LINC01354/microRNA-216b/KRAS Axis Promotes the Occurrence and Metastasis of Endometrial Cancer

Objective LINC01354 has been defined as a tumor driver in several cancers. Nevertheless, whether LINC01354 involves in endometrial cancer (EC) has been little navigated. Thus, the mechanism of LINC01354 was explored in the disease. Methods Measurements of LINC01354, microRNA (miR)-216b and kirsten rat sarcoma viral oncogene (KRAS) levels in EC tissues and cells were performed. LINC01354 low expression and miR-216b overexpression vectors were introduced into EC cells (lshikawa), thereby their effects on cell viability, apoptosis, migration and invasion were manifested. Rescue experiments were also carried out by down-regulating LINC01354 and miR-216b spontaneously. Tumorigenesis in vivo was also assessed. The relationships of LINC01354/miR-216b/KRAS were analyzed. Results Increased LINC01354 and KRAS and reduced miR-216b levels were measured in EC. Silencing LINC01354 or overexpressing miR-216b retarded EC cellular development. LINC01354 counteracted with miR-216b to target KRAS. Suppression of miR-216b antagonized silenced LINC01354-induced impacts on EC cell development. LINC01354/miR-216b/KRAS axis enhanced tumorigenesis in mice with EC. Conclusion It is testified that silencing LINC01354 inhibits KRAS by up-regulating miR-216b, thereby discouraging cell malignant phenotype in EC.


Introduction
Endometrial cancer (EC), derived from the epithelium of the uterine cavity, is a prevalent female pelvic malignant tumor, accounting for 4% lifetime incidence [1]. EC patients mainly present well-differentiated cancer in the early stage with favorable prognosis, but aggressive disease subtype remains the great challenge to overcome [2]. Menarche, anovulation, obesity and late menopause can increase estrogen levels and enlarge endometrium, eventually causing endometrial hyperplasia or EC [3]. Surgery (total laparoscopic or laparoscopic hysterectomy and bilateral salpingo-oophorectomy) is the top 1 treatment for EC [4]. However, for recurrent or metastatic EC, very limited treatment is available [5]. For better control of EC, more effective biomarkers and therapies are urgently required.
Dysregulated long non-coding RNAs (lncRNAs) are believed to connect with tumorigenesis and metastasis in EC [6]. For example, aberrant overexpression of lncRNA RHPN1 antisense RNA 1 [7] and SNHG14 [8] are linked with histological grade and cancer progression in EC. Importantly, lncRNAs-mediated networks are merged as the regulator of EC progression, such as lncRNA RP11-395G23.3-mediated microRNA (miR)-205-5p/ phosphatase and tensin homolog axis [9]. LINC01354 is considered as a tumor activator in osteosarcoma, lung cancer and colorectal cancer (CRC) through promoting cell progression [10][11][12]. Nevertheless, the defined role of LINC01354, along with its regulatory network has been scarcely ever discussed in EC. miR-216b is the regulated gene by lncRNA that can repress EC cell growth and metastasis [13]. miR-216 has been identified as a key modifier in cervical intraepithelial neoplasia [14] while miR-216-5p in part regulates tumorigenesis of cervical cancer (CC) [15]. Kirsten rat sarcoma viral oncogene (KRAS) is a frequently mutated gene in endometrioid ovarian carcinoma, gastric-type mucinous carcinoma and endometrial mesonephric-like adenocarcinoma [16][17][18]. KRAS can predict the transition from proliferative endometrium to well-differentiated EC, from further tumor invasion to advanced disease [19]. Actually, KRAS mutation mechanistically mediates the tumorigenesis of EC [20]. KRAS can assess the benignity of precursor or malignant mucinous lesions and distinguish endometrial lesions from cervical lesions [21]. Constructed on the reported researches, this study was initiated to decode the axis of LINC01354/miR-216b/KRAS in EC cell progression.

Ethics Statement
Our project has been approved by the Ethics Committee of The Fourth Affiliated Hospital of China Medical University. Each patient has issued an informed consent. Procedures and operations performed on animals were consistent with Guidelines for the Care and Use of Laboratory Animals.

