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Long intergenic non-coding RNA 1939 inhibits proliferation and migration of human renal cell carcinoma (RCC) cells by down-regulation of miR-154

Background: Renal carcinoma (RCC) is widely accepted as a malignant tumor of urinary system. Long intergenic non-coding RNA 1939 (LINC01939) is a novel lncRNA which was found to be down-regulated in RCC. Thus, we set out to explore the effect and regulation mechanism of LINC01939 in RCC. Methods: LINC01939 and miR-154 in RCC tissues and cell lines were detected using qRT-PCR assay. Cell counting kit-8 (CCK-8) assays was exploited to examine cell viability. Flow cytometric analysis was conducted to examine apoptosis. Cell mobility was valued through wound healing assays. Western blotting was applied for examination of proteins related to proliferation, apoptosis, migration and Wnt/β-catenin/Notch. Results: LINC01939 was down-regulated in RCC tissues. LINC01939 overexpression impeded proliferation and migration, and induced apoptosis. Further study found that the overexpression of LINC01939 strongly suppressed miR-154 expression. Then, the inhibiting effect of overexpressed LINC01939 on proliferation and mobility and the promoting role of LINC01939 in apoptosis were abolished by the combination of miR-154 mimic. Finally, we found that the overexpressed LINC01939 inactivated Wnt/β-catenin and Notch through suppressing miR-154. Conclusion: The up-regulation of LINC01939 inhibited proliferation and migration of RCC cells by down-regulating miR-154.


Introduction
As one of the 10 most common malignant obesity tumors, renal cell carcinoma (RCC) is a common chemotherapeutic drug-resistant disease. The global annual incidence of RCC is about 20,000 cases, with 102,000 deaths [ 1]. Among them, clear cell renal cell carcinoma (ccRCC) is the most common type, accounting for 80-90%, with the characteristics of local invasion, distant metastasis and high lethal rate [ 2]. Thus, the prognosis of patients with ccRCC is worse and the incidence of ccRCC is gradually increasing with a mortality rate about 40% [ 3]. At present, the main treatment of RCC is surgical treatment. However, even after early radical surgery, there are still many tumors recurrence and metastasis cases. Thus, it is of great significance to investigate the molecular mechanisms of RCC with multi-gene and multi-level diagnostic and therapeutic tools, aiming to get in-depth understanding of RCC.
Long non-coding RNA (lncRNA), defined as RNAs with no protein coding potential, is longer than 200 nt. LncRNA was initially thought to be merely transcriptional noise, which has no biological function [ 4]. But due to the growing depth of sequencing technology, more and more new lncRNAs have been found to be involved in various physiological and pathological processes [ 5]. Studies have shown that a number of lncRNAs are abnormally expressed in many tumors and may be involved in multiple processes of tumorigenesis and development, including cell differentiation and proliferation, cell cycle regulation, invasion and metastasis [6][7][8]. Thus, lncRNAs have been regarded as new biomarkers in various cancers including RCC. Recently, Chen et al. revealed that long intergenic non-coding RNA 1939 (LINC01939) is downregulated in gastric cancer tissues and is associated with clinical stage of gastric cancer patients [ 9]. However, the biological function in RCC has never been explored as well as the underlying mechanisms. microRNAs (miRNAs or miRs) are recognized as the small non-coding RNAs essentially with a length of 18-25 nt, which are widely distributed in eukaryotic organisms. They are mainly responsible for regulating gene and protein expression and participating in many physiological and pathological processes, for instance, cell growth, differentiation and apoptosis [ 10]. These single-stranded mature miRNAs mediate protein coding by binding to 3'untranslated region (3'-UTR) homologous sequence of their target gene mRNA [ 11]. miRNAs are very important regulator in urinary tumors and the expression and function of miRNAs can be observed in most urinary tumors. For example, up-regulation of miR-21 and miR-221 has been observed in tumor specimens relative to normal tissue, which is associated with the pathological status of patients [ 12]. Besides, Lin et al. found that miR-154 upregulation impedes apoptosis process and facilitates proliferation and migration and invasion in RCC cells [ 13]. However, the regulating effects between miR-154 and lncRNAs have never explored before.
In our present study, we noticed the down-regulation of LINC01939 in RCC tissues. Further study revealed that overexpressed LINC01939 suppressed proliferation and migration and promoted apoptosis by down-regulating miR-154 with bluntness of Wnt/β-catenin and Notch, providing novel diagnostic and therapeutic targets for RCC.

