Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.


Background
Colorectal cancer (CRC) ranks 3rd among the most frequent cancers globally [1]. Despite remarkable advances, the molecular basis of CRC progression is not fully understood and more effective therapeutic strategies are needed [2].
ZNF561-AS1 is a novel lncRNA whose function in CRC is unknown. Here, we find that ZNF561-AS1 overexpressed in CRC samples and this promotes the proliferative rates and survival of CRC cells in vivo, as well as in vitro. Mechanically, ZNF561-AS1 upregulates SRSF6 levels by sponging miR-26a-3p and miR-128-5p. Additionally, reduced SRSF6 levels upon ZNF561-AS1 depletion were restored by miR-26a-3p or/and miR-128-5p inhibition. Exogenous SRSF6 expression rescued proliferation of ZNF561-AS1-depleted CRC cells. The present results underscores the potential of ZNF561-AS1 to be targeted as an anti-CRC target.

Clinical tissues
Six human colorectal cancer tissues along with matched adjacent normal tissues were collected from the Second Affiliated Hospital of Harbin Medical University with patients' consents. The ethics committee of Harbin Medical University approved the study. Matched pre-treated, post-treated and recurrent samples from one patient, in total six patients were collected. The samples obtained at surgery refers to pre-treated samples. Post-treatment samples were collected after finishing chemotherapy by colonoscope. In 1-2 years after chemotherapy, the patients were suffered from relapsed tumors which refer to recurrent samples. Upon collection, tissues were snapfrozen in liquid nitrogen for further use.
Patients information is shown on Supplementary Table S1.

Western blot analysis
A RIPA buffer supplemented with protease inhibitors cocktail (Roche, Switzerland) was used to extract protein samples from cells. Equal amounts of proteins were resolved on SDS-PAGE and electro-transferred to PVDF membranes (Thermo Scientific, USA) and then blocked with 5% skim mile. After blocking, the blots were probed with following primary antibodies to SRSF6 (

TUNEL assay
Click-iT TUNEL Alexa Fluor Imaging Assay Kit (Invitrogen, USA) was employed to explore cells apoptosis.
In brief, control siRNA or ZNF561-AS1 siRNA transfected HCT-116 cells were fixed with 4% PFA for 15 min and permeabilized with 0.25% Triton X-100 for 10 min at room temperature. Then cells were washed and incubated with TdT reaction buffer for 10 min and incubated with Click-iT reaction cocktail for 30 min at room temperature. Cell nuclei were counterstained with DAPI after washing with PBS. Stained coverslips were mounted using prolong® diamond antifade mountant (Applied Biosystems, USA). Cells were imaged using a Zeiss Axiovert 200 microscope.

Nude mice experiments
Athymic BALB/c nude mice (6-weeks old) were purchased from Vital River Laboratory, Beijing, China to establish tumor xenograft models. Mice were maintained and housed under specific pathogen-free conditions and handled using aseptic procedures. HCT-116 cells (5 × 10 6 ) transfected with ZNF561-AS1 siRNA or control siRNA were then subcutaneously administered into the right shank of the mice (3 mice/ group). Tumor size (length; L, and width; W) determined with a caliper every 7 days and tumor volume given by the formula: ½LW 2 . Three weeks later, mice were killed. Isolated xenograft tumors were weighed and subjected to the further experiments.

Immunohistochemistry staining
Harvested tumors were fixed in 4% PFA for 24 h at room temperature, followed by permeabilization with PBST for 20 min and inactivation of endogenous peroxidases by incubating tissues in 0.3% H 2 O 2 for 20 min. After washing, tissues were blocked with 5% normal goat serum (Invitrogen, USA) for 30 min and incubated with anti-Ki-67 antibody (Cell Signaling Technology, USA) overnight at 4°C. Subsequently, tissues were rinsed and probed with a biotin-labeled secondary antibody for 30 min and washed. After washing, the specimens were developed with 0.05% DAB (Sigma-Aldrich, Oakville, ON, Canada) and 0.03% H2O2, and counterstained with hematoxylin, dehydrated in increasing ethanol concentrations, cleared with xylene. Images of stained specimens were captured using an Olympus BX51 microscope. Digital images were analyzed using Image-Pro Plus 6.0 software.

