MicroRNA-101-5p Suppresses the Expression of the Ras-Related Protein RAP1A

Abstract

MicroRNAs (miRs) are small, non-coding RNAs that negatively regulate gene expression. The stem-loop sequence miR-101-1 generates mature miR-101-5p and miR-101-3p. The function and target mRNA of miR-101-5p have not yet been elucidated in detail. Here, we demonstrate that miR-101-5p inhibits the expression of RAP1A, a member of the RAS gene family. Transfection of a miR-101-5p mimic significantly inhibited the expression of RAP1A mRNA in HeLa, HEK293, A549, and COLO201 cells. The same treatment significantly inhibited cell proliferation. The cytostatic effect with transfection of miR-101-5p was antagonized by treatment with the RAP inhibitor salirasib. These results suggested that miR-101-5p inhibits RAP1A, and thus, the expression levels of miR-101-5p regulate cell proliferation.

INTRODUCTION

RAS gene family members mediate cell signaling that controls cell proliferation, migration, and gene activation. RAS family proteins are small guanosine 5′-triphosphatases (GTPases) that undergo changes in their activities depending on whether they are bound to GTP or guanosine 5′-diphosphate (GDP). RAP1A is a distinct subfamily of RAP G proteins, a member of the RAS family. Rap plays an important role in signaling pathways that affect cell proliferation and adhesion.^1,2) Focal adhesion kinase (FAK) is an important contributor to the growth factor receptor and integrin signals in normal and cancer cells. FAK has a crucial role in signaling at cell adhesion points, adherens junctions, endosomes, and the nucleus.³⁾ Rac is a member of the Rho family GTPases, Rac also plays a critical role as a regulator of cellular functions that play important roles in cancer progression.⁴⁾ It is known that silencing of Rap1a reduced the phosphorylation of FAK on tyrosine 397.⁵⁾

MicroRNAs (miRs) have been shown to negatively regulate gene expression by binding to the mRNAs for protein-coding genes, thereby degrading or blocking their translation. miRs affect cancer biology, including cell proliferation, adhesion, invasion, and apoptosis.⁶⁾ Previous studies reported that RAP1A expression was suppressed by miRs, such as miR-202, miR-203, miR-205, miR-149, miR-196a, and miR-337-3p.^7–12) miR-101-3p (Previous ID: miR-101, Accession: MIMAT0000099) is known to play important roles in various physiological processes, and miR-101-3p has been shown to inhibit cell proliferation, migration, invasion, and apoptosis.^13–18) Mature miR-101-3p is generated from the stem-loop sequence miR-101-1 (accession No. MI0000103). Although miR-101-1 also generates mature miR-101-5p (Previous ID: miR-101*, accession No. MIMAT0004513), its function currently remains unclear. A previous study demonstrated that miR-101-5p levels were significantly higher in patients with relapsing-remitting multiple sclerosis than in healthy controls and neuromyelitis optica spectrum disorders. Chen et al. suggested a possible relationship among miR-101-5p, immune-mediated inflammation, cell survival, and differentiation.¹⁹⁾ It was estimated that miR-101-5p has possible targets using bioinformatics analysis.²⁰⁾ A bioinformatics resource, microRNA.org, predicted that miR-101-5p targets RAP1A. However, the inhibitory effects of miR-101-5p on RAP1A expression have yet to be clarified. We herein showed that miR-101-5p inhibited RAP1A expression. Next, we determined whether transfection with a miR-101-5p mimic affected RAP1A signaling or not. The effects of a RAP G protein inhibitor, salirasib, on cell proliferation as well as its downstream signaling molecules FAK and Rac were also examined.

