Overexpression of TPT1-AS1 and SAMMSON and down expression of LINC00961 associated with Advanced Grades of Gastric Cancer CURRENT

Background One of the deadliest cancers in the world is gastric cancer. Long non-coding RNAs play prominent roles in cancer. LINC00961, TPT1-AS1, and SAMMSON have recently been discovered, which significantly contribute in various cancers and can affect the tumor size, grade of tumors and the metastasis condition. The aim of this study was to determine LINC00961, SAMMSON and TPT1-AS1 expression in gastric cancer tissues in comparison with healthy adjacent tissues. Methods The number of cancerous tissues and control groups was calculated to be at 40 (n = 40) and were analyzed by Quantitative real-time polymerase chain reaction. We found that overexpression of TPT1-AS1 and SAMMSON, and downexpression of LINC00961 in cancerous tissues in comparison with healthy adjacent tissues. A positive association between TPT1AS1 and SAMMSON expression and tumor grade was observed. The level of mRNA folding change increased in cancer group compared to control group and *P < 0.05 is considered for mRNA folding change. Finally, we found that overexpression of TPT1-AS1 and SAMMSON, and downexpression of LINC00961 were observed significantly in gastric cancer tissues in comparison with adjacent non-cancerous tissues. These lncRNAs were suggested as potential tumor markers for the diagnosis and treatment of gastric cancer.


Abstract Background
One of the deadliest cancers in the world is gastric cancer. Long non-coding RNAs play prominent roles in cancer. LINC00961, TPT1-AS1, and SAMMSON have recently been discovered, which significantly contribute in various cancers and can affect the tumor size, grade of tumors and the metastasis condition. The aim of this study was to determine LINC00961, SAMMSON and TPT1-AS1 expression in gastric cancer tissues in comparison with healthy adjacent tissues.

Methods
The number of cancerous tissues and control groups was calculated to be at 40 (n = 40) and were analyzed by Quantitative real-time polymerase chain reaction.

Results
We found that overexpression of TPT1-AS1 and SAMMSON, and downexpression of LINC00961 in cancerous tissues in comparison with healthy adjacent tissues. A positive association between TPT1-AS1 and SAMMSON expression and tumor grade was observed. The level of mRNA folding change increased in cancer group compared to control group and *P < 0.05 is considered for mRNA folding change.

Conclusion
Finally, we found that overexpression of TPT1-AS1 and SAMMSON, and downexpression of LINC00961 were observed significantly in gastric cancer tissues in comparison with adjacent non-cancerous tissues. These lncRNAs were suggested as potential tumor markers for the diagnosis and treatment of gastric cancer.

Background
Gastric cancer is one of the most dangerous malignancies in the world. Especially, in the eastern regions of Asia. Each year, the death toll is estimated to be around 950,000 in the world, accounting for almost half of the prevalence in the East Asia [1].Although the prevalence and mortality rate is reduced by this malignancy, but gastric cancer is the third cause of cancer-associated deaths worldwide [2]. Most patients with gastric cancer are identified in advanced stages of the disease.
Although specific diagnostic and treatment methods for gastric cancer have been used, the survival rate of these patients is about 5 years, which is a very low survival rate [3][4][5][6][7]. Today, certain tumor 3 markers are used such as CA19-9 and carcinoma embryonic antigen (CEA) to diagnose gastric cancer, but they have no specificity to diagnose this malignancy [8].
Recently, non-coding RNAs such as long non-coding RNAs (lncRNAs) have been used as diagnostic tumor markers as well as therapeutic targets for malignancies [9,10]. Today, it has been discovered that TPT1-AS1 can involve in various cellular processes, such as cellular differentiation, proliferation, migration, apoptosis, invasion and stem-cell biology [9]. For example, in people with non-small cell lung cancer (NSCLC), lncRNAs can play important roles in NSCLC tumorigenesis and progression [11].
LncRNA tumor protein translationally controlled 1 (TPT1) antisense RNA 1 (TPT1-AS1) is one of the latest LncRNAs that has recently been investigated in gliomas and cervical cancer. It has been observed that increased expression of this lncRNA can increase the tumor size, proliferation, metastasis, and more difficult treatment [9].TPT1-AS1 is associated with one of transcription factors which are associated with the transcription of some mitochondrial genes, and thus TPT1-AS1 can affect the regulation of the expression of these genes [9,12]. However, there are few studies on this lncRNA. The next lncRNA, which was measured in this study, is SAMMSON. This lncRNA is associated with the Wnt / B Catenin pathway, and it has been shown that by increasing the expression of this lncRNA, the pathway in cancer cells can be activated and leads to the growth, metastasis and self-renewal of these cells [13,14]. According to the research, this lncRNA can make an important contribution in other cellular pathways.
Another lncRNA, which has been considered previously, is LINC00961. This lncRNA also plays a role in various cancer processes and this lncRNA affects a polypeptide called SPAR, which can inhibit mTORC1 [15]. It has been shown that the increase of LINC00961 expression can inhibit the metastasis and proliferation of the cancerous cells [11].

