LncRNA HOXA11‐AS promotes hepatocellular carcinoma progression by repressing miR‐214‐3p

Abstract Accumulating studies supported that lncRNAs played important roles in tumorigenesis. LncRNA HOXA11‐AS was a novel lncRNA that has been proved to involved in several tumours. However, the role of HOXA11‐AS in the development of hepatocellular carcinoma (HCC) remains to be explained. In our study, we showed that HOXA11‐AS expression was up‐regulated in the HCC tissues, and the higher expression of HOXA11‐AS was associated with the advanced stage in the HCC samples. In addition, we indicated that the expression of HOXA11‐AS was up‐regulated in HCC cell lines (Hep3B, SMMC‐7721, MHCC97‐H and BEL‐7402) compared with normal liver cell lines (HL‐7702). Overexpression of HOXA11‐AS promoted HCC proliferation and invasion and induced the epithelial‐mesenchymal transition (EMT) and knockdown of HOXA11‐AS suppressed the HCC cell proliferation and invasion. However, we showed that miR‐214‐3p expression was down‐regulated in the HCC tissues and cell lines. Ectopic expression of miR‐214‐3p suppressed HCC cell proliferation and invasion. Furthermore, we indicated that overexpression of HOXA11‐AS decreased the miR‐214‐3p expression and the expression of miR‐214‐3p was negatively related with the HOXA11‐AS expression in HCC samples. Ectopic expression of HOXA11‐AS increased HCC proliferation and invasion and induced EMT through inhibiting miR‐214‐3p expression. These data suggested that HOXA11‐AS/miR‐214‐3p axis was responsible for development of HCC.


| INTRODUCTION
Hepatocellular carcinoma (HCC) is one of most common tumour worldwide and is the 2nd leading cause of cancer-related death. [1][2][3][4][5] The high mortality rate of this disease is due to lack of impactful treatments. [6][7][8] Despite interventional therapy, liver transplantation, chemotherapy and surgery could cure HCC cases successfully; the 5-year survival rate was still dissatisfied. [9][10][11] Hepatocarcinogenesis was one complicated procedure and that several factors were involved in the initiation, development and progression of this disease. [12][13][14] However, the detail molecular mechanism of HCC remains largely unknown. 15 Therefore, it is crucial to unravel the molecular mechanism and find the diagnostic markers for HCC.
Long non-coding RNAs (lncRNAs) are one type of non-coding RNA that modulates gene expression at post-transcriptional or transcriptional level with the length more than 200 nucleotides. [16][17][18] Emerging studies indicated that lncRNA acts critical roles in a wide range of cell processes such as cell cycle, differentiation, proliferation, migration, metabolism and apoptosis. 19,20 LncRNAs can serve as oncogenes or tumour suppressor genes via crosstalk with other RNA or by several chromatin-based mechanisms. [21][22][23] The expression of lncRNAs was found to be deregulated in a large number of tumours including lung cancer, osteosarcoma, ovarian cancer, breast cancer, gastric cancer, colorectal cancer and HCC. [24][25][26][27][28][29][30] LncRNA HOXA11-AS was a novel lncRNA that has been proved to involved in development of some tumours such as gastric cancer, lung cancer and osteosarcoma. [31][32][33] However, the role of lncRNA HOXA11-AS in the HCC remains largely unknown.
In this study, we investigated the expression and the functional role of HOXA11-AS in HCC. We demonstrated that the expression of HOXA11-AS was down-regulated in HCC cell lines and tissues and overexpression of HOXA11-AS promoted HCC growth, invasion and epithelial-mesenchymal transition (EMT).

| Samples
A total of 40 pairs of HCC samples and the adjacent noncancerous samples were selected from cases undergone surgery at the Guangdong Second Provincial General Hospital. All tissues were obtained with written informed consent of each patient, and our study was approved by Review Board of Guangdong Second Provincial General Hospital. Tissues were snapping frozen in the liquid nitrogen until protein or RNA extraction. Both non-tumour and tumour samples were histologically confirmed.

| RNA extraction and quantitative RT-PCR
Total sample and cell RNA were extracted with TRIzol kit (Invitrogen), and then cDNA was synthesized following to manufacturer's information.

| Western blot analysis
Protein from HCC cells or samples was extracted using RIPA buffer with the proteinase inhibitor. The concentration of protein was evaluated by the Bradford kit (Bio-Rad, CA). Twenty micrograms protein lysate was resolved by 10% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride (PVDF). The membrane was blotted with the primary antibodies (anti-MELK, anti-E-cadherin, Ncadherin, vimentin and Snail antibodies, Abcam). The secondary antibody was IgG-HRP, and immunoreactive band was visualized by enhanced chemiluminescence detection system.

| Statistical analysis
The data were expressed as the mean AE standard deviation (SD). Statistical analysis was conducted by SPSS 17.0 (IBM, NY, USA). The Student's t test was performed to assess significance of differences between 2 groups. ANOVA was used assess significance of differences between more than 2 groups. P < .05 was determined as significant difference.

| HOXA11-AS was up-regulated in HCC samples
The expression of HOXA11-AS was examined using qRT-PCR in tumorous samples and compared normal samples of 40 patients.
As shown in the Figure 1A, HOXA11-AS level was higher in tumorous samples than that of compared normal tissues. In addition, expression of HOXA11-AS was lower in initial clinical stage (I-II phase) cases than that advanced clinical stage (III-IV phase) cases ( Figure 1B). Of 40 HCC samples, HOXA11-AS was up-regulated in 20 patients (20/30, 67%) compared with adjacent normal tissues ( Figure 1C). Moreover, we found that the expression of HOXA11-AS was up-regulated in the HCC tissues compared with in normal tissues using RT-PCR ( Figure 1D).

| miR-214-3p was down-regulated in HCC samples
The expression of miR-214-3p was measured using qRT-PCR in  Figure 5D). In addition, we confirmed that ectopic expression of HOXA11-AS decreased the protein expression of E-cadherin and increased the N-cadherin, vimentin and Snail expression in the SMMC-7721 cell ( Figure 5E).

| HOXA11-AS overexpression promoted HCC proliferation and invasion and induced EMT through inhibiting miR-214-3p expression
To determine the role of miR-214-3p in the HOXA11-AS-promoting HCC progression, we transfected miR-214-3p mimic in HOXA11-AS overexpressing SMMC-7721 cells. We found that ectopic expression of miR-214-3p attenuated the proliferation effect of HOXA11-AS in the SMMC-7721 cells ( Figure 8A). Furthermore, we indicated that From this project, we indicated that the expression of HOXA11-AS was up-regulated in the HCC samples and cell lines. Overexpression of HOXA11-AS promoted HCC cell proliferation, invasion and EMT partly through inhibiting miR-214-3p expression. These data suggested that HOXA11-AS may be a potential target for HCC treatment.

CONFLICT OF INTEREST STATEMENT
There is no conflict of interest statement.