Exploring the role of miR-361-3p in gastric cancer therapeutics and tumor progression

Abstract

Background: Gastric cancer (GC) remains a leading cause of cancer-related deaths globally. Even with advancements in treatment, it is not known precisely which molecular pathways cause GC to proceed. MicroRNAs, often referred to as small noncoding RNAs, have a crucial role in regulating gene expression, which impacts cancer growth, metastasis, and treatment resistance. Among these, miR-361-3p has drawn interest due to its possible role in the development of human cancer. While accumulating evidence highlights miR-361-3p involvement in various cancers, its precise biological function in gastric cancer remains largely unclear. Methodology: The expression levels of miR-361-3p in gastric cancer tissues were compared with those of adjacent non-cancer tissues using real-time quantitative PCR (qRT-PCR). The investigation involved a thorough examination of the expression of miR-361-3p in GC samples in order to identify any possible correlations with clinicopathological characteristics. Results: In 64.86 % of gastric cancer, expression levels of miR-361-3p mRNA were significantly reduced, which indicates that it may play a potential role in the pathophysiology of GC. However, no significant association has been found between miR-361-3p expression levels and clinical pathological features, such as tumor size, phase, or involvement of lymph nodes. This suggests that although miR-361-3p may contribute to GC progress, it is not related to traditional clinical markers.

Headnote

ARTICLE INFO

Keywords:

Gastric cancer

miR-361-3p

miRNA

Expression

ABSTRACT

Background: Gastric cancer (GC) remains a leading cause of cancer-related deaths globally. Even with advancements in treatment, it is not known precisely which molecular pathways cause GC to proceed. MicroRNAs, often referred to as small noncoding RNAs, have a crucial role in regulating gene expression, which impacts cancer growth, metastasis, and treatment resistance. Among these, miR-361-3p has drawn interest due to its possible role in the development of human cancer. While accumulating evidence highlights miR-361-3p involvement in various cancers, its precise biological function in gastric cancer remains largely unclear.

Methodology: The expression levels of miR-361-3p in gastric cancer tissues were compared with those of adjacent non-cancer tissues using real-time quantitative PCR (qRT-PCR). The investigation involved a thorough examination of the expression of miR-361-3p in GC samples in order to identify any possible correlations with clinicopathological characteristics.

Results: In 64.86 % of gastric cancer, expression levels of miR-361-3p mRNA were significantly reduced, which indicates that it may play a potential role in the pathophysiology of GC. However, no significant association has been found between miR-361-3p expression levels and clinical pathological features, such as tumor size, phase, or involvement of lymph nodes. This suggests that although miR-361-3p may contribute to GC progress, it is not related to traditional clinical markers.

1. Introduction

With over a million new cases and over 659,853 fatalities recorded in 2022, gastric cancer (GC) is still a significant global health concern. It is the fifth most prevalent malignancy worldwide and the fifth largest contributor to cancer-related mortality." The underlying molecular pathways driving the progression of GC remain poorly understood, despite advances in diagnostic and therapeutic techniques. This highlights the critical need for new treatment approaches to improve patient outcomes.

MicroRNAs, which are short non-coding RNAs of 18-24 nucleotides, have become important regulators of gene expression in recent years. Numerous biological processes, such as cell division, proliferation, apoptosis, and carcinogenesis, are coordinated by these molecules." By attaching to complementary sequences on target messenger RNAs (mRNAs), miRNAs mostly function as negative regulators by causing mRNA degradation or translation inhibition. Since miRNAs are essential for maintaining cellular homeostasis, they have been linked to the etiology of a number of malignancies, including stomach cancer. By modifying the expression of mRNAs that encode either tumor suppressors or oncogenes, dysregulation of miRNAs can aid in the formation of tumors."

Several investigations have shown that the expression of particular miRNAs differs between adjacent non-cancerous tissues and stomach cancer tissues. The observed variation in expression implies that miRNAs possess considerable promise as therapeutic targets for gastric cancer." Of all the miRNAs that have been researched, miR-361-3p has lately come to light due to its newly discovered function in controlling carcinogenesis and cancer metastasis.® Although miR-361-3p has been demonstrated to be essential in a number of human malignancies," its exact significance in the context of gastric cancer is yet unknown.

The objective of this work is to examine the expression patterns and biological relevance of miR-361-3p in gastric cancer, in light of the mounting evidence of miR-361-3p involvement in pathways linked to cancer. We aim to shed light on its role in gastric carcinogenesis in order to develop future therapeutic approaches that target miR-361-3p in the treatment of gastric cancer.

