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Abstract

OBJECTIVE:

Breast cancer is a common malignancy in women and long non-coding RNAs (lncRNAs) have been shown to play key roles in the development and progression of breast cancer. In the present study, we examined the biological role of lncRNA gastric carcinoma highly expressed transcript 1 (GHET1) in breast cancer.

METHODS:

The expression of GHET1 was determined by qRT-PCR assay; CCK-8, colony formation, Transwell invasion and migration assays detected breast cancer cell proliferation, invasion and migration; cell apoptosis and cell cycle were determined by flow cytometry; protein levels were determined by western blot assay.

RESULTS:

GHET1 was up-regulated in breast cancer tissues and cell lines, and the up-regulation of GHET1 was positively correlated with larger tumor size, advanced clinical stage, lymph node metastasis and shorter overall survival. Knockdown of GHET1 suppressed cell proliferation, invasion and migration, and induced apoptosis and G ${}_{0}$ /G ${}_{1}$ cell cycle arrest in MCF-cells. Knockdown of GHET1 also suppressed the protein levels of N-cadherin, vimentin, and decreased the protein level of E-cadherin in MCF-7 cells. On the other hand, overexpression of GHET1 promoted cell proliferation, invasion and migration, and inhibited cell apoptosis and increased cell population at S phase in BT-20 cells. Overexpression of GHET1 also promoted epithelial mesenchymal transition (EMT) in BT-20 cells. Furthermore, knockdown of GHET1 also suppressed in vivo tumor growth of MCF-7 cells, and also decreased the protein levels of N-cadherin and vimentin, and increased the protein levels of E-cadherin in the tumor tissues from the nude mice.

CONCLUSIONS:

Our results demonstrated that GHET1 was up-regulated in breast cancer tissues and cell lines, and promoted breast cancer cell proliferation, invasion and migration by affecting EMT. Our study for the first time revealed the biological functions of GHET1 in breast cancer.

Keywords

Breast cancer GHET1 cell proliferation invasion and migration apoptosis epithelial mesenchymal transition

1. Introduction

Breast cancer is a common malignancy in women and is one of leading cause of cancer-related deaths annually [1]. Though early detection and standard of care have improved survival, the heterogeneity of a single tumor, multiple disease subtypes, and the factors contributing to recurrence have been intensely examined to identify novel therapies for breast cancer [2, 3, 4]. Recent decades, growing evidence has shown that long non-coding RNAs (lncRNAs) played key roles in breast cancer development and progression [5, 6]. Unfortunately, the exact functional roles of most lncRNAs in breast cancer are largely unknown.

LncRNAs are a new class of RNA transcripts with more than 200 nucleotides and can not code proteins. Up to date, the roles of lncRNAs have been elucidated in various studies, and lncRNAs were found to play important roles in the development and the physiological activity of the cells [7]. In the cancer studies, dysregulation of lncRNAs has been identified in numerous tumors, and lncRNAs can function as either oncogenes or tumor suppressor genes. As far as we know, several lncRNAs have been identified to play key roles in breast cancer development and progression. For example, the lncRNA BORG drives breast cancer metastasis and disease recurrence [7]; lncRNA CRNDE promotes breast cancer progression via acting as a molecular sponge of miR-136 and activating Wnt/ $\beta$ -catenin [8]; lncRNA HOTAIR is an independent prognostic marker of metastasis in estrogen receptor-positive primary breast cancer [9]. Recently, the lncRNA gastric carcinoma highly expressed transcript 1 (GHET1) was identified as a new lncRNA and functioned as oncogene in gastric cancer, bladder cancer, colorectal cancer, pancreatic cancer, liver cancer and lung cancer [10, 11, 12, 13, 14, 15]. However, the role of GHET1 in breast cancer was not identified.

In this study, we identified the up-regulation of GHET1 in breast cancer tissues and was correlated with poor prognosis of breast cancer patients. Further, in vitro loss-of-function and gain-of-function assays mechanistically showed that GHET1 promoted breast cancer cell proliferation, invasion and migration. Our study showed the pivotal functions of GHET1 in breast cancer progression, which provide new insights into understanding the development and progression of breast cancer.

