Knockdown of long non-coding RNA GHET1 suppresses cervical carcinoma in vitro and in vivo

Abstract

INTRODUCTION:

The present study evaluated the effects of the long non-coding RNA (lncRNA) gastric carcinoma high-expressed transcript (GHET1) in cervical carcinoma development.

METHODS:

The expression levels of GHET1 and PTEN were measured using in situ hybridisation, immunohistochemistry (IHC) and quantitative reverse transcription PCR assay to investigate their correlations. In an in vitro study, the effects of GHET1 knockdown on the biological activities of SiHa and HeLa cells were evaluated by MTT, flow cytometry, transwell and wound-healing assays and relative protein expression was measured using western blotting. In an in vivo experiment, cell apoptosis and relative protein expression were measured in nude mice using TUNEL and IHC assays, respectively.

RESULTS:

The expression levels of lncRNA GHET1 and PTEN protein differed significantly between cancer and adjacent normal tissues ( $P<$ 0.05) and were negatively correlated in the clinical data. In vitro, proliferation rateswere significantly down-regulated in SiHa and HeLa cells. The GHET1 knockdown (si-GHET1) groups showed significantly higher G ${}_{1}$ phase and apoptosis rates and significantly suppressed invasion and migration abilities compared with the normal control (NC) group ( $P<$ 0.05 for all). The expression levels of PTEN, PI3 K, AKT, P53, matrix metalloproteinase (MMP)-2 and MMP-9 proteins differed significantly between the si-GHET1 and NC groups ( $P<$ 0.05 for all). In vitro, the lncRNA group showed significantly suppressed tumour volume and weight, increased cell apoptosis and different relative protein expression levels compared with the NC group ( $P<$ 0.05 for all).

CONCLUSION:

GHET1 knockdown suppressed cervical carcinoma development via the PTEN/PI3 K/AKT signalling pathway.

Keywords

GHET1 PTEN PI3 K AKT SiHa HeLa

1. Introduction

Cervical cancer is the second most serious malignancy in women that poses a significant threat to health [1]. However, its pathogenesis remains unclear. Understanding the pathogenesis of cervical cancer is crucial to its treatment. Long non-coding RNAs (lncRNAs) are a class of RNA with a transcription length of $>$ 200 nucleotides that do not encode proteins. They were initially considered transcriptional noise with no biological function [2]. However, recent studies have shown that they play important regulatory roles in gene expression, closely related to the occurrence and development of tumours [3, 4]. In cervical cancer, the involvement of MALAT1, MEG3, HOTAIR, XLOC-010588 and EBIC lncRNAs has been demonstrated [5, 6, 7, 8, 9]. A previous study showed that the lncRNA gastric carcinoma high-expressed transcript 1 (GHET1) was closely correlated with tumour size, invasion and migration [10]. However, studies demonstrating the correlation between lncRNA GHET1 and cervical cancer are limited. Previous studies have shown that lncRNAs play important roles in the tumourigenesis of leukaemia, glioblastoma, liver cancer and colorectal cancer. Furthermore, lncRNAsare similar to protein-coding genes and are involved in a variety of cell signalling pathways [11]. It was reported that lncRNA GHET1 could promote osteoblast proliferation, invasion and migration abilities via suppression of phosphatase and tensinhomologue deleted on chromosome ten (PTEN) [12].

The present study measured the expression ofGHET1 in cervical cancer and adjacent normal tissues and evaluated the effects of GHET1 knockdown in SiHa and HeLa cervical cancer cell lines as well as the effects of lncRNA GHET1 in vivo.

