MiR-145 Regulates the Chemoresistance of Hepatic Carcinoma Cells Against 5-Fluorouracil by Targeting Toll-Like Receptor 4.

BACKGROUND: 5-fluorouracil (5-FU) is a common drug for hepatic carcinoma (HCC), but the drug resistance of clinical chemotherapy restricts its use. Studies have demonstrated that miRNA molecules can act as a chemoresistance regulator in drug resistance of tumors, whereas the role of miR-145 in the 5-FU-resistant HCC remains unclear.
OBJECTIVE: To explore the prognostic value of miR-145 in HCC and its molecular mechanism in 5-FU-resistant HCC cells.
METHODS: A qRT-PCR assay was conducted to quantify miR-145 in HCC tissues and 5-FU-resistant HCC cells. The Cell Counting Kit-8 (CCK-8) and flow cytometry were adopted to analyze the proliferation and apoptosis of 5-FU-resistant HCC cells. The Western blot was adopted to quantify toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and apoptosis-related proteins. Moreover, an in vivo tumor xenotransplantation of nude mice was conducted to determine the effect of miR-145 on 5-FU-resistant HCC cells.
RESULTS: MiR-145 was expressed lowly in HCC tissues and cells, and linked to high TNM staging and lymph node metastasis of HCC patients. Down-regulation of miR-145 indicated a poorer prognosis and it promoted drug resistance of HCC cells and inhibited cell apoptosis. In contrast, miR-145 overexpression improved the sensitivity of HCC cells to 5-FU and enhanced the inhibition of 5-FU on tumor growth. The luciferase reporter gene assay showed that TLR4 was the direct target of miR-145, and the Western blot assay revealed that overexpression of TLR4 reversed the inhibitory effect of miR-145 overexpression on TLR4 and MyD88 protein and the effects of it on apoptosis-related proteins.
CONCLUSION: MiR-145 is an inhibiting factor in HCC and can target TLR4 to mediate the chemoresistance of HCC, which may provide novel ideas for treating HCC.

Introduction

Hepatic carcinoma (HCC) is a common fatal tumor in human beings, with lethality ranking third.1 Its treatment is extremely complicated. Although hepatectomy and liver transplantation are relatively effective for HCC, the recurrence rate of patients after them is still terribly high.2 Clinically, HCC is usually treated with platinum-based chemotherapy or platinum-based chemotherapy combined with radiotherapy. Since the introduction of 5-fluorouracil (5-FU) into the treatment y of HCC, the prognosis of patients with HCC has been significantly improved.3 However, acquired or inherent chemoresistance compromises the efficacy on HCC.4 Therefore, we aimed to investigate the molecular mechanism of therapeutic resistance of HCC cells to search for possible therapeutic targets for HCC.

MiRNAs are able to change gene expression by specifically binding to an mRNA or protein.5 They play a key regulatory role in the development and progression of tumor cells, and also take a crucial part in the tumor biosynthesis, growth, and drug resistance.6 Recent studies have found that miRNA expression can enhance the anti-tumor effect of 5-FU on tumor cells.7 According to reports, miR-145 acts as a tumor suppressor in various tumors.8 For example, miR-145 inhibits colorectal cancer tissues and cells and can act as an inhibitor of the DNA damage-related gene RAD18, which plays a crucial role in reversing chemoresistance against 5-FU.9 In addition, in esophageal squamous cell carcinoma, miR-145 is significantly down-regulated and can directly target the PI3K/AKT pathway to further lower the expression of multidrug resistance-related proteins and enhance the sensitivity to cisplatin.10 However, the effects of miR-145 on the evolution of chemoresistance of HCC against 5-FU remain unclear.

Toll-like receptors (TLRs) are essential receptors in the body’s immunity, and TLR4 is one of the important members of TLR, which can recognize the lipopolysaccharide of Gram-negative bacteria.11 TLR4 is widely expressed in tumor tissues and involved in the chemoresistance of tumors.12,13 In colorectal cancer cells, TLR4 can be induced by 5-FU and can promote apoptosis under the synergistic effect of lipopolysaccharide and 5-FU.14 In HCC, TLR4 can result in the poor prognosis of patients after being up-regulated and can be used as a marker of cancer stem cells. In addition, it can promote malignant phenotypes of HCC cells.15 TLR4 is overexpressed in various tumors including HCC,16 but its relationship with drug resistance of HCC is still under investigation.

