Down-regulated long non-coding RNA SNHG1 inhibits tumor genesis of colorectal carcinoma

Abstract

BACKGROUND:

Colorectal carcinoma (CRC) is the vital cancer mortality worldwide and the long noncoding RNAs (lncRNAs) is considered as an important biomarker. The aim of this study was to examine the influence of lncRNA-SNHG1in CRC, and explore the relationship of lncRNA-SNHG1 and CRC, and consequently find a new therapeutic target for CRC patients.

METHODS:

This study used 80 CRC patients and several cancer cell lines, with RNA interference technology to find the function of SNHG1.

RESULTS:

SNHG1 expression was higher in CRC tissue lines other than the cancer adjacent tissues. Moreover, down-regulated SNHG1 resulting in smaller tumor size and lighter tumor weight. Additionally, down-regulated SNHG1 inhibited cell migration, proliferation and colony formation, but promoted cell apoptosis.

CONCLUSION:

Our findings revealed that down-regulated SNHG1 could inhibit CRC tumor genesis and SNHG1 might act as an important potential therapeutic target in CRC treatment.

Keywords

Colorectal carcinoma SNHG1 proliferation apoptosis

1. Introduction

Colorectal carcinoma (CRC), one of the most important cancers which threaten human’s life and health seriously worldwide. According to the epidemiological investigation, CRC morbidity places the fourth among all the cancers [1]. Despite the enormous therapies including physical and chemical methods have been used over the past years, the rate of 40%–45% overall mortality still being there [2]. Researchers have shown that the occurrence and development of CRC were very complex multistep biologic processes, which were affected by many factors with none of the effective method has been explored to solve the problem [3, 4]. Thus, it is necessary to comprehensively study the molecular mechanism and to identify new colon therapy against CRC metastasis.

Long non-coding RNA (lncRNA) is a set of RNA transcripts, with more than 200 nucleotides in length and no potential capability of protein-coding, but can regulate gene expression based on superficial modification, intranuclear transport, transcription and reverse transcription, and almost involved in all the pathophysiological performance or the body [5, 6, 7]. Previous study has been demonstrated that the abnormal expression of lncRNA plays an important role in cancer, which actd as tumor suppressor gene or carcinogenic gene [8], for example: research has reported that lncRNA H19 and miR-675 has been expressed in human colon carcinoma cell line and primary tumor tissue, and never in adjacent tumor tissues [9]; another researcher Yang et al. found that lncRNA GAPLING could promote Colorectal cancer migration in combining protein PSF with NONO and targeting adjustment SNAI2 [10]; Chen et al. reported that lncRNA-SNHG15 could promote the gastric cancer cell proliferation and migration in regulating MMP2/MMP9 [11].

It follows that lncRNA has functional importance on regulating cancer cell proliferation and migration. LncRNA-SNHG1 has been demonstrated to play an important role on regulating the proliferation and migration of hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC) [12, 13]. However, the role of SNHG1 in CRC was not been legibly documented. Therefore, it is urgent to reveal whether SNHG1 to regulate CRC, and the relationship between them.

Herein, this study was undertaken to examine the expression of SNHG1 in cancer or non-cancer tissues, and then evaluate the role of lncRNA SNHG1 on cell proliferation, apoptosis, migration and invasion, and we also investigated the tumor growth in vivo in mice.

2. Materials and methods

2.1 Patients

All patients ( $n=$ 80) with the average age of 53.6 (ranged from 42 to 76) in this study were enrolled from The First Affiliated Hospital of Zhengzhou University. The patients were diagnosed with CRC for the first time and had not been treated before. The CRC tissues were obtained from the patients with CRC undergoing therapy. Tissue samples were confirmed by pathological examination. All patients in this study did not have received preoperative treatments and all signed informed consent. The study was approved by the Ethics Committee of The First Affiliated Hospital and Institute of Clinical Medicine, Zhengzhou University.