2. Materials and methods

2.1 Clinical data

A total of 36 pairs of cervical cancer and adjacent normal tissues were collected from patients diagnosed and treated for cervical cancer at the Second Hospital of Jilin University. Adjacent normal tissues were taken $>$ 5 cm from the cancer tissues. Fresh cancer and adjacent normal tissues were fixed in 10% neutral formaldehyde, dehydrated and made transparent, embedded in paraffin and continuously cut into 4 $\mu$ m slices. Diagnosis and routine pathological data for cervical cancer were confirmed by two experienced pathologists after reading the film. Staging was performed using the standards of the International Federation of Gynecology and Obstetrics. The age range of the 36 patients with cervical cancer was 30–61 years, with a median age of 41 years (17 patients were aged $<$ 40 years, and 19 patients were aged $\geqslant$ 40 years). There were seven cases of stage I, 10 cases of stage II, 16 cases of stage III, three cases of stage IV, 31 cases of squamous cell carcinoma, five cases of adenocarcinoma, 14 cases of no lymph node metastasis and 12 cases of lymph node metastasis. A total of 36 patients received postoperative radiotherapy and/or chemotherapy. All patients or a close relative gave written informed consent to take part in the study.

2.2 In situ hybridisation

The in situ hybridisation (ISH) probe used for detecting GHET1-labelled digoxin was designed and synthesised by Sanon Biotech Co., Ltd. (Shanghai, China). Tissue slices were processed using an Enhanced Sensitive ISH Detection Kit (POD) (Boster, Wuhan, China) according to the manufacturer’s protocol. Tissue chip de-waxing was performed at 37 ${}^{\circ}$ C using protease K for digestion for 15 min. After rinsing, gradient ethanol was dehydrated. After hybridisation for 30 min at 55 ${}^{\circ}$ C, the digoxigenin-labelled MEG3 probe was incubated for 1 h at a constant temperature and stained, using phosphate-buffered saline (PBS) as negative control. Phosphatase-labelled sheep anti-digoxin antibody was incubated at room temperature for 1 h. Slides were photographed using an Olympus BX51 microscope (Olympus, Japan).

2.3 Immunohistochemistry

Immunohistochemistry (IHC) was performed as follows. After de-waxing, samples were hydrated and rinsed with PBS, pH 7.4, three times for 5 min each. Peroxidase blocking solution (50 $\mu$ L) was added, and samples were incubated at room temperature for 10 min and then washed with PBS three times for 5 min each. Non-immune animal serum (50 $\mu$ L) was added, and samples were incubated at room temperature for 5 min and then washed with PBS. Primary PTEN antibody (50 $\mu$ L) was added and incubated at room temperature for 60 min before washing with PBS three times for 5 min each. Biotin-labelled secondary antibody (50 $\mu$ L) was added and incubated at room temperature for 10 min and then washed with PBS three times for 5 min each. Streptavidin peroxidase solution (50 $\mu$ L) was added and incubated at room temperature for 10 min and then washed with PBS three times for 5 min each. Two drops of fresh DAB solution was added and then rinsed with tap water, stained with haematoxylin and sealed using neutral gum.

2.4 RT-PCR assay

Cervical cancer and adjacent tissues were added to TRIzol reagent before being cut and milled separately. Total cellular RNA was extracted using the PureLink RNA Mini Kit. Genomic contamination was removed using DNase I during extraction. Reverse transcription of total RNA was performed using a reverse transcription kit to generate cDNA. GHET1 and PTEN expression levels were measured using SYBR Green real-time fluorescent quantitative PCR. GAPDH was stably expressed among the different tissues (data not shown) and used as a reference. The primer sequences were as follows: GHET1, R: 5 ${}^{\prime}$ -C C C C A C A A A T G A A G A C A C T-3 ${}^{\prime}$ and F: 5 ${}^{\prime}$ -T T C C C A A C A C C C T A T A A G A T-3 ${}^{\prime}$ ; PTEN, R: 5 ${}^{\prime}$ -C T C C T C T C C C A C C T A A A C C T-3 ${}^{\prime}$ and F: 5 ${}^{\prime}$ -T C A C C A C A C A-C A C C T A A C C C-3 ${}^{\prime}$ ; GAPDH, R: 5 ${}^{\prime}$ -A C C C A G A A G A C T G T G G A T G G-3 ${}^{\prime}$ and F: 5 ${}^{\prime}$ -T T C T A G A C G G C A G G T C A G G-3 ${}^{\prime}$ .