MATERIALS AND METHODS

Reagents

The human epithelial-like cell line HeLa (JCRB9004), human transformed embryonal kidney cell line HEK293 (JCRB1533), human lung adenocarcinoma cell line A549 (JCRB0076), and human colon adenocarcinoma cell line COLO201 (JCRB0266) were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). These cell lines were tested and authenticated by the JCRB Cell Bank. Dulbecco’s modified Eagle’s medium - high glucose (DMEM) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Synthetic RAP1A small interfering RNA (siRNA) (sense: 5′-GCA GGG ACA GAG CAA UUU ACT T, anti: 5′-GUA AAU UGC UCU GUC CCU GCT T), PCR primers for RAP1A (sense: 5′-GAA GAA CGG CCA AGG TTT TGC, anti: 5′-GCC AAC CAA AAT CAT TGG AAC A), and PCR primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, sense: AAC AGC CTC AAG ATC ATC AGC, anti: GGA TGA TGT TCT GGA GAG CC)²¹⁾ were obtained from Fasmac Co., Ltd. (Atsugi, Japan). Real-time PCR master mix, THUNDERBIRD SYBR qPCR Mix, and a SuperPrep Cell Lysis & RT kit were purchased from Toyobo Co., Ltd. (Osaka, Japan). Synthetic miRNA mimic and AllStars Negative Control siRNA were purchased from Qiagen (Valencia, CA, U.S.A.). Cell proliferation enzyme-linked immunosorbent assay (ELISA), bromodeoxyuridine (BrdU), and protease inhibitor cocktail tablets (Complete, ethylenediaminetetraacetic acid (EDTA) free) were purchased from Roche Diagnostics GmbH (Buckinghamshire, U.K.). Rabbit polyclonal antibodies (E20-60739) specific for FAK (phospho Tyr397) were purchased from EnoGene Biotech Co., Ltd. (NY, U.S.A.). Mouse monoclonal antibodies specific for RAP1A (4C8/1) and FAK (2C3) were purchased from Novus Biologicals (CO, U.S.A.), and Abnova (Taipei, Taiwan), respectively. Mouse monoclonal antibodies specific for GAPDH (5A12), an anti-mouse immunoglobulin G (IgG), peroxidase-conjugated rabbit polyclonal antibody, the ScreenFect siRNA Transfection Reagent, ImmunoStar LD, RIPA buffer, and all other reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). A Rac1 Pull-down Activation Assay Biochem kit (bead pull-down format) was obtained from Cytoskelton Inc. (CO, U.S.A.). The RAP inhibitor salirasib (farnesylthiosalicylic acid) was purchased from Abcam (Cambridge, U.K.). Salirasib inhibits RAS proteins, exerts antitumor effects, and induces autophagy.²²⁾ Salirasib also inhibits RAP G proteins.²³⁾

Cell Culture and Transfection Assays

Human epithelial-like cell line, HeLa; human transformed embryonal kidney cell line, HEK293; human lung adenocarcinoma cell line, A549; and human colon adenocarcinoma cell line, COLO201 were grown in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and 100 units/mL of penicillin at 37°C in a 5% CO₂ humidified atmosphere. Synthetic RAP1A siRNA and the miR-101-5p mimic were transfected with the ScreenFect siRNA Transfection Reagent in accordance with the manufacturer’s protocol. Cell proliferation levels were evaluated after 48 h’ incubation using the cell proliferation ELISA, BrdU, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The inhibitory effects of RAP1A on cell proliferation were assessed using incubation with salirasib for 48 h at a concentration of 3.5 µM.

Real-Time Quantitative PCR Analysis

Single-stranded cDNA was synthesized by reverse transcriptase using a SuperPrep Cell Lysis & RT kit in accordance with the manufacturer’s instructions. Real-time quantitative PCR methods were described in a previous study.²⁴⁾ GAPDH was used as a reference gene for mRNA. Comparisons between 3 groups of expression levels were performed with the Tukey–Kramer test.