This study focused on the correlation between miR-145 and the resistance of HCC cells against 5-FU to investigate the role of miR-145/TLR4 in chemoresistance of HCC.

Materials and Methods

A total of 102 HCC tissue specimens and corresponding normal paracancerous tissue specimens were sampled from patients undergoing surgery in The first Hospital of Jilin University from March 2011 to June 2014. All the patients were confirmed with HCC17 and had not received any related treatment before admission, and their expected survival time was longer than one month. This study was carried out with the permission from the ethics committee of the First Hospital of Jilin University and informed consent forms from all participants and it was in accordance with the Helsinki Declaration.

Cell Source and Culture

HCC cells (SNU449 and Huh7) and normal hepatic cell strains (LO2) purchased from Shanghai Xin Yu Biotech Co., Ltd. were cultured in RPMI 1640 medium (Gibco Company, USA) containing 50 U/mL penicillin, 50μg/mL streptomycin, as well as 10% fetal bovine serum (FBS) under 5％CO2 at 37°C. After 22–25 passages, the SNU449 and Huh7 cells were incubated in RMPI 1640 with 2.0 μg/mL 5-FU (Sigma-Aldrich, USA) that was replaced each day to make the cells resistant to 5-FU, thus establishing SNU449/5-FU and Huh7/5-FU cells.

Cell Transfection

MiR-145 overexpression sequence (miR-145-mimics), miR-145 knockout sequence (miR-145-inhibitor) and corresponding miR negative control (miR-NC) were purchased from MBL Beijing Biotech Co., Ltd. (China). The full-length TLR4 sequence was introduced into pcDNA3.1 vector (Zeye Biological Technology Co., Ltd., Shanghai, China) to obtain overexpressed TLR4 (TLR4-pcDNA3.1), and blank vector was adopted as control (TLR4-NC). After the cell confluency reached 70%-80%, the cell plasmids were transfected with Lipofectamine 2000 reagent (Huijia Biotechnology Co., Ltd., Xiamen, China) under manufacturer’s instructions, and the final concentration of miRNA was 50nM. Finally, a qRT-PCR assay was carried out to determine the transfection efficiency of cells, and the assay was repeated three times.

qRT-PCR

Total RNA was extracted from cells through TRizol reagent (Huijia Biotechnology Co., Ltd., Xiamen, China) under manufacturer’s instructions, and its purity and concentration were detected by a Multiskan Sky ultraviolet spectrophotometer (Thermo Fisher Scientific, Shanghai, China). The integrity of extracted total RNA was determined using the agarose gel electrophoresis. In addition, the extracted RNA was reversely transcribed using a reverse transcription kit. The reaction solution consisted of 4 μL 5 ×PrimeScript Buffer, RNase Inhibitor (40U/μL), 1 μL PrimeScript RTase (200U/μL), and 4.5 μL RNase-free ddH2O. Reverse transcription reaction was carried out under reaction at 42°C for 45 min, incubation at 95°C for 5 min, and cooling on ice, and then the generated cDNA was stored at −20°C. The amplification experiment was carried out using PrimeScript RT Master Mix reagent (Applied Biosystems Company, USA) on an ABI 7500 fluorescence ration PCR instrument (Thermo Fisher Scientific, Shanghai, China) with pre-denaturation at 95°C for 5 min, followed by denaturation at 95°C for 30 min, annealing at 60°C for 30 s, and extension at 72°C for 30 s. WithU6 or GAPDH as internal reference, the obtained data were analyzed using 2-ΔΔCt, and the assay was repeated three times.

Cell Viability Determination

Cell viability was detected using a CCK-8 Kit (Sigma-Aldrich, USA) as follows: The cells were seeded into a 96-well plate at 2×104 cells/well, and cultured at 37°C. After 24 hrs of transfection, the cells were added with 5-FU at different concentrations, and after 48 hrs of culturing, the optical density of cells at 450 nm was determined using a Multiskanmicroplate reader (Thermo Fisher Scientific, Shanghai, China).