2.5 Cell culture and transfection

HeLa and SiHa cells were seeded at a density of 1 $\times$ 10 ${}^{5}$ /mL in six-well plates and cultured in DMEM/F12 medium in an incubator containing 5% CO ${}_{2}$ at 37 ${}^{\circ}$ C. Cells were transfected using Lipofectamine 2000. HeLa and SiHa cells were transfected for 48 h with non-specific siRNA (BL group) or GHET1 siRNA (si-GHET1 group; si-GHET1 sense, 5 ${}^{\prime}$ -C G G C A G G C A U U A G A G A U G A A C A G C A-3 ${}^{\prime}$ and antisense, 5 ${}^{\prime}$ -U G C U G U U C A U C U C U A A U G C C U G C C G-3 ${}^{\prime}$ ).

2.6 CCK-8 cell proliferation assay

Cell proliferation rates were measured in the different groups by CCK-8 assay. After transfection, cells were seeded in 96-well plates at a density of 2 $\times$ 10 ${}^{3}$ /100 $\mu$ L and cultured for 24 h before performing the CCK-8 assay. Serum-free medium was replaced at the time of examination. CCK-8 solution (10 $\mu$ L) was added to each welland incubated at 37 ${}^{\circ}$ C in an atmosphere containing 5% CO ${}_{2}$ for 2 h. Optical density values were measured at 450 nm. All experiments were conducted in duplicate. This experiment repeated for 3 times.

2.7 Flow cytometryanalysis of cell apoptosis and cell cycle

2.7.1 Measurement of cell apoptosis

Cells from different groups were washed twice in pre-cooled PBS and then adjusted to a concentration of 1 $\times$ 10 ${}^{6}$ /mL. A total of 100 $\mu$ L of the cell suspension was transferred to a 5-mL flow tube, followed by the respective additions of 5 $\mu$ L of annexin V-FITC and 10 $\mu$ L of propidium iodide (PI) and gently vortexed. Cells were then incubated at room temperature for 15 min, and then, 400 $\mu$ L of binding buffer was added before analysing using flow cytometry. This experiment repeated for 3 times.

2.7.2 Measurement of cell cycle distribution

Cells from the different groups were washed twice in pre-cooled PBS before being fixed in 70% ice-cold ethanol for 4 h at 12 ${}^{\circ}$ C. The ethanol was removed, and cells were rinsed with PBS and then digested using RNAse. After adding 20 pg/mL of PI to the cell suspension, DNA staining was performed for 30 min to analyse the distribution of DNA content by flow cytometry. The percentage of each cell cycle phase was calculated using ModFit software. This experiment repeated for 3 times.

2.8 Transwell assay

Transwell chambers were placed into 24-well plates. The upper surface of the basement membrane of the transwell chamber was covered with Matrigel packets, dried at 4 ${}^{\circ}$ C and incubated for 30 min. After collecting the cells from the different groups, cell suspensions without foetal bovine serum were prepared at a density of 2 $\times$ 10 ${}^{5}$ cells/mL, and 200 $\mu$ L of the cell suspension was added to the upper chamber of the transwell. A volume of 500 $\mu$ L of cell suspension containing 10% foetal bovine serum was added to the chamber containing RPMI, and the medium was added to the lower chamber. After incubation for 24 h, the transwell chamber was removed. Cells at the bottom of the upper chamber were collected using cotton swabs, rinsed three times in PBS, fixed in formaldehyde solution for 20 min, stained using polycrystalline violet 20 min and rinsed two to three timesusing PBS. Cells were air-dried and then observed under an inverted microscope, and the number of invasive cells was recorded in five fields of view. The average number of invasive cells reflected the invasion ability of the tumour cells. Experiments were repeated three times.

2.9 Wound-healing assay

HeLa and SiHa cells were seeded in six-well plates. Cells were transfected when they reached 80% confluence. After 6 h, the medium was replaced, and a scratch was made in the cell layer using a 200- $\mu$ L pipette tip angled perpendicular to the plane of cells. Residual cells were washed three to five times using PBS, and the medium was replaced. Images were taken at 0 and 48 h after making the scratch. This experiment repeated for 3 times.