Electrophoresis and Western Blotting

Cells were lysed in RIPA buffer with a protease inhibitor. Protein concentrations were measured using a Protein Assay kit (Nacalai Tesque, Kyoto, Japan). Whole cell homogenates (GAPDH: 5 µg; RAP1A, FAK and phospho-(Tyr397) FAK: 20 µg) were separated using 12% sodium dodecyl sulfate (SDS) polyacrylamide gels, and proteins were then transferred onto nitrocellulose membranes. These membranes were then incubated as described in a previous study.²⁴⁾ Rac activity was assessed using the Rac1 pull-down activation assay kit. Optical densities were measured using the public domain program ImageJ.

RESULTS

Bioinformatics Analysis

A bioinformatics analysis was performed to estimate the complementary miRs of RAP1A. RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) is a comprehensive resource for miR target predictions with extreme value statistics of length-normalized minimum free energies (MFE).²⁵⁾ RNAhybrid estimated that the optimized form miR-101-5p binds to 3 sites (positions: 604, 855, and 276) in the 3′ UTR of RAP1A. The predicted alignment forms and MFE are shown in Fig. 1.

Fig. 1. Bioinformatics Analysis of Alignment: Possible Forms of miR-101-5p/RAP1A

The target sites in RAP1A were estimated using the bioinformatics resource, RNAhybrid. Target positions in RAP1A and minimum free energy (MFE) values were as follows: A, 604, MFE = −20.5 kcal/mol; B, 855, MFE = −19.6 kcal/mol; C, 276, MFE = −18.7 kcal/mol.

miR-101-5p Down-Regulated RAP1A

The miR-101-5p mimic or RAP1A siRNA was transfected into the human carcinoma cell lines, HeLa, HEK293, A549, and COLO201. Transfection of the miR-101-5p mimic significantly decreased RAP1A mRNA expression levels, whereas positive control siRNA also down-regulated the expression of RAP1A (Table 1). Transfection of the miR-101-5p mimic did not significantly affect GAPDH protein expression levels (control, 1.00 ± 0.15; RAP1A siRNA, 0.91 ± 0.07; miR-101-5p mimic, 1.17 ± 0.13, n = 4, Fig. 2A). Transfection of the miR-101-5p mimic also significantly decreased phosphorylation of FAK compared with control treatment (control, 1.00 ± 0.05; RAP1A siRNA, 0.46 ± 0.03; miR-101-5p mimic, 0.68 ± 0.12: p = 0.008, n = 4, Fig. 2B), but not the expression levels of FAK. Rac1 activity was evaluated using a pulldown assay kit. Transfection of the miR-101-5p mimic significantly decreased Rac1 activity (control, 1.00 ± 0.07; RAP1A siRNA, 0.51 ± 0.11; miR-101-5p mimic, 0.76 ± 0.06, p = 0.04, n = 4, Fig. 2C).

Table 1. Relative Expression Levels of RAP1A mRNA in Cells after Transfection of RAP1A siRNA or the miR-101-5p Mimic

Cell line	Control	RAP1A siRNA	miR-101-5p mimic
HeLa	1.00 ± 0.07	0.34 ± 0.05***	0.29 ± 0.03***
HEK293	1.00 ± 0.20	0.10 ± 0.03***	0.20 ± 0.07***
A549	1.00 ± 0.20	0.17 ± 0.09***	0.17 ± 0.04***
COLO201	1.00 ± 0.18	0.33 ± 0.02***	0.29 ± 0.01***

Values represent the relative ratio of the target gene per GAPDH, mean ± standard error of the mean (S.E.M.), to the control from independent experiments, n = 10. The vehicle group was transfected with negative control RNA. Statistical analyses were performed using the Tukey–Kramer test; *** p < 0.001. Cells were incubated for 48 h in accordance with the protocol for the siRNA transfection reagent.

