{'fraction_non_alphanumeric': 0.03843579692131179, 'fraction_numerical': 0.03131274500430251, 'mean_word_length': 5.104172745841844, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
LncRNA BLACAT1/miR-519d-3p/CREB1 Axis Mediates Proliferation, Apoptosis, Migration, Invasion, and Drug-Resistance in Colorectal Cancer Progression

Background Colorectal cancer (CRC) is a common severe disease around the world. The merging papers reported that long noncoding RNAs (lncRNAs) took part in the diversified pathological processes of CRC. This study aimed to uncover the role and the potential mechanism of lncRNA bladder cancer-associated transcript 1 (BLACAT1) in CRC progression. Methods LncRNA BLACAT1, micro-519d-3p (miR-519d-3p), and cAMP-responsive element binding protein 1 (CREB1) levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in CRC tissues and cells. The bio-functional effects were examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry assay, and transwell assay. The susceptibility testing was determined by oxaliplatin (OXA) administration. The potential binding sites between miR-519d-3p and BLACAT1 or CREB1 were predicted by online software starBase and confirmed by dual-luciferase reporter analysis. The relative proteins expression in CRC cells was determined by Western blot analysis. Xenograft tumor model was used to evaluate biological function of BLACAT1 in vivo. Results The expression of BLACAT1 was promoted in CRC tissues and cells, and correlated to the TNM (tumor, node, metastasis) stage, distant metastasis, and overall survival rate. Silencing of BLACAT1 limited the proliferation, migration, and invasion, facilitated the apoptosis, and re-sensitized OXA-resistance in CRC cells. MiR-519d-3p was a target of BLACAT1. Furthermore, miR-519d-3p deletion reversed the positive effects of BLACAT1 deletion on CRC cells. Moreover, our data showed that miR-519d-3p directly targeted CREB1 and BLACAT1 sponged miR-519d-3p to regulate CREB1 expression. Besides, CREB1 disrupted the bio-functional results above from BLACAT1 suppression. Additionally, BLACAT1 knockdown promoted CRC cells sensitivity to OXA in vivo. Conclusion BLACAT1 mediated the progression of CRC and OXA-resistance by miR-519d-3p/CREB1 axis.


Introduction
Colorectal cancer (CRC), an intractable malignancy, ranks the third leading cause in cancer-related death. 1 Although the therapeutic method for CRC has achieved improvements, the overall survival rate was still low due to the high recurrence rate and drug resistance after chemotherapy treatment. 2,3 Accumulating evidence indicated that CRC was considered as gene-related disease accompanied with distinctly pathological physiology and molecular features. Moreover, the dysfunction of long non-coding RNAs (lncRNAs) was found in CRC in recent years. 4,5 Hence, a better understanding of the molecular mechanisms of CRC may provide the target for CRC treatment.
LncRNAs are a class of long RNAs (more than 200 nucleotides (nts) in length) without translation capacity and could affect gene expression at the transcriptional stage. 6 Emerging evidence suggested that lncRNAs acted as tumor-suppressors or promoters, and widely participated in the processes of tumorigenesis. 7,8 LncRNA bladder cancer-associated transcript 1 (lncRNA BLACAT1) was reported to be involved in various human cancers, such as glioma, 9 papillary thyroid carcinoma, 10 cervical cancer, 11 colorectal cancer, 12 and non-small cell lung cancer. 13 Moreover, the high expression of lncRNA BLACAT1 in the serum of patients could work as a new supplementary biomarker for the diagnosis of CRC. 14 Su et al showed that BLACAT1 repressed p15 expression via binding with EZH2 and enhancing the regulation of cell cycle and proliferation in CRC cells. 12 However, the role and underlying mechanism of BLACAT1 in CRC progression need further investigation.
MicroRNAs (miRNAs) are small non-protein-coding RNAs with 18~22 nucleotides, and they exhibit crucial effects on a series of cell biological behaviors, such as cell growth, survival, metastasis, and differentiation. 15,16 Multiple researches indicated that the dysregulation of miRNAs was found in types of human diseases. 17 MicroRNA-519d-3p (MiR-519d-3p) was confirmed to be dysregulated in some human cancers, such as non-small cell lung cancer, 18 pancreatic ductal adenocarcinoma, 19 and gastric cancer. 20 Moreover, Ye et al reported that miR-519d-3p was downregulated in CRC tissues and cells. 21 However, the molecular mechanism of miR-519d-3p in CRC progression remains largely unclear.
In this study, we aimed to explore the role and the underlying mechanism of lncRNA BLACAT1 in human CRC, as well as investigating the effect of BLACAT1/ miR-519d-3p axis on the progression of CRC.

