Transthyretin Suppressed Tumor Progression in Nonsmall Cell Lung Cancer by Inactivating MAPK/ERK Pathway

Abstract

Background:

Lung malignancy is a main source of disease passing all throughout the planet, whereas the transthyretin (TTR) is a specific biomarker for clinical diagnosis. However, its role in lung malignancy stays to be obscure.

Materials and Methods:

In the current study, the authors made an endeavor to research impact of abnormal expression of TTR on nonsmall cell lung carcinoma (NSCLC) by overexpression or knockdown of TTR. To further explore the instruments' fundamental mechanism part of TTR in NSCLC, several signal pathways were searched and verified. To confirm the effect of TTR overexpression on tumors, in vivo experiments were conducted.

Results:

It was found that upregulated TTR clearly stifled cell proliferation, migration, invasion, and expanded apoptosis. Significant suppression of phosphor-extracellular signal-regulated kinase (ERK) was observed in TTR-treated NSCLC cells, implying that TTR was important for cellular progress by regulating mitogen-activated protein kinase/ERK signaling pathway. In in vivo experiment, overexpression of TTR promoted cell apoptosis and inhibited tumor growth.

Conclusion:

Overall, the results suggest that TTR has a potential antitumor effect in human NSCLC progression, which provides theoretical basis for the diagnosis and treatment of NSCLC. Above all, further understanding of TTR was useful for clinical care. Clinical Trial Registration Number: 2016-08.

Keywords

TTR proliferation apoptosis migration invasion MAPK/ERK pathway

Introduction

Lung cancer is the most well-known harmful tumor on the planet and is an essential explanation of disease-related demise, in this manner representing an incredible danger to human health.¹ In China, the dreariness and mortality due to lung cancer rank first in harmful tumors.² Of note, nonsmall cell lung carcinoma (NSCLC) is the main type of lung cancer.³ Although great progresses have been made in early detection and treatment strategies in the course of recent many years, the endurance rate actually stays low level.⁴ Therefore, further investigation on the molecular mechanisms on NSCLC will be essential for diagnosis and therapy.

Transthyretin (TTR) is a 55-kDa homotetrameric protein that helps transport thyroid hormones through the bloodstream^5,6 and plays a role in retinol digestion. It is halfway created by the liver and extrahepatic tissues, such as choroids plexus, retinal shade, epithelium cells, and islet A and B cells.^7–9 TTR is a profoundly plentiful protein that ties to coursing retinol binding protein (RBP) to forestall low subatomic mass RBP.^10–14 The degrees of TTR could be diminished in extreme liver illness, hunger, and inflammation.^15–17 TTR levels were also lower in the serum of patients with ovarian cancer, cervical cancer, and endometrial cancer.¹⁸ As a result, TTR has proven to be a useful biomarker for identifying lung cancer. Notwithstanding, the impact and mechanism of TTR on the lung cancer cells actually stay subtle.

In this study, the authors researched the mechanism underlying abnormal expression of TTR on NSCLC cells, and try to search for efficient signaling pathway in TTR-mediated NSCLC cells. By further investigation into effect of TTR on NSCLC, the authors may provide proof-of-principle evidence for clinical application of TTR.

Materials and Methods

Clinical tissue examples

The tumor tissues and combined contiguous nondisease tissues were gathered from an aggregate of 40 patients determined to have NSCLC at Tianjin Hospital, Tianjin Medical University. The convention of this examination was affirmed by the ethics committee of Tianjin Hospital, Tianjin Medical University (2016-08), and the patients who were enrolled gave their written informed consent.

Transfection and cell culture

The Chinese Academy of Science Cell Bank provided A549 and H1975 NSCLC cell lines, as well as 16HBE standard lung cell lines (Shanghai, China). Cells were refined in RPMI-1640 medium (Hyclone, South Logan, UT) +10% FBS (fetal bovine serum; Biowest, Barcelona, Catalonia, Spain) at 37°C and 5% CO₂. TTR overexpression and inhibitor were achieved from GenePharma (Shanghai, China). Sh-TTR were constructed to knockdown TTR, and sh-NC (negative control) were used as control. For TTR overexpression, the full length of TTR coding domain sequence was embedded into pcDNA3.1 vectors (Invitrogen) and the unfilled plasmids filled in as NC. Lipofectamine 2000 (Invitrogen) was used to transfect cells according to the item manuals.