Clinical specimens
Kidney cancer tissue specimens were acquired from patients (n = 18) who had tumor resection at the

Western Blot
The CAKI-1 and ACHN cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China). The extracted protein content was quantified using the BCA TM Protein Assay Kit (Pierce). After separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transference onto polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA), the proteins were blocked with 5% nonfat milk overnight at 4°C. Next, the proteins were probed with primary antibodies at 4°C overnight. After washed, the membranes were incubated with secondary antibody for 1 h at room temperature.
Finally, protein signaling was captured with an ECL system (Amersham Pharmacia, Piscataway, NJ, USA).

Migration assay
The migration activity of ACHN and CAKI-1 cells was valued by using a modified two-chamber (with a pore size of 8 mm) migration assay. Firstly, the cells were plated in the top chamber in medium without serum, and the complete medium (600 μl), FBS (with a final concentration of 10%) and epidermal growth factor (25 ng/ml) (Sigma-Aldrich) were supplemented into the lower compartment. Statistical analysis SPSS 19.0 statistical software (Chicago, IL, USA) was applied to analyze the data which were computed as the mean ± stand deviations (SD). Either one-way analysis of variance or student's t test was used to compare the differences, and P-values less than 0.05 was accepted and suggested the statistical difference.

LINC01939 was down-regulated in renal carcinoma tissues
The expression of LINC01939 in renal carcinoma tissues in the non-tumor tissues was firstly examined through qRT-PCR. As shown in Figure 1A (Fig.1A), the level of LINC01939 was suppressed in renal carcinoma tissues compared with the para-carcinoma tissues.
Overexpression of LINC01939 facilitated apoptosis process in renal carcinoma cell lines Specific pc-LINC01939 was transduced into ACHN and CAKI-1 cells respectively to up-regulate the level of LINC01939 as shown in Fig.1B and the empty vector was used as a control. Then we found that cell viability was obviously suppressed in LINC01939-transfected (Fig.1C). Further, results from Western blotting assay showed that p53 was elevated and cyclinD1 was suppressed through transfecting with LINC01939 in both CAKI-1 and ACHN cells, identifying that cell proliferation was suppressed by overexpression of LINC01939 (Fig.1D). In addition, we found that LINC01939 overexpression obviously induced the apoptosis progress of ACHN and CAKI-1 cells (Fig.1E). At the same time, the expression of Bax and the cleavage of caspase-3 were also elevated by LINC01939 (Fig.1F). Thus, we concluded that overexpression of LINC01939 promoted the apoptosis of renal carcinoma cell lines.

Migration activity of ACHN and CAKI-1 cells was weakened by LINC01939 overexpression
The effect of overexpression of LINC01939 on the migration of renal carcinoma cell lines was further examined through wound healing assay and Western blot, respectively. The statistical results suggested that the migration behavior was largely suppressed by the overexpression of LINC01939 ( Fig.2A). Besides, LINC01939 overexpression caused the abundance of MMP-2 and MMP-9, while the production of TIMP-1 was elevated by overexpressed LINC01939 (Fig.2B). Thus, we concluded that overexpression of LINC01939 suppressed the migration of renal carcinoma cell lines.
LINC01939 overexpression-caused down-regulation of miR-154 functioned as a mediator to induce apoptosis Results from Fig.3A indicated that miR-154 was obviously suppressed by overexpressed LINC01939, suggesting that there may be a targeting relationship between LINC01939 and miR-154. In order to explore the connection between LINC01939 and miR-154, specific miR-154 mimic was used in our following experiments to elevate the accumulation of miR-154 as shown in Fig.3B. Then we observed that the viability inhibited by overexpressed LINC01939 was elevated by the combination of miR-154 mimic (Fig.3C). Besides, the inhibiting effect of LINC01939 on cell proliferation was also abolished by the adding of miR-154 mimic through suppressing the level of p53 and elevating the level of cyclin D1 (Fig.3D). In addition, elevated cell apoptosis rate by LINC01939 was then suppressed by overexpressed miR-154 (Fig.3E). At the same time, elevated expression of Bax and cleaved caspase-3 induced by LINC01939 was both suppressed by the combination of miR-154 mimic in CAKI-1 and ACHN cells (Fig.3F). Thus, we conclude that overexpression of LINC01939 accelerated the apoptosis process by repressing the abundance of miR-154.