Clinical Data
The experiment was authorized by the Ethics Committee of Taizhou Hospital of Zhejiang Province (201924- 13) and executed according to the Declaration of Helsinki Principles. Paired colorectal cancer tumors and adjacent normal mucosa tissues were obtained from 55 CRC patients (23 males and 32 females; age: 56-70 years old) were collected at the Department of General Surgery, Taizhou Hospital of Zhejiang Province between September 2012 and December 2014. CRC patients who received neoadjuvant chemotherapy/radiotherapy were excluded. The tumor stage was classified according to the 7th edition of the AJCC Cancer Staging Manual (2010) and CRC patients were divided into two groups: T1/T2 stage (n=25) and T3/T4 stage (n=30). Written informed consents were provided by all participants. All samples were preserved at −80°C for storage.

Flow Cytometry
Cells were collected by digesting with pancreatin and centrifuging. Then, cells were washed by iced phosphatebuffered saline (PBS) and resuspended with 1×binding buffer. Next, Annexin V-fluorescein isothiocyanate propidium iodide (Annexin V-FITC/PI) kit (BD Pharmingen, San Diego, CA, USA) was conducted to steps subsequently following the manufacturer's instructions. Apoptotic cells were examined using flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and then the apoptosis rate was calculated.

Transwell Assay
Cell migration was detected by transwell chamber (Corning Life Sciences, Corning, NY, USA) without matrigel matrix, while cell invasion experiment was conducted with matrigel matrix (Corning). The lower chamber was added with RPMI-1640 medium with 10% FBS, while the transfected HCT116 and HT29 cells were injected into the upper one with 100 μL of serum-free medium. Cells on the lower surface of chambers were fixed by methanol and then stained with 0.1% crystal violet. Finally, a microscope was used to quantify the migrated or invaded cells.

OXA Sensitivity Assay
The cell viability of HT29/OXA and HCT116/OXA cells was determined by MTT assay kit (Sigma). OXA sensitivity was detected using the IC50 value (half maximal inhibitory concentration).

Xenograft Tumor Model
The animal experiments were approved by the Ethic Committee of Taizhou Hospital of Zhejiang Province (201924-13) and performed in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. BALB/c-nude mice (four-weekold) were purchased from Charles River Laboratories (Beijing, China). HT29/OXA cells (2.0×10 7 ) transfected with sh-BLACAT1 or sh-NC were subcutaneously injected into the dorsal flanks of each nude mouse. One week later, the mice were intraperitoneally administered with 3 mg/kg OXA every 5 days, and PBS was used as the control. 22 All mice were divided into 4 groups (n=6 per group): sh-NC, sh-BLACAT1, sh-BLACAT1+PBS, sh-BLACAT1+OXA, sh-NC group was used as a control group. The tumor volume was measured once per week with a digital caliper. The tumor volume was calculated with the equation: Volume = (length × width 2 )/2. After 5 weeks, the mice were euthanized via CO2 asphyxia method using the flow rate of CO2 to displace 50% air of cage volume per min in line with the current guideline of American Veterinary Medical Association (AVMA). Tumor tissues were separated for tumor weighing, qRT-PCR, and Western blot assay.

Statistical Analysis
All data were expressed as mean ± standard deviation (SD) and analyzed by the SPSS 17.0 software. Comparisons among different groups were analyzed using paired Student's t-test and one-way analysis of variance (ANOVA) followed by Tukey's test. A P value less than 0.05 was regarded as statistically significant.