2.2 Cell lines and cell culture

Human CRC cell lines M5, LOVO, HCT116, SW480 and human normal cell lines NCM460 (Control), were obtained from ATCC cell bank (America). Cell lines were then cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, NY, USA), 100 mg/mL streptomycin and 100 U/ml penicillin (Invitrogen, Carlsbad, CA, USA), in an incubator at 37 ${}^{\circ}$ C with 5% CO ${}_{2}$ .

2.3 Cell transfection

According to the experiment instruction, the sequence of SNHG1 was cloned into vector. Human SW480 cell was transfected with si-SNHG1 using Lipofectamine 2000 (Invitrogen) according to the manufacture’s instruction. The transfected cells were grown on six-well plates for culturing. After 24 h, cells were collected for real-time PCR to determine the transfect efficiency. The sequence of si-SNHG1 was: 5’-GACCUAAGCUUGUUGCCAAUTT-3’.

2.4 RNA extraction and real-time PCR

Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, USA), according to the manufacturer’s instruction. RNA quality was tested by nanodrop with the OD value of A260/A280. Subsequently, 2 $\mu$ l RNA was taken out and reversely transcribed into cDNA with PCR Reverse Transcriptase instrument (Eppendorf). The target genes expression was determined using a standard SYBR-Green method on DA7600 Real-Time PCR System. All reactions were performed in a 96-well plate, and ran with specific primers for three independent times based on a preheated real-time instrument (ABI 7500/7900HT, invitrogen, USA). The primers sequences were shown in the following: Forward: 5’-AGGCTGAAGTTACAGGTC-3’, Reverse: 5’-TTGG CTCCCAGTGTCTTA-3’.

2.5 Cell viability

The MTT was used to evaluate cell viability at 24, 48, 72 and 96 h after transfection according to the manufacturer’s instruction. A total of 1 $\times$ 10 ${}^{5}$ cells were planted in a 96-well plate and maintained in the DMEM (Dulbecco Modified Eagle Medium) including 10% of NCS (Newborn Calf Serum) for 12 h, and then 20 $\mu$ l of 5 mg/ml MTT solution were placed in each well for 4 h. Next, OD value was tested with the wave light of 490 nm. Finally, cell growth curve was drawn based on the OD values.

2.6 Cell invasion

Transwell chamber (8- $\mu$ m pore size; Millipore, MA, USA) was used for detecting of cell invasion. A total of 1 $\times$ 10 ${}^{5}$ cells were planted in the upper chamber all around. The upper chamber filled in 200 $\mu$ L SFM(serum-free), and the lower chamber filled in 800 $\mu$ L of the medium with 20% FBS. After incubation for 24 h, the cells were embathed, fixed and stained, finally, a microscope was used for the visualization.

2.7 Flow cytometric analysis of cell apoptosis

Transfected human CRC cells were obtained and stained by Annexin V-FIT and propidium iodide, and then evaluated by flow cytometer equipped with a Cell Quest software (BD Biosciences) according to the instructions. Finally, the apoptotic cells were assessed.

Figure 1.

The expression level of SNHG1 in CRC. A: SNHG1 expression significantly increased in tissue with CRC compared with the normal tissue. B: Higher expression of SNHG1 in 3/4 cancer stage than other cancer stages. C: Higher expression of SNHG1 in metastasis than non-metastasis. D: Relative expression level of SNHG1 in CRC cell lines, including NCM460 (Control), M5, LOVO, HCT116 and SW480 cell lines. ** $P<$ 0.01, *** $P<$ 0.001.

Figure 2.

Down-regulation of SNHG1 inhibits CRC cells colony formation and promotes cell apoptosis. A: SW480 cell line transfected with si-RNA1 or si-RNA1 $+$ 2 significantly decreased SNHG1 expression. B and C: SW480 cells transfected with si-RNA significantly suppressed cells proliferation. D and E: Annexin V assay revealed cell apoptotic ability with cells transfected with si-RNA. * $P<$ 0.05, ** $P<$ 0.01.

