissues was negatively correlated with the expression of RMRP. Ectopic expression of miR‐206 suppressed NP cell proliferation and suppressed the expression of Type II collagen and aggrecan and enhanced the expression of MMP13 and ADAMTS4. Furthermore, we demonstrated that overexpression of RMRP increased NP cell growth and regulated ECM expression through targeting miR‐206. These results suggested that lncRNA‐RMRP promoted the progression of IDD through targeting miR‐206, providing an attractive new therapeutic approach for the treatment of IDD disease.

found to play important roles in diverse human diseases and biological processes. [16][17][18][19][20][21] Mounting evidence has suggested that many lncRNAs are beregulated in diverse diseases such as tumours, diabetes, coronary heart disease, osteoarthritis and also IDD. [22][23][24][25][26][27][28][29] For example, Lan and colleagues 30 profiled lncRNA expression in degenerate disc tissues and normal adults' lumbar discs by microarray and found a total of 3081 differentially expressed lncRNAs. Wang et al 31 showed that knockdown of RP11-296A18. 3  In this study, we first measured the expression of RMRP in the intervertebral disc tissues and we found that RMRP expression was up-regulated in degenerated NP tissues compared to normal NP samples, and higher RMRP expression was associated with the disc degeneration grade.

| Tissues
Degenerated IVD tissues were collected from IDD patients undergoing discectomy surgery. Nondegenerated IVD tissues were obtained from cases with scoliosis undergoing surgery. Routine MRI of spine was performed for all these cases before operation and the modified Pfirrmann classification was used to determine the degree of disc degeneration according to T2-weighted images. Sample was washed three times in the physiological saline and IVD was separated using stereotaxic microscope and then the tissue was immediately frozen in liquid nitrogen. Informed consent was obtained from each patient and this study was approved by the ethics committee of QingDao Central Hospital.

| Cell cultures and cell transfection
Human NP cell was isolated from human NP tissues following to previous standardized method. NP samples were washed in PBS for three times and NP was separated from IVD samples using stereotaxic microscope and then cut to pieces. Then, these tissues were incubated in Type II collagenase in DMEM medium. After isolation, cells were cultured in the DMEM medium with penicillin, streptomycin and 10% FBS. pcDNA-RMRP and pcDNA-control vector were purchased from the Biosune biotechnology (Shanghai, China) and then transfected to the cell using DharmaFECT1 kit (Dharmacon, TX, USA) following to instruction.

| Cell proliferation and cell cycle assays
Cell growth of NP cell was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Cells were cultured in 96-well plate and MTT medium was added to each well.

| Statistical analysis
Data were measured by SPSS Data (version 18, Inc., Chicago, IL, USA) and were represented as mean ± SD. The comparison among multiple groups was determined with one-way ANOVA and the difference between two groups was measured with Student's t test.
P < 0.05 was considered to be statistically significant.

| The expression of RMRP was up-regulated in degenerated NP tissues
We first determined the expression level of RMRP in NP tissues.

RMRP expression in the 5 normal NP tissues and 30 degenerated
NP tissues was shown in Figure 1A,B. Furthermore, we indicated that the expression of RMRP was up-regulated in degenerated NP tissues compared to normal NP samples ( Figure 1C). Moreover, we showed that RMRP expression was higher in advanced the disc degeneration grade ( Figure 1D).

| The expression of miR-206 was downregulated in degenerated NP tissues
Then, we determined the expression level of miR-206 in NP tissues. miR-206 expression in 5 normal NP tissues and 30 degenerated NP tissues was shown in Figure 2A,B. Furthermore, we indicated that the expression of miR-206 was down-regulated in degenerated NP tissues compared to normal NP samples ( Figure 2C). Moreover, we showed that miR-206 expression was lower in early disc degeneration grade ( Figure 2D). In addition, we indicated that miR-206 expression in NP tissues was negatively correlated with the expression of RMRP ( Figure 2E).

expression in the NP cell
We confirmed that the expression of RMRP was significantly upregulated in NP cell after transfected with pcDNA-RMRP (Figure 3A). Overexpression of RMRP suppressed miR-206 expression in NP cell ( Figure 3B). In addition, we showed ectopic expression of RMRP promoted HDAC6 expression in NP cell ( Figure 3C). We also indicated that RMRP overexpression enhanced HDAC6 protein expression in NP cell ( Figure 3D).

