Study of Long Non-Coding RNA Tug 1 Expression in Egyptian Colorectal Adenocarcinoma Patients

ABSTRACT

Purpose:

Colorectal cancer (CRC) is one of the most fatal tumors worldwide. In Egypt, most CRC cases occur in individuals > 40 years old. TUG1 has been proved to be disrupted in different malignancies and may have a critical role in tumor progression, invasion, and metastasis. However, its role in CRC has not been adequately studied.

Materials / Methods:

Quantitative real-time polymerase chain reaction (PCR) was used to evaluate the expression levels of long non-coding RNA (LncRNA) taurine upregulated gene 1 (TUG1), in nonmetastatic and metastatic CRC tissues and adjacent noncancerous tissues as control.

Results:

LncRNA TUG1 expression was significantly upregulated in both nonmetastatic and metastatic CRC tissues, in comparison with the adjacent noncancerous tissue. It was found that TUG1 could have a possible prognostic role in CRC, by comparing the sensitivity and specificity of TUG1 with those of CEA and CA19–9.

Conclusion:

The results of the current study suggest that the LncRNA TUG1 participates in the malignant behaviors of CRC cells.

Keywords:
colorectal adenocarcinoma; CRC; LncRNA; TUG1; PCR

Introduction

Colorectal cancer (CRC) is ranked as the third most common cancer globally and second in cancer-related mortality. A total of 90% of CRC cases are diagnosed over the age of 55 years old.¹ It is a multistep malignancy in which genetic and epigenetic alterations accumulate.² Genetic diseases are familial adenomatous polyposis (FAP), hereditary non-polyposis CRC (HNPCC) and Gardner syndrome, while post-translational histone acetylation and methylation are epigenetic factors.³ Obesity, smoking, and chronic alcoholism are environmental risk factors.⁴

Noncoding RNAs have been widely studied as biomarkers in the context of many diseases with a focus on lncRNAs and miRNAs. LncRNAs are nonprotein coding transcripts > 200 nucleotides, that have an epigenetic effect in CRC.⁵

Taurine upregulated gene 1 (TUG1) is a lncRNA (~ 7.1 kb in length) located on chromosome 11. Previous studies expected TUG1 to be a new diagnostic biomarker and therapeutic target of certain cancers. TUG1 has been proved to act as a miRNA sponge (ceRNA) to regulate mRNA expression of the target gene and govern the progression of different cancers such as breast, bladder, and renal cell carcinoma (RCC).⁶,⁷,⁸,⁹

Several studies demonstrated that TUG1 knockdown in different cancer tissues such as urinary bladder carcinoma and hepatocellular carcinoma (HCC) suppressed cell proliferation, invasion and EMT.¹⁰,¹¹,¹² Moreover, LncRNA TUG1 has been shown to potentiate cancer metastasis and tumor progression in gastric carcinoma in ovarian mucinous adenocarcinomas.¹⁰ The previous findings show the regulatory roles of the TUG1 cancer progression. Our work suggests that TUG1 could have a potential role in the epithelial to mesenchymal transition of cancer in CRC tissue samples.

Materials and Methods

The present study included 65 subjects divided into 3 groups. Group I: 25 nonmetastatic colorectal adenocarcinoma tissue samples; group II: 25 metastatic (locoregional or blood born) colorectal adenocarcinoma tissue samples; and group III: 15 matched adjacent noncancerous healthy tissues. Pathological diagnosis of CRC is performed by biopsy of the mass suspected of tumor development. Disease extent is determined by imaging. Staging is done based on the TNM system. All samples were obtained from the Department of General Surgery, colorectal surgery unit, Alexandria main university hospital.

Samples (~ 0.4cm × 0.2 cm of tissue) were excised from cancer tissue and adjacent noncancerous tissue as control. Each sample was then divided into 2 sections; 1 was submerged in RNA later (Rnase inhibitor) (ThermoFisher Scientific)¹³; and was kept frozen at - 80°C until use. The samples were fixed in 10% formalin solution for 24 hours. they were processed for light microscopic study to obtain paraffin blots (Formalin Fixed and Paraffin Embedded [FFPE]). Five microns of sample thickness were cut and mounted on glass slide then stained using Hematoxylin and eosin stain (►Fig. 1).

Fig. 1
Pathological examination (formalin fixed and paraffin embedded [FFPE]).