RNA binding protein immunoprecipitation (RIP)
RIP assay was done using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) as described by the manufacturer protocol. Briefly, HCT-116 cells were lysed in complete RIP lysis buffer. 100 μl whole cell lysate from each group were incubated with RIP buffer containing magnetic beads conjugated to mouse anti-Ago2 antibody (Millipore, USA), or negative control normal mouse IgG (Millipore, USA). Next, proteins were digest by incubated with proteinase K and immunoprecipitated RNA was harvested. The RNA concentration was quantified on NanoDrop spectrophotometer (Thermo Scientific, USA). Purified RNA was reverse transcribed into cDNA and subjected to qPCR to examine the presence of ZNF561-AS1, miR-26a-3p, and miR-128-5p using indicated primers.

Data analysis
For biological experiments, data were obtained from at least 3 independent experiments and presented as the mean ± SD. The data were subjected to F-test, and then subjected to two-tailed Student's t test (comparison between two groups) or ANOVA (Comparisons among three groups or more) using Statistical Program for Social Sciences (SPSS) version 17.0 (Chicago, IL, USA). GEPIA web server was utilized to analyze the data of expression of ZNF561-AS1 and SRSF6 in CRC tumor and adjacent normal tissues from TCGA database. The data were subjected to ANOVA analysis for differentially expressed genes based on the comparison of tumors and matched normal samples, and Pearson correlation analysis for correlation between ZNF561-AS1 and SRSF6.

Overexpression of ZNF561-AS1 in CRC positively regulates SRSF6 expression
To examine the ZNF561-AS1 expression in CRC, analysis of TCGA CRC datasets revealed an elevated ZNF561-AS1 levels in CRC patients (Fig. 1a). Consistently, significantly higher ZNF561-AS1 level was found in our collected CRC patient samples compared to their matched adjacent normal tissue samples (Fig.  1b). Analysis of ZNF561-AS1 level in various CRC cell lines consisting of SW620, HCT-116, HT-29, SW480, SW48 and LoVo cells showed that ZNF561-AS1 expression was increased in all CRC cell lines compared to non-malignant human colon epithelial cell line, CCD841 CoN (Fig. 1c), with HCT-116 expressing the highest ZNF561-AS1 levels. Moreover, we found that compared to CRC samples from untreated patients, samples from patients treated with chemotherapy exhibited lower ZNF561-AS1 levels (Fig. 1d). However, ZNF561-AS1 rose in recurrent CRC samples (Fig. 1d). Together, these data suggest a critical role for ZNF561-AS1 in CRC.
Surprisingly, TCGA CRC dataset analysis found that Serine and arginine-rich splicing factor 6SRSF6 (SRSF6), an oncogenic factor, was also elevated in CRC and showed a positive association with ZNF561-AS1 expression (Fig. 1e-f). Elevated SRSF6 levels were also detected in CRC patients samples (Fig. 1g). To assess the correlation between ZNF561-AS1 and SRSF6 expression in CRC, we used siRNA to silence ZNF561-AS1 or SRSF6 in HCT-116 and then evaluated SRSF6 or ZNF561-AS1 expression. Interestingly, we found that compared to control group, ZNF561-AS1 silencing correlated with reduced SRSF6 mRNA and protein levels ( Fig. 1 h, j).