2. Methodology

2.1. Characteristics of the study subjects

The thirty-seven (n = 37) GC cases included in this study were recruited between October 2016 and December 2018. The SKIMS, Srinagar, India performed surgical resection in all cases, which were patients with histologically confirmed stomach cancer. The study did not include any individuals who underwent radiation therapy or chemotherapy. Age, sex, histology, or stage limits did not apply, however, GC patients who also had another cancer at the same time were not allowed to participate in the study.

2.2. Sample collection

Patients with histologically confirmed gastric cancer who provided informed consent underwent surgery in the General Surgery and Surgical Gastroenterology Department at SKIMS Tertiary Care Hospital. Both gastric cancer tissues and adjacent normal tissues were collected during the procedure. Following resection, the samples were immediately preserved in RNAlater to prevent RNA degradation. They were incubated overnight in RNAlater for optimal tissue preservation prior to being kept in storage until RNA extraction at -80 °C.

2.3. RNA extraction and complementary DNA synthesis

Using TRIzol (Invitrogen Waltham, MA), total RNA was isolated from tissues (n = 37) and quantified. The quality of isolated RNA was then examined. Following the manufacturer's instructions, Thermo Fisher Scientific Ltd (Waltham, MA) produced complementary DNA (cDNA) using a cDNA synthesis kit. Reverse transcription of 1-2 g of RNA is carried out in a final volume of 20 1 using the AMV reverse transcription enzyme and random hexamers. The cycle's conditions are 5 min at 25 °C, 60 min at 42 °C, and 5 min at 70 °C.

2.4. Quantitative real-time PCR for miR-361-3p

Using PikoReal Real-Time PCR equipment (Thermo Fisher Scientific Ltd.) and SYBR Green Master Mix, quantitative real-time PCR was carried out. Each sample was tested three times and GAPDH was used as an internal reference to normalize the expression values. The following primer sequences were used: Forward U6P: 5-ATTGG AACGA TACAG AGAAG ATT-3'; Reverse U6P: 5-GGAAC GCTTC ACGAA TTTG-3'; Forward miR-361-3p: 5-ATAAA GTGCTG ACAGT ССАСА TAGTG-3'; Reverse miR-361-3p: 5-TCAAG TACCC ACAGT GCGGT-3". The cycle threshold (Ct) defines miR-361-3p expressions. The 2-AACt method was used to calculate the relative expression levels." The reaction conditions for qRT-PCR were denaturation at 95 °C for 7 min, 40 cycles of denaturation (95 °C for 5 min), annealing (58 °C for 50 s), and extension (60 °C for 30 s). Melting curve analysis was also performed to check the specificity of the PCR product. 2 % agarose gel was used to check PCR products.

2.5. Statistical analysis

Using Chi-square/Fisher's exact test, categorical variables were analyzed, and additional odds ratios (OR) and 95 % confidence intervals (CI) were calculated. SPSS version 26 was used for statistical analysis.

3. Results

A total of 37 GC cases were included in the current research. There were 26 (70.27 %) men and 11 (29.72 %) women among the cases. Of the 37 subjects, 20 (54.05 %) were older than 60 years old, and 17 (45.94 %) were younger than 60, with a mean age of 62.81 + 7.60. Of the patients, 32 (86.48 %) were smokers and 5 (13.51 %) were non-smokers. Of the total, 34 (91.89 %) had stage III or IV disease, whereas 03 (8.10 %) had stage I or II disease. Patients in this study were also grouped according to the degree of distinction. Of the instances, 35 (94.59 %) were moderately/poorly differentiated, while 02 (5.40 %) were highly differentiated. According to the histopathological studies, 02 patients (5.40 %) did not develop lymph node metastasis, whereas 35 patients (94.59 %) did so. The clinicopathological aspects of these patients are fully described in Table 1.

3.1. mRNA expression of miR-361-3p in gastric cancer

Employing real-time qPCR, we examined the expression of miR-361P mRNA (n = 37) in GC tissues and the histologically verified adjacent normal tissues. MiR-361P mRNA expression in (n = 37) GC tissues and the surrounding normal samples was examined by qRT-PCR using certain primers. Comparing the miR-361P mRNA expression in the 37 GC cases to the adjacent normal tissues, 24 out of 37 cases (64.86 %) exhibited under expression.