Determination of Apoptosis and Cell Cycle

The cells were seeded into a 6-well plate at 2×105 cells/well, and cultured until their confluency reached 90%, followed by later analysis. Subsequently, the cells were cultured with 5 μg/mL 5-FU for 48 hrs, and then their apoptosis was determined via an Annexin V-FITC Kit (Huijia Biotechnology Co., Ltd., Xiamen, China) under the kit instructions. The treated cells were prepared into 1×106 cells/mL cell suspension, followed by incubation with Annexin V-FITC and propidium iodide in the dark. Finally, the cells were determined using a CyFlow Cube6 flow cytometer (Jiyuan Biotechnology Co., Ltd., Guangzhou, China).

Western Blot (WB) Assay

The cells were lysed through RIPA solution (Thermo Scientific, USA), and their concentration was determined using a Bicinchoninic Acid Kit (Bio-Rad, USA), and regulated to 4 μg/μL. Subsequently, the total protein was separated through SDS-PAGE and transferred to a PVDF membrane. The membrane was immersed in 5% skimmed milk for 2 h and added with TLR4, myeloid differentiation factor 88 (MyD88), cysteinyl aspartate specific proteinase 3 (Caspase3), Caspase9, B-cell lymphoma/leukemia-2 (Bcl-2), and Bcl-2-associated X protein (Bax) primary antibody, at a dilute ratio of 1:1000 each, and cultured at 4°C overnight, with GAPDH (1:1000) as internal control. Afterwards, the cells were reacted with secondary antibody (1:5000) at room temperature for 2 hrs. The protein was visualized with electrochemiluminescence (ECL) reagent and the band was quantitatively analyzed with ImageJ. Antibody reagents were all purchased from ShanghaiBeyotime Biotechnology Co., Ltd. (China).

Luciferase Determination

Based on Targetscan7.2 and miRDB database, it was predicted that TLR4might be a target of miR-145. Wild type (WT) TLR4 and mutant (Mut) TLR4 were cloned into pmirGLO dual luciferase expression vector, and sequenced downstream of luciferase reporter gene. Subsequently, TLR4-WT or TLR4-MUT was co-transfected with miR-145-mimics or miR-NC into SNU449 cells using Lipofectamine 2000 reagent. Finally, the luciferase activity of cells was determined with a dual-luciferase reporter (DLR) assay system (Yingbio Technology, Co., Ltd., Shanghai, China) in accordance with the manufacturer’s instructions.

In vitro Xenotransplantation

Lentivirus-mediated stably expressed miR-145 and miR-NC were purchased from Shanghai Gene Pharma Co., Ltd. (China). Cells were placed in a culture medium, and when their density reached 50%, they were incubated in complete medium supplemented with 1 mL lentivirus solution and 15 μg polybrene. After 48 h of cell transfection, stably transfected cell strains were screened using complete culture solution with 2 μg/mL puromycin.BALB/c nude mice (5-week-old) were purchased from Changzhou Cavens Experimental Animal Co., Ltd. (China), and 1×106 HCC cells with stable miR-NC, miR-145 mimics, miR-NC+5-FU, or miR-145 mimics+5-FU were suspended in PBS solution, and 0.2 mL suspension cells were injected subcutaneously into nude mice. Eight days after continuous feeding, the tumor in each mouse was measured every 4 days. After 24 days, the nude mice were euthanized, and their tumor tissues were taken out and the tissue mass was detected.

All the experiments involving animals in the study were carried out with approval from the Animal Ethics Committee of the First Hospital of Jilin University and carried out under the guidance of the Animal Ethics Committee and in line with the institutional ethics guidelines for animal experiments.

Statistical Analyses

Data in experiments were expressed as the mean ± standard deviation (mean ± SD), compared between groups using the t test, and compared among groups using the one-way variance. Post hoc pairwise comparison of data was carried out using the Bonferroni method. The Kaplan-Meier method was adopted to draw survival curves of HCC patients, and the Log rank test was adopted for analysis of the survival curves. Furthermore, Cox model was used to analyze the prognostic factors, and Pearson correlation coefficient was adopted for correlation analysis. Enumeration data were expressed by %, and compared between groups using the chi-square test. P<0.05 indicates a significant difference. Statistical analyses and figure drawing were carried out using SPSS 17.0 and GraphPad 6, respectively.