LINC01939 overexpression impeded migration behavior by repressing the enrichment of miR-154
The targeting relationship was also verified in cell migration. As shown in Fig.4A, overexpressed LINC01939 suppressed cell migration rate but the inhibiting effect was largely weakened by the combination of miR-154 mimic. At the same time, suppressed level of MMP-2 and MMP-9 was elevated and elevated expression of TIMP-1 was suppressed by the adding of miR-154 mimic compared with the effect of LINC01939 separately (Fig.4B). Thus, we concluded that overexpression of LINC01939 suppressed cell migration by down-regulating miR-154.
Overexpression of LINC01939 retarded the activation of Wnt/β-catenin and Notch signal pathways via downregulating miR-154 Related signal pathways were further investigated through Western blotting assay. As shown in

Discussion
RCC originates from the renal parenchymal urinary tubular epithelial system, and it is widely accepted as a common malignant tumor in urinary system, accounting for nearly 2%-5% of adult malignant tumors worldwide [ 14]. With the advancement of imaging technology, many small RCC less than 3 cm were found, but many patients had already been in the advanced stage of the disease when they were found. Therefore, it is urgent to improve the diagnosis of RCC and to find new therapeutic targets in RCC treatment.
In recent years, lncRNAs have become a research hotspot and they are found to play a more and more important role in tumorigenesis and development. Studies have shown that lncRNAs play an important role in differentiation, proliferation processes, invasion and metastasis processes, and its abnormal expression causes the functional changes which might be a causal association with malignant tumors and other diseases [ 15]. Also, up to now, numerous lncRNAs have been found to participate in the tumorigenic and tumor-suppressive regulatory network in RCC. As an example, lncRNA termed metastatic renal cell carcinoma-associated transcript 1 (MRCCAT1) is ectopic up-regulated in metastatic ccRCC tissues, which might cause the metastatic capacity of ccRCC via mediating associated signaling transduction systems [ 16]. Besides, lncRNA UCA1 is overexpressed in ccRCC and the overexpression of UCA1 has been regarded as a poor prognostic biomarker in ccRCC diagnosis [ 17]. In our present study, we found LINC01939 was obviously down-regulated in RCC tissues. Further study found that overexpressed LINC01939 through exogenous transfection effectively repressed proliferation, accelerated apoptosis and blocked migration process of RCC cell lines. Consequently, we concluded that LINC01939 could be regarded as a novel diagnostic and therapeutic interest lncRNA for RCC.

Conclusion
Taken together, we found that LINC01939 was down-regulated in RCC tissues. Overexpression of LINC01939 suppressed proliferation and migration and induced apoptosis by down-regulating miR-154. Further study also revealed that Wnt/β-catenin and Notch were inactivated by overexpression of LINC01939 via suppressing miR-154. Our present study for the first time revealed the regulating effect and associated regulation mechanism of LINC01939 in RCC, providing novel diagnostic and therapeutic targets for RCC.

Declarations
Ethics approval and consent to participate

Consent for publication
Not applicable.

Availability of data and material
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no conflict of interest.   LINC01939 overexpression-caused blockade of Wnt3a/β-catenin and Notch might be mediated by the down-regulation of miR-154. ACHN and CAKI-1 cells were transfected with NC (negative control) mimic or miR-154 mimic as indicated. Western blotting assay of signaling mediators of (A) Wnt3a/β-catenin (Wnt3a and β-catenin) and (B) Notch (Notch1 and Notch2) pathways was performed. Quantifications were expressed as means of three independent experiments ± SD. *P < 0.05, **P < 0.01.