Fig. 2. The miR-101-5p Mimic Inhibited RAP1A Expression and Decreased Phosphorylation of FAK and RAC1 Activity

(A) The miR-101-5p mimic inhibited RAP1A protein expression in HeLa cells. Values represent the relative ratio of the target gene per GAPDH. (B) The miR-101-5p mimic inhibited the phosphorylation levels of FAK in HeLa cells. Values represent the relative ratio of the target gene per GAPDH. (C) The miR-101-5p mimic inhibited RAC1 activity in HeLa cells. The values represent mean ± S.E.M., to the control from independent experiments, n = 4. The control group was transfected with negative control RNA. Statistical analyses were performed using the Tukey–Kramer test; *** p < 0.001, ** p < 0.01, * p < 0.05. (D) Typical Western blotting images of RAP1A, FAK, phospho-(Tyr397) FAK, and GAPDH. Whole cell homogenates (GAPDH: 5 µg; RAP1A, FAK, and phospho-(Tyr397) FAK: 20 µg) were separated using SDS polyacrylamide gels. RAP1A, FAK, and GAPDH proteins were detected as 21-, 120-, and 36-kDa bands, respectively. Transfection of the miR-101-5p mimic did not affect the expression levels of FAK (control, 1.00 ± 0.19; RAP1A siRNA, 0.94 ± 0.15; miR-101-5p mimic, 0.84 ± 0.19).

miR-101-5p Inhibited Cell Proliferation

The inhibitory effects of the miR-101-5p mimic and RAP1A siRNA on cell proliferation were assessed using BrdU ELISA and MTT assays. The miR-101-5p mimic or RAP1A siRNA was transfected into HeLa and HEK293 cells. Transfection of the miR-101-5p mimic or RAP1A siRNA significantly decreased the incorporation of BrdU, and RAP1A siRNA also decreased its incorporation. Transfection of the miR-101-5p mimic or RAP1A siRNA significantly decreased cellular MTT reductions in HEK293 and HeLa cells, similar to the incorporation of BrdU (Table 2).

Table 2. Relative Cell Proliferation Levels after Transfection of RAP1A siRNA or the miR-101-5p Mimic

BrdU ELISA
Cell line	Control	RAP1A siRNA	miR-101-5p mimic
HeLa	1.00 ± 0.05	0.74 ± 0.02***	0.83 ± 0.03*
HEK293	1.00 ± 0.06	0.76 ± 0.06**	0.74 ± 0.05**
MTT assay
Cell line	Control	RAP1A siRNA	miR-101-5p mimic
HeLa	1.00 ± 0.02	0.82 ± 0.06**	0.90 ± 0.08**
HEK293	1.00 ± 0.03	0.83 ± 0.03***	0.84 ± 0.02***

Values represent the relative ratio of the vehicle treatment, mean ± S.E.M., to the control from independent experiments, n = 10. The vehicle group was transfected with negative control RNA. Statistical analyses were performed using the Tukey–Kramer test; *** p < 0.001, ** p < 0.01, * p < 0.05, significantly different from the control treatment. Cells were incubated for 48 h in accordance with the protocol for the siRNA transfection reagent.

Inhibitory Effects of Salirasib on Cell Proliferation

To determine the contribution of RAP1A signaling to the inhibition effect of cell proliferation by miR-101-5p mimic treatment, cells were transfected with the miR-101-5p mimic, RAP1A, and negative control siRNA for 48 h. Salirasib was added to the culture medium at a concentration of 3.5 µM. This concentration of salirasib was the 50% inhibitory concentration, as determined using the MTT assay. Salirasib treatment significantly decreased cell proliferation in negative control siRNA-treated cells, whereas the same treatment did not cause reductions in RAP1A-siRNA or miR-101-5p mimic-treated cells (Fig. 3).

Fig. 3. The RAP G Protein Inhibitor Salirasib Inhibited Cell Proliferation, but Not in miR-101-5p Mimic- or RAP1A siRNA-Transfected Cells