| Ectopic expression of RMRP enhanced NP cell growth
We next showed that ectopic expression RMRP promoted NP cell proliferation ( Figure 4A). The expression of ki-67 was up-regulated in NP cell after transfected with pcDNA-RMRP ( Figure 4B). Overexpression of RMRP enhanced PCNA expression in NP cell ( Figure 4C).
Moreover, ectopic expression of RMRP promoted the expression of cyclin D1 in NP cell ( Figure 4D).

| RMRP overexpression increased Type II collagen and aggrecan expression and suppressed MMP13 and ADAMTS4 expression
We next indicated that overexpression of RMRP increased Type II collagen and aggrecan expression in the NP cell ( Figure 5A). In addition, we showed that ectopic expression of RMRP decreased the expression of MMP13 and ADAMTS4 ( Figure 5B).

| Ectopic expression of miR-206 suppressed NP cell proliferation and regulated the extracellular matrix (ECM) expression
The expression of miR-206 was significantly up-regulated in NP cell after transfected with miR-206 mimic ( Figure 6A). We found that overexpression of miR-206 suppressed NP cell proliferation ( Figure 6B). The expression of ki-67 was down-regulated in NP cell F I G U R E 1 The expression of RMRP was up-regulated in the degenerated NP tissues. A,B, The RMRP expression in the 5 normal NP tissues and 30 degenerated NP tissues was measured by qRT-PCR. U6 was used as the internal control. C, The expression of RMRP was up-regulated in degenerated NP tissues compared to normal NP samples. D, RMRP expression was higher with the disc degeneration grade. **P < 0.01 and ***P < 0.001 after transfected with miR-206 mimic ( Figure 6C). Overexpression of miR-206 suppressed PCNA expression in NP cell ( Figure 6D). Moreover, ectopic expression of miR-206 decreased the expression of cyclin D1 in NP cell ( Figure 6E). Furthermore, we showed that overexpression of miR-206 suppressed Type II collagen and aggrecan expression in NP cell ( Figure 6F). In addition, we showed that ectopic expression of miR-206 promoted the expression of MMP13 and ADAMTS4 in NP cell ( Figure 6G).

| Ectopic expression of RMRP increased NP cell growth and regulated ECM expression through targeting miR-206
To study the biological function association between RMRP and miR-206, we employed loss and gain-of-function approaches. We showed that the cell proliferation was down-regulated in the group of pcDNA-RMRP and scramble compared to pcDNA-RMRP and miR-206 mimic group ( Figure 7A). In addition, we showed

| DISCUSSION
In our study, we studied the expression and biological function of lncRNA-RMRP in the degenerated intervertebral disc. We showed that RMRP expression was up-regulated in degenerated NP tissues compared to normal NP samples and RMRP expression was associ- A growing number of studies indicated that lncRNAs were correlated with the development of several diseases. 23,[37][38][39] The role of lncRNAs in the development of IDD has also been explored. 29,31,40 For example, Wang et al 41  Recently, increasing evidence has suggested that lncRNAs function as competing endogenous RNAs (ceRNAs) to target miRNA expression. 44 F I G U R E 7 Ectopic expression of RMRP increased NP cell growth and regulated ECM expression through targeting miR-206 expression. A, Cell proliferation was determined by MTT method. B, The cyclin D1 expression in the NP cell was detected by qRT-PCR. C, The ki-67 expression in the NP cell was detected by qRT-PCR. D, The PCNA expression in the NP cell was detected by qRT-PCR. E, Ectopic expression of RMRP increased Type II collagen and aggrecan expression in the pcDNA-RMRP-overexpressing NP cell. F, The expression of MMP13 and ADAMTS4 was measured by qRT-PCR. *P < 0.05 and ***P < 0.001 NP tissues compared to normal NP samples, and lower miR-206 expression was correlated with the disc degeneration grade. Interestingly, we indicated that miR-206 expression in NP tissues was negatively correlated with the expression of RMRP. Ectopic expression of miR-206 suppressed NP cell proliferation and suppressed the Type II collagen and aggrecan expression and enhanced the expression of MMP13 and ADAMTS4. Furthermore, we demonstrated that overexpression of RMRP increased NP cell growth and regulated ECM expression through targeting miR-206.
Taken together, our study showed that RMRP expression was up-regulated in degenerated NP tissues compared to normal NP samples, and higher RMRP expression was associated with the disc degeneration grade. Ectopic expression of RMRP increased NP cell growth and regulated ECM expression through targeting miR-206.
These results suggested that lncRNA-RMRP promoted the progression of IDD through targeting miR-206, providing an attractive new therapeutic approach for the treatment of IDD disease.

CONF LICT OF I NTEREST
There is no conflict of interest statement.