RNA isolation and quantitative reverse transcription-polymerase chain reaction

All RNA was extricated utilizing TRIzol reagent (Invitrogen). Equivalent measures of RNA were interpreted into cDNA utilizing the cDNA first strand synthesis kit. Absolute cDNA was utilized as a beginning material for ongoing polymerase chain reaction (PCR) utilizing the Step One real-time PCR system (Life Technologies Corp), and each example was estimated in threefold. Complete response framework is as follows (20 μL): cDNA 1 μL, SYBR Premix EX Taq 10 μL, each of 1 μL (10 μM) the primers 1 μL, and ddH₂O 7 μL. The procedure of PCR was as follows: 94°C for 3 min followed by 35 cycles of 94°C for 10 s, and 63°C for 30 s. Segment overlay changes were determined utilizing the 2-ΔΔCT similar strategy utilizing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for standardization. The primers used were as follow: TTR: F-TGGGAGCCATTTGCCTCTG, R-AGCCGTGGTGGAATAGGAGTA; Wnt: F-CGATGGTGGGGTATTGTGAAC, R-CCGGATTTTGGCGTATCAGAC; transforming growth factor-β (TGF-β): F-GGCCAGATCCTGTCCAAGC, R-GTGGGTTTCCACCATTAGCAC; extracellular signal-regulated kinase (ERK): F-TACACCAACCTCTCGTACATCG, R-CATGTCTGAAGCGCAGTAAGATT; nuclear factor kappa B (NF-κB): F-AACAGAGAGGATTTCGTTTCCG, R-TTTGACCTGAGGGTAAGACTTCT; GAPDH: F-CTGGGCTACACTGAGCACC, R-AAGTGGTCGTTGAGGGCAATG.

Western blotting

In total, 50 μg all out protein per path was isolated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and smeared onto polyvinylidene fluoride membranes. The membrane was impeded in phosphate buffered saline with Tween 20 and examined with primary antibodies for Bcl-2 (1: 1000; ab32124), Bax (1: 1000; ab32503), caspase-3 (1: 1000; ab13847), caspase-9 (1: 1000; ab13847), matrix metal proteinases-2 (MMP-2) (1: 1000; ab37150), and MMP-9 (1: 1000; ab73734), these antibodies were bought for Abcam (Cambridge, MA). Place Phosphor-ERK (1:2000, no. 9106; Danvers, MA), ERK (1:1000, no. 9102; Danvers, MA), and GAPDH (1: 5000, G8795; Sigma-Aldrich, St. Louis, MOA) at 4°C overnight. Subsequently, appropriate secondary antibodies were used to incubate the membranes (1: 5000, ab6858: Abcam), protein bindings were visualized by ECL kit (Beyotime Institute of Biotechnology, Beijing, China). The groups were checked utilizing a densitometer, and the dark worth of the groups was determined naturally by the ImageJ software version k 1.45 (NIH, Bethesda, MD).

Cell proliferation tests

5-Ethynyl-2′-deoxyuridine (EdU) measure was applied to recognize cell proliferation as indicated by the producers' convention. For colony formation test, cells were cultivated in a six-well plate at a thickness of 1000 cells/well for ∼14 d, after which clones were fixed with methanol and stained with crystal violet (0.1%).

Cell apoptosis tests

FITC-Annexin V/PI apoptotic identification kit (BD ingen, San Diego, CA) was utilized to recognize apoptotic cells by flow cytometry. The cells (1 × 10⁵ cells) were stained with propidium iodide (4.5 mL) and Annexin V-FITC (4.5 mL), trailed by fluorescence assurance by flow cytometer (Beckman, Miami, FL). Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was also applied to detect the apoptosis in cells with the In Situ Cell Death Detection Kit (Roche). The cells were washed with 1 × phosphate-buffered saline (PBS) and fixed in paraformaldehyde solution (500 μL, 4%) for 15 min at 37°C. All examinations were performed in threefold.

