MicroRNA-200b suppresses the invasion and migration of hepatocellular carcinoma by downregulating RhoA and circRNA

Abstract

MicroRNAs could mediate the targeted coding gene and the targeted non-coding RNA to form endogenous competition, which have an important regulatory role in tumorigenesis of many types of cancer, including hepatocellular carcinoma. The goal of this study was to characterize the role of miR-200b in the pathogenesis of hepatocellular carcinoma. We identified miR-200b that was predicted to regulate RhoA and circ_000839. Our data establish that miR-200b is expressed at a relatively low level in hepatocellular carcinoma (p < 0.001). RhoA and circ_000839 are expressed at a relatively high level in hepatocellular carcinoma (p < 0.001, respectively). Our mechanistic data indicate that RhoA is a direct target of miR-200b (p < 0.001), binding of which affects the expression of invasion and migration in hepatocellular carcinoma cell lines (p < 0.05). And correlation analysis showed that miR-200b was inversely correlated with RhoA and circ_000839 (p = 0.012, p = 0.002, respectively), while RhoA was positively correlated with circ_000839 (p < 0.001). Taken together, our data suggest that miR-200b could mediate RhoA gene and circ_000839 to form endogenous competition. And this is a direction for the association study of miR-200b and RhoA in the future.

Keywords

Non-coding RNA micro RNA 200b RhoA Circ_000839 hepatocellular cancer

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, with an annual incidence rate approaching approximately 0.626 million over the world. HCC ranks the sixth among malignant tumors and the third among the causes for cancer death.¹ Invasion and metastasis are important factors affecting survival and quality of life of patients with hepatocellular carcinoma.² The high rate of metastasis and recurrence is a major obstacle to improving the survival rate of patients. There are many factors affecting the invasion and metastasis of HCC, in which RhoA (Ras homologue A) is worth concerning.

RhoA is an important member of the Rho gene family, and it is homolog of Ras. The RhoA expression was increased in a variety of tumor tissues and was associated with tumor malignancy, suggesting an important role in tumorigenesis and metastasis.^3,4 Currently, the relationship between RhoA and invasion and metastasis of malignant tumors has become one of the hot researches. The regulation of RhoA on tumor invasion and metastasis is through the activation of cytoskeletal proteins to promote interaction of myosin with actin filaments leading into contractility as to enhance the ability of movement and migration of tumor cells.⁵ RhoA can improve vascular endothelial growth factor expression in tumor cells to promote tumor invasion and metastasis by promoting tumor angiogenesis.⁶ A variety of factors were involved in the regulation of RhoA expression, in which microRNA (miRNA) was worth a further study.⁷

MiRNA is a class of small RNA molecules composed of 19-23bp nucleotide acids.⁸ So far, more than 2000 kinds of miRNA have been identified in various species, including vertebrates, fruit flies, and nematodes.⁹ MiRNA can promote the degradation of target messenger RNA (mRNA) or inhibits its translation by recognizing specific binding sites on the 3′-untranslated region (UTR) of the target mRNA in either a completely or partially complementary fashion.¹⁰ Through its direct targeting, miRNA serves as an important post-transcriptional regulator and plays an important role in a variety of physiological and pathological processes, such as proliferation, differentiation, apoptosis, and tumorigenesis of cells.¹¹ The expression of miRNAs in cancers has tissue specificity, and miRNAs’ expression spectrum in different tumors is various. Researches show that miR-200b inhibits the progress of HeLa cell cycle by targeting recombinant human Rho family.¹² Downregulation of miR-200b expression in bronchial epithelial cells can promote epithelial–mesenchymal transition (EMT), leading to tumorigenesis.¹³ The role of miR-200b is bidirectional. However, whether miR-200b in hepatocellular cancer can influence the invasion of HCC through regulation of RhoA requires a further study.

Circular RNA (circRNA) is a class of endogenous RNA molecular which is formed by non-colinear reverse splicing and plays an important role in the regulation of gene expression at post-transcriptional level.^14,15 The major function of circRNA is to regulate gene expression and to act as competing endogenous RNA (ceRNA) which is also known as miRNA sponge.¹⁶ CircRNA could also sequester miRNA to terminate regulation of the miRNA target genes.^17,18 Thus, we speculate that miR-200b could regulate some circRNAs except its target coding genes which causes the endogenous competing for the circRNAs and coding genes. But which circRNAs could co-express with miR-200b in the hepatocellular cancer tissues? We used bioinformatics tools to predict these circRNAs and verified the expression of these circRNAs in hepatocellular cancer tissues.

