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. 2022 Jan 2;21(4):340–351. doi: 10.1080/15384101.2021.2020431

Nc886 promotes renal cancer cell drug-resistance by enhancing EMT through Rock2 phosphorylation-mediated β-catenin nuclear translocation

Weiyin Gao a,*, Shouhua Zhang b,*, Li Guorong c, Queling Liu d, Anyi Zhu e, Fu Gui e, Yan Zou e, Yiguo Wu e, Yang Luo a, Zhengdong Hong e,✉
PMCID: PMC8855853  PMID: 34974812

ABSTRACT

Drug resistance is a significant challenge in the present treatment regimens of renal cell carcinoma (RCC). Our previous study confirmed that nc886 functions as an oncogene in RCC. Nevertheless, the role and underlying mechanism of nc886 in RCC drug resistance are unclear. In the present study, Sunitinib and Everolimus treatment, respectively, downregulated nc886 expression in a dose-dependent manner in all four renal cancer cell lines. Nc886 overexpression in 786-O cells and ACHN cells significantly reduced the sensitivity of cancer cells to both Sunitinib and Everolimus treatment, respectively, by promoting cell viability and inhibiting cell apoptosis, whereas nc886 silencing increased cancer cell sensitivity. In renal cancer cell line with the highest drug-resistance, 786-O cells, Sunitinib, or Everolimus treatment enhanced the cellular EMT and was further enhanced by nc886 overexpression while attenuated by nc886 silencing. In 786-O cells, nc886 overexpression significantly promoted EMT, ROCK2 phosphorylation, and β-catenin nucleus translocation under Sunitinib or Everolimus treatment. Moreover, ROCK2 silencing significantly reversed the effects of nc886 overexpression on EMT, ROCK2 phosphorylation, and β-catenin nucleus translocation, as well as drug-resistant renal cancer cell viability and apoptosis. In conclusion, it was demonstrated that nc886 promotes renal cancer cell proliferation, migration, and invasion, as demonstrated previously. nc886 also promotes renal cancer cell drug-resistance to Sunitinib or Everolimus by promoting EMT through Rock2 phosphorylation-mediated nuclear translocation of β-catenin.

KEYWORDS: Renal cell carcinoma (RCC), nc886, drug-resistance, EMT, ROCK2 phosphorylation, β-catenin nucleus translocation

Introduction

Renal cancer, also known as renal cell carcinoma (RCC) or renal adenocarcinoma, is one of the most common renal parenchymal malignancies [1]. Despite the curable nature of localized RCC through surgery, numerous patients present incurable metastatic RCC [2]. Multi-targeted tyrosine kinase inhibitors (such as Sunitinib) [3] and mammalian target of rapamycin inhibitors (such as everolimus) [4] have recently shown higher efficiency in treating metastatic RCC over interferon-α or placebo. However, the acquisition of drug resistance still remains a significant challenge. Therefore, there is a pressing need to investigate promising diagnostic markers and therapeutic targets.

Our previous study analyzed the differentially-expressed genes and demonstrated the oncogenic role of nc886, a short non-coding RNA first identified in cord blood cells and named CBL-3 [5], in RCC. Numerous studies have demonstrated that nc886 is ubiquitously expressed in nonmalignant tissues, but suppressed in many cancer cells of various tissue origins [6–8]. The abnormal upregulation of nc886 in human RCC tissues compared with normal tissues was observed in our previous study, and its expression level was found to be positively associated with the RCC stage [9]. Moreover, nc886 overexpression in the RCC A‑498 cell line enhances cell proliferative and invasive capacities and suppresses cell apoptosis, while nc886 silencing exerts opposite effects [9]. These previous findings confirmed that nc886 functions as an oncogene in RCC; however, the role and underlying mechanism of nc886 in RCC drug-resistance remain unclear as of yet.