Scratch assay

The cells were grown in six-well plates. Inexact 80% of the cells were placed into the divider and washed in 1 × PBS twice to eliminate gliding cells. Wounds were gotten from sterile pipette tips. Subsequently, cells were washed with 1 × PBS twice, and RPMI-1640 culture medium (2 mL, 10% FBS) was added into the culture plate. Photos were taken 24 and 48 h after the wound was formed.

Transwell invasion measurement

Transwell invasion measure was estimated according to the maker's guidelines. To sum things up, a sum of 3.5 × 10⁴ cells/well in Dulbecco's modified Eagle's medium (DMEM) (100 μL, 0.5% FBS) were put in the upper Transwell chamber (Corning Incorporated, NY), which was precovered with matrigel (Growth factor reduced; BD Biosciences, MD). The bottom chambers were filled with DMEM containing 20% FBS. Once passed through the membranes, the cells were fixed and stained after 24 h. Cells in nine haphazardly chosen fields were checked under a modified magnifying lens (200 × magnification; Carl ZEISS, Jena, German) and a normal worth was utilized as the quantity of invaded cells. For the cell transmembrane relocation examination, except for the method covered by Matrigel (BD Biosciences, Franklin lakes, NJ), each method is completed relative to the method in the invasion test.

Tumor xenograft model in naked mice

The Beijing Vital River Laboratory Animal Technology sold BALB/c nude mice (male, 5 weeks old; 18–20 g) (Beijing, China). Human A549 and H1975 cells were used to create a xenograft model. A549 and H1975 cells transfected with sh-NEAT1 or sh-NC were infused subcutaneously into the back fossa of naked mice (six per group). After 5 d, when normal tumor volume was ∼50 mm³, mice were intraperitoneally infused with paclitaxel (3.5 mg/kg body weight) or PBS every 3 d. The tumor volumes were estimated each week by utilizing calipers: volume (cm³) = width² (cm²) × length (cm)/2. Mice were sacrificed at 32nd day after infusion and tumors were eliminated, gauged, and analyzed by quantitative RT-PCR. All animal experiments were performed with the approval of the institutional Ethics Committee.

Statistical analysis

All outcomes were performed in any event three independent replicates and revealed as means ± standard deviation. All measurable examinations were performed utilizing GraphPad software 5.0. Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test was used to assess the differences between the two groups or three gatherings, respectively. Statistical significance was defined as a p < 0.05.

Results

The level of TTR expression in NSCLC tissues and cells is reduced

By examining at 40 pairs of human NSCLC and normal tissues, a prominent decrease in TTR expression in NSCLC was found than in the normal tissues (Fig. 1A). At the same time, the authors observed that compared with human bronchial epithelial cells, the TTR expression level in two NSCLC cell lines was significantly reduced (Fig. 1B). The overall survival time of patients with high-TTR expression was significantly higher than those with low-TTR expression (Fig. 1C). These results indicate that TTR may be negatively correlated with NSCLC progression.

FIG. 1.

TTR expression in tissues and cell lines of NSCLC. (A) TTR expression was lower in NSCLC tissues than in normal tissues. (B) TTR expression was lower in A549 and H1975 than in 16HBE, (A, B) all were calculated by qRT-PCR. (C) Overall survival of patients. ***p < 0.001 versus normal tissues. **p < 0.01 versus 16HBE. NSCLC, nonsmall cell lung carcinoma; qRT-PCR, quantitative RT-polymerase chain reaction; TTR, transthyretin. Color images are available online.

TTR curbs in vitro NSCLC cell expansion and advances its apoptosis

To analyze the part of TTR in cell proliferation, the authors overexpressed TTR (TTR overexpression) or transfected with TTR inhibitors in A549 cells and H1975 cells, respectively (Fig. 2A). EdU tests indicated that both A549 cells and H1975 cells proliferation capacity were reduced in TTR-overexpressed group compared with NC group, while were increased in the TTR inhibitor-treated group compared with NC group (Fig. 2B).

FIG. 2.

