Long non-coding RNA SNHG1 protects human AC16 cardiomyocytes from doxorubicin toxicity by regulating miR-195/Bcl-2 axis

Doxorubicin (DOX) is a highly effective anti-tumor drug, but its cardiotoxicity largely restricts its clinical application. The present study was designed to explore whether in vitro DOX toxicity in AC16 cardiomyocytes can be regulated by long non-coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) and to elucidate the underlying mechanisms. We found that DOX treatment led to severe damage in AC16 cells through decreasing cell viability and increasing cell apoptosis. DOX treatment also reduced the expression of SNHG1 in AC16 cells, and overexpression of SNHG1 alleviated the increased apoptosis in DOX-treated AC16 cells. Moreover, we found that SNHG1 could counteract the inhibitory effect of miR-195 on Bcl-2, and miR-195 restoration blocked the beneficial effect of SNHG1 against DOX toxicity in AC16 cells. In short, the present study provided convincing evidence that SNHG1 protects human AC16 cardiomyocytes from DOX toxicity partly by regulating miR-195/Bcl-2 axis.


Introduction
Cancer is the main cause of deaths worldwide. Doxorubicin (DOX), an anthracycline antibiotic derived from Streptomyces, is one of the most powerful and widely used anti-tumor drugs [1]. However, the clinical application of this drug is largely limited by the toxic side effects on healthy human organs, specifically heart [2]. Approximately 10% of patients treated with DOX or its derivatives will develop cardiac complications [3]. Accordingly, there is a growing need to develop effective therapies against DOX cardiotoxicity.
Long non-coding RNAs (lncRNAs), a group of RNA molecules greater than 200 nucleotides in length with limited protein-coding potential, have been recently identified as a critical player in many human diseases, including cardiovascular disorders [4]. LncRNA small nucleolar RNA host gene 1 (SNHG1), located in human chromosome 11, was found to be aberrantly expressed in a wide variety of human cancers [5]. In addition, Zhang et al. [6] reported that SNHG1 attenuated the effects of H 2 O 2 on the viability and apoptosis of human cardiomyocytes. In this study, using in vitro DOX-treated AC16 cardiomyocytes, our major purpose was to explore whether DOX toxicity can be regulated by SNHG1 and to elucidate the underlying mechanisms.
For SNHG1 overexpression, the full-length cDNA of SNHG1 was synthesized and cloned into the expression vector pcDNA3.1 (Invitrogen). An empty vector served as a negative control (NC). The short interfering RNA targeting SNHG1 (si-SNHG1) and NC (si-NC) were chemically synthesized by GenePharma Co., Ltd. (Shanghai, China), and their sequences were listed as follows: si-SNHG1, 5 -GAAACAGCAGUUGAGGGUUUGCUGU-3 ; si-NC, 5 -GAUCAUACGUGCGAUCAGA-3 . The specific mimics, inhibitor and the corresponding NC for miR-195 were chemically synthesized by RiboBio Co., Ltd. (Guangzhou, China). Two hours before DOX stimulation, cells were seeded in six-well plates and the transfection was performed by using Lipofectamine 2000 reagent (Invitrogen).

Cell counting kit-8 assay
Cell viability was determined by Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Briefly, after treatments, CCK-8 solution (10 μl) was added to each well, and the plates were incubated for additional 1 h. The absorbance of each well was measured at 450 nm using a microplate reader (MultiskanEX, Lab systems, Helsinki, Finland).

Dual-luciferase reporter assay
The fragment of SNHG1 or Bcl-2 carrying a putative miR-195-binding site was amplified by PCR and inserted into the psiCHECK-2 luciferase reporter vector (Promega, Madison, WI, U.S.A.). The binding sites were mutated using a Mut Express II Fast Mutagenesis kit (Vazyme, Piscataway, NJ, U.S.A.). Cells were seeded in 24-well plates and co-transfected with the luciferase reporter plasmid and miR-195 mimics or NC using Lipofectamine 2000. After 48 h, the cells were lysed, and the luciferase activities were measured using the dual-luciferase reporter assay system (Promega).

Statistical analysis
All statistical analyses were performed using GraphPad Prism (version 6.01) software (GraphPad Software, Inc., La Jolla, CA, U.S.A.). Data were expressed as mean + − standard deviation (SD) of three repeated experiments, each consisting of three culture plates (n=9). The difference between groups was determined by Student's t test or one-way ANOVA. All P-values were two sided, and P<0.05 was statistically significant.

DOX reduces SNHG1 expression in AC16 cells
First, as shown in Figure 1A, the exposure of AC16 cells to DOX for 24 h induced remarkable cytotoxicity, leading to a decrease in cell viability. In addition, the apoptosis rate of AC16 cells was strikingly increased after DOX treatment ( Figure 1B). Western blot analysis also showed that the expression of pro-apoptotic proteins (Bax and cleaved caspase-3) was increased, whereas the expression of anti-apoptotic protein (Bcl-2) was decreased in DOX-treated AC16 cells ( Figure 1C). Moreover, as indicated by RT-qPCR analysis, DOX treatment remarkably decreased SNHG1 expression in AC16 cells ( Figure 1D).