Sample Collection
EC tissues and normal tissues (68 pairs) were collected in The Fourth Affiliated Hospital of China Medical University. Tumor pathology and Federation of Gynecology and Obstetrics stage were analyzed by two pathologists. The samples were frozen in liquid nitrogen and preserved at -80 °C [22].

Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA isolated from tissues and cells with Trizol reagent (Life Technologies, Gaithersburg, MD, USA) were quantified by a SmartSpec Plus spectrophotometer (Bio-Rad, Hercules, USA). The target gene was amplified by GoTaq 2-Step RT-qPCR kit (Promega, Madison, USA) and analyzed in Mx3005P qPCR system (Taratagne, CA, USA). Gene expression normalized to GAPDH and U6 was assessed by 2 −ΔΔCt method [23]. Table 1 showed the primer sequences.

Scratch Test
Cells cultured in 6-well plate at 5 × 10 5 cell/mL for 48 h were scratched by a 10-μL pipette tip. With removal of the suspended cells, the remaining cells were cultured in serum-free medium for another 48 h, viewed by a MOTOC inverted microscope (Thermo Fisher Scientific, Waltham, USA) and analyzed by IPP (Media Cybernetics, Bethesda, MD, USA) [27].

Transwell Assay
A transwell incubator (Corning Costar Corp. Corning, USA) was coated with matrix gel, in which cells were cultured (3 × 10 4 cells/well) in the upper side. A medium containing 20% FBS was set in the lower side. At 24 h post culture, cells that not passed through the filter were removed while those passed were fixed with 4% paraformaldehyde solution (Sigma-Aldrich), stained with 0.01% crystal violet and photographed under a microscope (Olympus BH-2, Tokyo, Japan) [28].

Dual Luciferase Reporter Gene Assay
Jefferson or Starbase website assessed the binding sites of LINC01354 and miR-216b, and miR-216b and KRAS. In dual luciferase reporter gene assay, LINC01354 or KRAS fragment containing the miR-216b target sequence was inserted into a PmirGLO (GenePharma), thereby LINC01354-WT, LINC01354-MUT, KRAS-WT and KRAS-MUT were obtained. Transfection with the above vector with miR-216b mimic or mimic NC into lshikawa cells was performed with the help of Lipofectamine 2000 (Invitrogen, USA). Cell luciferase activity was measured by a dual luciferase detection kit (GeneCopoeia, Rockville, USA). Relative luciferase activity = firefly/Renilla luciferase activity [11].

Tumor Xenografts
BALB/c nude mice (female, 4-5 weeks old) provided by Zhejiang University were injected with the stablytransfected lshikawa cells (1 × 10 6 cells/mL, 0.2 mL). The injection was performed at the subscapular area of mice at 8 weeks old. Five mice were utilized in each group. The largest length (L) and the width (W) perpendicular to the L were measured every 5 d. Volume = 0.5 × L × W 2 . All mice were euthanized 30 d later and tumors were weighed and photographed [24].

Immunohistochemistry
The tumor tissue sections obtained from mice were embedded in paraffin, dissected and baked at 68 °C. Then, the tissues was deparaffinized in conventional xylene, dehydrated with ethanol, and blocked with goat serum. The sections having been incubated with KRAS antibody (1:100; Abcam) were added with diaminobenzidine and hematoxylin successively and observed through a microscope (Nikon, Tokyo, Japan) [31].

Statistical Analysis
Analyzed SPSS 18.0 software (IBM, NY, USA), the data were presented as mean ± standard deviation (repetition = 3). Data calculation utilized t-test or one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant [27].