Epithelial-mesenchymal transition (EMT) is a complicated transformation process whereby cells acquire mesenchymal morphology and lose polarity, resulting in the detachment and elongation of the cells [10,11]. Furthermore, the expression of epithelial markers, such as E-cadherin, are commonly downregulated, while mesenchymal markers, such as Vimentin and N-cadherin, are upregulated in cells undergoing EMT [10,11]. Notably, EMT is a preceding step of invasion and metastasis cascades of renal cancer [12–16]. TNF-α induced EMT in RCC cells by downregulating E-cadherin, promoting invasiveness and enhancing matrix metalloproteinase (MMP) 9 activity [14]. Hypoxia-induced miR-30 c inhibition in RCC cells led to the overexpression of Slug and inhibition of E-cadherin, thereby enhancing EMT and cell migration [16]. Moreover, epidermal growth factor receptor activation-induced EMT has been reported to contribute to sunitinib resistance in human RCC cell lines [17]. Since EMT plays a critical role in RCC development and metastasis, it was therefore hypothesized that nc886 might exert its functions in an EMT-related way.

Transforming growth factor (TGF)-β1 has been recognized as a central regulator of EMT, involving the Smad, RhoA/ROCK and p38/MAPK signaling pathways as per studies based on in vitro and in vivo animal systems [18–22]. ROCK phosphorylates GSK-3β, leading to β-catenin activation [23,24]. Tian et al. [22] demonstrated the role of TGF-β1-induced RhoA activation in renal proximal tubular epithelial cells. Zhang et al. [25] revealed that TGF-β1-induced EMT in rat peritoneal mesothelial cells (RPMCs) was mediated by the RhoA/ROCK signaling, and a ROCK inhibitor, Y-27632, significantly reversed the changes in EMT characteristics. The complete reversal of EMT requires inhibition of both ZEB expression and the Rho pathway in renal tubular epithelial cells [26]. Regarding cancers, arginine-specific ADP-ribosytransferases 1-mediated regulation of the RhoA/ROCK/AKT/β-catenin pathway promotes the migration and EMT in colon carcinoma [27]. However, ROCK and β-catenin signaling pathways have not yet been proved to be involved in nc886 role in renal cancer cell EMT and drug-resistance.

The expression levels of nc886 in four renal cancer cell lines were detected, 786-O, Caki-1, A498, and ACHN, under the treatment of 0, 1, 2, 5, and 10 μM Sunitinib or Everolimus treatment, overexpressed or knocked down nc886 in 786-O and ACHN cells and examined the effects of nc886 on cell viability and apoptosis under Sunitinib or Everolimus treatment. The specific effects of nc886 on EMT markers, ROCK2 phosphorylation, and β-catenin nucleus translocation were subsequently examined under Sunitinib or Everolimus treatment. Finally, 786-O cells were transfected with nc886-overexpressing vector and sh-ROCK2 and examined for the dynamic effects of nc886 overexpression and ROCK2 silencing on cell viability and apoptosis under Sunitinib or Everolimus treatment. A novel mechanism whereby nc886 promotes the drug-resistance of renal cancer through EMT and ROCK2-mediated β-catenin nucleus translocation has been propounded.

Materials and methods

Cell lines and cell transfection

Renal cancer cell lines A498 (ATCC® HTB-44™), 786-O (ATCC® CRL-1932), ACHN (ATCC® CRL-1611), and Caki-1 (ATCC® HTB-46™) were procured from ATCC (Manassas, VA, USA). A498 and ACHN cells were cultured in Eagle’s Minimum Essential Medium (ATCC) supplemented with 10% FBS (Gibco, Waltham, MA, USA). 786-O cells were cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS (Gibco). Caki-1 cells were cultured in McCoy’s 5a Medium Modified (ATCC) supplemented with 10% FBS (Gibco). All cells were cultured at 37°C in 5% CO2. Regarding anti-cancer drug treatment, renal cancer cell lines were treated with varying concentrations of Sunitinib or Everolimus (0,1,2,5,10 μM) for 24 h. The cells were then harvested pending further investigations.

To generate nc886 silencing or overexpression, target cells were transfected with a si-nc886 or nc886-overexpressing vector (GenePharma, Shanghai, China). All the transfections were performed using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). 24 h after transfection, cells were further treated with 10 μM of Sunitinib or Everolimus for 48 h. The cells were then harvested pending further investigations.

RNA extraction and PCR-based analyses

Total RNA was obtained from treated/transfected cells using Trizol reagent (Invitrogen). mRNA expressions were subsequently determined using an SYBR Green qPCR assay (Takara, Dalian, China) as per a previously-mentioned method [16] taking GAPDH expression as an endogenous control. The 2−ΔCT method was applied for data processing.

Cell viability determined by CCK-8 assay

Cells were treated, transfected and plated into 96-well culture plates at a density of 5 × 103 cells/well. The cell viability was then assessed using a CCK-8 kit (Beyotime, Shanghai, China). The absorbance was measured at 450 nm.

