LncRNA ACTA2-AS1 suppress colon adenocarcinoma progression by sponging miR-4428 upregulation BCL2L11

Background Long non-coding RNA is considered to be essential to modulate the development and progression of human malignant cancers. And long non-coding RNA can act as crucial modulators by sponging the corresponding microRNA in tumorigenesis. We aimed to elucidate the function of ACTA2-AS1 and its molecular mechanism in colon adenocarcinoma. Materials and methods The expression of ACTA2-AS1, miR-4428 and BCL2L11 in colon adenocarcinoma tissues were detected via qRT-PCR. SW480 and HT29 cells were transfected with shRNA ACTA2-AS1, OE ACTA2-AS1, miRNA mimics of miR-4428, miR-4428 inhibitor, si-BCL2L11 and over-expression of si-BCL2L11. Cell proliferation, colony formation and apoptosis were respectively assessed using CCK-8 assay, colony assay and flow cytometry. Luciferase reporter assay was performed to verify the targets of ACTA2-AS1 and miR-4428. Tumor subcutaneous xenograft mode was constructed to explore tumor growth in vivo. Results ACTA2-AS1 was obviously downregulated in human colon adenocarcinoma tissues and colon adenocarcinoma cell lines. Silence or over-expression of ACTA2-AS1 promoted or inhibited cell proliferation and colony formation abilities, and regulated apoptosis. The silence of ACTA2-AS1 resulted in the decrease of Bax and increase of Bal2, while restored in OE ACTA2-AS1 group when compared with the control transfected cells. In addition, luciferase reporter assay revealed that ACTA2-AS1 interacted with miR-4428 and suppressed its expression. miR-4428 could bind to 3ʹ untranslated region of BCL2L11 and modulated the expression of BCL2L11 negatively. Knockdown of ACTA2-AS1 and over-expression of BCL2L11 reversed the biological function that ACTA2-AS1 mediated by knockdown ACTA2-AS1 alone. Conclusion Our data demonstrated that ACTA2-AS1 could suppress colon adenocarcinoma progression via sponging miR-4428 to regulate BCL2L11 expression.


Introduction
Colorectal cancer (CRC) is one of the most frequently diagnosed tumors with poor prognosis and the most common CRC is colon adenocarcinoma (COAD) [1]. The development and progress of COAD is a multistep process in which accumulating genetic changes can play an important role. Although great progression has been made in surgery, chemotherapy, radiotherapy, and targeted drugs, there is no actual achievements in the Pan et al. Cancer Cell Int (2021) 21:203 overall survival rate of COAD patients [2]. Hence, the investigation of promising therapeutic targets and molecular mechanism involved in the carcinogenesis of COAD remains especially crucial for the early diagnosis, timely treatment, and prognosis.
It is known that long non-coding RNAs (lncRNAs) are RNA molecules with more than 200 nucleotides [3,4]. Generally, lncRNAs modulate the expression level of targeted genes at the post-transcriptional period [5]. More and more lncRNAs have been found to involve in many aspects of cellular homeostasis, such as angiogenesis, metastasis, cell proliferation, immunity adjustment, genomic stability, and so on [6,7]. Previous studies also suggest that lncRNAs are involved in the tumorigenesis through multiple mechanisms, such as transcriptional regulation, protein post-translational regulation miRNA regulation and so on [8]. Previous studies also proved that a growing number of lncRNAs play crucial roles in COAD tumorigenesis and development, such as ZDH-HC8P1, FOXD3-AS1, and ZEB1-AS [9][10][11]. Therefore, lncRNAs could be regarded as potential diagnostic and prognostic biomarkers for human cancers.
LncRNA ACTA2-AS1 (ACTA2 Antisense RNA 1) is located at 10q23.31 with five exons [12]. Recent studies revealed that dysregulation of ACTA2-AS1 has been found to be closely related to poor prognosis of several cancers, such as cervical cancer, hepatocellular carcinoma, liver cancer, breast cancer and lung adenocarcinoma [13][14][15]. According to previous studies, ACTA2-AS1 may play an important role involved in the development of human cancers. However, the role of ACTA2-AS1 in COAD and its underlying molecular mechanisms remains unclear. Our previous studies found an obvious decrease of ACTA2-AS1 expression in both COAD cell lines and COAD tissues, and we presumed that ACTA2-AS1 may act as a crucial regulator in COAD. Therefore, this study is aimed to explore the specific function of ACTA2-AS1 in COAD and the molecular mechanisms involved.