2.8 Wound scratch assay

For the migration assay, about 1 $\times$ 10 ${}^{5}$ cells were planted into 96-well plate for growing. The samples were taken out at 24 h and scratched, then generated with a 200 $\mu$ l disposable pipette tip. A Nikon inverted microscope (Nikon, Tokyo, Japan) was used for photographing the scratch wounds, and the ImageJ software (rsbweb.nih.gov/ij) was used to quantify the scratch widths. Finally, the data was plotted and analyzed.

2.9 Soft-agar colony-forming assays

The upper agar was prepared by 0.7% agars with 20% NBCS. The lower agar was prepared by 1% agars with 20% NBCS and added the cell suspension. The cells were incubated at 37 ${}^{\circ}$ C with 5% CO ${}_{2}$ for 21 days. Finally, the cells were stained by 0.5 mL of 12% crystal violet, and colony number was calculated.

2.10 Western blot assay

Protein extracted from cells were separated by 10% SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis), an equal amount of protein extracts were transferred onto polyvinyl lidenedifluoride membranes. Concentration of proteins was determined by a Bio-Rad protein assay kit. The membranes were incubated with primary antibodies at 4 ${}^{\circ}$ C for 24 h. Then the blots were incubated with fluorescence-labeled secondary antibodies in 5% bovine serum albumin for 1 h at 37 ${}^{\circ}$ C. Next, the protein was observed with electrochemiluminescence. Finally, the intensity of the bands was quantified by time-lapse imaging.

2.11 Transplanted tumor model establishment

All female mice were purchased with 4 $\sim$ 6 weeks old, and randomly divided into two groups ( $n=$ 20 of each group). SW480 cell line stably knocked down for SNHG1 expression were implanted in the mice subcutaneously, and the mock group were implanted without si-SNHG1. The tumor volume was assessed every 4 days. After 24 days, all mice were sacrificed and the tumors were taken out for the next experiment. The tissues were fixed and stained for microscopic examination. The terminal-deoxynucleoitidyl transferase mediated nike end labling (TUNEL) was used for apoptosis analysis. The experiments were approved by Animal Ethics Committee of The First Affiliated Hospital and Institute of Clinical Medicine, Zhengzhou University. All experiments in this study were abided the guidance of Laboratory Animal Care and Use of The First Affiliated Hospital and Institute of Clinical Medicine, Zhengzhou University.

2.12 Statistical analysis

Data were expressed as means $\pm$ SD. All the Statistics were performed using the SPSS 13.0 software with the ANOVA (analysis of variance). * $P<$ 0.05 was considered as statistically significant difference.

Figure 3.

Down-regulation of SNHG1 inhibits cell migration and invasion of CRC. A and B: Down-regulation of SNHG1 inhibits cell migration. C and D: Down-regulation of SNHG1 inhibits cell invasion. ** $P<$ 0.01, *** $P<$ 0.001.

3. Results

3.1 SNHG1expression in tissues and cell lines of CRC

To identify the relationship between tumor growth and SNHG1, we examined different expression level of SNHG1 in patients’ tissues of CRC and matched tissues of non-cancer. The expression of SNHG1 was obviously higher in CRC tissues than non-cancer tissues (Fig. 1(A)). As is presented in Fig. 1(B), the expression of SNHG1 was markedly higher in 3/4 stage of cancer than other stages. Moreover, the expression of SNHG1 was also analyzed in CRC tissues with or without metastasis. We found that, compared with the non-metastasis, a significantly higher SNHG1expression in metastasis group (Fig. 1(C)).

Next, M5, LOVO, HCT116, SW480 and Control were applied to evaluate the expression of SNHG1, as shown in Fig. 1(D), the higher expression of SNHG1 in SW480 cell line compared with the other 4 cell lines. Thus SW480 cell line was chosen for the subsequent experiments.