In vitro impact of TTR on cell proliferation of NSCLC in vitro. (A) qRT-PCR showed TTR expression level in H1975 and A549 cell lines with different treatment. (B) EdU-positive nuclei indicated proliferating cells in H1975 and A549 cell lines in different groups. (C) Colony formation assay showed a higher proliferative capacity in H299 and A549 cell lines in TTR inhibitor group. ***p < 0.001 versus NC. EdU, 5-ethynyl-2′-deoxyuridine; NC, negative control; NSCLC, nonsmall cell lung carcinoma; qRT-PCR, quantitative RT-polymerase chain reaction; TTR, transthyretin. Color images are available online.

Reliably, these outcomes were additionally affirmed by colony formation assay (Fig. 2C).

Furthermore, cell apoptosis by flow cytometry and TUNEL assays was estimated, suggesting that proliferation capacity of both A549 and H1975 cells was decreased in the TTR-overexpressed group (Fig. 3A, B). At the molecular level, we found that proapoptotic protein Bcl2 was downregulated and Bax and caspase-3/9 (antiapoptosis proteins) was up-regulated in TTR overexpression group were determined using Western blot analysis compared with NC group (Fig. 3C). The results indicated that the TTR overexpression group advanced A549 cells and H1975 cells apoptosis.

FIG. 3.

In vitro impact of TTR on cell apoptosis of NSCLC. Cell apoptosis capacity of H1975 and A549 cells was detected by (A) TUNEL analysis and (B) flow cytometry. (C) Western blot assay was utilized to investigate the apoptosis-related proteins expression level in A549 and H1975 cells. **p < 0.01 versus NC. ***p < 0.001 versus NC. NC, negative control; NSCLC, nonsmall cell lung carcinoma; TTR, transthyretin; TUNEL, terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling. Color images are available online.

TTR restrains in vitro NSCLC cell migration and invasion

The impacts of TTR on cell migration and invasion were studied by Transwell chamber and scratch methods. Transwell chamber assay that less cells were moved and attacked to the lower medium in TTR overexpression group compared with TTR inhibitor group (Fig. 4A, B). As shown in Figure 4C, a scratch test revealed that the migrated distance between the control and TTR inhibitor groups differed significantly. Then, Western blotting was performed to detect Matrix metal proteinases (MMP2/9). The MMP-2 and MMP-9 expression levels were downregulated in the TTR overexpression group (Fig. 4D). According to these results, upregulated TTR suppressed NSCLC cell migration and invasion in vitro.

FIG. 4.

In vitro TTR suppressed NSCLC cell migration and invasion. Transwell migration (A) and invasion (B), wound healing (C) migration assays were applied to detect cell migration and invasion ability of cells A549 and H1975. (D) MMP-2 and MMP-9 protein levels were explored using Western blot assay. **p < 0.01 versus NC. ***p < 0.001 versus NC. MMP-2, matrix metal proteinases-2; NC, negative control; NSCLC, nonsmall cell lung carcinoma; TTR, transthyretin. Color images are available online.

TTR represses the in vitro mitogen-activated protein kinase/ERK pathway activation

To further gain insights into the downstream signaling pathways regulation by TTR in NSCLC cells, the functional effects of TTR in several important cancer pathways were analyzed including Wnt, TGF-β, NF-κB, and ERK by RT-PCR. TTR overexpression altogether hindered the declaration of ERK in A549 cells and H1975 cells (Fig. 5A). Western blot results showed phosphor-ERK was observed to be significantly suppressed in the TTR overexpression group (Fig. 5B), demonstrating that the mitogen-activated protein kinase (MAPK)/ERK flagging pathway was fundamental for tumor growth in NSCLC cells. TTR overexpression inhibited ERK phosphorylation, thereby stimulating cell proliferation and cell survival.

FIG. 5.

In vitro TTR suppressed tumor progression through ERK pathway. (A) TTR overexpression significantly suppressed ERK in A549 and H1975 cells. (B) ERK signaling pathway-related proteins were explored by Western blot. ERK, extracellular signal-regulated kinase; TTR, transthyretin.