Results

MiR-145 is Lowly Expressed in Cases with HCC and the Low Expression Indicates Poor Prognosis of Patients

First, we carried out a qRT-PCR assay to quantify miR-145 in HCC tissues, finding that miR-145 was significantly down-regulated (Figure 1). We further analyzed the relationship between miR-145 and pathological parameters of HCC patients, finding that low miR-145 expression was linked to high TNM staging and the existence of lymph node metastasis. Additionally, we analyzed the relationship between miR-145 and patients’ 5-year overall survival (OS) by defining high and low miR-145 expression according to the median miR-145 expression in tissues (1.26) and found that patients with low miR-145 expression showed poorer 5-year OS. Cox regression analysis revealed that lymph node metastasis, TNM staging, and low miR-145 expression were independent prognostic indicators of patients’ 5-year OS (Figure 1, Table 1–2).

Figure 1

MiR-145 is lowly expressed in HCC and low expression of it indicates poor prognosis of patients. (A) Expression of miR-145 in HCC tissues and corresponding paracancerous tissues according to the qRT-PCR assay. (B) Expression of miR-145 in HCC patients at different TNM stages. (C) Expression of miR-145 in HCC patients with different lymph node metastasis. (D) Relationship between miR-145 expression in tissues and 5-year OS of the HCC patients.

Note: *** indicates P<0.001.

Abbreviations: miR, microRNA; TNM, tumor node metastasis.

Table 1

Relationship Between MiR-145 Expression and Clinicopathological Features of HCC Patients

Clinicopathological Parameters	n	MiR-145		χ2	P-value
		Low Expression	High Expression
Age (Y)				0.362	0.548
<60	59	28 (54.90)	31 (60.78)
≥60	43	23 (45.10)	20 (39.22)
Sex				1.259	0.262
Female	27	11 (21.57)	16 (31.37)
Male	75	40 (78.43)	35 (68.63)
TNM staging				5.755	0.016
I–II	58	23 (45.10)	35 (68.63)
III–IV	44	28 (54.90)	16 (31.37)
Histological grading				0.648	0.421
G1/G2	60	28 (54.90)	32 (62.75)
G3	42	23 (45.10)	19 (37.25)
Lymph node metastasis				4.865	0.027
No	59	24 (47.06)	35 (68.63)
Yes	43	27 (52.94)	16 (31.37)
Tumor size				0.177	0.674
<5cm	68	33 (64.71)	35 (68.63)
≥5cm	34	18 (35.29)	16 (31.37)

Abbreviations: miR, microRNA; HCC, hepatic carcinoma; TNM, tumor node metastasis.

Table 2

Univariate and Multivariate Regression Analyses of Relevant Prognostic Parameters of HCC Patients

Factors	Univariate		Multivariate
	HR (95%CI)	P-value	HR (95%CI)	P-value
Age (Y)	1.060 (0.683–1.645)	0.795
Sex	1.236 (0.746–2.048)	0.410
TNM staging	1.647 (1.063–2.553)	0.026	1.612 (1.040–2.499)	0.033
Histological grading	1.103 (0.709–1.714)	0.664
Lymph node metastasis	1.939 (1.244–3.022)	0.003	1.774 (1.131–2.783)	0.013
Tumor size	1.390 (0.886–2.182)	0.152
miR-145	1.755 (1.127–2.734)	0.013	1.574 (1.004–2.469)	0.048

Abbreviations: HCC, hepatic carcinoma; HR, hazard ratio; CI, confidence interval; TNM, tumor node metastasis; miR, microRNA.

MiR-145 is Lowly Expressed in 5-FU-Resistant HCC Cells

We acquired that miR-145 was lowly expressed in HCC cells (SNU449 and Huh7), and then we constructed SNU449/5-FU cell strains and Huh7/5-FU cell strains. It was found that after 5-FU treatment, SNU449/5-FU cell strains and Huh7/5-FU cell strains showed significantly stronger proliferation and significantly weaker apoptosis than SNU449 and Huh7 cells. Quantification of miR-145 in drug-resistant cell strains and parental cell strains revealed that SNU449/5-FU cell strains and Huh7/5-FU cell strains showed significantly lower miR-145 expression than SNU449 and Huh7 cells. These results imply that miR-145 plays a role in the progression of HCC and drug resistance of it (Figure 2).