Increased LINC01354 Level is Measured in EC
LINC01354 has been revealed to overexpress in nonsmall cell lung cancer (NSCLC) [11]. As to its role in EC, we firstly measured its level in tissues (normal tissues and EC tissues) and cell lines (normal endometrial cells and EC cell lines lshikawa, HHUA, KLE and ECC-1). Exactly, LINC01354 level was augmented in EC tissues and cells (Fig. 1a, b). Taking the average of LINC01354 relative expression as the boundary, EC samples were divided into LINC01354 high expression group (n = 46) and low expression group (n = 22). Clinical analysis demonstrated the connections between LINC01354 expression and tumor differentiation, tumor node metastasis (TNM) stage and lymph node metastasis (LNM) ( Table 2).

Silencing LINC01354 Retards EC Cellular Development
Lshikawa cells with highest LINC01354 expression were applied to cell experiments to further study the role of LINC01354 in EC. RT-qPCR ensured LINC01354 level was inhibited by transfection with sh-LINC01354 in lshikawa cells (Fig. 2a). Subsequently, LINC01354 down-regulation-induced effects on the biological processes of lshikawa cells were studied by MTT assay, flow cytometry, scratch test and Transwell. In lshikawa cells with knocked down LINC01354, cell viability, invasion and migration were depressed, apoptotic rate was raised (Fig. 2b-e). In summary, silencing LINC01354 can suppress EC cell progression.

Reduced miR-216b is Tested in EC; Overexpressing miR-216b Suppresses EC Cell Progression
Next, the downstream targets of LINC01354 involved in EC were explored. miR-216b has been previously discussed to reduce in glioma and breast cancer tissues, and can inhibit cancer cell proliferation, migration and invasion [32,33]. In EC, miR-216b level was inhibited in cancer tissues and cell lines (Fig. 3a, b). miR-216b mimic was transfected into lshikawa cells, after which miR-216b expression was augmented (Fig. 3c). Then, lshikawa cells with elevated miR-216b expression showed repressed cell progression ( Fig. 3d-g). Collectively, miR-216b overexpression limited EC cell malignant phenotype.

LINC01354 Counteracts with miR-216b to Target KRAS
miR-216b is negatively regulated by lncRNAs [34,35]. RT-qPCR measured an increment in miR-216b expression after down-regulating LINC01354 in cells (Fig. 4a). Therefore, a targeting relation may exist between LINC01354 and miR-216b. Jefferson searched the potential binding sites between LINC01354 and miR-216b (Fig. 4b). Subsequently, dual luciferase report analysis further verified the targeting relationship between LINC01354 and miR-216b, as evident by impaired luciferase activity in cells after co-transfection of LINC01354-WT and miR-216b mimic (Fig. 4c). Next, miR-216b-mediated downstream genes regulating EC were studied. KRAS expression was enhanced in EC tissues and cell lines (Fig. 4d). After silencing LINC01354 or overexpressing miR-216b in lshikawa cells, KRAS expression was reduced (Fig. 4e, f ). Starbase website showed that miR-216b and KRAS had a targeting relationship (Fig. 4g), and dual luciferase report test manifested that the luciferase activity of KRAS-WT and miR-216b mimic was destructed (Fig. 4h), verifying miR-216b targeting KRAS.

Suppression of miR-216b Antagonizes Silenced LINC01354-Induced Impacts on EC Cell Development
The regulation of the LINC01354/miR-216b/KRAS axis on the biological development of EC cells was surveyed. sh-LINC01354 and miR-216b inhibitor  were co-transfected into lshikawa cells. Then, assays revealed that miR-216b inhibition could negate depleted LINC01354-induced suppression on KRAS expression (Fig. 5a), as well as on lshikawa cell development (Fig. 5b-e). In brief, LINC01354/miR-216b/KRAS axis regulated EC cell fate.