Cell apoptosis determined by flow cytometry

After treatment and transfection, the cell apoptosis was determined using an Annexin V-FITC apoptosis detection kit (Keygen, China) as per a previously-mentioned method [28]. The nuclei staining was performed using propidium iodide (PI). The excitation wavelength (Ex) was 488 nm and the emission wavelength (Em) was 530 nm.

Protein levels determined by immunoblotting

Proteins extracted from the target cells were isolated by SDS- PAGE, transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA), and subsequently incubated with proper primary antibodies followed by another incubation with anti- rabbit or anti-mouse IgG conjugated with horseradish peroxidase (Abcam, Cambridge, MA, USA). The primary antibodies used are as follows: anti-Vimentin (60330-1-lg; ProteinTech, Wuhan, China), anti-E-cadherin (20874-1-AP;ProteinTech), anti-N-cadherin (ab18203; Abcam, Cambridge, UK), anti-p-ROCK2 (ab228008; Abcam), anti-ROCK2 (CSB-PA020059DA01HU; CUSABIO, Houston, TX, USA), anti-Histone (CSB-PA166887, CUSABIO), anti-β-catenin (51067-2-\AP, ProteinTech), and anti-GAPDH (T0004; Affinity, Cincinnati, OH, USA).

Data analysis and statistics

All data were analyzed using SPSS 13.0 software (SPSS, Chicago, IL, USA) and presented as means ± SD of three independent experiments. The differences between groups were analyzed by one-way ANOVA (normal distribution), and multiple comparisons were performed by the Bonferroni correction. P < 0.05 was considered statistically significant.

Results

Downregulation of nc886 is correlated with the drug-resistance of renal cancer

First, the expression levels of nc886 in renal cancer cell lines and the correlation between nc886 expression and renal cancer drug-resistance were examined. Four renal cancer cell lines, 786-O, Caki-1, A498, and ACHN, were treated with 0, 1, 2, 5, and 10 μM Sunitinib or Everolimus, respectively, and examined for nc886 expression and cell viability. Both Sunitinib and Everolimus treatment dose-dependently inhibited nc886 expression in all four cell lines (Figure 1(a,c)). Meanwhile, both Sunitinib and Everolimus treatment could inhibit renal cancer cell viability in a dose-dependent manner (Figure 1(b,d)). Among the four cancer cell lines, 786-O cell viability was the least inhibited, while ACHN cell viability was the most strongly inhibited (Figure 1(b,d)).

Figure 1.

Figure 1.

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Downregulation of nc886 is correlated with the drug-resistance of renal cancer Four renal cancer cell lines, 786-O, Caki-1, A498, and ACHN, were treated with 0, 1, 2, 5, and 10 μM Sunitinib for 24 h and examined for (a) the mRNA expression of nc886 by real-time PCR and (b) the cell viability by CCK-8 assay. Then, these four cell lines were treated with 0, 1, 2, 5, and 10 μM Everolimus for 48 h and examined for (c) the mRNA expression of nc886 by real-time PCR and (d) the cell viability by CCK-8 assay. *P < 0.05, **P < 0.01 compared to 0 μM group; #P < 0.05, ##P < 0.01 compared to 786-O cell group. (e) The silencing or overexpression of nc886 was generated in 786-O and ACHN cells by the transfection of si-nc886 or nc886-overexpressing vector (OE nc886), as confirmed by the real-time PCR. (f) 786-O and ACHN cells were transfected with si-nc886 and/or nc886 upon Sunitinib or Everolimus treatment and examined for the cell viability by CCK-8 assay. (g) 786-O and ACHN cells were transfected with si-nc886 and/or nc886 upon 10 μM Sunitinib or Everolimus for 48 h and examined for cell apoptosis by flow cytometry assay. *P < 0.05, **P < 0.01, compare to Control group; #P < 0.05, ##P < 0.01, compared to the respective group.