Figure 2

MiR-145 is lowly expressed in HCC and 5-FU-resistant cells. (A) Expression of miR-145 in LO2, SNU449, and Huh7 cells according to the qRT-PCR assay. (B) Proliferation of designated cell strains treated through 5-FU according to the CCK-8 assay. (C) Apoptosis of designated cell strains treated through 5-FU according to the flow cytometry and apoptosis pattern. (D) Expression of miR-145 in SNU449/5-FU cell strains and Huh7/5-FU cell strains according to the qRT-PCR assay.

Notes: *Indicates P<0.05; **indicates P<0.01, and ***indicates P<0.001.

Abbreviations: miR, microRNA; 5-FU, 5-fluorouracil.

MiR-145 Plays a Role in Drug Resistance of HCC Cells

In order to verify the effect of miR-145 on resistance of HCC cells against 5-FU, we transfected miR-145 mimics, miR-145 inhibitor, and corresponding miR-NC into drug-resistant cell strains and parental cell strains. It was turned out that compared with the transfection of miR-NC, transfection of miR-145 mimics significantly up-regulated miR-145 in drug-resistant cell strains and parental cell strains, while transfection of miR-145 inhibitor exerted opposite effects. The CCK-8 assay revealed that compared with transfection of miR-NC, knock-down of miR-145 promoted the growth of SNU449 cells and SNU449/5-FU cells and inhibited their apoptosis, while up-regulation of miR-145 inhibited the growth of Huh7 cells and Huh7/5-FU cells and induced their apoptosis, which implied that miR-145 plays a crucial role in regulating the sensitivity and drug resistance to 5-FU. In order to evaluate the effect of miR-145 on drug resistance against 5-FU in vivo, we carried out an in vitro nude mouse formation assay by injecting SNU449/5-FU cells with stably expressed miR-145 into nude mice. As expected, miR-145 overexpression significantly inhibited tumor growth, and it also enhanced the sensitivity to 5-FU. These studies imply that miR-145 can make HCC cells sensitive to 5-FU in vivo (Figure 3).

Figure 3

MiR-145 plays a role in drug resistance of HCC cells. (A) HCC cells transfected with miR-145-mimics, miR-145-inhibitor, or corresponding miR-NC. (B) Proliferation ability of SNU449 cells and SNU449/5-FU cells according to the CCK-8 assay. (C) Proliferation ability of Huh7 cells and Huh7/5-FU cells according to the CCK-8 assay. (D) Apoptosis rate of SNU449 cells and SNU449/5-FU cells according to the flow cytometry. (E) Apoptosis rate of Huh7 cells and Huh7/5-FU cells according to the flow cytometry. (F) Xenotransplantation tumor growth curves and representative figures. (G) Weight of tumor mass resected.

Notes: *Indicates P<0.05; **indicates P<0.01, and ***indicates P<0.001.

Abbreviations: miR, microRNA; 5-FU, 5-fluorouracil.

TLR4 is a Direct Target of MiR-145 in HCC

For the purpose of studying the role of miR-145 in regulating the sensitivity of drug-resistant HCC cells to 5-FU, we carried out an analysis according to the public database (TargetScan), finding that TLR4 was a target gene of miR-145. Then, we carried out a DLR assay, finding that miR-145 mimics inhibited the luciferase activity of WT-TLR4 3ʹ-UTR by binding to its 3ʹ-UTR, but exerted no effect on that of MUT-TLR4. We further quantified TLR4 in cells. The WB assay revealed that knock-down of miR-145 up-regulated TLR4 in SNU449 cells and SNU449/5-FU cells, while overexpression of miR-145 down-regulated TLR4 in Huh7 and Huh7/5-FU cells. Consistently, HCC cells showed higher TLR4 expression than normal tissues. Furthermore, miR-145 was negatively correlated with TLR4 expression in HCC cells (Figure 4).

Figure 4

TLR4 is a direct target of miR-145 in HCC. (A) There were targeted binding sites between miR-145 and TLR4. (B) Interaction between miR-145 and TLR4 according to the DLR assay. (C) Expression of TLR4 protein in cells transfected with miR-145-mimics, miR-145-inhibitor or corresponding miR-NC according to the WB assay, and protein bands. (D) Expression of TLR4 protein in HCC tissues and corresponding paracancerous tissues according to the qRT-PCR assay. (E) Expression of TLR4 protein in HCC tissues and corresponding paracancerous tissues according to the WB assay, and protein bands. (F) Negative correlation between miR-145 and TLR4 in HCC tissues.