TTR ameliorates in vivo NSCLC progression

In light of the capacity of TTR in vitro, the authors further examined whether overexpression of TTR could suppress NSCLC progression in vivo. The expression of TTR was distinguished by qRT-PCR (Fig. 6A). Accordingly, tumors from TTR overexpression developed clearly more slow than the NC group. The mice were killed 5 weeks after the injection and the tumors were taken out. The tumors were obviously smaller than those in other groups on size and weight (Fig. 6B). These results implied that TTR could inhibit tumor progression. TUNEL assay revealed that the TTR overexpression group had more apoptosis than the NC group. Inhibiting TTR expression, on the other hand, can significantly reduce cell apoptosis (Fig6C).

FIG. 6.

In vivo upregulated TTR suppressed NSCLC progression. (A) TTR expression was detected by qRT-PCR. (B) Tumor size and tumor weight of different groups. (C) TUNEL staining in the xenograft tissues of nude mice was used to explore the apoptosis ability in different groups. **p < 0.01 versus NC. ***p < 0.001 versus NC. NC, negative control; NSCLC, nonsmall cell lung carcinoma; qRT-PCR, quantitative RT-polymerase chain reaction; TTR, transthyretin; TUNEL, terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling. Color images are available online.

Discussion

Lung cancer is the most frequent cause of cancer-related death all over the world, and the 5-year survival rate is less than 15%.^19,20 Although there have been numerous studies on the resistance, discovery, and treatment of lung cancer, the expectations for lung cancer patients remain pessimistic. In this research, it was demonstrated that TTR suppressed tumor progression in NSCLC by inactivating the MAPK/ERK pathway.

Abnormal expression levels of TTR have been accounted for to assume a significant part in tumor formation and development.^21,22 For example, Shimura et al. found a significant negative correlation between TTR levels and tumor size and the number of involved lymph nodes.²³ However, there is still limited understanding of the effect of TTR abnormal expression levels on tumor progression. It was discovered that human NSCLC tissues had lower TTR expression than normal tissue, and that TTR expression was associated with clinic pathological features in this study. Similar results have reported that the expression of TTR in patients with cervical cancer was significantly lower than that in healthy controls.²⁴ Segment TTR expression in NSCLC cells cultured in vitro can boost cell proliferation, migration, and invasion while decreasing cell apoptosis. Overexpression of TTR, in contrast, is sufficient to inhibit cell proliferation and invasive potential in NSCLC cells, as well as increase in apoptosis. To further investigate mechanisms of TTR on tumor progression in NSCLC, several signaling pathways were searched, and MAPK/ERK signals were found to respond to the TTR manipulation in NSCLC cells. In in vivo experiment, overexpression of TTR obviously inhibited tumor growth and promoted cell apoptosis, implying that TTR was important for regular cellular progression.

The tumor-related pathways, such as Wnt, PI3K/AKT, or NF-kB, may assume a significant part in the advancement of tumorigenesis.^24–30 Furthermore, there is expanding proof that common MPAK/ERK flagging pathway is a crucial flagging pathway associated with the improvement of a few tumors.^31–36 To additionally investigate the likely atomic system of TTR in A549 cells and H1975 cells, the authors zeroed in on the MAPK/ERK flagging pathway. Past investigations have detailed that a few genes influenced the MAPK/ERK signal pathway.³⁷ Segment actuation of the MAPK/ERK pathway promoted cell proliferation, migration, and differentiation, and induced cell cycle arrest.^38–41 The results suggested that the expression of phosphor-ERK was markedly suppressed in the TTR overexpression group, indicating that the MAPK/ERK signaling pathway was fundamental for tumor development in NSCLC cells. It shows similar results as the previous study, which found that phosphor-ERK expression was significantly reduced in the TTR overexpression group. Therefore, further investigation on the molecular mechanisms of TTR on NSCLC will be essential for diagnosis and therapy.

Taken together, our study exhibited that TTR assumed an imperative part in NSCLC tumorigenesis. TTR stifled cell proliferation, migration, and invasion, and advanced apoptosis at any rate somewhat through hindering MPAK/ERK flagging pathway, which was demonstrated to be an effective diagnostic and predictive biomarker for diagnosis and treatment in NSCLC.

Footnotes

Funding Information

This work was supported by Tianjin Health Industry Key Project (no:16KG138).