Cell lines and COAD tissues
Normal human colon mucosal epithelial cell line (CCD-18Co) and six COAD cell lines (SW480, HT29, LS174T, HCT116 and DLD-1) were procured from ATCC. The above cells were cultured in RPMI 1640 medium or DMEM (Invitrogen, USA) with 10% fetal bovine serum (Invitrogen, USA) and 1% Penicillin/Streptomycin (Sigma-Aldrich, USA) at 37 °C incubator containing 5% CO 2 . 82 newly diagnosed patients with COAD in The Fifth Hospital of Wuhan were included in the present study. All the experiments were carried out according to the principles of The Fifth Hospital of Wuhan.

Cytoplasm and nuclear localization
The NE-PER ™ Cytoplasmic and Nuclear Extraction Reagents Kit (Thermo Fisher Scientific) was done to confirm the cytoplasmic localization of ACTA2-AS1 in COAD cells. Following the manufacturer's instructions, the COAD cells nuclear and cytoplasmic constituents were sorted and collected. Afterward, qRT-PCR was used to evaluate the ACTA2-AS1 expression in the nucleus and cell cytoplasm, respectively. GAPDH was used as the cytoplasm localization control while U6 was for the nucleus localization control.

Cell viability and colony assay
The

Apoptotic assay
Apoptotic assay was performed using V-FITC Annexin and PI Apoptosis Detection Kit (Beyotime, China). SW480 and HT29 were fixed in 70% cooled ethanol and stained with Annexin V-FITC and PI for 20 min at room temperature according to the protocol, and then cell apoptosis was detected by flow cytometer.

Dual luciferase reporter assay
Through starbase 2.0, we found the miR-4428 binding site in ACTA2-AS1 and the downstream target gene of miR-4428 is BCL2L11. The 3ʹ-UTR of BCL2L11 and ACTA2-AS1 containing wild type (wt) and mutant type (mut) reporter vectors were purchased from Beijing TransGen Biotech Co., (Beijing, China). miR-4428 mimics binding sequence was inserted downstream of the firefly luciferase gene in psi-CHECK2 vector to synthesis the BCL2L11-wt or ACTA2-AS1-wt and psi-CHECK2-BCL2L11-mut or ACTA2-AS1-mut plasmids, respectively. The wt and mut plasmids subsequently were co-transfected into in SW480 and HT29 cells cells with negative control and miR-4428 mimics. After transfection for 48 h, the cells were lysed and the relative luciferase activity was measured via the Dual-luciferase reporter Assay System (Promega, Madison, WI, USA).

RNA pull-down assay
For RNA pull-down assay, the streptavidin-coated magnetic beads (Life Technologies, CA, USA) were covered by biotinylated ACTA2-AS1 (Bio-ACTA2-AS1) and Bio-Oligo according to its instruction and transfected into l × 10 6 SW480 and HT29 cells at 50 nM as a final concentration for 48 h. Subsequently, 0.7 mL lysis buffer (5 mM MgClz, 100 mM KCl, 20 mM Tris (pH 7.5), 0.3% NP-40) and complete protease inhibitor cocktail (Roche Applied Science, IN) were added into the cell pellets, then the cell lysates were incubated together with the RNA-tagged beads for the co-immunoprecipitation (Invitrogen, Carlsbad, CA, USA). The RNA-RNA complexes were subsequently collected by centrifugation at 10,000 r for 10 min and then the miR-4428 enrichment level was detected with qRT-PCR analysis.

Western blotting
Protein was extracted using Radioimmunoprecipitation assay (RIPA, Beyotime, China). Protein samples were separated through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF (polyvinylidene fluoride) membranes (Thermo Fisher Scientific, USA). Membranes were blocked in 5% bovine serum albumin (BSA) for 2 h and then incubated with primary antibodies, including anti-Bcl-2, anti-Bax and GAPDH (Cell Signaling Technology, USA) overnight at 4 °C. Next day, after washed with PBST for three times, the membranes were incubated with the HRP-conjugated secondary antibodies (Cell Signaling Technology, USA) at 37 °C for 1 h. Finally, the intensity of the bands was visualized by using enhanced chemiluminescence (ECL).

Tumor xenografts in nude mice
BALB/c nude mice (16-20 g, 5 weeks of age) were purchased from Shanghai Animal Laboratory Center (Shanghai, China). Cells transfected with sh-NC or sh-ACTA2-AS1 were digested with 0.25% trypsin, diluted in PBS, counted by trypan blue staining, adjusted to a concentration of 1.0 × 10^7 cells/mL, and 0.1 mL (1.0 × 10^6 cells) of this solution was injected hypodermically into the back flank of each mice. Tumor size was calculated every 7 days. Additionally, the present investigation was approved by the ethics committee of The Fifth Hospital of Wuhan.