In this study, we analyzed the relationship of miR-200b and RhoA by detecting the expression of miR-200b and RhoA in HCC. We further observed effects of miR-200b on the invasion and metastasis of hepatocellular cancer cell by transfection. By luciferase reporter gene detection technology, we validated whether miR-200b affected the invasion and metastasis of hepatocellular cancer cells by directly binding to RhoA. And then, we used bioinformatical tools to predict the target circRNAs for miR-200b. circRNA in HCC tissues in the same group is also detected, as well as the correlation analysis for the relationship between miR-200b, RhoA, and circRNA. This research is helpful for the understanding of the HCC etiology and also supplied the new molecular target for the treatment of HCC.

Materials and methods

Cell lines and cell culture

The Huh7 and HepG2 HCC cell lines used in this study were obtained from the American Type Culture Collection. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified 5% CO₂ atmosphere at 37°C. HCC cells were stimulated with 1 µg/mL of eHSP70, rinsed with phosphate-buffered saline (PBS), and collected at the designated experimental time point. For the assessment of apoptosis, luciferase activity, and western blot assays, cells were harvested 48 h after transfection with miRNA mimics.

Patient information and tissue specimens

This research project was approved by the Ethical Committee of the First Affiliated Hospital of the China Medical University and written informed consent was obtained. The hepatocellular cancer tissues were obtained during surgical resection of the liver. The participants who underwent surgical operation were diagnosed by pathological confirmation with HCC according to World Health Organization (WHO) classification. The inclusive criteria are pathological diagnosed as hepatocellular cancer, and cholangiocellular carcinoma and sarcoma are excluded. The medical histories (including age, sex, smoking, and alcohol consumption) were obtained by questionnaire and the records were computerized. Individuals who smoked at least once a day for more than 1 year were defined as ever smokers, included current smokers and former smokers who had quit smoking for more than 1 year, and the others were defined as never smokers. Individuals who consumed one or more alcoholic drinks per week for at least 1 year were considered drinkers, and the rest were defined as nondrinkers. The immediate family members who had death because of cancer defined as having family history.

Real-time reverse transcription polymerase chain reaction

Total RNA was extracted using TRIzol (Life Technology, California, USA). An amount of 1.5 µg of isolated total RNA was converted into complementary DNA (cDNA) using QuantScript RT Kit (TIANGEN Biotech Co., Ltd, Beijing, China), and simultaneously, 1.5 µg of another total RNA was converted into miRNA cDNA using One Step Prime Script miRNA cDNA (Perfect Real Time; TIANGEN Biotech Co., Ltd). The RhoA expression level was detected and an internal-control gene GAPDH was examined using SYBR Premix Ex Taq II (TaKaRa Biotech, Dalian, China) in an Eppendorf Mastercycler Gradient System (Eppendorf AG, Hamburg, Germany). The miRNA levels were examined using miRcute miRNA qPCR detection kit (SYBR; TIANGEN Biotech Co., Ltd) in an Eppendorf Mastercycler Gradient System (Eppendorf AG) according to the manufacture’s protocol. The relative quantification of gene mRNA expression was calculated using the 2^−ΔΔCt method.¹⁹ To calculate the expression levels of the target miRNA (miR-200b), the synthetic miRNA oligonucleotides (commercial miR-16; TaKaRa Biotech) at known concentrations were also reverse-transcribed and amplified. The concentration of each miRNA was then calculated according to the standard curve. For each assay, the resulting CT values were plotted versus the log10 of the amount of miR-16.^20–22 Each reaction was performed in duplicate repeats, and controls with no template were included in each experiment. The primers used for the detection of RhoA, GAPDH, and miR-200b were summarized in Supplementary Table S1.

Vectors, retroviral infection, and transfection

The cells were plated at 5 × 10⁵ cells/well in six-well plates approximately 24 h before transfection. After the cells reached 30%–50% confluence, transient transfection of miRNA mimics (RiboBio, Guangzhou, China) and/or plasmids were carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Transfection efficiency was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) 24 h after transfection to ensure that cells were successfully transfected.