The proliferation levels of HEK293 cells were assessed after 48 h incubation using a cell proliferation ELISA with bromodeoxyuridine (BrdU). Values represent the relative ratio of the control siRNA treatment. Salirasib treatment significantly inhibited the proliferation of negative control siRNA-transfected cells (control: 1.00 ± 0.06, salirasib-treated: 0.76 ± 0.09, n = 7, mean ± S.E.M.). Proliferation levels were significantly decreased in miR-101-5p mimic-transfected cells (miR-101-5p: 0.64 ± 0.04, miR-101-5p with salirasib: 0.63 ± 0.06, n = 7). Salirasib treatment did not inhibit the proliferation of miR-101-5p-transfected cells. Proliferation levels were significantly lower in RAP1A siRNA-transfected cells than in negative control siRNA-transfected cells (RAP1A siRNA: 0.55 ± 0.09, RAP1A siRNA with salirasib: 0.50 ± 0.11, n = 7). The addition of salirasib did not exert inhibitory effects on the proliferation of RAP1A siRNA-transfected cells. Statistical analyses were performed using the Tukey–Kramer test; *** p < 0.001, * p < 0.05. significantly different from negative control siRNA-treated cells (open bar).

DISCUSSION

Here, we demonstrated the inhibitory effects of miR-101-5p on the expression of RAP1A. Transfection of the miR-101-5p mimic significantly decreased the expression of RAP1A mRNA and protein, and the same treatment also significantly inhibited cell proliferation (Tables 1, 2; Fig. 2). A positive control treatment, transfection of synthetic RAP1A siRNA, exerted similar effects. A comprehensive resource for predicting microRNA/target duplexes, RNAhybrids estimated possible forms and MFE (Fig. 1). Estimated MFE values were similarly low. For example, the MFE of synthetic RAP1A siRNA and the target mRNA duplex were estimated to be −40.3 kcal/mol. These results suggested that miR-101-5p negatively regulated RAP1A gene expression by binding to mRNA.

RAP1A is a member of the RAS family of proteins, which plays an important role in cell proliferation. Alterations in cell proliferation after miR-101-5p mimic transfection were assessed in order to estimate the down-regulatory effects of RAP1A. Cell proliferation levels were evaluated using BrdU ELISA and MTT assays. Treatment with the miR-101-5p mimic or RAP1A siRNA significantly decreased the incorporation of BrdU in HeLa and HEK293 cells. The same treatment significantly decreased MTT reductions in HEK293 cells. BrdU assays reflect BrdU incorporation correlated with DNA synthesis. Tetrazolium salts, such as MTT, are metabolized by nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenases in mitochondria; therefore, the MTT assay reflects energy metabolism. This result suggested that the transfection of miR-101-5p may have different effects on energy metabolism depending on the cell type.

Downstream of RAP1a signaling molecules are FAK encoded by the protein tyrosine kinase gene PTK2^5,26) and Rac.²⁷⁾ It was reported that silencing of Rap1a reduced the phosphorylation of tyrosine 397 in FAK.⁵⁾ Treatment with the miR-101-5p mimic or RAP1A siRNA significantly decreased the phosphorylation levels of FAK. The same treatment also significantly decreased Rac activity. These results provide support for the downregulation effect of RAP1A by transfection of the miR-101-5p mimic.

The alternative effects of salirasib on the proliferation of HEK293 cells were examined. Salirasib treatment significantly decreased the proliferation of negative control siRNA-treated cells. However, salirasib did not affect miR-101-5p mimic- or RAP1A siRNA-treated cells (Fig. 3). These results suggested that the inhibitory effects of salirasib on RAP1A decreased with reductions in RAP1A after transfection of RAP1A siRNA or the miR-101-5p mimic. Salirasib is an oral RAS inhibitor that has potential as a drug to treat hematological malignancies.²⁸⁾ The present results suggest that the expression levels of miR-101-5p and RAP1A affect the efficacy of salirasib.

We herein demonstrated that miR-101-5p negatively regulated the gene expression of RAP1A, which plays a crucial role in cell proliferation. Furthermore, miR-101-5p may influence cell adhesion signaling through FAK. The present results indicate that miR-101-5p controls cell proliferation via the expression of RAP1A.