To further investigate the correlation of nc886 expression and renal cancer drug-resistance, nc886 was silenced or overexpressed in 786-O and ACHN cell lines through the transfection of si-nc886 or nc886-overexpressing vector (OE nc886), respectively, as confirmed by the real-time PCR (Figure 1(e)). Next, si-nc886 and/or nc886-transfected 786-O and ACHN cells were treated with Sunitinib and Everolimus and the cell viability was assessed. The suppressive effects of Sunitinib and Everolimus on 786-O and ACHN cell viability were further enhanced by nc886 silencing while attenuated by nc886 overexpression; the effect of nc886 repression on 86-O and ACHN cell viability could be offset by nc886 overexpression. Moreover, the suppressive effects of Sunitinib and Everolimus were more conspicuous on ACHN cells (Figure 1(f)). Regarding cell apoptosis, Sunitinib or Everolimus treatment promoted the cell apoptosis of 786-O and ACHN cells; nc886 silencing enhanced while nc886 overexpression attenuated the promotive effects of Sunitinib and Everolimus treatment on cell apoptosis; the role of nc886 silencing and nc886 overexpression on cell apoptosis offset each other (Figure 1(g)). These data indicate that nc886 expression could be inhibited by Sunitinib and Everolimus treatment, respectively. Nc886 overexpression enhances the drug-resistance of renal cancer cells to Sunitinib and Everolimus.

Effects of nc886 on the epithelial-mesenchymal transition (EMT) of cancer cells upon drug treatment

EMT is a crucial event during cancer progression that could enhance cancer cell motility, leading to cancer metastatic potential [29,30]. To investigate the mechanism. Through which nc886 affects renal cancer cell drug-resistance, 786-O cells were treated, which were the most resistant to drugs among the four renal cancer cell lines, with Sunitinib or Everolimus. The protein levels of EMT markers, including Vimentin, E-cadherin, and N-cadherin were examined. The mesenchymal marker Vimentin and N-cadherin protein levels were significantly increased while epithelial marker E-cadherin significantly decreased upon both Sunitinib and Everolimus treatment (Figure 2(a)). These data suggest that along with the acquisition of drug resistance by 786-O cells, EMT is enhanced in drug-resistant renal cancer cells upon Sunitinib or Everolimus treatment.

Figure 2.

Figure 2.

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Effects of nc886 on the epithelial-mesenchymal transition (EMT) of cancer cells upon drug treatment (a) 786-O cells were treated with 10 μM Sunitinib or Everolimus for 48 h and examined for the protein levels of EMT markers, including Vimentin, E-cadherin, and N-cadherin by Immunoblotting. **P < 0.01, compared to Control group. (b) 786-O cells were transfected with si-nc886 and/or nc886 for 24 h and then treated with 10 μM Sunitinib or Everolimus for 48 h and examined for the protein levels of Vimentin, E-cadherin, and N-cadherin by Immunoblotting. **P < 0.01, compared to the si-NC group; ##P < 0.01, compared to the respective group.

786-O cells were subsequently transfected with si-nc886 and/or nc886 upon Sunitinib or Everolimus treatment and the EMT proteins were examined. Upon both Sunitinib and Everolimus treatment, nc886 silencing significantly reduced Vimentin and N-cadherin while increasing E-cadherin protein levels. Contrariwise, nc886 overexpression further enhanced the promotive effects of Sunitinib or Everolimus treatment on Vimentin and N-cadherin protein levels and the suppressive effects of Sunitinib or Everolimus treatment on E-cadherin protein levels, and the effect of nc886 silencing on EMT proteins could be neutralized by nc886 overexpression (Figure 2(b)). These data suggest that nc886 silencing could inhibit the EMT in drug-resistant renal cancer cells upon Sunitinib or Everolimus treatment.

Nc886 promotes cancer cell EMT and ROCK2 phosphorylation-mediated β-catenin nucleus translocation

ROCK phosphorylates GSK-3β, which leads to β-catenin activation [23,24]. It was investigated whether nc886 could affect ROCK2 phosphorylation and β-catenin nucleus translocation when nc886 influences cancer cell EMT. 786-O cells were transfected with nc886 upon Sunitinib or Everolimus treatment. The phosphorylation of ROCK2 or total ROCK2 protein and the cytoplasm and nucleus levels of β-catenin protein were subsequently determined. Under Sunitinib or Everolimus treatment, respectively, nc886 overexpression significantly promoted the p-ROCK2/ROCK2 protein and the nucleus translocation of β-catenin (Figure 3(a-b)).

Figure 3.

Figure 3.