Notes: **Indicates P<0.01, and ***indicates P<0.001.

Abbreviations: TLR4, toll-like receptor 4; miR, microRNA; WT, wild type; MUT, mutant type; 5-FU, 5-fluorouracil.

TLR4 is a Functional Target of MiR-145 in Drug-Resistant HCC Cells

In order to verify the effects of TLR4 on miR-145-mediated chemical resistance, we transfected miR-NC, miR-145 mimics, miR-NC+TLR4-NC, and miR-145 mimics+TLR4-pcDNA3.1 into SNU449/5-FU cells and Huh7/5-FU cells, finding that overexpression of TLR4 enhanced the cell proliferation ability caused by miR-145 overexpression and lowered the apoptosis rate caused by it. In addition, WB assay revealed that up-regulation of TLR4 reversed the inhibitory effect of miR-145 overexpression on TLR4 and MyD88 proteins, and quantification of apoptosis-related proteins revealed that miR-145 overexpression increased the expression of Caspase3, Caspase9, and Bax and lowered Bcl-2 expression, while up-regulation of TLR4 reversed the effect of miR-145 overexpression on the apoptosis-related proteins. Additionally, overexpression of TLR4 prevented the inhibition of the up-regulation of miR-145 on drug-resistant HCC cells, which indicated that the action of miR-145 on drug resistance of HCC cells may depend on TLR4 (Figure 5).

Figure 5

TLR4 is a functional target of miR-145 in drug-resistant HCC cells. (A) Proliferation ability of SNU449/5-FU cells and Huh7/5-FU cells according to the CCK-8 assay. (B) Apoptosis rate of SNU449/5-FU cells and Huh7/5-FU cells according to the flow cytometry and apoptosis map. (C) Expression of TLR4 and MyD88 proteins according to the WB assay and protein bands. (D) Expression of Caspase3, Caspase9, Bcl-2, and Bax proteins according to the WB assay and protein bands.

Notes: *Indicates P<0.05; and ***indicates P<0.001.

Abbreviations: miR, microRNA; TLR4, toll-like receptor 4; 5-FU, 5-fluorouracil; MyD88, myeloid differentiation factor 88; Caspase3, cysteinyl aspartate specific proteinase 3; Caspase9, cysteinyl aspartate specific proteinase 9; Bcl-2, B-cell lymphoma/leukemia-2; Bax, Bcl-2-associated X protein.

Discussion

Growing studies show that miRNA plays a crucial role in malignant tumors and may be important for the development, prognosis, and treatment of cancer.18,19 In this study, the loss of miR-145 expression was linked to the progression of HCC, and low miR-145 expression indicated an unfavorable prognosis of patients. In addition, miR-145 expression in drug-resistant HCC cells decreased, and importantly, overexpression of miR-145 could suppress the proliferation of drug-resistant HCC cells and promoted their apoptosis. Our in vivo experiments verified that up-regulation of miR-145 could strongly hinder the growth of tumors and enhance the sensitivity to 5-FU, and rescue experiments revealed that up-regulation of TLR4 could prevent overexpression of miR-145 from sensitizing drug-resistant HCC cells, which suggested that the action of miR-145 on drug resistance of HCC cells may depend on TLR4. Our study confirmed for the first time that the function of miR-145 in chemoresistance of HCC against 5-FU.