Each 30-mg tissue sample was homogenized in a 2 ml sterile tube with 700 uL Qiazol solution using an electric homogenizer at 3,000 rpm.¹⁴

Total RNA isolation from tissue samples was conducted by using the Qiagen miRNeasy Mini Kit (Qiagen, CA) according to the instructions of the manufacturer. (ID: 217004).¹⁴

Single-stranded cDNA was synthesized from purified samples of RNA using High-Capacity cDNA Reverse Transcription Kit. (Applied Biosystems, USA) (Cat. No. 4368814 Archive).¹⁵ The purity and the concentration of RNA were measured at 260, 280 and 230 nm by using the Thermo Scientific, Nano-Drop 2000/2000c Spectrophotometer (USA) Ratios of A₂₆₀/ A₂₈₀ and A₂₆₀/A₂₃₀ = 1.8–2.1 indicates the high purity of RNA.

Relative quantification of tissue lncRNAs TUG1 genetic expression: real-time polymerase chain reaction (PCR) was performed using Thermo Scientific Maxima SYBR Green qPCR Master Mix (2X) (Thermo Scientific), and specific primers for lncRNA TUG1. Sequences used are TUG1 forward primer (5'-CTGAAGAAAGGCAACATC-3') and reverse (5'-GTAGGCTACTACAGGATTTG-3').¹⁶ Primers were revised using primer blast system. GAPDH was used as internal control to normalize the expression of TUG1. The used GAPDH forward primer is 5'-GTCTCCTCTGACTTCAACAGCG -3' and reverse primer is 5'-ACCACCCTGTTGCTGTAGCCAA – 3'.¹⁷

Real time PCR was done using Applied Biosystems Ste-pOne Real-time PCR System. (Cat. No. 4376357).¹⁸

Reagents were purchased from Applied Biosystems, USA. The calculation of RNAs’ expression was done using the comparative cycle threshold (CT) method (2-^ΔΔCT).

Results

For statistical analysis, SPSS Statistics for Windows, Version 17.0 (SPSS Inc, Chicago, IL, USA) was used. The significance of the differences between the two groups was estimated with the Student t-test. Multiple group comparisons were analyzed by one-way analysis of variance (ANOVA). The age of the patients ranged from 40 to 83 years old. Thirty patients were males with a percentage of 46.15% and 35 were females with percentage 53.85% (►Table 1).

Table 1
Sex distribution of the 3 studied groups

	Group I (n = 25)		Group II (n = 25)		Group III (n = 15)		Total
	No.	%	No.	%	No.	%	No	%
Sex
Male	14	56.0	10	40.0	6	40.0	30	46.15
Female	11	44.0	15	60.0	9	60.0	35	53.85

The correlation between sex and tissue TUG1 expression in patients of groups I and II was statistically insignificant (►Table 2).

Table 2
Relation between Sex and different measurements in group I and II

	Sex
Group I	Male (n = 14)	Female (n = 11)	p-value
TUG1
Median (Min – Max.)	2.83 (1.62–4.40)	2.04 (1.74–4.88)	0.344
	Sex
Group II	Male (n = 10)	Female (n = 15)	p-value
TUG1
			0.605
Median (Min. – Max.)	5.34 (2.09–8.03)	4.46 (2.04–7.98)

• p-value for comparing between Male and Female.

Analysis of LncRNA TUG1

To discover the role of TUG1, its level was measured in CRC tissues and corresponding non-cancerous tissues and results showed that TUG1 was upregulated in CRC tissues with a strong significant difference between patients of group I and II and the control group (p < 0.001) (►Table 3 ►Fig. 2A). Also, LncRNA TUG1 expression was significantly higher in patients of group I in comparison with samples of group II. (p₁ = 0.002). LncRNA TUG1 expression was significantly higher in patients of stage I in comparison with the control group (Group III) (p₂ < 0.001). A meaningful change was found when comparing the TUG1 expressions in cases of stage II and the control group. (p₃ < 0.001).

Table 3
Comparison between the 3 studied groups according to TUG1 expression

	Group I (n = 25)	Group II (n = 25)	Group III (n = 15)	p-value
TUG1
Min. – Max.	1.62–4.88	2.04–8.03	0.04–3.33	< 0.001*
Median (IQR)	2.71 (1.97–3.38)	4.46 (2.96–6.44)	0.73 (0.49–2.28)
Sig. bet. groups.	P1 = 0.002*, P2 = 0.010*, P3 < 0.001 *

• Abbreviation: IQR, interquartile range.
• ^*
  Statistically significant at P ≤ 0.05.
• Group I: Nonmetastatic colorectal adenocarcinoma.
• Group II: Metastatic (locoregional and blood born) colorectal adenocarcinoma.
• Group III: Noncancerous control colon tissue.