2. Materials and methods

2.1 Clinical samples

In the present study, 60 pairs of breast cancer tissues and matched adjacent normal tissues were obtained from breast cancer patients who underwent surgical resection at the First Affiliated Hospital of Xi’an Medical University from January 2014 to June 2017. All the samples were confirmed as breast cancer by histopathological examination. The collected tissues were immediately snap-frozen in liquid nitrogen and store in $-$ 80 ${}^{\circ}$ C for further use. All the patients had not received any radiotherapy or chemotherapy before the surgery. The clinicopathological data of the breast cancer patients were show in Table 1. The study was approved by the Ethics Committee of the First Affiliated Hospital of Xi’an Medical University, and the informed consent was obtained all the patients from this study.

Table 1
The association between GHET1 expression level and clinical parameters with cancer patients

Parameters	GHET1		P value
	Low	High
Age (years)
$<$ 60	13	10	0.5959
$\geqslant$ 60	17	20
Menopausal status
Premenopausal	18	13	0.3015
Postmenopausal	12	17
Tumor size (cm)
$<$ 2	20	11	0.0379
$\geqslant$ 2	10	19
Clinical stage
I–II	19	9	0.0191
III–IV	11	21
Lymph node metastasis
No	21	12	0.037
Yes	9	18

2.2 Cell culture

The immortalized breast epithelial cell line MCF-10A and breast cancer cell lines (SKBR3, ZR-75-1, BT-20 and MCF-7) were obtained from American Type Culture Collection (ATCC, Manassas, USA). MCF-10A cells were cultured with M-171 medium supplemented with mammary epithelial growth factors (Invitrogen, Carlsbad, USA). SKBR3 and MCF-7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, USA), and ZR-75-1 and BT-20 cells were cultured in RMPI 1640 medium supplemented with 10% FBS (Thermo Fisher Scientific). All the cells were kept in a humidified incubator with 5% CO ${}_{2}$ at 37 ${}^{\circ}$ C.

2.3 Oligonucleotides and cell transfection

Small interfering RNAs (siRNAs) of GHET1 (siGHET1#1 and siGHET1#2) and the negative control (NC) siRNA (siNC) were obtained from Ribobio (Guangzhou, China). The GHET1-overexpressing plasmid (pcDNA-GHET1) and the control plasmid (pcDNA3.1) were purchased from GenePharma Co, Ltd (Shanghai, China). All the cells were transfected by using Lipofectamine 2000 reagent (Invitrogen) according to manufacturer’s instruction.

2.4 RNA extraction and quantitative real-time PCR (qRT-PCR) analysis

Total RNAs from tissues and cells were isolated by using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The cDNA synthesized from RNA were performed by using the PrimeScript RT reagent Kit (Takara, Dalian, China). The real-time PCR was performed in an ABI 7900 real-time PCR system (Applied Biosystems, Foster City, USA) by using SYBR Premix Ex Taq reagent (Takara). GAPDH was used as internal controls for GHET1. The relative expression of GHET1 was calculated by comparative Ct method.

2.5 Cell proliferation assay

CCK-8 assay was used to determine cell proliferation. Cells were seeded in the 96-well plate at a density of 1 $\times$ 10 ${}^{4}$ cell/well and cultured until cell confluent rate reached about 70%. At 24, 48 and 72 h after transfection with different siRNAs or plasmids, cells were incubated with CCK-8 solution (Beyotime, Beijing, China) according to the manufacturer’s protocol. The cell proliferative ability was measured by detecting absorbance at a wavelength of 450 nm.

2.6 Colony formation assay

Colony formation assay was performed to determine cell growth. The transfected cells were seeded into the 6-well plates at 1000 cells/well, and the culture medium was changed every 4 d, and after 14 d, cells were fixed and stained by crystal violet. The number of colonies were counted under a light microscope.

