Long non‐coding RNA CRNDE promotes the proliferation, migration and invasion of hepatocellular carcinoma cells through miR‐217/MAPK1 axis

Abstract Hepatocellular carcinoma (HCC) is an invasive malignant tumour and the second major cause of cancer‐related deaths over the world. CRNDE and miR‐217 are non‐coding RNAs which play critical roles in cell growth, proliferation, migration. Mitogen‐activated protein kinase 1 (MAPK1) also participates in cancer cell process. Hence, this study aimed at investigating the effect of CRNDE on migration and invasion of HCC and figuring out the role of miR‐217 and MAPK1 in this process. The overexpression of CRNDE was demonstrated by a microarray‐based lncRNA profiling study. CRNDE expression in HCC was verified by qRT‐PCR. MTT assay and BrdU staining were applied to detect cell proliferation level. Transwell assay was utilized to examine cell migration and invasiveness abilities. Wound healing assay was performed for further exploration of cell migration capacity. MiR‐217 was predicted by bioinformatics. The dual luciferase reporter assay was performed to corroborate the targeting relationship between CRNDE, miR‐217 and MAPK1. MAPK1, the downstream target of miR‐217, was predicted using bioinformatics and was further confirmed by qRT‐PCR and Western blot. The interaction between CRNDE, miR‐217 and MAPK1 was studied by qRT‐PCR, Western blot, MTT, BrdU, transwell assay and wound healing assay. CRNDE was up‐regulated in HCC tissues and HCC cell lines. The high expression of CRNDE facilitated cell proliferation, migration and invasion, while the inhibited one affected on the contrary. MiR‐217, negatively correlated with CRNDE expression, was the target of CRNDE and was more lowly expressed in HCC. With the high expression of miR‐217, HCC cell proliferation, migration and invasion were suppressed. MAPK1, the possible target of miR‐217, was negatively correlated with miR‐217 but positively correlated with CRNDE and had the same effect in HCC formation process as CRNDE. Long non‐coding RNA CRNDE promotes the proliferation, migration and invasion of HCC cells through miR‐217/MAPK1 axis.


| INTRODUCTION
As the predominant liver malignancy, hepatocellular carcinoma (HCC) is the second major cause of cancer death in the world. 1 In spite of remarkable efforts to discover novel therapeutic strategies against HCC, the long-term survival rate remains dismal. Cancer metastasis and poor prognosis are two major obstacles in HCC therapy. 2 Most of HCC patients still suffer from metastasis and recurrence after treatment. The molecular mechanism underlying HCC has not been clearly characterized. Thus, it is extremely critical to promote researches on molecular biomarkers which regulate metastatic behaviour and prognosis evaluation of HCC. 3 Recently, an increasing number of studies have revealed the crucial role of non-coding RNAs in HCC, including long non-coding RNA (lncRNAs) and micro-RNAs (miRNAs). 4,5 LncRNAs are a class of poor conserved endogenous RNAs longer than 200 nucleotides that do not encode proteins but regulate gene expression. 6 In recent years, accumulating evidences have revealed the importance of lncRNAs on cancer metastasis and development, suggesting the involvement of lncRNAs in cancer progression. 7,8 The lncRNA Colorectal Neoplasia Differentially Expressed (CRNDE), which was initially found to increase in colorectal cancer, was also overexpressed in various malignant diseases including HCC. 9 Current evidences have suggested that CRNDE exert influence on the propagation and metastasis of malignant carcinoma. 10 However, the expression profiles and precise biological functions of CRNDE in HCC remain largely unknown.
On the other hand, miRNAs, a class of endogenous non-coding RNAs with 20-25 nucleotides length, have been proved to modulate mRNA stability and protein translation. 11 MiRNAs play a crucial role in cancer development process such as differentiation, proliferation and metastasis. 12 According to previous studies, microRNA 217 (miR-217) was linked with cell progression as a tumour suppressor. 13,14 But, the mechanism of miR-217 is still not clear in HCC.
Epithelial-to-mesenchymal transition (EMT), the process by which epithelial cells gain migratory and invasive properties, has been identified as an important process in cancer progression, especially in HCC. 15 Dysregulated expression of lncRNAs and miRNAs were reported to mediate the EMT progression in HCC cells. 16,17 Consequently, lncRNAs and miRNAs are considered as potential novel therapeutic targets against the poor prognosis and metastasis of HCC. Meanwhile, mitogen-activated protein kinase 1 (MAPK1) is involved in a variety of cancer cell processes such as proliferation and migration. 18 MAPK1 has been proved to mediate EMT as an intracellular signalling molecule, and some signalling molecules can also affect EMT progression through MAPK1 pathway. 19 Zhang  Recently, some studies revealed that one potential function of lncRNAs was to directly interact with miRNAs, regulating their expression and activity. 21 In recently described mechanism, lncRNAs might function as competitive endogenous RNAs to sponge specific miRNAs, thereby mediating the de-repression of miRNAs targets. 22 For instance, lncRNA MALAT1 facilitated migration and invasiveness by modulating miR-1 in breast cancer. 23 LncRNA H19 regulated cancer cell propagation by regulating miR-194-5p. 24 LncRNA UCA1 exerted oncogenic effects by targeting mir-193a-3p in lung cancer. 25 We therefore hypothesized that CRNDE might also directly interact with some particular miRNAs.
Herein, we reported that CRNDE and miR-217 had different expression in HCC. Our results elucidated that CRNDE could modulate MAPK1 pathway by competitively inhibiting miR-217, thereby promoting HCC cells migration and invasiveness. Our findings exhibited that CRNDE might serve as a potential therapeutic target against HCC.