LncRNA BLACAT1 Expression Was Upregulated in CRC Tissues and Cells, and Correlated with TNM Stage, Distant Metastasis, and Overall Survival Rate
Firstly, we examined the BLACAT1 level in the CRC tumor tissues. The expression of BLACAT1 was higher in CRC tissues (n=55) relative to the adjacent normal tissues (n=55) ( Figure 1A). Next, we explored the correlation between BLACAT1 expression level and the clinicopathological parameters in CRC patients. The expression of BLACAT1 in T1/T2 stage patients (n=25) and T3/T4 stage patients (n=30) was measured by qRT-PCR. The results demonstrated that the BLACAT1 level was remarkably correlated with TNM stage, and the advanced TNM stage was significantly positively related to the high BLACAT1 expression ( Figure 1B). Next, we assessed the role of BLACAT1 in CRC patients without distant metastasis (n=37) and CRC patients with distant metastasis (n=18). The data showed that high BLACAT1 level was significantly correlated with distant CRC metastasis (nonmetastasis, n=37 versus metastasis, n=18) ( Figure 1C). To further evaluate the value of BLACAT1 in the prognosis of patients with CRC, we used Kaplan-Meier survival analysis and Log-rank tests. We divided the samples into high (above the mean, n=28) and low (below the mean, n=27) BLACAT1 expression groups according to the mean value of the BLACAT1 level. As shown in Figure 1D, the high BLACAT1 level was associated with shorter overall survival. Subsequently, BLACAT1 levels in four CRC cell lines (HCT116, HT29, LOVO, and SW620) and the normal human intestinal epithelial cell-6 (HIEC-6) cell line were determined using qRT-PCR. We observed that BLACAT1 expression was higher in CRC cell lines than in HIEC-6 ( Figure 1E). To further investigate the bio-functional effect of lncRNA BLACAT1 on CRC cells, we knocked down the BLACAT1 expression of HCT116 and HT29 cells using three different synthesized siRNA, si-BLACAT1#1, si-BLACAT1#2, si-BLACAT1#3 individually. The expression level of BLACAT1 was downregulated by transfection of si-BLACAT1 ( Figure 2A). Si-BLACAT1#1 was used to conduct for further loss-functional experiments. MTT assay indicated that cell activity was significantly reduced by BLACAT1 deletion in HCT116 and HT29 cells ( Figure 2B and C). Then, flow cytometry was used to detect the apoptosis, and the number of apoptotic cells was significantly increased in HCT116 and HT29 cells transfected with si-BLACAT1 ( Figure 2D). Moreover, the abilities of migration and invasion were obviously suppressed by BLACAT1 knockdown ( Figure 2E). Then, the expression of cell apoptosis-related protein Cleaved cas3 and cell metastasis-related protein MMP9 was detected by Western blot. Our data showed that BLACAT1 knockdown elevated Cleaved cas3 expression and suppressed MMP9 expression in HCT116 and HT29 cells ( Figure 2F). MTT assay was performed to determine the IC50 of OXA in HT29/OXA and HCT116/OXA cells transfected with si-BLACAT1 or si-NC. Notably, the results showed that BLACAT1 knockdown resensitized HCT116/OXA and HT29/OXA cells to OXA ( Figure 2G-J). These results indicated that knockdown of BLACAT1 repressed the abilities of proliferation, migration, and invasion, and promoted the rate of apoptosis cells, and re-sensitized CRC cells to OXA.