2.7 Cell apoptosis and cell cycle

For the cell apoptosis assay, the cell apoptosis was measured by using the FITC Annexin V/propidium iodide (PI) Apoptosis Detection Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, the transfected cells were trypsinized and washed with cold phosphate buffered saline (PBS) twice, and were doubled stained with Annexin V and PI at room temperature in the dark for 15 min. The cell apoptotic rate was analyzed by using a FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA). For the cell cycle analysis, the transfected cells trypsinized and washed with PBS for three times, and were then fixed with 70% ethanol, and the fixed cells were incubated with RNase A for 30 min at 37 ${}^{\circ}$ C and then incubated with PI for 20 min at room temperature. The cell cycle was analyzed by using FACSCalibur Flow Cytometer (BD Biosciences).

2.8 Transwell invasion and migration assay

Transwell invasion and migration assays were performed to determine cell invasion and migration. For the Transwell invasion assay, transfected cells were re-suspended in serum-free medium in the Matrigel-coated upper insert chamber (24-well insert, 8 $\mu$ m pore size, Corning Costar, Lowell, MA, USA); for the Transwell migration assay, the upper insert had no Matrigel coating, and the lower chamber was filled with medium supplemented with 20% FBS. The cells adhering to the lower surface of the membrane were fixed and stained with crystal violet, and the number of invaded cells and migrated cells were counted under a light microscope.

2.9 Western blot assay

Proteins were extracted from transfected cells by using the RIPA buffer, and the protein concentrations were measured by a BCA kit. Equal amount of proteins (50 $\mu$ g protein per lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% denaturing, and the separated proteins were transferred to the PVDF membranes (Sigma-Aldrich, St. Louis, MO, USA). The PVDF membranes were blocked with 5% skimmed milk for 1 h at room temperature, and the blocked membranes were incubated with primary antibodies including polyclonal rabbit to N-cadherin antibody, polyclonal rabbit to E-cadherin antibody, polyclonal rabbit to vimentin antibody and monoclonal rabbit to $\beta$ -actin antibody (Abcam, Cambridge, UK). Then the membranes were incubated with corresponding secondary antibody for 1 h at room temperature. The western blot bands were visualized by using the enhanced chemiluminescence Western blot blotting detection system (Bio-Rad, Hercules, CA, USA).

Figure 1.

The expression of lncRNA GHET1 in breast cancer tissues and cell lines. (A) Relative expression of GHET1 was determined by qRT-PCR in breast cancer tissues ( $n=$ 60) and normal adjacent tissues ( $n=$ 60). (B) The low expression of GHET1 and high expression of GHET1 were divided according to the median expression of GHET1 in breast cancer tissues. (C) Kaplan-Meier survival curve according to GHET1 expression levels. (D) The expression GHET1 was determined by qRT-PCR in breast cancer cell lines. All in vitrodata are representative of three independent experiments and expressed as mean $\pm$ SD. ${}^{**}P<$ 0.01 and ${}^{***}P<$ 0.001 vs. control group.

Figure 2.

The effects of GHET1 knockdown on MCF-7 cell proliferation, cell apoptosis, cell cycle, cell invasion and migration, and EMT in vitro. (A) The expression of GHET1 in MCF-7 cells transfected with siNC, siGHET1#1, or siGHET#2 was determined by qRT-PCR. (B) The effect of GHET1 knockdown on MCF-7 cell proliferation was determined by CCK-8 assay. (C) The effect of GHET1 knockdown on MCF-7 colony formation ability was determined by colony formation assay. (D) Cell apoptosis and (E) cell cycle in MCF-7 cells transfected with siNC or siGHET1#2 were determined by flow cytometry. (F) The effect of GHET1 knockdown on MCF-7 cell invasion was determined by Transwell invasion assay. (G) The effect of GHET1 knockdown on MCF-7 cell migration was determined by Transwell migration assay. (H) The effect of GHET1 knockdown on protein levels of N-cadherin, E-cadherin and vimentin in MCF-7 cells transfected with siNC or siGHET1#2 was determined by western blot assay. All in vitro data are representative of three independent experiments and expressed as mean $\pm$ SD. ${}^{*}P<$ 0.05, ${}^{**}P<$ 0.01 and ${}^{***}P<$ 0.001 vs. control group.