Patients And Tissue Specimens
In this study, tissue samples have been taken from the Cancer Institute of Imam Khomeini Hospital in Tehran. Patients' information and the written informed consent from participation were prepared by the center. Our exclusion criteria include any chemotherapy, radiotherapy, other cancers with gastric cancer and chronic or acute inflammatory diseases, and its progression confirmed by the pathologist by the relevant protocols. Cancer tissues and adjacent healthy ones were taken by the surgeon from 40 patients, and each of these specimens was divided into two parts. One part was immediately placed in liquid nitrogen and used for RNA extraction. cDNA generation and QRT-PCR technique were used to determine the expression of the related lncRNAs genes, and the other part was laid down in formalin for pathological examinations. Demographic information such as age, gender, site of primary neoplasm, date of diagnosis, race, marital status, family history of cancer, history of disease, specific drug use, were recorded for each patient.

Data Analysis
Statistical analysis was carried out using the Statistical Package for Social Sciences version 16 (SPSS Inc., Chicago, IL). In order to compare the expression ratio of these genes in two groups, the distribution of data using the Kolmogorov-Smirnov test was examined and according to the proportions of the results of parametric and non-parametric tests (Student t tests and Mann Whitney).
In this study, the significance level p was less than 0.05. The Pearson and Spearman tests were used to examine the relationship between variables in terms of parametric and non-parametric proportions.

Characteristics Of The Participants
Properties of the patients are showed in Table 1. All study participants were Iranian, and the average age was 62 years (33 to 85). Twenty-nine (72.5%) were male patients, and 11 (27.5%) were female patients. In TNM staging, patients with low stage (I & II) were 5 (12.5%) and with high stage were 35 (87.5%). In this study, patients with adeno-carcinoma were 29 (72.5%) and non-adenocarcinoma was 11 (27.5%). In addition, primary site of neoplasm was determined in these patients and include 10 (25%) in Cardia, 10 (25%) in Antrum and 20 (50%) in the body of the stomach. Table 1 Clinicopathological characteristics of the participants. Quantitative Rt-pcr Analysis TPT1-AS1 expression In this study, to demonstrate the importance of Lnc RNA TPT1-AS1 in cancerous tissues of gastric samples, we evaluated TPT1-AS1 expression in 40 cancerous tissues (n = 40) and observed that the TPT1-AS1 RNA levels significantly increased compared to the adjacent non-cancerous tissues (n = 40) (p < 0.05 Fig. 1-a).

Sammson Expression
We measured SAMMSON expression in cancer tissues (n = 40) and observed that the RNA levels enhanced compared to non-cancer tissues (n = 40) (p < 0.05 Fig. 1-b).

Linc00961 Expression
According to the LINC00961 expression and our findings, the RNA levels in gastric cancer tissues (n = 40) decreased significantly in comparison to the adjacent non-cancerous tissues (n = 40) (p < 0.05