Statistical analysis
The data in in study were presented as mean ± standard deviation (SD). The data were visualized through the GraphPad Prism 7.0 software. All statistical analysis was

LncRNA ACTA2-AS1 was down-regulated in COAD tissue samples and cells
To investigate the role of lncRNA ACTA2-AS1 in COAD tissue samples, we firstly downloaded data from TCGA database and exhibited that the expression of ACTA2-AS1 was obviously upregulated in normal samples compared with COAD samples (Fig. 1a). Then, 82 pairs of human COAD tissue samples and their corresponding adjacent non-tumor samples were detected by qRT-PCR. As shown as Fig. 1b, the expression of ACTA2-AS1 was remarkably low in COAD tissues. In addition, this study detected the expression level of ACTA2-AS1 in six COAD cell lines (SW480, HT29, LS174T, HCT116 and DLD-1) and normal human colon mucosal epithelial cell line CCD-18Co, finding that ACTA2-AS1 expression was extremely lower in COAD cells than in CCD-18Co cell (Fig. 1c). ACTA2-AS1 expression was detected in different clinical stages of COAD, suggesting that the expression of ACTA2-AS1 was negatively correlated with advanced TNM stage. We also observed that ACTA2-AS1 expression was further significantly down-regulated in COAD patients that had lymph node or distal metastasis compared with those without metastasis ( Fig. 1e and f ). Furthermore, the median expression value of ACTA2-AS1 in COAD tissues was the cut-off value and then divided the tissues into high expression group (n = 41) and low expression group (n = 41), Kaplan-Meier survival analysis suggested that high-ACTA2-AS1 expression was associated with longer survival time in patients with COAD (Fig. 1g). Next, we further demonstrated that ACTA2-AS1 was mainly localized in cytoplasm (Fig. 1h). These results revealed that prominent low-expression of ACTA2-AS1 was detected in COAD samples and low expression of ACTA2-AS1 was related to poor prognosis in patients with COAD ( Table 1).

Effect of ACTA2-AS1 on the proliferation and apoptosis of COAD cells
In order to examine the effect of ACTA2-AS1 in COAD, we silenced ACTA2-AS1 expression in SW480 using shRNAs and over-expressed ACTA2-AS1 in HT29 cell lines, respectively. The knockdown and over-expression efficiency results demonstrated that ACTA2-AS1 expression was significantly decreased or increased by sh-ACTA2-AS1 or OE ACTA2-AS1 transfection, respectively (Fig. 2a), suggesting that the effect of ACTA2-AS1 was inhibited or promoted. The results of CCK-8 assay disclosed that knockdown of ACTA2-AS1 obviously increased the proliferative ability of SW480 cells in contrast to the sh-NC groups, while cell proliferation was obviously inhibited in the OE ACTA2-AS1 group compared with vector group (Fig. 2b). Similarly, the clone formation assay revealed that inhibition of ACTA2-AS1 increased the number of colony formation in SW480 cells lines compared with the sh-NC group, while the ability of colony formation was inhibited in the OE ACTA2-AS1 group (Fig. 2c). Next, the apoptosis role of ACTA2-AS1 in COAD cell was detected via flow cytometric analysis assay, indicating that the cell apoptosis rate was obviously decreased or increased after ACTA2-AS1 down-regulation or up-regulation (Fig. 2d). Taken together, lncRNA ACTA2-AS1 could inhibit COAD cell proliferation, and meanwhile promote apoptosis.

LncRNA ACTA2-AS1 sponged miR-4428 in COAD cells
To verify the potential molecular target of ACTA2-AS1 involved in COAD cells, an online bioinformatics tool lncBASE was detected. As shown as Fig. 3a, there was a potential complementary sequence between ACTA2-AS1 and miR-4428. Luciferase reporter assay was assessed to detect and verify the interaction of ACTA2-AS1 and miR-4428, finding that the relative luciferase activities were reduced in ACTA2-AS1-wt transfected cells while that in ACTA2-AS1-mut groups displayed no change (Fig. 3b). Moreover, RNA pull-down assay also exhibited that miR-4428 were enriched in bio-ACTA2-AS1 group (Fig. 3c). Transfection of sh-ACTA2-AS1 markedly  increased the expression of miR-4428, while transfection of OE ACTA2-AS1 decreased the expression of miR-4428 (Fig. 3d). In addition, qRT-PCR assay also disclosed that miR-4428 was increased in 82 pairs of human COAD tissue comparing with adjacent non-tumor tissues (Fig. 3e). Meanwhile, a negative relationship was observed between the expression of ACTA2-AS1 and miR-4428 in COAD tissues (Fig. 3f ). The above results confirmed that ACTA2-AS1 could serve as a sponge of miR-4428 in COAD cells.