2.10 Western blot assay

A total of 5 $\times$ 10 ${}^{6}$ cells from each of the different groups were collected and washed in PBS three times. Total protein in the cell lysate was extracted, and the concentration measured using the BCA method and isometric protein line SDS-PAGE was performed. Constant concentration gel electrophoresis was performed at 80 V for 40 min, and separation was performed at 120 V. Proteins were transferred to a film at 170 V at a low temperature for 30 min to 1 h depending on the molecular weight. The film was then incubated in 5% skimmed milk powder for 2 h at 37 ${}^{\circ}$ C, followed by incubation overnight for 4 h. After washing with TPBS, the film was incubated with horseradish-peroxidase-labelled secondary antibody (1:5000 dilution) at 37 ${}^{\circ}$ C for 1.5 h. The film was rinsed using solution B and reacted with the membrane for 1 min in a transparent plastic bag. The PVDF membrane and X-ray film were exposed in a dark room to be developed and fixed. After scanning, quantitative analysis of the western blot bands was performed using Image Lab 2 software (BioRad Corporation, Japan). The ratio of the integral optical density of the target and GAPDH bands was used as the final result. This experiment repeated for 3 times.

2.11 Grouping and treatment in the in vivo study

Balb/c nude mice (male, 4–6 weeks age) were purchased from Nanjing Medicine University. HeLa cells, at a concentration of 5 $\times$ 10 ${}^{5}$ /mL, were injected subcutaneously into the right upper limb and axilla of the nude mice. To confirm successful tumour formation after 1 week, mice were randomly divided into three groups: normal control (NC) group, BL group, which received caudal vein injection of non-specific siRNA plus vector; and si-GHET1 group, which received caudal vein injection of si-GHET1 plus vector. After feeding for 4 weeks, mice were sacrificed, and the tumour tissues were collected to measure tumour volume and weight. This experiment repeated for 3 times.

2.12 TUNEL assay

Tumour tissues were fixed in 4% paraformaldehyde, followed by conventional dehydration, embedding, slicing and de-waxing in water, and then washed using PBS. After protease K digestion at 37 ${}^{\circ}$ C for 10 min, the cells were washed in PBS. After incubation at room temperature for 10 min in H ${}_{2}$ O ${}_{2}$ , TUNEL reaction mixture was added, and the cells were cultured at 37 ${}^{\circ}$ C for 1 h. POD was added, and the cells were cultured at 37 ${}^{\circ}$ C for 30 min. Samples were washed in PBS and then stained using DAB, dehydrated, made transparent and sealed using neutral gum. Under light microscopy, cells containing brown and yellow particles were deemed to be positive. The presence of apoptotic cells was established by combining morphological characteristics. Grey Image-ProPlus6 image analysis software was used to quantify the data. This experiment repeated for 3 times.

2.13 Statistical analysis

All data were analysed using SPSS 19.0 software. The measured data were shown as mean $\pm$ standard deviation. One-way analysis of variance was used for comparisons among the groups. Dunnett T3 test was used to determine variance. Relationships among variables were evaluated using Pearson correlation analysis. $P$ -values $<$ 0.05 indicated statistical significance.

3. Results

3.1 Clinical data and analysis

ISH and RT-PCR analyses revealed that GHET1 expression was significantly up-regulated in cervical cancer tissue compared with that in adjacent normal tissue ( $P<$ 0.05, Fig. 1A and $P<$ 0.05, Fig. 1C, respectively). PTEN protein expression was localised in the nucleus and cytoplasm and was significantly down-regulated in cervical cancer tissue compared with that in adjacent normal tissue ( $P<$ 0.05, Fig. 1B). PTEN gene expression was significantly suppressed in cervical cancer tissue compared with that in adjacent normal tissue ( $P<$ 0.05, Fig. 1D). Furthermore, GHET1 expression was negatively correlated with PTEN expression (Fig. 1E).