Wound-healing assay

A lesion was created using a plastic pipette tip 24 h after extracellular HSP70/HSP70–PCs treatment, and cells were washed twice with PBS to remove debris. The monolayer was then maintained in serum-free DMEM and cultured for 24 h. Then, five randomly selected fields at the border of the lesion were viewed under an inverted microscope (IX71; Olympus, Tokyo, Japan).

Transwell invasion assays

Cell invasion and migration assays were performed using a Transwell system (Corning, Grand Island, NY, USA) according to the manufacturer’s protocol. To assess invasion ability, membranes were precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Approximately 5 × 10⁴ cells in serum-free media were added to the top chamber; the bottom chamber was filled with DMEM containing 10% FBS. After 24-h incubation, cells on the upper surface of the membrane were gently removed with a cotton swab, the membrane fixed in 4% methanol for 30 min, and stained with 0.1% crystal violet for 30 min. Cells that migrated to the bottom surface of the membrane were captured and the number of cells counted. The same experimental design was used for the migration assay, but the membranes were not precoated with Matrigel.

Immunofluorescence staining

Cells grown on coverslips were fixed, blocked, and permeabilized. Cells were then stained with fluorescein isothiocyanate (FITC)-conjugated phalloidin (5 μg/mL) for 60 min. Actin filaments (F-actin) were visualized and photographed with an Olympus IX71 digital inverted microscope. More than 200 cells from three independent experiments were analyzed for each condition. The average number of dot- or fan-like protrusions around a cell was counted as an index for lamellipodia formation.

Western blot analysis

Cells were lysed at the designated time points and equal amounts of protein lysate underwent 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the cells were transferred to polyvinyledene fluoride membranes (Millipore, Billerica, MA, USA) and probed with the primary and secondary antibodies. Bound antibodies were detected with Pierce Enhanced Chemiluminescence (ECL) Western Blotting Substrate (Thermo Fisher Scientific, Fair Lawn, NJ, USA). Band densities were analyzed using ImageJ software (NIH, Bethesda, MA, USA).

Luciferase assay

Luciferase reporter assays were carried out using the pmiR-REPORTTM vectors (RiboBio) containing wild-type (WT) RhoA 3′-UTR sequence or mutant (MUT) RhoA 3′-UTR sequences. Cells were transiently cotransfected with miR-2

00b mimics/miR-negative control (NC) mimics and WT-RhoA 3′-UTR vector/MUT-RhoA 3′-UTR vectors. Luciferase activity was measured using a dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions 48 h after transfection.

Transient transfection of RhoA plasmid

Transfection of RhoA plasmid was performed using Lipofectamine™ 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. The RhoA plasmid was purchased from Genchem, Shanghai, China.

The prediction of target circRNAs

We adopted starBase v2.0 (http://starbase.sysu.edu.cn/) software to predict the target circRNAs for miR-200b, and we selected the maximization of these three parameters including target sites, biocomplex, and clip read numbers as the inclusive criteria. As a result, circ_000839 and circ_001289 were selected as the target circRNAs.

The expression of circRNAs

The primers used for the detection of circ_000839 and circ_001289 were also summarized in Supplementary Table S1. The circRNAs’ expression level was detected and the internal-control gene GAPDH was examined using the same method described above.

Statistics

A paired-sample t-test was used to compare RhoA mRNA expression, miRNA, and circRNA expression levels in hepatocellular cancer with that of their paired adjacent normal liver tissue samples. The relationship of RhoA, miR-200b, and circRNA was analyzed by the bivariate correlation. The correlation of RhoA, miR-200b, circRNA, and HCC clinical features was assessed using χ² test. Other statistical analysis in cellular experiments was measured using two-independent sample t-test. All analyses were performed using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was defined as p < 0.05.

Results

The expression of miR-200b and RhoA in HCC tissues

A total of 75 pairs of specimens from hepatocellular cancer and adjacent normal liver tissues were analyzed by the expression of miR-200b and RhoA by real-time RT-PCR. The expressions of miR-200b mRNA in HCC tissue were significantly lower than in normal liver tissue (p < 0.001; Figure 1(a)). But, the expressions of RhoA mRNA in HCC tissue were significantly higher than in normal liver tissue (p < 0.001, Figure 1(b)). Correlation studies showed that miR-200b levels were inversely correlated with the expression of RhoA in all the samples (r = −0.204, p = 0.012, Table 1). And the correlation in the subgroup of the hepatocellular cancer and adjacent normal liver tissues was also shown in Table 1.