In Fig. 1, box and whisker graphs representing the ACt values of miR-361-3p mRNA in the normal tissues and tumors of individuals with gastric cancer are displayed. Every experiment also included a melt curve analysis, which showed no non-specific product production at all. Age, gender, lymph node metastasis, staging, grading, and other clinicopathological features were all linked with the expression of miR-361-3p. No significant correlation was found between any of the investigated clinicopathological characteristics and our study (p > 0.05) according to Table 2.

4. Discussion

In our study, we observed that 64.86 % of cases exhibited decreased expression of miR-361-3p, indicating a potential tumor suppressor role for this microRNA. This finding is particularly noteworthy, as it highlights the possible involvement of miR-361-3p in the mechanisms underlying gastric cancer progression." Our results are consistent with those of Chen et al., who concluded that miR-361 inhibits prostate cancer cell invasion by targeting GLI1. This supports the notion that miR-361 may function as a tumor suppressor in various malignancies."

Furthermore, our findings are in concordance with a study conducted by Liu et al., which also identified a possible tumor suppressor gene role for miR-361-3p in the development and progression of prostate cancer. Liu et al.'s research further emphasizes the significance of miR-361-3p in cancer biology and its potential therapeutic implications. ' Similarly, in another investigation, Chen et al. found that miR-361-3p expression was significantly decreased in non-small cell lung cancer (NSCLC) tissues and cell lines, reinforcing the idea that this microRNA plays a critical role in suppressing tumor development."

Moreover, miR-361-3p has been implicated as a tumor-suppressive miRNA in colorectal carcinoma and gastric cancer, where it functions through direct binding to SND1, suggesting a multifaceted role for this microRNA family in different cancer types. · In a related context, Sun et al. discovered that miR-361-3p acts as a tumor suppressor in hepatocellular carcinoma, further solidifying the evidence for its role in cancer suppression across various tissues. ·

Interestingly, however, contrasting results have been reported by Wu et al., who identified miR-361-3p as an oncogene in the progression of cervical cancer, enhancing cell proliferation and invasion.'® This disparity raises important questions regarding the context-dependent roles of miR-361-3p, suggesting that its function may vary significantly depending on the specific cellular environment or tissue type.

While our study provides evidence for the tumor-suppressive role of miR-361-3p, it is essential to note that we observed no significant association between miR-361-3p expression and any relevant clinicopathological parameters. This lack of correlation may be influenced by the distinct population studied and the relatively small sample size of our research. These factors could contribute to variability in results and highlight the necessity for further investigations with larger cohorts to elucidate the precise role of miR361-3p in gastric cancer. Overall, our findings contribute to the growing body of literature on the significance of miR-36-3p in cancer biology, underscoring the need for continued research in this area.

5. Conclusion

Overall, the results indicate that miR-361-3p can act as a tumor suppressor and as a new therapeutic target for the treatment of GC patients. Therefore, further study with larger sample sizes and more techniques is needed to further validate our results.

CRediT authorship contribution statement

Kulsum Akhtar: Writing - original draft, Methodology. Gowhar Rashid: Writing - review & editing, Visualization, Data curation. Tahseen Bilal Rather: Visualization. Irfan Magbool: Visualization. Ishrat Parveiz: Visualization. Gulzar Ahmad Bhat: Writing - review & editing. Fazl Q. Parray: Supervision, Resources, Project administration. Syed Besina Yasin: Resources, Project administration. Syed Mudassar: Validation, Supervision, Software, Resources, Conceptualization.

Ethical approval

The study received ethical approval from the SKIMS Ethical Clearance Committee (Approval No. SIMS 1131/IEC-SKIMS/2017-18177).

Funding

This study was financially supported by an Intramural grant of SKIMS, Srinagar, India, under order no. SIMS/Acad/28 of 2017.

Conflict of interest statement

The authors declare that there are no conflicts of interest regarding the publication of this manuscript.

All authors have no financial, personal, or other relationships with individuals or organizations that could inappropriately influence (bias) the work presented in this study.

Acknowledgement

The authors are grateful to the Department of General Surgery and Surgical Gastroenterology's technical staff for their invaluable assistance with sample collection. The authors also extend their warmest regards to the research scientists of the Department of Multidisciplinary Research Unit, SKIMS, for their support through timely scientific inputs and assistance with manuscript writing.

Sidebar

Received 17 October 2024; Received in revised form 2 November 2024; Accepted 4 November 2024

Available online 5 November 2024

2543-1064/© 2024 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).

Footnote

* Corresponding author.

E-mail address: syed. [email protected] (5. Mudassar).

1 Equally contributed as Ist author.

References

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