Figure 1.

Clinical data and analysis. (A) Expression of lncRNA GHET1 in cervical cancer and normal tissues measured by ISH (200 $\times$ magnification). *** $P<$ 0.05, (B) PTEN protein expression in cervical cancer and normal tissues by IHC (200 $\times$ magnification). *** $P<$ 0.05,(C) GHET1 gene expression in cervical cancer and normal tissuesmeasured by RT-PCR. *** $P<$ 0.05, (D) PTEN gene expression in cervical cancer and normal tissuesmeasured by RT-PCR. *** $P<$ 0.05, (E) Correlation between PTEN and GHET1 in cancer tissues.

3.2 Cell proliferation rates

To evaluate the effects of GHET1 knockdown in cervical cancer cells, CCK-8 assay was used to measure the proliferation rates of HeLa and SiHa cells transfected with either si-GHET1 or non-specific siRNA (Fig. 2A and B). Proliferation of HeLa and SiHa cells was significantly reduced in the si-GHET1 groups compared with that in the NC groups ( $P<$ 0.05 for both cell lines).

Figure 2.

Cell proliferation rates in the knockdown and control groups measured by CCK-8 assay. HeLa cell (A) and SiHa cell (B) proliferation rates in the two groups. *** $P<$ 0.05.

Figure 3.

Cell apoptosis rate in the knockdown and control groups measured by flow cytometry. HeLa cell (A) and SiHa cell (B) apoptosis rates in the two groups. *** $P<$ 0.05.

Figure 4.

Cell cycle in the knockdown and control groups measured by flow cytometry. HeLa cell cycle (A) and SiHa cell cycle in the two groups. *** $P<$ 0.05.

Figure 5.

Cervical cancer cell invasion in the knockdown and control groups measured by transwell assay. HeLa cell (A) and SiHa cell (B) invasion in the two groups. *** $P<$ 0.05.

Figure 6.

Wound-healing rate in the knockdown and control groups in HeLa and SiHa cells measured by wound-healing assay. HeLa cell (A) and SiHa cell (B) wound-healing rate in the two groups. *** $P<$ 0.05.

3.3 Cell apoptosis rates

Flow cytometry analysis showed that the apoptosis rates of HeLa and SiHa cells were significantly higher in the GHET-siRNA groups compared with those in the NC groups ( $P<$ 0.05 for both cell lines; Fig. 3A and B).

3.4 Analysis of the G ${}_{1}$ phase

To investigate the role of GHET1 in cervical cancer development, we assessed the cell cycle status among the different groups using flow cytometry (Fig. 4A and B). The G ${}_{1}$ phase of the HeLa and SiHa cells was significantly enhanced in the si-GHET1 groups compared with that in the NC groups ( $P<$ 0.05 for both).

Figure 7.

Relative protein expression in HeLa and SiHa cells measured by western blotting. Relative protein expression in the knockdown and control groups in HeLa cells (A) and SiHa cells (B). *** $P<$ 0.05.

3.5 Invasion ability of cervical cancer cell lines

Transwell assay analysis showed that the number of invasive HeLa and SiHa cells was significantly lower in the si-GHET1 groups compared with that in the NC groups ( $P<$ 0.05 for both; Fig. 5A and B).

3.6 Migration ability of cervical cancer cell lines

Wound-healing assays revealed that the wound-healing rate was significantly suppressed in the si-GHET1 groups compared with that in the NC groups of HeLa and SiHa cells ( $P<$ 0.05 for both; Fig. 6A and B).

3.7 Relative protein expression

To further elucidate the mechanisms of GHET1 in cervical cancer cell lines, we measured relative protein expression by western blot assay. Compared with the NC groups, the si-GHET1 groups showed significant stimulation of PTEN and P53 protein expression ( $P<$ 0.05 for both) but significant suppression of PI3 K, AKT, matrix metalloproteinase (MMP)-2 and MMP-9 protein expression ( $P<$ 0.05 for all) in both HeLa and SiHa cells (Fig. 7A and B).