Figure 1.

Expression of miR-200b and RhoA in HCC. (a) The expression levels of miR-200b were further downregulated in HCC samples of patients. (b) The expression levels of RhoA were further upregulated in HCC samples of patients. A paired sample t-test was used to compare miR-200b and RhoA expression levels in hepatocellular cancer with that of their paired adjacent normal liver tissue samples. To calculate the expression levels of the target miRNA (miR-200b), the synthetic miRNA oligonucleotides (commercial miR-16) at known concentrations were also reverse-transcribed and amplified. The concentration of each miRNA was then calculated according to the standard curve. For each assay, the resulting CT values were plotted versus the log10 of the amount of miR-16. The relative quantification of RhoA mRNA expression was calculated using the 2^−ΔΔCt method which is compared with GAPDH (**p < 0.001).

Table 1.

The correlation of RhoA–miR-200b–circ_000839.

	RhoA		MiR-200b		Circ_000839
	p	r	p	r	p	r
Total
RhoA			0.012	−0.204	<0.001	0.460
MiR-200b	0.012	−0.204			0.106	−0.133
Circ_000839	<0.001	0.460	0.106	−0.133
HCC
RhoA			0.135	−0.174	<0.001	0.429
MiR-200b	0.135	−0.174			0.174	−0.159
Circ_000839	<0.001	0.429	0.174	−0.159
Normal
RhoA			0.520	−0.075	0.001	0.370
MiR-200b	0.520	−0.075			0.974	−0.004
Circ_000839	0.001	0.370	0.974	−0.004

HCC: hepatocellular carcinoma.

Mir-200b suppressed HCC cell migration and invasion

To examine the role of miR-200b in HCC cell migration and invasion, cells were transfected with miR-200b. HCC cell motility was then investigated using a wound-healing assay. At 24 h after injury, the wound in the miR-200b transfected group had healed less compared to that in the NC group (Figure 2(a)). Migration and invasion are widely considered two closely interrelated processes. As miR-200b suppressed HCC cell migration, we next tested the effect of miR-200b on cell invasion using Matrigel-precoated Transwell chambers. The number of invasive cells decreased markedly following miR-200b transfection (Figure 2(b)).

Figure 2.

The effect of miR-200b on HCC cell migration and invasion. (a) Overexpression was achieved by transfection with RhoA cDNA. (b) Effect of miR-200b on HepG2 cell migration as examined by a wound-healing assay. Representative digital pictures were taken at 0 and 24 h. (c1) The figures for the Transwell invasion assay. (c2) The count for the cells in different groups.

MiR-200b inhibited RhoA expression

Previous studies reported that knockdown or inhibition of the small guanosine triphosphatase (GTPase) RhoA resulted in the process of elongation and suppressed HCC cell migration.²³ We measured RhoA expression using western blotting and real-time RT-PCR after miR-200b tarnsfection. The results indicate that both RhoA mRNA and protein were significantly decreased following miR-200b tarnsfection (Figure 3(a) and (b)).

Figure 3.

MiR200b inhibited RhoA expression and lamellipodia formation. (a) and (b) The effect of miR-200b on the expression of RhoA as evaluated by real-time RT-PCR and western blot, respectively. The results are presented as the mean ± SD from three independent experiments. (c) Lamellipodia formation was visualized by F-actin staining after miR-200b transfection. Cells were photographed with an Olympus IX71 digital inverted microscope. (C1) Control group, (C2) NC group, and (C3) miR-200b group (*p < 0.05).

RhoA expression and activity are important for cytoskeletal regulation in HCC cells, including F-actin stress fiber and lamellipodia formation.²⁴ We therefore examined whether miR-200b affects F-actin reorganization using FITC-conjugated phalloidin staining. Dot- or fan-like protrusions, that is, lamellipodia, representing a form of concentrated F-actin, were detected at the cell periphery in control cells. Following miR-200b transfection, the numbers of lamellipodia at the cell margins were markedly decreased (Figure 3(c)) and were consistent with our observation that cellular RhoA expression was decreased after extracellular HSP70/HSP70–PCs treatment.