Availability of Data and Material

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Disclosure Statement

There are no existing financial conflicts.

References

Torre

, Bray

, Siegel

, et al. Global cancer statistics. CA Cancer J Clin, 2012; 65:87.

Wood

, Pernemalm

, Crosbie

, et al. Molecular histology of lung cancer: From targets to treatments. Cancer Treat Rev, 2015; 41:361.

Zhou

, Huang

, Zhang

, et al. MicroRNAs: A novel potential biomarker for diagnosis and therapy with non-small cell lungcancer. Cell Prolif, 2017; 50.

Siegel

, Miller

, Jemal

: Cancer statistics. CA Cancer J Clin, 2018; 68:7.

Power

, Elias

, Richardson

, et al. Evolution of the thyroid hormonebinding protein, transthyretin. Gen Comp Endocrinol, 2000; 119:241.

Fung

, Yip

, Lomas

, et al. Classification of cancer types by measuringvariants of host response proteins using SELDI serum assays. Int J Cancer, 2005; 115:783.

Jacobsson

, Collins

, Grimelius

, et al. Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic islets. J Histochem Cytochem, 1989; 37:31.

Itoh

, Hanafusa

, Miyagawa

, et al. Transthyretin (prealbumin) in the pancreas and sera of newly diagnosed type I (insulin-dependent) diabetic patients. J Clin Endocrinol Metab, 1992; 74:1372.

Thomas

JGL

, Jeffrey

, Victoria

, et al. Transthyretin (prealbumin) immunoreactivity in renal cell carcinoma and other neoplasms. Int J Surg Pathol, 1996; 4:1.

10.

Menard

, Johann

, Lowenthal

, et al. Discovering clinical biomarkers of ionizing radiation exposure with serum proteomic analysis. Cancer Res, 2006; 66:1844.

11.

Lowenthal

, Mehta

, Frogale

, et al. Analysis of albumin-associated peptides and proteins from ovarian cancer patients. Clin Chem, 2005; 51:1933.

12.

Liotta

, Petricoin

. Serum peptidome for cancer detection: Spinning biologic trash into diagnostic gold. J Clin Invest, 2006; 116:26.

13.

Geho

, Liotta

, Petricoin

, et al. The amplified peptidome: The new treasure chest of candidate biomarkers. Curr Opin Chem Biol, 2006; 10:505.

14.

Raghu

, Sivakumar

. Interactions amongst plasma retinol-binding protein, transthyretin and their ligands. implications in vitamin A homeostasis and transthyretin amyloidosis. Biochim Biophys Acta, 2004; 1703:1.

15.

Imanishi

. Clinical and experimental studies on the profiles of serum proteins in acute hepatic injury. Gastroenterol Jpn, 1981; 16:493.

16.

Marten

, Sladek

, Straus

. Effect of dietary protein restriction on liver transcription factors. Biochem J, 1996; 317:361.

17.

Ritchie

, Palomaki

, Neveux

, et al. Reference distributions for the negative acute-phase serum proteins, albumin, transferrin and transthyretin: A practical, simple and clinically relevant approach in a large cohort. Clin Lab Anal, 1999; 13:273.

18.

Kozak

, Su

, Whitelegge

, et al. Characterization of serum biomarkers for detection of early stage ovarian cancer. Proteomics, 2005; 5:4589.

19.

Zhao

, Zhang

, Liu

, et al. MiR-185 inhibits cell proliferation and invasion of non-small cell lung cancer by targeting KLF7. Oncol Res, 2018; 27:1015.

20.

Masters

, Johnson

, Temin

. Systemic therapy for stage IV non-small-cell lung cancer: American Society of Clinical Oncology clinical practice guideline update. J Oncol Pract, 2017; 33:832.

21.

Raveche

, Salerno

, Scaglione

, et al. AbnormalmicroRNA-16 locus with synteny to human 13q14 linked to CLLin NZB mice. Blood, 2007; 109:5079.

22.

Hatley

, Patrick

, Garcia

, et al. Modulation of K-Rasdependentlung tumorigenesis by MicroRNA-21. Cancer Cell, 2010; 18:282.