Figure 3.

The effects of GHET1 overexpression on BT-20 cell proliferation, cell apoptosis, cell cycle, cell invasion and migration, and EMT in vitro. (A) The expression of GHET1 in BT-20 cells transfected with pcDNA3.1 (pcDNA) or GHET1-overexpressing plasmid (pcDNA-GHET1) was determined by qRT-PCR. (B) The effect of GHET1 overexpression on BT-20 cell proliferation was determined by CCK-8 assay. (C) The effect of GHET1 overexpression on BT-20 colony formation ability was determined by colony formation assay. (D) Cell apoptosis and (E) cell cycle in BT-20 cells transfected with pcDNA or pcDNA-GHET1 were determined by flow cytometry. (F) The effect of GHET1 overexpression on BT-20 cell invasion was determined by Transwell invasion assay. (G) The effect of GHET1 overexpression on BT-20 cell migration was determined by Transwell migration assay. (G) The effect of GHET1 overexpression on protein levels of N-cadherin, E-cadherin and vimentin in BT-20 cells transfected with pcDNA or pcNDA-GHET1 was determined by western blot assay. All in vitro data are representative of three independent experiments and expressed as mean $\pm$ SD. ${}^{*}P<$ 0.05, ${}^{**}P<$ 0.01 and ${}^{***}P<$ 0.001 vs. control group.

Figure 4.

The effects of GHET1 knockdown on in vivo tumor growth. (A) Tumor volumes were measured every 7 d up to 28 d, and knockdown of GHET1 suppressed in vivo tumor growth. (B) Representative tumor images were taken and tumor weight was measured at the end of the experiment. (C) The protein levels of N-cadherin, E-cadherin and vimentin in tumor tissues were determined by western blot assay.

2.10 In vivo tumor growth assay

Female nude (nu/nu) mice (5–6 weeks old) were ob- tained from Charles River (Wilmington, USA). All the procedures of the animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Xi’an Medical University. MCF-7 cells were transfected with siGHET#2 or scrambled siRNA, respectively. The cells at the exponential stage were harvested and were then mixed with 50% Matrigel (BD Biosciences, San Jose, CA, USA). Cells (2 $\times$ 10 ${}^{6}$ cells) were subcutaneously injected into axillary fossae of female nude mice, six mice per group. Tumor volume (volume $=$ length $\times$ width $\times$ width/2) was measured every 7 d. At 28 d after injection, mice were killed and tumors were dissected for further experimental analysis.

2.11 Statistical analysis

All the statistical analyses were performed by using GraphPad Prism and SPSS 15.0 (Chicago, IL, USA). Categorical data were analyzed by Chi-square test. Student’s t-test and one-way ANOVA were used to analyze two or multiple groups for statistical significance, respectively. The overall survival curves were calculated with the Kaplan-Meier method and were analyzed with the log-rank test. $P<$ 0.05 was considered to be statistically significant.

3. Results

3.1 GHET1 is up-regulated in breast cancer tissues and cell lines

By performing qRT-PCR assay, we first compared the expression of GHET1 in 60 breast cancer tissues and 60 normal adjacent tissues, and the expression of GHET1 was highly expressed in breast cancer tissues (Fig. 1A). Furthermore, the low expression of GHET1 and high expression of GHET1 in breast cancer tissues were classified based on the median expression of GHET1 (Fig. 1B). The correlation analysis showed that high expression of GHET1 significantly correlated with larger tumor size, advanced clinical stage and lymph node metastasis (Table 1). The Kaplan-Meier analysis showed that breast cancer patients with high expression level of GHET1 had poorer overall survival (Fig. 1C). Consistently, the up-regulation of GHET1 was also identified in breast cancer cell lines compared to normal breast epithelial cell line MCF-10A (Fig. 1D).