Acknowledgment

This work was supported by JSPS KAKENHI Grant No. 15K08088.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Minato N. Rap G protein signal in normal and disordered lymphohematopoiesis. Exp. Cell Res., 319, 2323–2328 (2013).
• 2) Gao L, Feng Y, Bowers R, Becker-Hapak M, Gardner J, Council L, Linette G, Zhao H, Cornelius LA. Ras-associated protein-1 regulates extracellular signal-regulated kinase activation and migration in melanoma cells: two processes important to melanoma tumorigenesis and metastasis. Cancer Res., 66, 7880–7888 (2006).
• 3) Kleinschmidt EG, Schlaepfer DD. Focal adhesion kinase signaling in unexpected places. Curr. Opin. Cell Biol., 45, 24–30 (2017).
• 4) Kazanietz MG, Caloca MJ. The Rac GTPase in cancer: from old concepts to new paradigms. Cancer Res., 77, 5445–5451 (2017).
• 5) Carmona G, Göttig S, Orlandi A, Scheele J, Bäuerle T, Jugold M, Kiessling F, Henschler R, Zeiher AM, Dimmeler S, Chavakis E. Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis. Blood, 113, 488–497 (2009).
• 6) Esquela-Kerscher A, Slack FJ. Oncomirs—microRNAs with a role in cancer. Nat. Rev. Cancer, 6, 259–269 (2006).
• 7) Xiang J, Bian C, Wang H, Huang S, Wu D. MiR-203 down-regulates Rap1A and suppresses cell proliferation, adhesion and invasion in prostate cancer. J. Exp. Clin. Cancer Res., 34, 8 (2015).
• 8) Skourti E, Logotheti S, Kontos CK, Pavlopoulou A, Dimoragka PT, Trougakos IP, Gorgoulis V, Scorilas A, Michalopoulos I, Zoumpourlis V. Progression of mouse skin carcinogenesis is associated with the orchestrated deregulation of mir-200 family members, mir-205 and their common targets. Mol. Carcinog., 55, 1229–1242 (2016).
• 9) Bischoff A, Huck B, Keller B, Strotbek M, Schmid S, Boerries M, Busch H, Müller D, Olayioye MA. miR149 functions as a tumor suppressor by controlling breast epithelial cell migration and invasion. Cancer Res., 74, 5256–5265 (2014).
• 10) Wang K, Li J, Guo H, Xu X, Xiong G, Guan X, Liu B, Li J, Chen X, Yang K, Bai Y. MiR-196a binding-site SNP regulates RAP1A expression contributing to esophageal squamous cell carcinoma risk and metastasis. Carcinogenesis, 33, 2147–2154 (2012).
• 11) Du L, Subauste MC, DeSevo C, Zhao Z, Baker M, Borkowski R, Schageman JJ, Greer R, Yang CR, Suraokar M, Wistuba II, Gazdar AF, Minna JD, Pertsemlidis A. miR-337-3p and its targets STAT3 and RAP1A modulate taxane sensitivity in non-small cell lung cancers. PLOS ONE, 7, e39167 (2012).
• 12) Fang B, Wei L, Dong K, Niu X, Sui X, Zhang H. miR-202 modulates the progression of neuropathic pain through targeting RAP1A. J. Cell. Biochem., 120, 2973–2982 (2019).
• 13) Friedman JM, Liang G, Liu CC, Wolff EM, Tsai YC, Ye W, Zhou X, Jones PA. The putative tumor suppressor microRNA-101 modulates the cancer epigenome by repressing the polycomb group protein EZH2. Cancer Res., 69, 2623–2629 (2009).
• 14) Jiang R, Zhang C, Liu G, Gu R, Wu H. MicroRNA-101 inhibits proliferation, migration and invasion in osteosarcoma cells by targeting ROCK1. Am. J. Cancer Res., 7, 88–97 (2017).