RhoA was involved in miR-200b-induced HCC cell migration

To examine the possibility that RhoA is involved in the effect of miR-200b on migration, we pretreated cells with PMIR-RhoA and then stimulated the cells with miR-200b. Overexpression of RhoA attenuated HCC cell migration (Figure 1). These data indicate that RhoA is involved in miR-200b-induced HCC cell migration.

RhoA is a direct target of miRNA-200b

To determine whether RhoA is the direct target gene for miR-200b, a dual-luciferase reporter system was employed. The luciferase reporter assay indicated that the luciferase activity of the reporter containing the RhoA gene’s WT 3′-UTR decreased (52%) following treatment with miR-200b mimics (p < 0.001 when compared with the other groups). By contrast, the inhibitory effect of the miR-200b mimics was abolished in the mutated construct (Figure 4(a)). The result indicates that miR-200b most likely suppresses gene expression through miR-200b-binding sequences at the 3′-UTR of RhoA. Taken together, these data suggest that miR-200b reduces RhoA expression by inhibiting translation and/or causing mRNA instability.

Figure 4.

RhoA is a direct target of miRNA-200b. (a) MiR-200b target sequences of RhoA 3′-UTR. (b) MicroRNA luciferase reporter assay. The NC control group: added negative miRNA mimics; the miR-200b group: added miR-200b mimics; the wild-type group: added the wild type of RhoA 3′-UTR luciferase reporter plasmid; and the mutant group: mutate the combination site of the RhoA 3′-UTR luciferase reporter plasmid for the nucleotide sequence miR-200b–RhoA 3′-UTR. Wild-type and mutant miR-200b target sequences were fused with luciferase reporter and cotransfected with miR-200b or control oligo into HEK293T cells. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. MiR-200b significantly suppressed the luciferase activity of wild-type RhoA 3′-UTR (**p < 0.001).

The expression of circRNAs which are predicted as the target of miR-200b

We found that the circ_001289 was not expressed in liver cancer and normal tissues. The expressions of circ_000839 in HCC tissue were significantly higher than in normal liver tissue (p < 0.001, Figure 5). Correlation studies showed that miR-200b levels were inversely correlated with the expression of circ_000839 (r = −0.252, p = 0.002), while the expression of circ_000839 was shown positive correlation with that of RhoA (r = 0.460, p < 0.001, Table 1).

Figure 5.

Expression of circ_000839 in HCC. The expression levels of circ_000839 were upregulated in HCC samples of patients. A paired-sample t-test was used to compare circ_000839 expression levels in hepatocellular cancer with that of their paired adjacent normal liver tissue samples. The relative quantification of gene mRNA expression was calculated using the 2^−ΔΔCt method which is compared with GAPDH (**p < 0.01).

The relationship of the RhoA gene, miR-200b, and circ_000839 expression and HCC clinical features

To clarify the correlation of RhoA, miR-200b, and circ_000839 expression and clinical feature, we divided the patients into two groups according to the median value of the expression. The expression level of RhoA was related to the family history (p = 0.044), and the expression of miR-200b was related to the differentiation and drinking (p = 0.027 and p = 0.035, respectively; Supplemental Table S2).

Discussion

Invasion and metastasis are the main biological characteristics of malignant tumors.²⁵ They are also major factors affecting the prognosis of patients. But the ability of tumor invasion depends on the ability of tumor cells to migrate. Studies have shown that abnormal expression of RhoA plays an important role in the development of tumors.³ The study found that through a variety of signal transduction pathways, RhoA was involved in tumor malignant transformation, invasion, metastasis, angiogenesis, and so on.²⁶ So, RhoA can act as a potential therapeutic target for treatment of cancer metastasis.

MiRNAs are a class of small, non-coding RNAs of ~22 nt, which exist in various eukaryotes.¹¹ MiRNAs, through incomplete complementary pairing to the mRNA, cause translational repression of targeting mRNAs and then inhibit the expression of targeting mRNAs.¹⁰ Target genes of miRNAs in tumor cells are closely related to the apoptosis, proliferation, and invasion of tumors. The study showed that downregulation of miR-200b expression in bronchial epithelial cells can promote EMT, leading to tumorigenesis.²⁷ Then, miR-200 is also associated with the invasion of liver cancer.