One previous study has demonstrated that the functional status of miRNA is strongly linked to the response of cancer patients to chemotherapy.20 Although the mechanism of miRNA resistance is still under investigation,21 there are evidence indicating that miRNA plays a pivotal role in treatment-induced tumor cell death, changes in drug targets, and regulation of multidrug resistance-related proteins.22,23 According to earlier studies, miR-145 mostly acts as a tumor suppressor in malignant tumors.24,25 Our study results demonstrated that miR-145 was lowly expressed in HCC, and was linked to high clinical staging and lymph node metastasis. In addition, follow-up for prognosis revealed that low miR-145 expression was linked to the unfavorable prognosis of the patients. Consistently, miR-145 has been reported to be lowly expressed in HCC tissues and cells.26 For instance, Liu et al have pointed out that up-regulation of miR-145 can inhibit the malignant phenotypes of cells in vitro experiments, and can target ROCK1 to further inhibit the metastasis and progression of HCC.27 It is worth noting that in one study by Noh et al, miR-145 expression in HCC cells is significantly down-regulated, and up-regulation of miR-145 can degrade the growth of HCC cells and directly target histone deacetylase 2 to further suppress tumor.28 In our study, we found that knockdown of miR-145 could enhance the proliferation of drug-resistant HCC cells and inhibit their apoptosis, and overexpression of miR-145 could restore the apoptosis of 5-FU-resistant cells and inhibit their proliferation. In addition, according to the in vivo experiments, the up-regulation of miR-145 could make HCC cells sensitive to 5-FU. These results imply that miR-145 plays a key role in regulating the sensitivity and resistance of cells to 5-FU and up-regulation of miR-145 can enhance the anti-tumor effect of 5-FU on HCC cells. However, the mechanism of miR-145 in 5-FU-resistant HCC cells is still under investigation.

The latest studies have concluded that the functions of miRNA and their targets are crucial to the development or chemical resistance of tumors.29,30 For example, in gallbladder carcinoma, up-regulation of miR-145 can improve the therapeutic effect of cisplatin, whereas down-regulation of it can lower the sensitivity to the drug, and can directly target multidrug resistance-related protein 1 in gallbladder cancer cells to cause cisplatin toxicity increase.31 Furthermore, one other study has revealed that miR-145 overexpression in drug-resistant ovarian cancer cells can reduce the expression of pRb and P-gp, making cells more sensitive to paclitaxel in vivo or in vitro experiments.32 Therefore, we carried out bioinformatics analysis and found potential binding sites between miR-145 and TLR4, and we also carried out a DLR assay, and confirmed that TLR4 was the target of miR-145.TLR4 is usually overexpressed in human tumors and is strongly linked to metastasis and chemical resistance.33 According to previous reports, in breast cancer, TLR4 promotes the tumor development, and activation of TLR4 pathway can improve the survival rate of tumor cells, while blocking TLR4 can improve the therapeutic effect of paclitaxel on breast cancer cells.34 However, there is no relevant research on the relationship between TLR4 and 5-FU resistance in HCC cells. Through detection, we found that TLR4 was overexpressed in HCC tissues. Consistently, TLR4 was expressed at high levels in patients with HCC, and the increase of its level indicates the early recurrence and poor survival rate of patients.35 Our study results further demonstrated that up-regulation of miR-145 could intensify the therapeutic sensitivity of 5-Fu-resistant HCC, and transfection of overexpressed miR-145 and overexpressed TLR4 into 5-Fu-resistant HCC cells could offset the sensitizing effect of miR-145 overexpression on 5-Fu-resistant cells, which implied that miR-145 acted as a tumor suppressor gene in HCC, and up-regulation of it can increase the sensitivity of 5-Fu-resistant HCC cells. We further investigated the effects of miR-145 on the drug resistance of HCC. The WB assay revealed that the up-regulation of TLR4 reversed the inhibitory action of miR-145 overexpression on TLR4 and MyD88 proteins, and could reverse the levels of apoptosis-related proteins. Silencing the TLR4 signaling pathway in cancer cells can reduce the risk of tumor formation.36 Previous studies have shown that TLR4-mediated signaling pathway takes a crucial part in malignant phenotypes of HCC cells, and knocking out TLR4 can suppress cell proliferation and promote cell apoptosis, and inhibit TLR4 pathway,37 which indicates that miR-145 may regulate the sensitivity of drug-resistant human HCC cells by mediating the TLR4/MyD88 pathway. However, we have not detected other factors related to the TLR4 signaling pathway, and the specific mechanism of action in the signaling pathway mediated downstream of TLR4 mediated by miR-145 is unclear. In addition, the effects of miR-145 on drug-resistant HCC cells still need further exploration.

Conclusion

MiR-145 may be adopted as a prognostic marker for HCC, and up-regulation of it can enhance the anti-tumor effect of 5-Fu on HCC cells, which may be achieved by mediating the chemical resistance of HCC by targeting TLR4.