Fig. 2
Expression levels of TUG1, TUG1 was upregulated in CRC tissues with strong significant difference between patients of group I and II and control group. TUG1 expression was significantly higher in patients of group I in comparison with samples of group II.

Correlation Studies

1. – Correlation between tissue TUG1 expression and of age, hemoglobin, CEA, and CA19.9 serum levels in patients of group I were statistically insignificant (p = 0.760, 0.473, 0.507, 0.493, respectively).

1. – Correlation between tissue TUG1 expression and age, hemoglobin, CEA, and CA19.9 serum levels in patients of group II was statistically insignificant (p = 0.322, 0.719, 0.565, 0.685, respectively) (►Table 4).

Table 4
Correlation between LncRNA TUG1 and age, hemoglobin, CEA and CA19.9 in Group I & II

	TUG1
	Group I		Group II
	rs	p-value	rs	p-value
Age (years old)	- 0.064	0.760	0.206	0.322
HB	0.150	0.473	- 0.076	0.719
CA19–9	0.139	0.507	0.121	0.565
CEA	0.144	0.493	- 0.085	0.685

• Abbreviations: HB, hemoglobin.
• r_s: Spearman coefficient.
• *: Statistically significant at p ≤ 0.05.
• Group I: Nonmetastatic colorectal adenocarcinoma.
• Group II: Metastatic (locoregional and blood born) colorectal adenocarcinoma.

Discussion

Colorectal cancer has become a main health problem and one of the most fatal malignancies, and its incidence is increasing worldwide. Colorectal cancer is usually accompanied by distal metastasis (liver or lung), and it is associated with increased mortality rate.¹⁹ Therefore, there is an urgent necessity to discover the molecular mechanisms of CRC progression.²⁰

Long noncoding RNAs (lncRNAs) are RNAs with a length > 200 nucleotides. They participate in various biological processes, such as chromatin remodeling, transcriptional activation or interference, and they are involved in the occurrence of CRC by acting as tumor suppressor genes and oncogenes.²¹

Several studies have documented a tumor-promotive function of TUG1 in different malignancies, especially CRC. However, the mechanisms by which it exerts its role in CRC remain undefined.²²

Our study revealed that TUG1 is upregulated in CRC. Wang et al.²³ reported that the LncRNA TUG1 upregulated in CRC promoted CRC progression and 5-fluorouracil (5-FU) resistance by sponging miR-197–3p. Also, Shen et al.²⁴ found that the decrease in LncRNA TUG1 inhibited CRC tumor cell migration, invasion, and EMT, and has a major role in reducing lung metastasis.

TUG1 plays a main role in regulating different cancer types by functioning as a ceRNA as in oral squamous cell carcinoma by sponging mir-593–3p as reported by Jiang et al.²⁵ and, in case of osteosarcoma, Farzaneh et al.²⁶ expressed that MALAT1 LncRNA has been found to regulate CDK9 expression through sponging miR-206 and it can also interact with miR-202 and promote lung metastasis.

Tian et al. declared that the increase in TUG 1 expression has been shown to enhance CRC cell proliferation, invasion, and EMT in vitro, through promoting SW620 cell motility by decreasing miR-26a-5p activity and upregulating MMP-14. Moreover, TUG1 promoted carcinogenesis and EMT in colon cancer by stimulating the P38MAPK/Hsp27 axis.²⁷

However, Barbagallo. et al analyzed via RT- PCR the expression of 17 lncRNAs in 20 CRC tissues compared with noncancerous adjacent tissues, and in serum exosomes of these 20 CRC patients compared with 20 healthy individuals identified 8 ncRNAs (including TUG1) differentially expressed in tissues while in serum exosomes of CRC patients was downregulated.²⁸

Conclusion

TUG1 was upregulated in CRC tissues and cells. Its effects are on proliferation and apoptosis of cancer cells. Collectively, the present study demonstrated that TUG1 overexpression induces proliferation and inhibits apoptosis in CRC. This possible molecular mechanism provides a theoretical basis for the research on lncRNA-directed therapeutics in CRC.

Availability of Data and Materials

The data supporting the conclusions are included within the article.

• Funding
  The researchers did not receive any specific grant from funding agencies in the public or not for profit sections.

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Publication Dates

• Publication in this collection
  23 Oct 2023
• Date of issue
  2023

History

• Received
  14 Apr 2023
• Accepted
  21 June 2023