3.2 The effects of GHET1 knockdown on MCF-7 cell proliferation, cell apoptosis, cell cycle, cell invasion and migration, and EMT in vitro

To determine the in vitro effects of GHET1 knockdown in breast cancer cells, we transfected MCF-7 cells with siNC, siGHET1#1 and siGHET#2, and MCF-7 cells transfected with siGHET1#1 and siGHET1#2 had significantly lower expression of GHET1 than that transfected with siNC (Fig. 2A). As siGHET1#2 had more profound effects on suppressing GHET1 expression, siGHET1#2 was selected from further experiments. CCK-8 assay showed that knockdown of GHET1 significantly suppressed MCF-7 cell proliferation at 48 h and 72 h post-transfection (Fig. 2B). In addition, knockdown of GHET1 also markedly reduced the number of colonies in MCF-7 cells (Fig. 2C). The flow cytometry experiments were used to determine MCF-7 cell apoptosis and cell cycle, and knockdown of GHET1 increased cell apoptotic rate (Fig. 2D); cycle analysis showed that cell population at G0/G1 phase was increased and cell population at S phase was decreased in MCF-7 cells transfected with siGHET1#2 (Fig. 2E). To determine the MCF-7 cell invasion and migration, we performed Transwell cell invasion and cell migration assays, respectively. Knockdown of GHET1 dramatically reduced the number of invaded cells and migrated cells (Fig. 2F and G). As epithelial mesenchymal transition (EMT) is an important process for tumor metastasis, we then measured the protein levels of EMT-related markers including N-cadherin, E-cadherin and vimentin, and the results showed that knockdown of GHET1 significantly decreased the protein levels of N-cadherin, vimentin, and increased the protein levels of E-cadherin (Fig. 2H).

3.3 The effects of GHET1 overexpression on BT-20 cell proliferation, cell apoptosis, cell cycle, cell invasion and migration, and EMT in vitro

To further determine the in vitro effects of GHET1 overexpression in breast cancer cells, we transfected with BT-20 cells with pcDNA or pcDNA-GHET1, and transfection with pcDNA-GHET1 significantly increased the expression of GHET1 in BT-20 cells (Fig. 3A). The cell proliferation was determined CCK-8 assay, and overexpression of GHET1 significantly promoted BT-20 cell proliferation (Fig. 3B). Similarly, colony formation results showed that overexpression of GHET1 dramatically increased the number of colonies of BT-20 cells (Fig. 3C). Flow cytometry analysis showed that overexpression of GHET1 significantly inhibited cell apoptosis, decreased cell population at G0/G1 phase and increased cell population at S phase in BT-20 cells (Fig. 3D and E). Furthermore, the Transwell invasion and migration results showed that overexpression of GHET1 markedly increased cell invasive and migratory abilities of BT-20 cells. The effects of GHET1 overexpression on EMT were also assessed by using western blot assay, and overexpression of GHET1 significantly promoted EMT in BT-20 cells (Fig. 3H).

3.4 The effects of GHET1 knockdown on in vivo tumor growth

To explore the role of GHET1 in in vivo tumor growth, we performed xenograft experiments in nude mice. The tumor volume of the nude mice from different groups were measured every other 7 d for 28 d, and the results showed that knockdown of GHET1 significantly reduced the tumor volume (Fig. 4A). The weight of the dissected tumors was also significantly lower in siGHET1#2 group than that in siNC group (Fig. 4B). Consistent with the in vitro findings, knockdown of GHET1 also suppressed the protein levels of N-cadherin, vimentin, and increased the protein level of E-cadherin as measured by western blot assay (Fig. 4C).

4. Discussion

Breast cancer is the most mortality-related malignancy in women and the incidence of breast cancer are rising during recent years [16, 17, 18]. Due to insufficient understanding of the molecular mechanisms that involve breast cancer development and progression, the diagnosis and prognosis of breast cancer are still challenging. In this study, we identified the up-regulation of GHET1 in breast cancer, and GHET1 functioned as oncogene to promote breast cancer cell proliferation, invasion and migration, and EMT, suggesting the potential role of GHET1 in breast cancer.

Due to the rapid development and application of second-generation sequencing technology, growing evidence has identified the dysregulation of lncRNAs in tumor tissues [19]. In recent decades, studies have demonstrated that lncRNAs are involved in various cell processes such as transcriptional activation, genome imprinting and histone modification [20, 21]. Though the functional roles of various lncRNAs have been elucidated in cancer, most lncRNAs have not been examined.