MiR-519d-3p Was a Target miRNA of BLACAT1
Next, we predicted the relationship between BLACAT1 and miR-519d-3p by starBase, and the result showed that miR-519d-3p contained complementary sequences with BLACAT1 ( Figure 3A). Then, we constructed dualluciferase reporter vectors and co-transfected miR-519d-3p or miR-NC with BLACAT1-WT or BLACAT1-MUT into HCT116 and HT29 cells. Dual-luciferase reporter assays showed that miR-519d-3p reduced the luciferase activity of BLACAT1-WT, but not BLACAT1-MUT ( Figure 3B and C). Furthermore, the expression level of miR-519d-3p was significantly upregulated by BLACAT1  Figure 3E). Besides, we also evaluated that the expression patterns of miR-519d-3p in CRC cells and tissues of CRC patients, and the data represented that the level of miR-519d-3p was decreased in both CRC cells and tissues, compared with its own corresponding controls ( Figure 3D and F). In addition, the expression relationship between miR-519d-3p and BLACAT1 kept a negative correlation ( Figure 3G). Therefore, we determined that miR-519d-3p was the target miRNA of BLACAT1.
Meanwhile, cell migration and invasion were suppressed by miR-519d-3p overexpression or BLACAT1 deletion, while the effect of BLACAT1 deletion on cell migration and invasion was blocked by miR-519d-3p inhibitor ( Figure 4E and F). Western blot results indicated that miR-519d-3p overexpression or BLACAT1 knockdown promoted the expression of Cleaved-cas3 and inhibited the expression of MMP-9, while these effects of BLACAT1 knockdown were reversed by miR-519d-3p inhibitor ( Figure 4G-I). Moreover, BLACAT1 deletion promoted the sensitivity of HCT116 and HT29 cells to OXA, which was abolished by miR-519d-3p inhibitor ( Figure 4J-K). These results indicated that miR-519d-3p inhibitor inverted the effects of si-BLACAT1 transfection, and suppressed the sensitized OXA-resistance in CRC cells.

Replenishment of CREB1 Reversed the Effect of BLACAT1 Downregulation on CRC Cells
To explore the functional relationship between BLACAT1 and CREB1, the si-NC, si-BLACAT1#1, si-BLACAT1#1 +vector or si-BLACAT1#1+CREB1 was transfected into HCT116 and HT29 cells, individually. Firstly, we examined the expression of CREB1 in CRC cells by Western blot and found that the level of CREB1 was significantly inhibited by BLACAT1 knockdown, which was abolished by CREB1 overexpression ( Figure 6A). Then, the promotion effect of BLACAT1 knockdown on cell apoptosis, and the inhibition effects on cell viability, migration, and invasion were reversed by CREB1 overexpression (Figure 6B-F). Our data showed that the promotion effect of BLACAT1 knockdown on Cleaved-cas3 expression, and the inhibition effect on MMP-9 expression were abolished by CREB1 overexpression (Figure 6G and H). Also, the sensitivity of HCT116/OXA and HT29/OXA cells was arrested when cells were transfected with si-BLACAT1#1 +CREB1 ( Figure 6I-J). The results indicated that overexpression of CREB1 reversed the effects of BLACAT1 downregulation on CRC cells in vitro.

BLACAT1 Knockdown Enhanced OXA-Sensitivity in CRC in vivo
To further determine the functional role of BLACAT1 in OXA resistance in vivo, HT29/OXA cells infected with sh-NC or sh-BLACAT1 were subcutaneously injected into the nude mice to generate xenograft, followed by treatment with OXA or PBS. The result indicated that BLACAT1 knockdown or OXA treatment significantly suppressed tumor growth, evidenced by the reduced tumor volume and tumor weight. Moreover, simultaneous BLACAT1 knockdown and OXA treatment resulted in a more obvious inhibition on tumor growth, suggesting that down-regulation of BLACAT1 facilitated the sensitivity of CRC cells to OXA in vivo ( Figure 7A and B). Furthermore, qRT-PCR analysis showed that a lower mRNA level of BLACAT1 and a higher expression of miR-519d-3p was found in tumors after sh-BLACAT1 introduction ( Figure 7C and D), especially after the combination of sh-BLACAT1 introduction and OXA treatment. Western blot assay revealed that BLACAT1 knockdown greatly weakened CREB1 and MMP-9 protein level, and promoted Cleaved-cas3 expression in tumor tissues ( Figure 7E). The combination of BLACAT1 knockdown and OXA exposure led to much lower CREB1 and MMP-9 protein expression and higher Cleaved-cas3 expression ( Figure 7E). All these data demonstrated that BLACAT1 depletion enhanced the OXA sensitivity of CRC cells in vivo.