Figure 8.

Tumour volumes and weights, and cell apoptosis. (A) Tumour volumes and weights in the knockdown and control groups. *** $P<$ 0.05; (B) Cell apoptosis in the knockdown and control groups in tumour tissues measured by TUNEL assay (200 $\times$ magnification). *** $P<$ 0.05.

Figure 9.

Tissue PTEN and PI3 K protein expression in the knockdown and control groups. PTEN (A) and PI3 K (B) protein expression in the different groups and tissues (200 $\times$ magnification). *** $P<$ 0.05.

Figure 10.

AKT and P53 protein expression in the knockdown and control groups in cervical cancer and normal tissues. AKT (A) and P53 (B) protein expression in the different groups and tissues (200 $\times$ magnification). *** $P<$ 0.05.

Figure 11.

MMP-2 and MMP-9 protein expression in the knockdown and control groups in cervical cancer and normal tissues. MMP-2 (A) and MMP-9 (B) protein expression in the different groups and tissues (200 $\times$ magnification) *** $P<$ 0.05.

3.8 Tumour volume and weight

Tumour volumes and weights were significantly suppressed in the si-GHET1 groups compared with those in the NC groups ( $P<$ 0.05 for both; Fig. 8A). TUNEL assay analysis revealed that the cell apoptosis rate was significantly up-regulated in the si-GHET1 groups compared with that in the NC groups ( $P<$ 0.05 for both; Fig. 8B).

3.9 Relative protein expression in the tumour tissues

IHC analysis showed that the relative protein expression of PTEN, PI3 K, AKT, P53, MMP-2 and MMP-9 differed significantly between the si-GHET1 and NC groups ( $P<$ 0.05 for all; Figs 9–11).

4. Discussion

lncRNAs are important non-coding RNAs that cannot be translated into protein. Although many studies have shown that lncRNAs play key roles in the development and progression of tumours [13], their precise mechanisms of action remain unclear. In particular, the clinical significance and use of lncRNAs as biomarkers for tumour resection and prognosis have not yet been definitely established. Some studies have shown that lncRNAs are closely correlated with cervical cancer [14, 15, 16, 17]. The functions of lncRNAs vary, and they may act as either tumour suppressor genes or oncogenes according to different tissues, indicating individualtissue-specific expression of lncRNAs. Since lncRNAs may have multiple target genes, regulation of the downstream target genes (with different functions) and their final role in tumour development may differ among organs and individuals.

In recent years, the lncRNA GHET1 has been shown to play a new role in cancer [18, 19]. However, the correlation between GHET1 and cervical cancer remains unclear. The present study evaluated the expression of lncRNA GHET1 in cervical cancer and adjacent normal tissue as well as the anti-tumour effects and mechanisms of GHET1 knockdown in HeLa and SiHa cervical cancer cell lines. We found that GHET1 knockdown suppressed biological activities such as proliferation, invasion and migration of the cervical cancer cell lines and improved apoptosis via regulation of the cell cycle.

We also measured relative protein expression. PTEN is the first tumour suppressor gene of the protein and lipid phosphatase family that blocks the signal transduction pathway mediated by cytokines, thereby inhibiting growth, invasion and metastasis of tumour cells and promoting tumour cell apoptosis [20, 21]. PTEN gene and protein expression varies betweenhaematopoietic malignancies, such as leukaemia, lymphoma, myeloma and other cancer cell lines. Although low expression, deletion, mutations and rearrangement of PTEN are rare, low expression is associated with poor prognosis. Inactivation of the PTEN gene occurs predominantly because of indirect mechanisms such as transcriptional silencing and post-transcriptional modification and methylation [22, 23, 24, 25]. Our clinical data showed that PTEN protein expression was significantly down-regulated in cervical cancer tissue compared with that in adjacent normal tissue and was negatively correlated with GHET1 expression. In the in vitro experiments, PTEN protein expression increased in response to GHET1 knockdown. On the basis of these results, we inferred that GHET1 could be a tumour factor in cervical carcinoma as knockdown of GHET1 suppressed cervical carcinoma with a concurrent increase in PTEN expression levels both in vitro and in vivo.