3.2 Down-regulation of SNHG1 inhibits cell proliferation

To further examine whether SNHG1 influence the gene expression, we performed SW480 cell lines to measure the function of SNHG1. For example, according to Annexin V-FITC dual staining, si-SNHG1 (si-RNA1, si-RNA2, si-RNA1+2) interference could significantly inhibit the expression of SNHG1, especially si-RNA2 and si-RNA1+2, which showed no difference on SNHG1 expression level (Fig. 2(A)). Cell proliferation, which commonly measured via MTT method and colony numbers, was examined in this study. We found that, down-regulated SNHG1 could significantly reduce the absorb value (Fig. 2(B)) and decrease the colony numbers (Fig. 2(C)). Meanwhile, the cell apoptosis was increased with the down-regulation of SNHG1 (Fig. 2(D) and (E)). All the data demonstrated that down-regulated SNHG1 could inhibit cell proliferation and promote cell apoptosis.

Figure 4.

Down-regulation of SNHG1 suppressed CRC cells in vivo. A: photographs of the tumor with injection si-SNHG1 or Mock. B: The volumes of the tumors were calculated every 4 days. C: The tumor weights were measured after the mice were sacrificed. *** $P<$ 0.001.

3.3 Down-regulation of SNHG1 inhibits cell migration and invasion

To further investigate the molecular mechanism of SNHG1 in CRC cell invasion and migration, we used Transwell and scratch assay to detect them. According to Fig. 3, we observed that down-regulation of SNHG1 could significantly decrease the cell migration (Fig. 3(A) and (B)) and invasion (Fig. 3(C) and (D)).

3.4 Down-regulation of SNHG1 inhibits tumor growth in vivo

We then examined whether SNHG1 promoted tumor growth in vivo. Firstly, SW480 cells transfected with si-SNHG1were injected into the nude mice, after 25 days, all the mice were sacrificed and the tumors were taken out, the volumes and weights of the tumors were measured. The tumor photographs were shown in Fig. 4(A). As a result, both the volumes and the weights were decreased with si-SNHG1 interference (Fig. 4(B) and (C)), which indicated that down-regulated SNHG1inhibits tumor growth in vivo.

4. Discussion

Colorectal carcinomas is the most important death-related cancer worldwide, the patients without nonspecific symptoms at early stage and most of them lost the best opportunity of surgical therapy when detect at an advanced stage [14]. However, to find molecular mechanism and therapy method early for its diagnosis are necessary. In recent years, lots of studies have reported that the expression of lncRNA is related to human cancer [15, 16]. Researchers have tried to explore the value of SNHG1 in CRC, but so far, little information has been documented. In this study, we figured out the relationship between SNHG1 and colorectal carcinoma.

SNHG1 was a gene that plays an important role in RNA regulation processes. Previous studies have reported that SNHG1 may be a therapeutic biomarker and therapeutic target for several cancers, such as hepatic cellular carcinoma (HCC) and lung cancer intervention [12, 13, 17]. In this study, SNHG1 expression was measured to evaluate the functional response of CRC. SNHG1 expression was found to be increased at a high level in CRC tissues and was also significantly associated with cancer stage as well as the cell metastasis and cell lines, indicating that SNHG1 might exhibit a vital role in CRC carcinogenesis.

Then the functions of CRC proliferation, apoptotic, migration and invasion were determined. We found that down-regulated SNHG1 significantly impaired cell proliferation, which related to tumor growth in vivo, but promoted cell apoptosis. The cell invasion and migration, which generally regard as a rule of measuring the tumor cells malignant capacity [18], were notably decreased in our research, revealing that down-regulation of SNHG1 would inhibit the malignant ability of tumor cells. In vivo experiments demonstrated that down-regulation of SNHG1 significantly reduced tumor sizes and tumor weights, indicating SNHG1 played an important role in tumor growth. Previous study has reported that SNHG15 plays an important role in many aspects of biology, including cell proliferation, differentiation, and other aspects as remodeling of the extracellular matrix and cell metastasis [19]. The findings might indicate that the series of SNHG are important biomarkers in tumor oncogenesis.

In summary, this report suggested that down- regulated SNHG1 might closely associate with CRC occurrence and development. We preliminarily suggested that SNHG1 might act as an important biomarker of CRC. Further therapy of CRC on effectively controlling of SNHG1 might be critical to prevention of oncogenesis.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

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