First, we examined the miR-200b expression in HCC tissues, and then, the results showed that the expression of miR-200b in adjacent tissues was higher than that in the tumor tissues, and the difference was statistically significant. By detecting RhoA expression in HCC and analyzing the relationship between the expression of miR-200b and RhoA, we discovered there was a negative correlation between miR-200b expression and RhoA (r = −0.209, p = 0.010). These results suggested that there was a certain correlation between miR-200b expression and RhoA.

We further validated the role of miR-200b in liver cancer cells by transfecting miR-200b. First, we detected the effect of miR-200b on the invasive ability of HepG2 cells through wound-healing assay and Transwell invasion assays. The results showed that the ability of invasion and metastasis with transfection of miR-200b cells was inhibited, and compared with the control group, the difference was statistically significant (p < 0.05). By phalloidin staining we found that cytoskeletal actin protein of cells with transfection of miR-200b decreased compared with the control group. This result suggested that miR-200b could reduce the formation of actin cytoskeletal protein and thus reduce the exercise capacity of cells. Is the effect of miR-200b on invasive ability of liver cancer cells achieved by regulating the expression of RhoA? By examining the expression of RhoA in each group with western blot, we discovered that the expression of RhoA protein with transfection miR-200b was significantly reduced. By detection of luciferase reporter gene, we found that miR-200b was capable of targeting at specific sequence 3′-UTR of the RhoA mRNA. These results suggest that, miR-200b, through combination with 3′-UTR sequence of RhoA mRNA, reduced the RhoA expression, and then reduced the formation of cytoskeleton microfilament, to affect the invasion and metastasis of hepatocellular carcinoma cells.

MiRNAs could mediate the targeted coding gene and the targeted non-coding RNA to form endogenous competition. And our results showed miR-200b was inversely correlated with RhoA and circ_000839, while RhoA was positively correlated with circ_000839. This is an evidence that miR-200b could mediate RhoA gene and circ_000839 to form endogenous competition. And this is a direction for the association study of miR-200b and RhoA in the future.

Our study still had some limitations. First, the role of circ_000839 in the mobility and invasion of HCC was not researched in this study, but the circRNA might be correlated with miR-200b, and RhoA was selected out and could be a reminding factor for further investigation. Second, we analyzed the relationship of the miR-200b, RhoA, and circ_000839 and clinical features in cancer tissues (Table S2), but the clinical significance and the possible reasons were needed to further investigate in the future study. Third, because the in silico prediction cannot elucidate all the potential targets of mir200b, and RhoA and circ_001289 were also not the only regulated genes after mir-200b transfection. Thus, the absence of gene expression analysis at whole transcriptome level was lacking and should be studied in the future.

In summary, our results confirmed that the expression level of miR-200b in HCC tissues was lower than that in adjacent tissues. The results also revealed that miR-200b affected the formation of cytoskeleton microfilament through combination with RhoA, thereby affected the invasion and metastasis of hepatocellular carcinoma cells. And the advantage of our study was to propose a direction for the association of miR-200b and RhoA, in which the circRNA_000839 may be involved.

Footnotes

Acknowledgements

Any underlying research materials related to this paper (for example, data, samples, or models) can be accessed.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the National Natural Science Foundation of China (No. 31200968); the Natural Science Foundation of Liaoning Province, China (20170541001); and Fund for Scientific Research of The First Hospital of China Medical University (FSFH201713).

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MicroRNA-200b suppresses the invasion and migration of hepatocellular carcinoma by downregulating RhoA and circRNA_000839

Abstract

Keywords

Introduction

Materials and methods

Cell lines and cell culture

Patient information and tissue specimens

Real-time reverse transcription polymerase chain reaction

Vectors, retroviral infection, and transfection

Wound-healing assay

Transwell invasion assays

Immunofluorescence staining

Western blot analysis

Luciferase assay

Transient transfection of RhoA plasmid

The prediction of target circRNAs

The expression of circRNAs

Statistics

Results

The expression of miR-200b and RhoA in HCC tissues

Mir-200b suppressed HCC cell migration and invasion

MiR-200b inhibited RhoA expression

RhoA was involved in miR-200b-induced HCC cell migration

RhoA is a direct target of miRNA-200b

The expression of circRNAs which are predicted as the target of miR-200b

The relationship of the RhoA gene, miR-200b, and circ_000839 expression and HCC clinical features

Discussion

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

References