miR-4428 directly interacted with BCL2L11
To predict the downstream direct target mRNA of miR-4428, an online bioinformatics tool Starbase was carried out. The results showed that an underlying miR-4428 binding sites was detected in the 3ʹ-untranslated region (3ʹUTR) of BCL2L11 (Fig. 4a). Then, luciferase reporter assay verified that the relative luciferase activities were reduced in BCL2L11-wt transfected cells while no significant change of relative luciferase activity was measured in the BCL2L11-mut group (Fig. 4b). Data from qRT-PCR and Western blot demonstrated that both mRNA and protein expression level of BCL2L11 decreased or increased in COAD cell lines transfected with miR-4428 mimics or miR-4428 inhibitor, respectively ( Fig. 4c  and d). The proteins of apoptosis markers Bcl-2 and Bax were assessed by Western blotting, suggesting that Bcl-2 were up-regulated in the sh-ACTA2-AS1 group in comparison with the control group. However, the level of Bax was suppressed. Next, OE ACTA2-AS1 was transfected into HT29 cells, the expression trend of the above proteins was reversed (Fig. 4e). Conversely, the downregulation of ACTA2-AS1 resulted in the decrease of Bax when compared with the control transfected cells, while increased in OE ACTA2-AS1 group in comparison with the vector group (Fig. 4e). Next, we identified that BCL2L11 was memorably down-regulated in 82 pairs of human COAD tissue compared to that in matched adjacent normal tissue (Fig. 4f ). And the expression of human COAD tissues via Spearman's rank-order analysis (Fig. 4g). These results illustrated that BCL2L11 directly interacts with miR-4428.
LncRNA ACTA2-AS1 suppressed COAD progression by sponging miR-4428 upregulation BCL2L11 MRNA expression of BCL2L11 was reduced in HT29 cells by transfection with si-BCL2L11 (Fig. 5a), and its mRNA and protein expression could be rescued in SW480 cells by sh-ACTA2-AS1 co-transfection with the miR-4428 inhibitor and BCL2L11, while the expression of BCL2L11 was increased by transfection with OE ACTA2-AS1, and its expression could be suppressed in the cells by co-transfection with the miR-4428 mimics and si-BCL2L11 in HT29 (Fig. 5b-d). Furthermore, both cell proliferation and colony formation were increased in the cells transfected of sh-ACTA2-AS1 in comparison with sh-NC group, while restored in sh-ACTA2-AS1 + miR-4428 inhibitor and sh-ACTA2-AS1 + BCL2L11 group. Correspondingly, cells transfected with OE ACTA2-AS1, both cell proliferation and colony formation were inhibited compared with sh-NC group and increased by cotransfection with OE ACTA2-AS1 and miR-4428 mimics or OE ACTA2-AS1 and over-expression of BCL2L11 ( Fig. 5e-g). In addition, flow cytometry assays manifested that silence of miR-4428 or over-expression of BCL2L11 could reverse the promoting apoptosis effect of ACTA2-AS1 (Fig. 5h). These results indicated that ACTA2-AS1 suppressed COAD progression by sponging miR-4428 upregulation BCL2L11.

The suppressive roles of LncRNA ACTA2-AS1 in COAD in vivo experiments
Then, in order to evaluate the effects of ACTA2-AS1 in vivo experiments, the BALB/c nude mice were employed to construct tumor subcutaneous xenografts, the results suggested that the volume and weight of the xenograft tumors were larger or smaller in the sh-ACTA2-AS1 group or in OE ACTA2-AS1 group comparing the sh-NC and vector group respectively ( Fig. 6a  and b). The proteins of apoptosis markers Bcl-2 and Bax were also assessed in subcutaneous xenograft tumor, the expression of Bcl-2 was showed to significantly increase in sh-ACTA2-AS1 group in comparison with the sh-NC group, while decreased in OE ACTA2-AS1 group in comparison with the vector group. Conversely, the downregulation of ACTA2-AS1 resulted in the decrease of Bax when compared with the control transfected cells, while restored in OE ACTA2-AS1 group in comparison with the vector group (Fig. 6c). A schematic representation of the ACTA2-AS1 mechanism of regulation of CRC progression is presented in Fig. 7.