Depleting LINC01354 Up-Regulates miR-216b to Slow Down Tumorigenesis in Mice with EC
In vivo growth of EC tumors was observed to further confirm the functional roles of LINC01354 and miR-216b in EC. Lshikawa cells carrying sh-LINC01354 and miR-216b mimic were transplanted into mice and then tumor Reduced miR-216b is tested in EC; Overexpressing miR-216b suppresses EC cell progression. a miR-216b expression was low in EC tissues versus normal tissues (n = 68) in RT-qPCR; b miR-216b expression was decreased in EC cell lines versus NE cells (RT-qPCR); c miR-216b expression was elevated by miR-216b mimic in lshikawa cells (RT-qPCR); d Cell viability was suppressed by miR-216b mimic (MTT assay); e Cell apoptosis rate was induced by miR-216b mimic (flow cytometry); f Cell migration was limited by miR-216b mimic (scratch test); g Cell invasion was weakened by miR-216b mimic (Transwell assay). The data were expressed as mean ± standard deviation (N = 3). # P < 0.05 versus the mimic NC group volume and weight were reduced (Fig. 6a-c). In addition, immunohistochemistry demonstrated that silencing LINC01354 or overexpression of miR-216b reduced KRAS expression in EC tumors (Fig. 6d). Shortly, LINC01354/miR-216/KRAS can regulate the growth of EC tumors in vivo.

Discussion
EC is the 6th common cancer in female globally whose mortality is largely dependent on tumor recurrencerelated poor prognostic factors [36]. As to EC cell progression, this research was pivoted on LINC01354meidated regulatory network. Firstly, LINC01354 level trended toward up-regulate in EC, which was connected with tumor differentiation, TNM and LNM. Then, silencing LINC01354 in EC cells was proved to be suppressive for cell growth. After that, decreased miR-216b expression was also investigated in EC and restoring miR-216b limited the acquisition of malignant phenotype of EC cells. Subsequently, inhibiting miR-216b abrogated silenced LINC01354-induced impacts on EC cell development. In a word, LINC01354 suppression elevated miR-216b expression to down-regulate KRAS, thereby restraining cell growth in both vivo and vitro. At one time, LINC01354 is studied as a competing endogenous RNA in regulating cancer-related pathways in CRC [37]. Incremental LINC01354 level has been once examined in osteosarcoma, and artificially eliminating LINC01354 is conducive for retarding cell invasion in vitro and metastasis in vivo [10]. In the field of NSCLC, the overexpressed LINC01354 is also measured, manifesting a correlation with advanced TNM, while knocking down LINC01354 restricts cancer cells to proliferate and invade [11]. LINC01354 expression goes to an Fig. 4 LINC01354 counteracts with miR-216b to target KRAS. a miR-216b expression was promoted in lshikawa cells by sh-LINC01354 transfection (RT-qPCR); b LINC01354 had binding sites with miR-216b on the Jefferson website; c LINC01354 bound to miR-216b (dual luciferase reporter gene assay); d KRAS expression was high in EC cells versus NE cells (RT-qPCR and Western blot); e KRAS expression was lowered by sh-LINC01354 transfection in lshikawa cells (RT-qPCR and Western blot); f KRAS expression was depressed by miR-216b mimic transfection in lshikawa cells (RT-qPCR and Western blot); g miR-216 had targeting sites with KRAS through the website http:// starb ase. sysu. edu. cn/; H. miR-216b targeted KRAS (dual luciferase reporter gene assay). The data were expressed as mean ± standard deviation (N = 3). *P < 0.05 versus the sh-NC group; # P < 0.05 versus the mimic NC group elevation in CRC, and up/down-regulating encourages/ discourages cells to form proliferative and migratory phenotypes [38]. As suggested by the aforementioned studies and the present study in combination, LINC01354 indeed is pro-tumor.
Though we identified the binding relation between LINC01354 and miR-216b in the experiments, their reciprocal in diseases needs further confirmation. Regarding to miR-216b, it is implicated to hamper tumors and its under-expression may relate to cancer biology [39]. Of importance, knocking down lncRNA XLOC_008466 is witnessed to retard proliferation, invasion and migration, as well as drive apoptosis through enhancing miR-216b in CC [40]. Announced in an innovative research, miR-216b expression is reduced and forced expression of miR-216b achieves to destruct cell viability, migration, invasion whereas aggravate apoptosis in EC [13]. Experimentally measured, down-regulated miR-216b showcases in CC while up-regulating miR-216b is the switch for proliferation limitation [41]. LINC00152-mediated miR-216b-5p restoration has been lately confirmed to induce G0/G1 phase cell entry and apoptosis of CC cells [42]. The regulatory mechanism of miR-216b has not only mentioned in gynecological cancers, but in other cancer types. For instance, restoring the suppressed level of miR-216b in osteosarcoma is promoting for apoptosis induction in vitro [43]. Other than that, in terms of gastric cancer and hepatocellular carcinoma, miR-216b level manifests a reduction in cancer tissues and cells, and forced miR-216b expression induces the restrictions on cancer cell biological activities [44,45]. In summary, miR-216b itself is the blocker for human cancer development, including but not limited to EC.
KRAS was suggested as the target gene of miR-216b in this EC-focused study, which was supported by reported study findings. Exactly, an inverse correlation exists between miR-216b level and KRAS protein in nasopharyngeal carcinoma, and miR-216b targets KRAS to obstruct cell aggressiveness and tumor formation [46]. Further proved currently, miR-216b-targeted KRAS down-regulation is the inhibitor for pancreatic cancer cell progression [47,48]. Notably, clear cell renal cell carcinoma cell proliferation and invasion, as well as tumor growth suppression are ascribed to KRAS Fig. 5 Suppression of miR-216b antagonizes silenced LINC01354-induced impacts on EC cell development. a KRAS expression was lowered in lshikawa cells after silencing both LINC01354 and miR-216b (RT-qPCR and Western blot); b Cell viability was enhanced after silencing both LINC01354 and miR-216b (MTT assay); c Cell apoptosis rate was reduced after silencing both LINC01354 and miR-216b (flow cytometry); d Cell migration was accelerated after silencing both LINC01354 and miR-216b (scratch test); e Cell invasion was augmented after silencing both LINC01354 and miR-216b (Transwell assay). The data were expressed as mean ± standard deviation (N = 3). *P < 0.05 versus the sh-LINC01354 + inhibitor NC group down-regulation induced by miR-216b [49]. Recurrent KRAS mutation is tested in mesonephric adenocarcinoma [50], and KRAS amplification and its mRNA expression are measured in early stage of recurrent endometrioid EC [51]. In tanshinone I-treated CC cells, KRAS overexpression can enhance cell proliferation [52]. Anyway, miR-216b-meidated KRAS has been implied to manage cancer development and silencing of KRAS restrains tumorigenesis.