Discussion
LncRNAs have been reported to be involved in the progression of colorectal cancer (CRC). In recent three years, several novel lncRNAs with abnormal expression have been defined in CRC. [23][24][25][26][27] Zhong et al found that MLK7 antisense RNA 1 (MLK7-AS1) was significantly upregulated in both CRC tissues and lines. Meanwhile, the increased MLK7-AS1 expression was positively correlated to a series of clinicopathological factors in patients with CRC. 28 Li et al reported that long noncoding RNA FGD5 antisense RNA 1 (FGD5-AS1) was increased in CRC cells9. 29 However, the molecular mechanisms of lncRNAs in CRC progression have not been fully understood yet.
Several studies showed that BLACAT1 participated in multiple cancers. High expression of lncRNA BLACAT1 was considered to be a new prognostic predictor of tumors. 14 For instance, the enhanced expression of BLACAT1 correlated with advanced stage, metastasis, and poor prognosis of participants with non-small cell lung cancer (NSCLC). 14,30 Previous studies documented that BLACAT1 exerted vital patterns in the progression of CRC. Enhanced expression of BLACAT1 was an independently poor prognostic indicator for CRC, and downregulation of BLACAT1 significantly inhibited tumor proliferation and invasion in vitro. 12,31 Furthermore, high expression of BLACAT1 contributed to resistance to tumor cells with OXA or cisplatin treatment, which suggested that BLACAT1 may participate in tumor progression and chemical resistance. 9,32 Overexpression of miR-519d-3p in vitro could significantly inhibit the abilities of proliferation, migration, and invasion in CRC cells, as well as increase the sensitivity of cells to 5-fluorouracil (5-Fu) exposure. [33][34][35] Our data showed that the high expression of BLACT1 positively correlated to TNM grade, distant metastasis, and overall survival, which was consistent with reported paper. 12 Moreover, BLACAT1 knockdown suppressed cell viability, migration, invasion, and promoted cell apoptosis. Meanwhile, identical results also could be seen in osteosarcoma. 36 Furthermore, downregulated BLACT1 re-sensitized OXA-resistance in vitro. Moreover, miR-519d-3p was a potential target of BLACAT1. Meanwhile, function assays revealed that miR-519d-3p knockdown reversed the regulatory effects of BLACAT1 silencing on CRC cells. These results above showed that BLACAT1 could play an essential role in proliferation, apoptosis, migration, invasion, and drug-resistance in CRC progression in vitro by sponging miR-519d-3p.
CREB1 is an oncogene that regulates various processes including cell proliferation and differentiation. Studies have shown that inhibition of CREB1 expression could reduce the resistance of colorectal cancer cells to chemical drugs (OXA, 5-Fu). 37,38 In our study, we identified that CREB1 was upregulated in CRC tissues and cells, and the similar results upon CREB1 in CRC were documented in the previous study. 39 Interestingly, CREB1 was a target of miR-519d-3p. Moreover, the CREB1 protein expression was upregulated in CRC cells treated with co-transfection of both si-BLACAT1 and anti-miR-519d-3p in comparison with transfection of si-BLACAT1 solely in vitro. Function assays and drug-sensitivity tests demonstrated that the biological function regulated by BLACAT1 silencing could be inverted by CREB1 ectopic expression. Finally, diminished BLACAT1 facilitated the OXA-sensitivity of CRC cells in xenograft model experiments.
There were some limitations in this study, the interaction between miR-519d-3p and BLACAT1 or CREB1 was initially detected by dual-luciferase reporter assay, and it should be confirmed by RNA immunoprecipitation or RNA pull-down. Besides, the results and conclusions obtained using commercial cell lines could not fully represent the actual situation in vivo. In conclusion, our study uncovered that BLACAT1 worked as an oncogene, suppressed cell apoptosis, and OXA-induced sensitivity, facilitated cell proliferation, migration, and invasion by targeting miR-519d-3p to regulate CREB1 expression in CRC progression in vitro. Additionally, this paper also revealed a novel axis of miR-519d-3p/CREB1, supplying a different therapeutic method for CRC treatment.

Funding
This work was approved by Basic public welfare research plan of Zhejiang Province (LGF19H160017).

Publish your work in this journal
Cancer Management and Research is an international, peer-reviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes, enhanced survival and quality of life for the cancer patient. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/testimonials.php to read real quotes from published authors.