Discussion
Gastric cancer is one of the most dangerous and fatal cancers in the world, which is ranked third in terms of the deadliest among all types of neoplasms. This type of neoplasm is increasing in the world today, and different cognitive ways have been expressed. However, extensive studies are required to identify and cure it [17,18].
Long non-coding RNAs (lncRNAs) contribute greatly in many diseases, for example cancer. Genomic studies of transcriptomes (a study of all coding and non-coding RNAs) have shown that transcription is abundant in the mammalian genome, which at least 80% of this transcription is exclusively related to non-coding RNAs (ncRNAs) [19]. ncRNAs are classified into two groups based on their sequencing length: short ncRNAs with less than 200 nucleotides (sncRNAs) and long non-coding RNAs with more than 200 nucleotides (lncRNAs). Until now, according to numerous studies, more than 58,000 lncRNAs important role of lncRNAs in many diseases, especially cancer, has been reported in numerous studies [20]. Recently, a number of lncRNAs have proven to play a significant part in the proliferation, cell cycle, apoptosis, invasion, migration, metastasis and tumorigenicity of gastric cancer cells [21]. As previously mentioned, less than thousands of lncRNA mechanisms have been discovered. Recently, three lncRNAs have been identified including TPT1-AS1, SAMMSON and LINC00961, which have been subjected to limited studies on their action mechanism in cancer.
TPT1-AS1 is one of the most recent lncRNAs that is located on the 13q14.13 gene. This lncRNA has recently been investigated in glioma and cervical cancer. It is observed that increased expression of this lncRNA can enhance the tumor size, proliferation, metastasis, and requires treatments that are more difficult. [9]. On the other hand, this lncRNA is associated with a protein called SP1 [9]. SP1 along with zinc finger protein 179 (ZnF 179) create a route which can affect the protection against oxidative stress and mitochondria-induced ROS. This protein is one of the factors involved in transcription and also regulates the expression of some subunits in the complex of electron transport chain [12]. It has been observed that overexpression of TPT1-AS1 can increase the expression of gene and SP1 protein, which eventually results in the disruption of the electron transfer chain, ultimately 8 damaging the mitochondria and cells and exacerbating oxidative stress condition [9].
SAMMSON locus is located on the chromosome 3p13. This lncRNA is associated with the Wnt/B Catenin pathway. It has been shown that increased expression of this lncRNA can activate the pathway in cancerous cells and accelerate the growth of the cells, metastasis, and self-renewal of these cells [14]. This lncRNA is also associated with a mitochondrial protein called P32, which is effective both in the integrity of the mitochondrial structure and in the production of energy through oxidative phosphorylation. It can also contribute to the progression of certain cancers and the transformation of the cell metabolism from oxidative phosphorylation to glycolysis and the increased oxidative stress, due to its ineffectiveness and lack of proper production [22]. SAMMSON, as mentioned above, is associated with this protein, and by deactivating P32, it can disrupt the mitochondrial function and structure and also leads to an oxidative stress condition[23] [14].
The ROC curve results showed a relatively appropriate specificity and sensitivity for SAMMSON and LINC00961 RNA levels in cancerous and non cancerous tissues, indicating that these two genes expression levels may be used for gastric cancer diagnosis.
As it is shown in our finding, overexpression of TPT1-AS1 and SAMMSON have a significant association with the advanced grades of cancerous tissues. According to the previous studies, increased expression of these lncRNAs may enhance the growth of the cancer cell and tumor tissue proliferation. It can be concluded that TPT1-AS1 and SAMMSON take a leading role in the development of gastric cancer, especially in the higher grades, and increased expression of these genes can be considered as a diagnosis pathway in gastric cancer. In the future, it can be used to treat this lethal cancer by inhibiting lncRNAs. On the other hand, increased expression of these genes can damage mitochondria and increase oxidative stress circumstance.
Another lncRNA, which has been investigated in this study, is LINC00961. This lncRNA, which is located on the 9p13.3 gene, significantly contribute in various cancer processes and has 1546 nucleotides. This lncRNA acts on a polypeptide called Small Regulatory Polypeptide of Amino Acid Response (SPAR), which can inhibit (mammalian Target Of Rapamycin Complex 1) mTORC1, but nevertheless this lncRNA can affect the activity of mTORC1, and this complex can also control mitochondria in electron transport chain complex proteins and ATP production and consumption [15].
It is expected to modify the toxicity of mitochondrial oxidative stress and contribute to the onset of cancer. It has been shown that increased expression of LINC00961 can have an inhibitory role in the metastasis and proliferation of cancerous cells [11].
Based on the findings of the present study, a significant overexpression of TPT1-AS1 and SAMMSON in cancerous tissues has been shown in comparison with healthy adjacent tissues. On the other way, (TSCC) and observed that the expression of LINC00961 has significantly decreased and this lncRNA, with its own function, inhibits the growth, proliferation and metastasis of cancerous cells by regulating the Wnt/β-Catenin signaling pathway [26].
Considering these functions of the lncRNAs mentioned above, if a mutation takes place in the expression of these lncRNAs, it could affect different signaling pathways. With the effect on various genes and proteins presented in these pathways, they can contribute to the progression, invasion and metastasis in cancerous cells, which is due to the increased expression of TPT1-AS1 and SAMMSON and reduced expression of LINC00961 [9,11,12]. In this study, using genetic techniques and biochemical tests, it has been found that overexpression of TPT1-AS1 and SAMMSON and down expression of LINC00961 could increase proliferation, invasion and metastasis in cancerous cells compared to healthy subjects. According to the previous studies and the present study, it can be concluded that these important lncRNAs may cause gastric cancer and can be used as tumor markers of this cancer. However, more research and large numbers of samples are required to reveal the exact mechanisms of this LncRNAs and better understanding of their potential use in gastric cancer.

Conclusion
Finally, according to our findings, we observed that in patients with Gastric Cancer compared to group control, the increased expression of TPT1-AS1 and SAMMSON genes and down expression of LINC00961 gene and changes in the different signaling pathways, which can ultimately cause gastric cancer to start and exacerbate. However, we can recognize one of the causes of the promotion and exacerbation of this fatal cancer as a disorder in expression and function of TPT1-AS1, SAMMSON and LINC00961 genes, and we introduce these lncRNAs as tumor markers for gastric cancer but in this study is required more researches to confirm it.