| Patients and samples
HCC tissues were obtained from 46 patients with informed consents of Tongji Hospital. None of these patients received chemotherapeutic treatment or radical surgical treatment. All adjacent tissues and tumour tissues were preserved in liquid nitrogen under −80°C.
This study was approved by the Institutional Ethics Committee of Tongji Hospital.

| Microarray
Ten fresh human HCC tissues and paired para-tumour tissues were acquired. Total RNA was extracted from these tissues and pooled. The collected RNA samples serve as templates for cDNA synthesis. Probe labelling and hybridization were carried out by Affymetrix GeneChip  Then, we employed whole genome microarray expression profiling as a discovery platform to identify differentially expressed genes (DEGs) between HCC and normal control. After the preprocessing of the raw expression data, the DEGs were analysed using limma package in R/ Bioconductor. The criteria for DEGs were based on fold change>2 combined with adjusted P value less than 0.05.

| Dual luciferase reporter assay
The wild-type CRNDE and MAPK1 3′UTR sequence were amplified,

| MTT assay
The cellular proliferation viability of HepG2 and Huh-7 cells was

| BrdU staining assay
Transfected cells were seeded on coverslips in 96-well plates and

| Statistical analysis
Mean ± standard deviation (SD) presented all quantitative values. Student's t test was utilized for comparison between two groups. Paired t test was used for CRNDE/miR-147 comparison between adjacent and tumour tissues, unpaired t test was applied for other comparison between two groups. One-way analysis of variance (ANOVA) was applied for comparison in multi-groups. Statistical analyses were conducted by GraphPad Prism v6.0 (Graphpad Software, La Jolla, CA, USA). The difference was statistically significant when P < 0.05.

| CRNDE was screened out by microarray analysis
Ten pairs of HCC tissues and adjacent tissues were used to perform microarray. Fold change > 2 and P < 0.05 were applied to explore the abnormal lncRNA expressions. The volcanic plot and the heat map of lncRNA expression reflected that compared with adjacent normal tissues, CRNDE was up-regulated in HCC tumour tissues.

| CRNDE highly expressed while miR-217 had a low expression in HCC cells and there was a targeted regulatory relationship between them
The overexpression of CRNDE was further confirmed by qRT-PCR in 46 HCC specimens (Figure 2A,

| MAPK1 was the target of miR-217 and was regulated by CRNDE/miR-217 axis
Bioinformatics analysis was utilized to predict that MAPK1 was the possible targeting genes of miR-217 ( Figure 5A)

| MiR-217/MAPK1 regulatory axis affected HCC cell proliferation, migration, invasion enhance proliferation, migration and invasion through EMT process
MTT assay showed that HepG2 cell proliferation level was increased CRNDE has been identified to promote tumour development, 9,29 and its function in HCC remains had been preliminarily revealed in some research, such as Chen et al revealed that CRNDE promotes hepatic carcinoma cell proliferation, migration and invasion by suppressing miR-384. 30 In this study, CRNDE expression was proved to be remarkably increased in HCC and the results are consistent with previous studies. Up till now, a number of lncRNAs have been characterized as regulators in HCC cells metastasis process. 31   Relative CRNDE expression Relative Vimentin expression F I G U R E 8 CRNDE/miR-217 were related to EMT process and MAPK1 signalling pathway. (A) Expression of EMT markers was related with CRNDE/miR-217 expression. (B, C) The protein expression levels of MAPK1 and down-stream c-Myc were regulated by CRNDE/miR-217 axis. **P < 0.01, compared with NC group. ## P < 0.01, compared with anti-miR-217+sh-MAPK1/miR-217+MAPK1 group the pathogenesis of HCC, some deficiencies in our study should be ameliorated in future researches. For example, in vivo experiment should be employed to verify the molecular mechanism of CRNDE/ miR-217/MAPK1 axis.
In conclusion, this study elucidated that CRNDE was increased in HCC and promoted migration, invasiveness and EMT progression of HCC cells via CRNDE/miR-217/MAPK1 axis. Specifically, CRNDE could promote HCC process as a molecular sponge of miR-217, which targeting MAPK1 pathway. We preliminarily clarified the relationship between CRNDE and miR-217. Promising therapeutic strategies against HCC might be established by targeting CENDE or repairing the balance between miR-217 and MAPK1.