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Nc886 promotes cancer cell EMT and ROCK2 phosphorylation-mediated β-catenin nucleus translocation (a-d) 786-O cells were transfected with nc886 overexpression vector for 24 h and then treated with 10 μM Sunitinib or Everolimus for 48 h and examined for (a) the protein levels of p-ROCK2 and ROCK2 by Immunoblotting and (b) the cytoplasm and nucleus protein levels of β-catenin by Immunoblotting. Then, 786-O cells were transfected with si-nc886 for 24 h and then treated with 10 μM Sunitinib or Everolimus for 48 h and examined for (c) the protein levels of p-ROCK2 and ROCK2 by Immunoblotting and (d) the cytoplasm and nucleus protein levels of β-catenin by Immunoblotting. **P < 0.01, #P < 0.05, compared to NC group. (e) 786-O cells were transfected with sh-ROCK2 and ROCK2 mRNA expression level was determined by real-time PCR assay. **P < 0.01, compared to sh-NC group. (f-g) Next, 786-O cells were co-transfected with nc886 and sh-ROCK2, then treated with 10 μM Sunitinib or Everolimus for 48 h and examined for (f) the protein levels of p-ROCK2, ROCK2, Vimentin, E-cadherin, and N-cadherin by Immunoblotting and (g) the cytoplasm and nucleus protein levels of β-catenin by Immunoblotting. *P < 0.05, **P < 0.01, compared to NC+sh-NC group; #P < 0.05, ##P < 0.01, compared to the respective group.

786-O cells were subsequently transfected with si-nc886 under Sunitinib or Everolimus treatment, and the above-described indexes were determined. As opposed to nc886 overexpression, nc886 silencing significantly blocked the p-ROCK2/ROCK2 protein and the subsequent β-catenin nucleus translocation under Sunitinib or Everolimus treatment (Figure 3(c-d)).

To explore the role of ROCK2 in 786-O cells, sh-ROCK2 was transfected into 786-O cells and the mRNA expression level of ROCK2 was determined. As illustrated in Figure 3(e), sh-ROCK2 notably mitigated ROCK2 mRNA expression. 786-O cells were subsequently co-transfected with nc886 and sh-ROCK2 upon Sunitinib or Everolimus treatment and examined for the above-mentioned proteins and EMT markers. Consistent with earlier results, nc886 overexpression significantly reduced E-cadherin protein levels, increased the Vimentin and N-cadherin protein levels, promoted the p-ROCK2/ROCK2 proteins, and promoted the nucleus translocation of β-catenin (Figure 3(f-g)). ROCK2 silencing increased E-cadherin protein level, decreased the Vimentin and N-cadherin protein levels, inhibited the p-ROCK2/ROCK2 protein, and blocked the nucleus translocation of β-catenin (Figure 3(f-g)). Furthermore, the effects of nc886 overexpression were significantly reversed by ROCK2 silencing (Figure 3(f-g)). These findings suggest that nc886 promotes EMT in renal cancer cells through ROCK2 phosphorylation-mediated β-catenin nucleus translocation.

Nc886 enhances renal cancer cell drug-resistance through ROCK2 phosphorylation-mediated β-catenin nucleus translocation

786-O cells were co-transfected with nc886 and sh-ROCK2 upon Sunitinib or Everolimus treatment and the modulation of renal cancer cell drug-resistance by nc886 through ROCK2 phosphorylation-mediated β-catenin nucleus translocation was examined. As revealed by CCK-8 assay, nc886 overexpression increased the IC50 values for 786-O cells from 12.42 and 10.72 to 24.12 and 21.68 under Sunitinib and Everolimus treatment, respectively (Figure 4(a,c)), while ROCK2 silencing reduced the IC50 values for 786-O cells to 8.92 and 7.633, respectively (Figure 4(a,c)). Consistently, nc886 overexpression inhibited, while ROCK2 silencing promoted the cancer cell apoptosis under Sunitinib and Everolimus treatment (Figure 4(b,d)). Furthermore, the effects of nc886 overexpression were significantly reversed by ROCK2 silencing under Sunitinib and Everolimus treatment, respectively (Figure 4(a-d)). These findings suggest that nc886 overexpression enhances renal cancer cell drug-resistance through promoting ROCK2 phosphorylation-mediated β-catenin nucleus translocation.

Figure 4.

Figure 4.