• 15) Fan P, Liu L, Yin Y, Zhao Z, Zhang Y, Amponsah PS, Xiao X, Bauer N, Abukiwan A, Nwaeburu CC, Gladkich J, Gao C, Schemmer P, Gross W, Herr I. MicroRNA-101-3p reverses gemcitabine resistance by inhibition of ribonucleotide reductase M1 in pancreatic cancer. Cancer Lett., 373, 130–137 (2016).
• 16) Chen X, Zhang N. Downregulation of lncRNA NEAT1_2 radiosensitizes hepatocellular carcinoma cells through regulation of miR-101-3p/WEE1 axis. Cell Biol. Int., 43, 44–55 (2019).
• 17) Wang L, Yao J, Sun H, He K, Tong D, Song T, Huang C. MicroRNA-101 suppresses progression of lung cancer through the PTEN/AKT signaling pathway by targeting DNA methyltransferase 3A. Oncol. Lett., 13, 329–338 (2017).
• 18) Tang XR, Wen X, He QM, Li YQ, Ren XY, Yang XJ, Zhang J, Wang YQ, Ma J, Liu N. MicroRNA-101 inhibits invasion and angiogenesis through targeting ITGA3 and its systemic delivery inhibits lung metastasis in nasopharyngeal carcinoma. Cell Death Dis., 8, e2566 (2017).
• 19) Chen J, Zhu J, Wang Z, Yao X, Wu X, Liu F, Zheng W, Li Z, Lin A. MicroRNAs correlate with multiple sclerosis and neuromyelitis optica spectrum disorder in a Chinese population. Med. Sci. Monit., 23, 2565–2583 (2017).
• 20) Yang X, Pang YY, He RQ, Lin P, Cen JM, Yang H, Ma J, Chen G. Diagnostic value of strand-specific miRNA-101-3p and miRNA-101-5p for hepatocellular carcinoma and a bioinformatic analysis of their possible mechanism of action. FEBS Open Bio., 8, 64–84 (2018).
• 21) Gao L, Feng Y, Bowers R, Becker-Hapak M, Gardner J, Council L, Linette G, Zhao H, Cornelius LA. Ras-associated protein-1 regulates extracellular signal-regulated kinase activation and migration in melanoma cells: two processes important to melanoma tumorigenesis and metastasis. Cancer Res., 66, 7880–7888 (2006).
• 22) Schmukler E, Grinboim E, Schokoroy S, Amir A, Wolfson E, Kloog Y, Pinkas-Kramarski R. Ras inhibition enhances autophagy, which partially protects cells from death. Oncotarget, 4, 145–155 (2013).
• 23) Berzat AC, Brady DC, Fiordalisi JJ, Cox AD. Using inhibitors of prenylation to block localization and transforming activity. Methods Enzymol., 407, 575–597 (2006).
• 24) Shibayama Y, Kondo T, Ohya H, Fujisawa S, Teshima T, Iseki K. Upregulation of microRNA-126-5p is associated with drug resistance to cytarabine and poor prognosis in AML patients. Oncol. Rep., 33, 2176–2182 (2015).
• 25) Krüger J, Rehmsmeier M. RNAhybrid: microRNA target prediction easy, fast and flexible. Nucleic Acids Res., 34 (Web Server), W451–W454 (2006).
• 26) Eppler FJ, Quast T, Kolanus W. Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion. PLOS ONE, 12, e0172443 (2017).
• 27) Fukuyama T, Ogita H, Kawakatsu T, Fukuhara T, Yamada T, Sato T, Shimizu K, Nakamura T, Matsuda M, Takai Y. Involvement of the c-Src-Crk-C3G-Rap1 signaling in the nectin-induced activation of Cdc42 and formation of adherens junctions. J. Biol. Chem., 280, 815–825 (2005).
• 28) Badar T, Cortes JE, Ravandi F, O’Brien S, Verstovsek S, Garcia-Manero G, Kantarjian H, Borthakur G. Phase I study of S-trans, trans-farnesylthiosalicylic acid (salirasib), a novel oral RAS inhibitor in patients with refractory hematologic malignancies. Clin. Lymphoma Myeloma Leuk, 15, 433–438.e2 (2015).