23.

Shimura

, Shibata

, Gonda

, et al. Serum transthyretin level is associated with prognosis of patients with gastric cancer. J Surg Res, 2018; 227:145.

24.

Keeratichamroen

, Subhasitanont

, Chokchaichamnankit

, et al. Identification of potential cervical cancer serum biomarkers in Thai patients. Oncol Lett, 2020; 19:3815.

25.

Qiao

, He

, Cai

, et al. MicroRNA-27a-3p modulates the Wnt/β-catenin signaling pathway to promote epithelial-mesenchymal transition in oral squamous carcinoma stem cells by targeting SFRP1. Sci Rep, 2017; 7:44688.

26.

, Liu

, Qu

, et al. Exosomal microRNA-32-5p induces multidrug resistance in hepatocellular carcinoma via the PI3K/Akt pathway. J Exp Clin Cancer Res, 2018; 37:52.

27.

Sun

, Tang

, Li

, et al. MiR-29c reduces the cisplatin resistance of non-small cell lung cancer cells by negatively regulating the PI3K/Akt pathway. Sci Rep, 2018; 8:8007.

28.

Hui

, Huang

, Jiao

, et al. MiR-494-3p promotes PI3K/AKT pathway hyperactivation and human hepatocellular carcinoma progression by targeting PTEN. Sci Rep, 2018; 8:10461.

29.

Liao

, Wu

, Wen

, et al. Taraxerol exerts potent anticancer effects via induction of apoptosis and inhibition of Nf-kB signalling pathway in human middle ear epithelial cholesteatoma cells. Trop J Pharm Res, 2018;17, DOI: 10.4314/tjpr.v17i6.5.

30.

Gao

, Chen

, Gao

, et al. MAP4K4 is a novel MAPK/ERK pathway regulator required for lung adenocarcinoma maintenance. Mol Oncol, 2017; 11:628.

31.

Wagner

, Nebreda

. Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer, 2009; 9:537.

32.

Zhu

, Qi

, Chen

, et al. PI3K/Akt and MAPK/ERK1/2 signaling pathways are involved in IGF-1-induced VEGF-C upregulation in breast cancer. J Cancer Res Clin Oncol, 2011; 137:1587.

33.

Chang

, Chen

, Chiang

, et al. Abstract 1405: Mesothelin enhances ovarian cancer invasion of by inducing MMP-7 through MAPK/ERK and JNK pathways. Cancer Res, 2011; 71(8 Supplement):1405.

34.

Dahlmann

, Okhrimenko

, Marcinkowski

, et al. RAGE mediates S100A4-induced cell motility via MAPK/ERK and hypoxia signaling and is a prognostic biomarker for human colorectal cancer metastasis. Oncotarget, 2014; 5:3220.

35.

Han

, Gu

, Ji

, et al. PBX3 promotes migration and invasion of colorectal cancer cells via activation of MAPK/ERK signaling pathway. World J Gastroenterol, 2014; 20:18260.

36.

Specific alterations of the microRNA transcriptome and global network structure in colorectal cancer after treatment with MAPK/ERK inhibitors. J Mol Med 2012;90:1421.

37.

Paroo

, Ye

, Chen

, et al. Phosphorylation of the human microRNA-generating complex mediates MAPK/Erk signaling. Cell, 2009; 139:0.

38.

Reddy

, Nabha

, Atanaskova

. Role of MAP kinase in tumor progression and invasion. Cancer Metastasis Rev, 2003; 22:395.

39.

Van Der Steen

, Honeywell

, Dekker

, et al. Resistance to crizotinib in a cMET gene amplified tumor cell line is associated with impaired sequestration of crizotinib in lysosomes. J Mol Clin Med, 2018; 1:99.

40.

Liu

, Zhao

, Lv

. Inhibitory effects of miR-101 overexpression on cervical cancer SiHa cells. Eur J Gynaecol Oncol, 2017; 38:236.

41.

, Zhang

, Tian

, et al. miRNA profiling reveals the upregulation of osteogenesis-associated miRNAs in ovariectomy osteoporosis mice. Clin Exp Obst Gynecol, 2018; 45:817.