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Nc886 enhances renal cancer cell drug-resistance through ROCK2 phosphorylation-mediated β-catenin nucleus translocation (a and c) 786-O cells were co-transfected with nc886 and sh-ROCK2 for 24 h, then treated with different concentrations of Sunitinib or Everolimus for 48 h examined for cell viability by CCK-8 assay; (b and d) 786-O cells were co-transfected with nc886 and sh-ROCK2 for 24 h, then treated with 10 μM Sunitinib or Everolimus for 48 h and the cell apoptosis was determined by Flow Cytometry assay. *P < 0.05, **P < 0.01, compared to NC+sh-NC group; #P < 0.05, compared to the respective group.

Discussion

In the present study, downregulated nc886 expression in a dose-dependent way in four renal cancer cell lines upon Sunitinib or Everolimus treatment has been observed. Nc886 overexpression in 786-O cells and ACHN cells significantly reduced the sensitivity of cancer cells to Sunitinib or Everolimus treatment by promoting cell viability and inhibiting cell apoptosis whereas nc886 silencing increased cancer cell sensitivity. In the renal cancer cell line with the highest drug-resistance, 786-O cells, Sunitinib or Everolimus treatment enhanced the cellular EMT, which was further enhanced by nc886 overexpression while it was attenuated by nc886 silencing. In 786-O cells, nc886 overexpression significantly promoted EMT, ROCK2 phosphorylation and β-catenin nucleus translocation under Sunitinib or Everolimus treatment. Moreover, ROCK2 silencing significantly reversed the effects of nc886 overexpression on EMT, ROCK2 phosphorylation and β-catenin nucleus translocation, as well as drug-resistant renal cancer cell viability and apoptosis.

Nc886 (also known as vtRNA2-1, miRNA886 precursor, or CBL3) is a recently discovered non-coding RNA that was first deemed a miRNA precursor or a steroidal ribonucleoprotein complex. However, numerous studies have demonstrated that progression into mature miRNAs is difficult, and is rare in carcass complexes. nc886 is silenced due to the CpG DNA methylation in many solid tumors [13–16]. Through controlling the activity of PKR (protein kinase RNA activated), it serves as a tumor suppressor in many types of cancers, such as cholangiocarcinoma. esophageal squamous cell carcinoma, and gastric cancer [17–19]. However, Kong et al. [20] reported opposite results in cervical cancer, that nc886 steroid 2-1-5p has a pro-carcinogenic effect and is closely related to cervical cancer progression. Our previous study also observed that significantly upregulated nc886 expression in renal cancer tissues and cells is closely related to renal cancer invasion and migration [5]. Thus, this study further investigated the specific role of nc886 in renal cancer drug-resistance. Upon Sunitinib or Everolimus treatment, nc886 expression was significantly downregulated in a dose-dependent manner; however, nc886 expression showed different patterns in different renal cancer cell lines, with highest expression in 786-O cells and lowest expression in ACHN cells. Notably, as shown by cell viability and apoptosis analyses, 786-O cells were more resistant to both Sunitinib and Everolimus treatments while ACHN cells were the most sensitive to both Sunitinib and Everolimus treatments. These data suggest that nc886 expression is higher in drug-resistant cells, and that its overexpression might be related to the drug resistance of renal cancer cells.

The EMT process in cancer cells has been widely recognized as closely related to conventional chemotherapy resistance [31–33]. E-cadherin expression is associated with a high sensitivity to gefitinib, an epidermal growth factor receptor (EGFR) inhibitor [34,35]. Another conventional chemotherapy agent, Sunitinib, is a receptor tyrosine kinase (RTK) inhibitor targeting vascular endothelial growth factor receptor-1 (VEGFR-1)/-2/-3 [36,37]. Different from Sunitinib, Everolimus is an mTOR inhibitor [38]. Interestingly, resistance to Sunitinib in renal cell cancer is related to EMT caused by the activation of EGFR [39]. Regarding Everolimus resistance, the mTOR pathway blockade in a mammary epithelial cells model could activate EMT [40]. In our study, 786-O cells treated with Sunitinib or Everolimus independently showcased significantly increased Vimentin protein levels and decreased E-cadherin protein levels, which was consistent with previous findings [41]. Moreover, nc886 overexpression enhanced, while nc886 silencing attenuated EMT in 786-O cells in response to Sunitinib or Everolimus treatment, indicating that nc886 might affect the drug-resistance of 786-O cells through EMT.