GHET1 is located in an intergenic region on chromosome 7, and is initially studied in gastric carcinoma [13]. Up to date, GHET1 was identified in various types of cancers including gastric cancer, bladder cancer, colorectal cancer, pancreatic cancer, liver cancer and lung cancer [10, 12, 13, 14, 15]. GHET1 was found to promote gastric carcinoma cell proliferation by increasing c-Myc mRNA stability [13], and to promote multidrug resistance in gastric cancer cells [22]; while knockdown of GHET1 suppressed cell activation of gastric cancer [23]. In bladder cancer, GHET1 was upregulated in bladder cancer tissues, and correlated with tumor size, advanced tumor and lymph node status and poor survival, and also promoted cell proliferation and invasion of bladder cancer cells [12]. Knockdown of GHET1 also inhibited cell proliferation and invasion in both colorectal cancer and lung cancer cells [10, 15]. In addition, GHET1 predicts poor prognosis in both liver cancer and pancreatic cancer [11, 14]. Consistently, our results showed up-regulation of GHET1 in breast cancer tissues was corrected with poor prognosis in breast cancer patients, and GHET1 promoted breast cancer proliferation, invasion and migration. Taken together, our results suggest the oncogenic role of GHET1 in breast cancer development and progression.

As far as we know, lncRNAs exerted its function in the development and progression of breast cancer via different mechanisms, and one of the important mechanisms is EMT, which is a key process for tumor metastasis [24]. For example, lncRNA HOXA11-AS promoted breast cancer invasion and metastasis by affecting EMT-related molecular markers (E-cadherin, N-cadherin and Vimentin) [25]. Li et al. showed that lncRNA ANCR down-regulation promoted tumor growth factor-beta-induced EMT and metastasis in breast cancer [26]. In addition, lncRNA H19 also mediated breast cancer cell plasticity during EMT by differentially sponging miR-200b/c and let-7b [26]. In our results, we found that GHET1 overexpression enhanced EMT; while knockdown of GHET1 had the opposite effects on EMT in breast cancer cells, suggesting that GHET1 may promote the invasion and migration of breast cancer cells via affecting EMT. However, lncRNA can also function as a molecular sponge for miRNAs [27], and in the future studies, identifying novel miRNAs that can interact with GHET1 may be of great significance to further illustrate the underlying mechanisms of GHET1 in regulating breast cancer progression.

In the present study, in vitro and in vivo experiments were performed to explore the biological role of GHET1 in breast cancer, and our results demonstrated that GHET1 was up-regulated in breast cancer tissues and cell lines, and promoted breast cancer cell proliferation, invasion and migration by affecting EMT. Further mechanistic studies may be performed to elucidate the biological role of GHET1 in breast cancer.

Footnotes

Conflict of interest

The authors declare no competing financial interests.

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Long non-coding RNA GHET1 promotes human breast cancer cell proliferation,invasion and migration via affecting epithelial mesenchymal transition

Abstract

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Clinical samples

Table 1 The association between GHET1 expression level and clinical parameters with cancer patients

2.3 Oligonucleotides and cell transfection

2.4 RNA extraction and quantitative real-time PCR (qRT-PCR) analysis

2.5 Cell proliferation assay

2.6 Colony formation assay

2.7 Cell apoptosis and cell cycle

2.8 Transwell invasion and migration assay

2.9 Western blot assay

2.11 Statistical analysis

3. Results

3.1 GHET1 is up-regulated in breast cancer tissues and cell lines

3.2 The effects of GHET1 knockdown on MCF-7 cell proliferation, cell apoptosis, cell cycle, cell invasion and migration, and EMT in vitro

3.3 The effects of GHET1 overexpression on BT-20 cell proliferation, cell apoptosis, cell cycle, cell invasion and migration, and EMT in vitro

3.4 The effects of GHET1 knockdown on in vivo tumor growth

4. Discussion

Footnotes

Conflict of interest

References

Table 1
The association between GHET1 expression level and clinical parameters with cancer patients