ZNF561-AS1 is critical for CRC cells proliferation and survival
Since we found that ZNF561-AS1 is dramatically increased in CRC and it positively regulates SRSF6 expression, we next examined its biological effects on CRC cells. As expected, we found ZNF561-AS1 silencing significantly suppressed HCT-116 growth and colony formation compared to controls (Fig. 4 a-b). Ki-67 Immunofluorescence staining exhibited significantly lower Ki-67 signal in ZNF561-AS1 depleted HCT-116 cells compared to controls, indicating that ZNF561-AS1 silencing suppressed HCT-116 cells proliferation (Fig.  4c). Additionally, we found that expression of the cell cycle promoting factors, PCNA, CDK4, and Cyclin D1 was reduced markedly in ZNF561-AS1 deficient HCT-116 cells (Fig. 4d). However, p21, a strong cell cycle inhibitor, was elevated (Fig. 4d). Interestingly, the level of cleaved caspase-3, an apoptotic factor, was increased upon ZNF561-AS1 knockdown (Fig. 4d). This observation led us to examine whether ZNF561-AS1 silencing would trigger cell apoptosis in HCT-116 cells. AO/EB staining revealed that knockdown of ZNF561-AS1 increased the proportion of apoptotic cells in ZNF561-AS1 depleted HCT-116 cells compared to control cells (Fig. 4e). This phenotype was further confirmed by TUNEL staining (Fig. 4f).
To study the role of ZNF561-AS1 in vivo, ZNF561-AS1 siRNA or control siRNA transfected HCT-116 cells were subcutaneously injected into the flanks of nude mice. After 3 weeks, the mice were sacrificed, and xenograft tumors were isolated. RT-qPCR analysis confirmed ZNF561-AS1 depletion in tumors from ZNF561-AS1 depleted cells (Fig. 4g), which also exhibited increased miR-26a-5p and miR-128-3p levels and reduced SRSF6 levels relative to controls (Fig. 4 g-h). Tumors from ZNF561-AS1 depleted cells were smaller in size and lower in weight compared to control tumors. (Fig. 4 i-j). Immunohistochemical staining revealed lower Ki-67 levels in ZNF561-AS1 depleted tumors (Fig. 4k). Together, these data indicate that ZNF561-AS1 is crucial for CRC cell proliferation, as well as survival both in vitro and in vivo.

Discussion
Herein, we investigated the role of ZNF561-AS1 in CRC and the reasonable mechanism. ZNF561-AS1 was upregulated in CRC compared to matched adjacent normal tissues and ZNF561-AS1 silencing suppressed CRC cells proliferation and survival, suggesting an oncogenic role in CRC. In contrast with our findings, another study found that ZNF561-AS1 downregulation promoted migration and invasion of laryngeal cancer cells (LSCC), indicating tumor suppressor function [19]. This difference was mostly considered from the different histological types [20]. The disparity in ZNF561-AS1 function in LSCC vs CRC suggests tissue-specific or cancer-specific effects.
SRSF6 is a member of the serine-arginine-rich splicing factor family. These proteins are essential for pre-mRNA splicing and mRNA stability, export, and translation [26]. SRSF6 is a proto-oncogene often overexpressed in human skin cancer [27]. By controlling exon skipping, SRSF6 is critical for leukemia cells survival [28]. SRSF6 is also overexpressed in CRC [29]. Evidence show that SRSF6-regulated alternative splicing of ZO-1 promotes CRC progression by directly binding its motif to ZO-1 exon23 [30]. This alternative splicing effect relies on SRSF6's RRM2 domain. Here, we find that ZNF561-AS1 promotes SRSF6 expression by acting as sponge of miR-26a-5p and miR-128-3p. SRSF6 overexpression rescued proliferation and survival in ZNF561-AS1 depleted cells. Indicating that ZNF561-AS1 regulates CRC cells proliferation and survival by modulating SRSF6 expression. Because SRSF6 knockdown did not alter ZNF561-AS1 expression, while ZNF561-AS1 knockdown suppressed SRSF6 expression, we speculate that ZNF561-AS1 is an upstream post-transcriptional regulator of SRSF6.

Conclusion
In summary, our study revealed an essential role of over-expression of ZNF561-AS1 for CRC cells proliferation and survival. ZNF561-AS1 servers as a ceRNA, competitively binding to miR-26a-5p and miR-128-3p to enhance the expression of SRSF6, an alternative splicing factor. These findings provide novel insight into our understanding of CRC progression and highlight ZNF561-AS1 as a potential therapeutic target against CRC.