Conclusion
To conclude, the present study makes it comprehensive that LINC01354 raises KRAS expression through binding to miR-216b, thereafter stimulating tumorigenic aggravation in EC. Supplemented by the present study, the mechanism of lncRNA-mediated networks in EC has been further understood. Studies are at wanting in larger scales to further develop the results obtained.

Authors' Contributions
ST finished study design, YZ, WZ, FN finished experimental studies, YZ, ML finished data analysis, YZ finished manuscript editing. All authors read and approved the final manuscript.

Funding
No funds, grants, or other support was received.

Availability of Data and Materials
The original contributions presented in the study are included in the article/ Supplementary Material, further inquiries can be directed to the corresponding author.

Declarations Ethics Approval and Consent to Participate
This study was approved and supervised by the animal ethics committee of The Fourth Affiliated Hospital of China Medical University. The treatment of animals in all experiments conforms to the ethical standards of experimental animals.

Consent for Publication
Patients signed informed consent regarding publishing their data and photographs.

Competing interests
The authors have no conflicts of interest to declare that are relevant to the content of this article.  Depleting LINC01354 up-regulates miR-216b to slow down tumorigenesis in mice with EC. a Tumor photos obtained after silencing LINC01354 or overexpressing miR-216b; b Tumor volume was decreased after silencing LINC01354 or overexpressing miR-216b; c Tumor weight was suppressed after silencing LINC01354 or overexpressing miR-216b; d KRAS expression was reduced after silencing LINC01354 or overexpressing miR-216b (IHC). The data were expressed as mean ± standard deviation (n = 5). *P < 0.05 versus the sh-NC group; # P < 0.05 versus the mimic NC group