Rho-associated coiled-coil forming protein kinase (Rock) is a relatively new signal protein and a key regulator in the Rho/Rock pathway. Many studies have confirmed that Rock2 is involved in malignant tumor formation and progression. Rock2 has played an important role in metastasis of gallbladder cancer, esophageal cancer, gastric cancer, liver cancer and other malignant tumors [42–44] and is also involved in many aspects of tumor progression. For instance, it accelerates the contraction of tumor cells and the separation of tails. It also regulates the progression of tumor cells by promoting the degradation of the extracellular matrix (ECM) and promoting microfilament-associated cellular activities [45]. The invasive performance of hepatoma cells is related to the overexpression of Rock2. Silencing Rock2 can significantly reduce the invasion and migration of tumor cells in vitro and in rat liver cancer models in vivo [45,46]. Zheng et al. [44] found that the overexpression of Rock2 is closely related to the EMT of liver cancer, which can transform epithelial cells to malignant cells with migratory capacity. Our previous studies demonstrated that Rock2 is highly expressed in renal cancer tissues compared with adjacent non-cancerous tissues, and renal cancer patients with higher expression of Rock2 have a relatively poorer prognosis. The overexpression of Rock2 can reduce the proliferative capacity of renal cancer cells; in vivo, knocking down Rock2 in the renal cancer rat model reduced the tumor growth [47]. This study observed that both Sunitinib and Everolimus treatment significantly enhanced Rock2 phosphorylation and subsequent β-catenin nuclear translocation. The overexpression of nc886 enhanced the above process and EMT in 786-O cells, whereas the silencing of Rock2 showed the opposite effect. Moreover, the silencing of Rock2 partially restored the effect of overexpression of nc886, suggesting that nc886 overexpression promotes EMT in 786-O cells by affecting Rock2 phosphorylation-mediated nuclear translocation of β-catenin. Regarding the cancer cell drug-resistance, nc886 overexpression enhances renal cancer cell drug-resistance to both Sunitinib and Everolimus by promoting cell viability and inhibiting cell apoptosis, while the knocking down Rock2 exerted opposite effects. Similarly, the effects of nc886 overexpression were significantly reversed by Rock2 silencing, suggesting that nc886 overexpression enhances renal cancer cell drug-resistance through the Rock2 phosphorylation-mediated nuclear translocation of β-catenin.

Wnt/β-catenin signaling has a profound influence on EMT during cancer progression [48]. The abnormal activation of the Wnt/β-catenin signaling is ubiquitous in tumor tissues, and β-catenin is the central protein in this signaling [49]. Under normal circumstances, the lack of Wnt signal, β-catenin and E-cadherin are attaching on the cell membrane, and very few are presenting in the cytoplasm. The loss of E-cadherin may abnormally activate the pathway, the degradation of β-catenin is inhibited, and β-catenin penetrates the nucleus to promote the expression of downstream target genes such as cyclin D1 and c-Myc, and finally promotes the malignant progression of cells [50,51]. In the nuclei, β-catenin combines with the TCF/LEF transcription factors to co-activate a series of target genes including EMT markers [52]. According to the previous reports [53,54], researchers revealed that the crucial role of the Wnt/β-catenin signaling pathway drives the EMT phenotype in the etiology of renal cancer. The current study demonstrated that nc886 regulates β-catenin nucleus translocation and EMT process under Sunitinib or Everolimus treatment in renal cancer cells.

In conclusion, it has been conclusively demonstrated that nc886 promotes renal cancer cell proliferation, migration, and invasion as proven in our previous study, and also promotes renal cancer cell drug-resistance to both Sunitinib and Everolimus by promoting EMT through Rock2 phosphorylation-mediated nuclear translocation of β-catenin.

Funding Statement

This study was supported by the National Natural Science Foundation of China [81760458] and Natural Science Foundtaion of Jiangxi Province [20181BAB215028].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Please contact the authors for data requests.

Author contributions

Conceptualization, Weiyin Gao, Shouhua Zhang and Zhengdong Hong; Funding acquisition, Zhengdong Hong; Investigation, Guorong Li; Methodology, Shouhua Zhang; Project administration, Yiguo Wu and Zhengdong Hong; Resources, Queling Liu and Yan Zou; Software, Guorong Li and Fu Gui; Validation, Anyi Zhu; Writing – original draft, Weiyin Gao; Writing – review & editing, Yang Luo.

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