SNHG1 alleviates DOX toxicity in AC16 cells
To further investigate the role of SNHG1 in DOX toxicity, SNHG1 was then overexpressed in DOX-treated AC16 cells, and the successful transfection was verified by RT-qPCR analysis ( Figure 2A). As shown in Figure 2B, SNHG1 overexpression significantly alleviated DOX toxicity by increasing the AC16 cell viability. Moreover, SNHG1 overexpression also markedly reduced the apoptosis rate of DOX-treated AC16 cells ( Figure 2C). As revealed by Western blot analysis, SNHG1 overexpression increased the Bcl-2/Bax expression ratio and suppressed the cleavage of caspase-3 in DOX-treated AC16 cells ( Figure 2D).

SNHG1 acts as a competing endogenous RNA for miR-195 in AC16 cells
By using the Starbase database (http://starbase.sysu.edu.cn/index.php), miR-195 was predicted to directly bind to SNHG1, with the binding sequences shown in Figure 3A. To verify the prediction, dual-luciferase reporter assay was then performed, and the results demonstrated that miR-195 mimics decreased the luciferase activity of SNHG1-WT but had no obvious effect on SNHG1-MUT in AC16 cells ( Figure 3B). We also found that SNHG1 overexpression decreased, whereas SNHG1 knockdown increased miR-195 expression in AC16 cells ( Figure 3C).
Utilizing the TargetScan database (http://www.targetscan.org), Bcl-2 was predicted as a potential target gene of miR-195 ( Figure 3D), and this prediction was confirmed by dual-luciferase reporter assay, as the luciferase activity was markedly reduced in AC16 cells by co-transfection with Bcl-2-WT and miR-195 mimics ( Figure 3E). Besides, as shown in Figure 3F, miR-195 overexpression decreased, whereas miR-195 inhibition increased Bcl-2 protein expression in AC16 cells.

miR-195 inhibition alleviates DOX toxicity in AC16 cells
Furthermore, as shown in Figure 4A, miR-195 is significantly up-regulated after DOX treatment in AC16 cells. We then found that the increased apoptosis rate of DOX-treated AC16 cells was also diminished by miR-195 inhibition ( Figure 4B), whereas CCK-8 assay indicated that miR-195 inhibition rescued the impaired viability of DOX-treated AC16 cells ( Figure 4C).

miR-195 blocks the beneficial role of SNHG1 in DOX-treated AC16 cells
We subsequently carried out rescue experiments, and the results showed that co-transfection with miR-195 mimics obviously blocked the beneficial role of SNHG1, through increasing the apoptosis ( Figure 5A) and decreasing the viability ( Figure 5B) of DOX-treated AC16 cells.

Discussion
DOX cardiotoxicity remains a tough problem for both cardiologists and oncologists, and the underlying mechanisms have been extensively studied since DOX was first used. Recently, lncRNAs have attracted increasing attention due to their regulatory role in DOX cardiotoxicity. For example, LINC00339 aggravates DOX-induced cardiomyocyte apoptosis [9], and FOXC2-AS1 was down-regulated in heart tissues of mice with DOX cardiotoxicity [10]. In this study, the effects of SNHG1 on DOX-treated AC16 cardiomyocytes were studied.
The heart is composed of various types of cells, and cardiomyocyte is a classical cellular target of DOX [11]. Apoptosis, a type of programmed cell death, plays an essential role in the pathogenesis of DOX cardiotoxicity [12]. Myocardial apoptosis causes loss of myocytes and impairment of heart function. Hence, identification of effective methods to suppress DOX-induced apoptosis of cardiomyocytes is imperative. In the present study, we also identified a significant increase in the apoptosis rates of AC16 cells after DOX stimulation, which was further diminished by SNHG1 overexpression.
We then explored the possible mechanisms underlying the beneficial role of SNHG1. MicroRNAs (miRNAs), another type of noncoding RNA, are well characterized. A wealth of studies indicate that lncRNAs can serve as competing endogenous RNAs (ceRNAs) to regulate the expression and activity of miRNA [13,14]. miR-195, a critical regulator in cardiac physiology, is overexpressed in STZ-induced diabetic mouse hearts and hypoxia/reoxygenation-treated H9c2 cells [15,16]. In the present study, we provided several lines of evidence to indicate that, in DOX-treated AC16 cells, miR-195 expression was increased, and SNHG1 might act as a ceRNA for miR-195 to enhance the expression of target gene Bcl-2, thereby alleviating the DOX toxicity.
In conclusion, the present study, for the first time, demonstrated that SNHG1 could protect human AC16 cardiomyocytes from DOX toxicity partly by regulating miR-195/Bcl-2 axis. Our findings provided a possible molecular mechanism underlying DOX cardiotoxicity, which will gives a new direction for its treatment.

Author Contribution
Sisi Chen conceived the study and designed the experiments. Sisi Chen, Jichun Wang and Yanli Zhou performed the experiments and analyzed the data. Sisi Chen supervised the study and wrote the manuscript. All authors read and approved the final manuscript.

Funding
The authors declare that there are no sources of funding to be acknowledged.