PI3 K/AKT is an important cell signal transduction pathway that is closely correlated with cell biological activities such as proliferation, apoptosis, invasion and migration [26, 27, 28, 29]. Our results implied that GHET1 knockdown may have exerted its anti-tumour effects via the PTEN/PI3 K/AKT signalling pathway. P53 is a tumour suppressor gene with a wide range of roles, including regulation of tumour formation, growth, development and progression, as well as regulation of tumour stem cell division and self-renewal and inhibition of tumour recurrence [30, 31, 32]. Under normal circumstances, p53 protein acts as a tumour suppressor and regulates all aspects of cell life activities [33]. In response to DNA damage, the DNA damage stress response is induced, p53 protein triggers cell cycle arrest, and complete DNA repair or cell apoptosis is induced to prevent the damaged DNA from continuing to expand and transcribe [34]. The present study revealed that GHET1 knockdown led to significant suppression of PI3 K/AKT pathway activity and induction of PTEN, as well as enhanced P53 protein expression in vitro and vivo. From our results, we inferred that the suppressed proliferation and enhanced apoptosis observed in the cervical cell lines were mediated by the PTEN/PI3K/AKT/P53 pathway and stagnation of the cell cycle in the G ${}_{1}$ phase.

MMPs comprise a large family of proteins that require calcium, zinc and metal ions as cofactors. They can degrade almost all protein components in the extracellular matrix as well as the basement membrane to promote tumour cell invasion and play a key role in tumour invasion and metastasis [35, 36]. MMP-2 and MMP-9 were shown to be involved in angiogenesis and basement membrane degradation, which are directly related to tumour metastasis and prognosis [37, 38]. In the present study, the invasion and migration abilities of the HeLa and SiHa cell groups were significantly suppressed in the si-GHET1 groups. Furthermore, MMP-2 and MMP-9 protein expression was significantly reduced in vitro and in vivo. Therefore, we hypothesised that down-regulation of GHET1 could inhibit cervical cancer cell invasion and migration via regulation of MMP-2 and MMP-9 in vitro and in vivo. In the cervical development, lncRNA GHET1 play a key role to promote cervical cancer occurrence.

5. Conclusion

GHET1 knockdown showed anti-tumour effects by suppressing cell proliferation, invasion and migration and by improving cell apoptosis via regulation of the PTEN/PI3 K/AKT/P53 pathway and MMP-2 and MMP-9.

Footnotes

Acknowledgment

This study was supported by the Natural Science Foundation of Liaoning Province, China (Grant no. 20170540570), and was a project of the Liaoning Clinical Research Center for Colorectal Cancer (Grant no. 2015225005), which was sponsored by the Liaoning BaiQianWan Talents Programme (2017, no. C13).

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Knockdown of long non-coding RNA GHET1 suppresses cervical carcinoma in vitro and in vivo

Abstract

INTRODUCTION:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Clinical data

2.2 In situ hybridisation

2.3 Immunohistochemistry

2.4 RT-PCR assay

2.5 Cell culture and transfection

2.6 CCK-8 cell proliferation assay

2.7 Flow cytometryanalysis of cell apoptosis and cell cycle

2.7.1 Measurement of cell apoptosis

2.7.2 Measurement of cell cycle distribution

2.8 Transwell assay

2.9 Wound-healing assay

2.10 Western blot assay

2.11 Grouping and treatment in the in vivo study

2.12 TUNEL assay

2.13 Statistical analysis

3. Results

3.1 Clinical data and analysis

3.4 Analysis of the G 1 phase

3.6 Migration ability of cervical cancer cell lines

3.7 Relative protein expression

3.9 Relative protein expression in the tumour tissues

4. Discussion

5. Conclusion

Footnotes

Acknowledgment

References

3.4 Analysis of the G ${}_{1}$ phase